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Sample records for calcitonin-gene-related peptide stimulates

  1. Calcitonin Gene-Related Peptide (CGRP)

    PubMed Central

    Russo, Andrew F.

    2015-01-01

    Migraine is a neurological disorder that manifests as a debilitating headache associated with altered sensory perception. The neuropeptide calcitonin gene-related peptide (CGRP) is now firmly established as a key player in migraine. Clinical trials carried out during the past decade have proved that CGRP receptor antagonists are effective for treating migraine, and antibodies to the receptor and CGRP are currently under investigation. Despite this progress in the clinical arena, the mechanisms by which CGRP triggers migraine remain uncertain. This review discusses mechanisms whereby CGRP enhances sensitivity to sensory input at multiple levels in both the periphery and central nervous system. Future studies on epistatic and epigenetic regulators of CGRP actions are expected to shed further light on CGRP actions in migraine. In conclusion, targeting CGRP represents an approachable therapeutic strategy for migraine. PMID:25340934

  2. Inflammatory mediators release calcitonin gene-related peptide from dorsal root ganglion neurons of the rat.

    PubMed

    Averbeck, B; Izydorczyk, I; Kress, M

    2000-01-01

    The interactions between the inflammatory mediators bradykinin, serotonin, prostaglandin E(2) and acid pH were studied in rat dorsal root ganglion neurons in culture. For this purpose, the cultures were stimulated by inflammatory mediators (bradykinin, serotonin, prostaglandin E(2), 10(-5)M each) or acid solution (pH 6.1) for 5 min and the content of calcitonin gene-related peptide was determined in the supernatant before, during and after stimulation, using an enzyme immunoassay. Acid solution resulted in a threefold increase of the basal calcitonin gene-related peptide release which was entirely dependent on the presence of extracellular calcium. The release could not be blocked by the addition of the capsaicin antagonist capsazepine (10(-5)M). Bradykinin (10(-5)M) caused a 50% increase of the basal calcitonin gene-related peptide release which was again dependent on the presence of extracellular calcium, whereas serotonin and prostaglandin E(2) were each ineffective at 10(-5)M concentration. The combination of bradykinin, serotonin and prostaglandin E(2) led to a fivefold increase of the calcitonin gene-related peptide release which could not be further enhanced by acidification. The competitive capsaicin receptor antagonist capsazepine (10(-5)M) significantly reduced the release induced by the combination of bradykinin, serotonin and prostaglandin E(2). It is suggested that the inflammatory mediators co-operate and together may act as endogenous agonists at the capsaicin receptor to cause calcium influx and consecutive neuropeptide release. PMID:10858619

  3. Calcitonin Gene-Related Peptide: Key Regulator of Cutaneous Immunity

    PubMed Central

    Granstein, Richard D.; Wagner, John A.; Stohl, Lori L.; Ding, Wanhong

    2014-01-01

    Calcitonin gene-related peptide (CGRP) has been viewed as a neuropeptide and vasodilator. However, CGRP is more appropriately thought of as a pleiotropic signaling molecule. Indeed, CGRP has key regulatory functions on immune and inflammatory processes within the skin. CGRP-containing nerves are intimately associated with epidermal LCs and CGRP has profound regulatory effects on Langerhans cell antigen-presenting capability. When LCs are exposed to CGRP in vitro, their ability to present antigen for in vivo priming of naïve mice or elicitation of delayed-type hypersensitivity is inhibited in at least some situations. Administration of CGRP intradermally inhibits acquisition of immunity to Th1-dominant haptens applied to the injected site while augmenting immunity to Th2-dominant haptens, although the cellular targets of activity in these experiments remains unclear. Although CGRP can be a pro-inflammatory agent, several studies have demonstrated that administration of CGRP can inhibit the elicitation of inflammation by inflammatory stimuli in vivo. In this regard, CGRP inhibits the release of certain chemokines by stimulated endothelial cells. This is likely to be physiologically relevant since cutaneous blood vessels are innervated by sensory nerves. Exciting new studies suggest a significant role for CGRP in the pathogenesis of psoriasis and, most strikingly, that CGRP inhibit the ability of LCs to transmit the human immunodeficiency virus 1 to T lymphocytes. A more complete understanding of the role of CGRP in the skin immune system may lead to new and novel approaches for the therapy of immune mediated skin disorders. PMID:25534428

  4. Calcitonin Gene-Related Peptide: Physiology and Pathophysiology

    PubMed Central

    Russell, F. A.; King, R.; Smillie, S.-J.; Kodji, X.; Brain, S. D.

    2014-01-01

    Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide. Discovered 30 years ago, it is produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP has two major forms (α and β). It belongs to a group of peptides that all act on an unusual receptor family. These receptors consist of calcitonin receptor-like receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for full functionality. CGRP is a highly potent vasodilator and, partly as a consequence, possesses protective mechanisms that are important for physiological and pathological conditions involving the cardiovascular system and wound healing. CGRP is primarily released from sensory nerves and thus is implicated in pain pathways. The proven ability of CGRP antagonists to alleviate migraine has been of most interest in terms of drug development, and knowledge to date concerning this potential therapeutic area is discussed. Other areas covered, where there is less information known on CGRP, include arthritis, skin conditions, diabetes, and obesity. It is concluded that CGRP is an important peptide in mammalian biology, but it is too early at present to know if new medicines for disease treatment will emerge from our knowledge concerning this molecule. PMID:25287861

  5. Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide

    PubMed Central

    Szklany, Kirsten; Ruiter, Evelyn; Mian, Firoz; Kunze, Wolfgang; Bienenstock, John; Forsythe, Paul; Karimi, Khalil

    2016-01-01

    The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body’s internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4+ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4+CD25+ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4+ T cell: neuron interaction through the release of neuropeptide CGRP. PMID:27022966

  6. Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis

    PubMed Central

    Sardi, Claudia; Zambusi, Laura; Finardi, Annamaria; Ruffini, Francesca; Tolun, Adviye A.; Dickerson, Ian M.; Righi, Marco; Zacchetti, Daniele; Grohovaz, Fabio; Provini, Luciano; Furlan, Roberto; Morara, Stefano

    2015-01-01

    Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null > heterozygote > wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing–remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocationof RCP. PMID:24746422

  7. A novel function of calcitonin gene-related peptide in body fluid Cl- homeostasis.

    PubMed

    Wang, Yi-Fang; Lafont, Anne-Gaëlle; Lee, Yi-Chun; Hwang, Pung-Pung

    2016-06-15

    Vertebrates need to maintain extracellular chloride (Cl(-)) concentrations to ensure the normal operation of physiological processes; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl(-) homeostasis in the face of fluctuating Cl(-) levels. Zebrafish calcitonin gene-related peptide (CGRP), unlike its splice variant calcitonin, does not respond to environmental Ca(2+) levels. This study aimed to test the hypothesis that CGRP is involved in the control of body fluid Cl(-) homeostasis. Acclimation to high-Cl(-) artificial water stimulated the mRNA expression of cgrp and the receptor (crlr1) when compared with low-Cl(-) CGRP knockdown induced upregulation of the Na(+)-Cl(-) co-transporter (ncc2b), while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl(-) uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC-expressing ionocytes. Elucidation of this novel function of CGRP in fish body fluid Cl(-) homeostasis promises to enhance our understanding of the related physiology in vertebrates. PMID:27306053

  8. Calcitonin gene-related peptide targeted immunotherapy for migraine: progress and challenges in treating headache.

    PubMed

    Peroutka, Stephen J

    2014-06-01

    A role for calcitonin gene-related peptide (CGRP) in the pathophysiology of migraine has been established over the past 25 years. There have now been at least five different small-molecule CGRP antagonists that have demonstrated statistical proof of efficacy in the acute treatment of migraine. At present, multiple clinical trials are underway that are assessing the ability of long-acting antibodies against CGRP to prevent frequent migraine attacks. This review summarizes the existing data concerning the role of CGRP in migraine and attempts to highlight some possible outcomes from the ongoing anti-CGRP antibody trials. PMID:24452707

  9. Distribution of calcitonin gene-related peptide in vertebrate neuromuscular junctions: relationship to the acetylcholine receptor.

    PubMed

    Csillik, B; Tajti, L; Kovács, T; Kukla, E; Rakic, P; Knyihár-Csillik, E

    1993-10-01

    Calcitonin gene-related peptide (CGRP), regarded by several authors to be involved in maintenance of the acetylcholine receptor, is present in the motor axons of various striated rat muscles. It is present, however, only in motor endplates of several selected striated muscles, where it is located in presynaptic axon terminals of neuromuscular junctions. No immunoreactivity could be seen within synaptic vesicles themselves. In the non-human primate Macaca fasciculata, neuromuscular junctions, including those in the diaphragm, display an intense CGRP reaction. The structure of the simian motor endplates is more elaborate than that of the rat. Amphibian motor nerve endings, both in tetanic and tonic muscles, display CGRP immunoreactivity. In tetanic muscles the CGRP reaction outlines "terminaisons en placque" (true motor end plates) and weakly reacting "terminaisons en grappe" (grape-like endings) in tonic muscles. On supramaximal stimulation of the motor nerve, CGRP is depleted from the affected neuromuscular junctions. Wallerian degeneration of the motor axon results in complete disappearance of CGRP. In most rat muscles in which motor endplates do not normally exhibit CGRP immunoreactivity, e.g., the diaphragm and buccinator muscles, the pre-terminal motor axons are CGRP-positive. After immobilization of such muscles by local bupivacaine injection to rats under brief chloral hydrate anesthesia, CGRP immunoreactivity of the neuromuscular junctions can be elicited because blockade of neuromuscular transmission results in accumulation of CGRP in the endplates. Even more striking is the appearance of CGRP immunoreactivity in normally non-reactive motor endplates during axon regeneration after an experimentally induced Wallerian degeneration of the motor axons. We conclude that CGRP is a regular, genotypically determined component of neuromuscular junctions, present either in a manifest or in a latent form. The latter can be elicited by various experimental approaches

  10. Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells

    PubMed Central

    Huang, Jing; Stohl, Lori L.; Zhou, Xi; Ding, Wanhong; Granstein, Richard D.

    2011-01-01

    This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP8-37 and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced IκBα degradation and NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-κB activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-κB pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation. PMID:21334428

  11. Calcitonin gene-related peptide immunoreactive (CGRP-IR) intradental nerves in the dog.

    PubMed

    Ngassapa, D; Narhi, M; Hirvonen, T; Markkula, I

    1998-03-01

    Calcitonin gene-related peptide (CGRP) is the most frequently occurring peptide in sensory neurons in the rat. Most of the Calcitonin Gene-Related Peptide Immunoreactive (CGRP-IP) nerves have been found to be Capsaicin-sensitive suggesting an involvement in certain types of pain. In the dental pulp CGRP-IR nerve fibres have been studied in the rat, guinea pig, cat, sheep, pig, cow and horse but not in the dog. Extensive sprouting of CGRP-IP intradental nerves has been demonstrated in the teeth with pulpal inflammation in rat molars. In the present investigation the occurrence and distribution of CGRP-IR intradental nerves both in the normal and the injured and inflamed teeth of the dog was studied. Immunolabelling was done by the Avidin-Biotin Complex (ABC) immunoperoxidase technique. CGRP-IR intradental nerves were demonstrated in the dog. The pattern of distribution was found to be similar to that in other animal species. However, the tip one third of the coronal pulp was sparsely innervated compared to that in the rat. Also much fewer fibres were seen to penetrate predentine and dentine, and this appears characteristic of the dog teeth. Sprouting phenomenon seen in the rat was not found in the dog teeth. It is suggested that there might be a species difference in the innervation pattern of CGRP-IR intradental nerves between the rat molar and the dog canine and incisor teeth. PMID:9640812

  12. Inhibition of Calcitonin Gene-Related Peptide Function: A Promising Strategy for Treating Migraine

    PubMed Central

    Durham, Paul L.

    2011-01-01

    The neuropeptide calcitonin gene-related peptide (CGRP) is implicated in the underlying pathology of migraine. Serum levels of CGRP, which are elevated during a migraine attack, have been reported to return to normal with alleviation of pain. In addition, CGRP administration has been shown to cause a migraine-like headache in susceptible individuals. Importantly, CGRP receptors are found on many cell types within the trigeminovascular system that are thought to play important roles in controlling inflammatory and nociceptive processes. Based on these findings, it was proposed that blockage of CGRP receptor function and, hence, the physiological effects of CGRP would be effective in aborting a migraine attack. This review will summarize key preclinical data that support the therapeutic potential of using CGRP receptor antagonists or molecules that bind CGRP within the context of current neurovascular theories on migraine pathology. PMID:18808507

  13. Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells.

    PubMed

    Malon, Jennifer T; Grlickova-Duzevik, Eliza; Vaughn, James; Beaulac, Holly; Vunk, Tyler R; Cao, Ling

    2015-02-15

    C57BL/6 (B6) mice develop peripheral neuropathy post-LP-BM5 infection, a murine model of HIV-1 infection, along with the up-regulation of select spinal cord cytokines. We investigated if calcitonin gene-related peptide (CGRP) contributed to the development of peripheral neuropathy by stimulating glial responses. An increased expression of lumbar spinal cord CGRP was observed in vivo, post-LP-BM5 infection. Consequently, in vitro CGRP co-treatments led to a microglial content-dependent attenuation of viral loads in spinal cord mixed glia infected with selected doses of LP-BM5. This inhibition was neither caused by the loss of glia nor induced via the direct inhibition of LP-BM5 by CGRP. PMID:25670002

  14. Peripheral amplification of sweating – a role for calcitonin gene-related peptide

    PubMed Central

    Schlereth, Tanja; Dittmar, Jan Oliver; Seewald, Bianca; Birklein, Frank

    2006-01-01

    Neuropeptides are the mediators of neurogenic inflammation. Some pain disorders, e.g. complex regional pain syndromes, are characterized by increased neurogenic inflammation and by exaggerated sudomotor function. The aim of this study was to explore whether neuropeptides have a peripheral effect on human sweating. We investigated the effects of different concentrations of calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and substance P (SP) on acetylcholine-induced axon reflex sweating in healthy subjects (total n = 18). All substances were applied via dermal microdialysis. The experiments were done in a parallel setting: ACh alone and ACh combined with CGRP, VIP or SP in various concentrations were applied. Acetylcholine (10−2m) always elicited a sweating response, neuropeptides alone did not. However, CGRP significantly enhanced ACh-induced sweating (P < 0.01). Post hoc tests revealed that CGRP in physiological concentrations of 10−7–10−9m was most effective. VIP at any concentration had no significant effect on axon reflex sweating. The duration of the sweating response (P < 0.01), but not the amount of sweat, was reduced by SP. ACh-induced skin blood flow was significantly increased by CGRP (P < 0.01), but unaltered by VIP and SP. The results indicate that CGRP amplifies axon reflex sweating in human skin. PMID:16931551

  15. Calcitonin gene-related peptide in migraine: intersection of peripheral inflammation and central modulation

    PubMed Central

    Raddant, Ann C.; Russo, Andrew F.

    2012-01-01

    Over the past two decades, a convergence of basic and clinical evidence has established the neuropeptide calcitonin-gene-related peptide (CGRP) as a key player in migraine. Although CGRP is a recognised neuromodulator of nociception, its mechanism of action in migraine remains elusive. In this review, we present evidence that led us to propose that CGRP is well poised to enhance neurotransmission in migraine by both peripheral and central mechanisms. In the periphery, it is thought that local release of CGRP from the nerve endings of meningeal nociceptors following their initial activation by cortical spreading depression is critical for the induction of vasodilation, plasma protein extravasation, neurogenic inflammation and the consequential sensitisation of meningeal nociceptors. Mechanistically, we propose that CGRP release can give rise to a positive-feedback loop involved in localised increased synthesis and release of CGRP from neurons and a CGRP-like peptide called procalcitonin from trigeminal ganglion glia. Within the brain, the wide distribution of CGRP and CGRP receptors provides numerous possible targets for CGRP to act as a neuromodulator. PMID:22123247

  16. Calcitonin gene-related peptide increases acetylcholine quantal size in neuromuscular junctions of mice.

    PubMed

    Gaydukov, Alexander E; Bogacheva, Polina O; Balezina, Olga P

    2016-08-15

    We used an intracellular microelectrode technique to study the mechanisms of action of two isoforms (human and rat) of calcitonin gene-related peptide (CGRP) on the evoked and spontaneous quantal secretion of acetylcholine (ACh) in mouse diaphragm motor synapses. Recordings of miniature endplate potentials (MEPPs) and evoked multiquantal endplate potentials (EPPs) in a cut neuromuscular preparation showed that CGRP increased the amplitude of EPPs without influencing their quantal content. Both isoforms of CGRP in a wide range of concentrations (1nM-1μM) provoked a similar considerable increase in MEPPs amplitude in a dose-dependent manner (up to 150-160% compared to control) without changing their frequency, rise-time, and decay. Inhibition of CGRP-receptors by truncated CGRP (CGRP8-37) completely prevented the potentiating effect of CGRP on the MEPPs amplitude. The effect of CGRP was not accompanied by changes in input resistance of muscle fiber membrane but was fully prevented by inhibition of vesicular ACh transport by vesamicol. Inhibition of protein kinase A (PKA) by H-89 also prevented CGRP action on the MEPPs amplitude. It is concluded that, in mammalian neuromuscular junctions, different isoforms of exogenously applied CGRP uniformly potentiate amplitudes of evoked and spontaneous postsynaptic potentials acting presynaptically via an increase in ACh quantal size. PMID:27288020

  17. Calcitonin gene-related peptide is a key neurotransmitter in the neuro-immune axis

    PubMed Central

    Assas, Bakri M.; Pennock, Joanne I.; Miyan, Jaleel A.

    2014-01-01

    The question of how the neural and immune systems interact in host defense is important, integrating a system that senses the whole body with one that protects. Understanding the mechanisms and routes of control could produce novel and powerful ways of promoting and enhancing normal functions as well as preventing or treating abnormal functions. Fragmentation of biological research into specialities has resulted in some failures in recognizing and understanding interactions across different systems and this is most striking across immunology, hematology, and neuroscience. This reductionist approach does not allow understanding of the in vivo orchestrated response generated through integration of all systems. However, many factors make the understanding of multisystem cross-talk in response to a threat difficult, for instance the nervous and immune systems share communication molecules and receptors for a wide range of physiological signals. But, it is clear that physical, hard-wired connections exist between the two systems, with the key link involving sensory, unmyelinated nerve fibers (c fibers) containing the neuropeptide calcitonin gene-related peptide (CGRP), and modified macrophages, mast cells and other immune and host defense cells in various locations throughout the body. In this review we will therefore focus on the induction of CGRP and its key role in the neuroimmune axis. PMID:24592205

  18. Structure–activity relationships for α-calcitonin gene-related peptide

    PubMed Central

    Watkins, Harriet A; Rathbone, Dan L; Barwell, James; Hay, Debbie L; Poyner, David R

    2013-01-01

    Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1–7); an α-helix (residues 8–18), a region incorporating a β-bend (residues 19–26) and the C-terminal portion (residues 27–37), that is characterized by bends between residues 28–30 and 33–34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. LINKED ARTICLES This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 PMID:23186257

  19. Adrenal responses to calcitonin gene-related peptide in conscious hypophysectomized calves.

    PubMed Central

    Bloom, S R; Edwards, A V; Jones, C T

    1989-01-01

    1. Right adrenal and various cardiovascular responses to an intra-aortic infusion of calcitonin gene-related peptide (CGRP; 4 micrograms min-1) have been investigated in the presence and absence of exogenous adrenocorticotrophin ACTH1-24 (2 or 5 ng min-1 kg-1, I.V.). The adrenal clamp technique was employed in conscious calves in which the pituitary stalk had been cauterized 3-7 days previously. 2. At the higher dose (5 ng min-1 kg-1) the I.V. infusion of ACTH raised mean plasma ACTH concentration by about 1000 pg ml-1 and mean right adrenal cortisol output by about 750 ng min-1 kg-1. Under these conditions the intra-aortic infusion of CGRP had no apparent effect on adrenal cortisol output by about 750 ng min-1 kg-1. Under these conditions the intra-aortic infusion of CGRP had no apparent effect on adrenal function, other than to produce moderate adrenal vasodilatation. In contrast, in the absence of exogenous ACTH, the same dose of CGRP produced a substantial rise in cortisol output, which rose steadily to a peak mean value of 409 +/- 31 pg min-1 kg-1 at 10 min. It also significantly inhibited the release of free, but not of total, met5-enkephalin-like immunoreactivity from the gland (P less than 0.001) together with a significantly greater fall in adrenal vascular resistance (P less than 0.001). 3. At the lower dose of ACTH (2 ng min-1 kg-1, I.V.) CGRP raised mean plasma cortisol output from 314 +/- 31 to 486 +/- 44 ng min-1 kg-1 (P less than 0.01) and this effect was not attributable to an increase in the adrenal presentation rate of ACTH. 4. It is concluded that this peptide exerts a steroidogenic action on the adrenal cortex which is manifest in the absence of exogenous ACTH in the functionally hypophysectomized calf. PMID:2555477

  20. Calcitonin/calcitonin gene-related peptide transcription unit: tissue-specific expression involves selective use of alternative polyadenylation sites.

    PubMed Central

    Amara, S G; Evans, R M; Rosenfeld, M G

    1984-01-01

    Different 3' coding exons in the rat calcitonin gene are used to generate distinct mRNAs encoding either the hormone calcitonin in thyroidal C-cells or a new neuropeptide referred to as calcitonin gene-related peptide in neuronal tissue, indicating the RNA processing regulation is one strategy used in tissue-specific regulation of gene expression in the brain. Although the two mRNAs use the same transcriptional initiation site and have identical 5' terminal sequences, their 3' termini are distinct. The polyadenylation sites for calcitonin and calcitonin gene-related peptide mRNAs are located at the end of the exons 4 and 6, respectively. Termination of transcription after the calcitonin exon does not dictate the production of calcitonin mRNA, because transcription proceeds through both calcitonin and calcitonin gene-related peptide exons irrespective of which mRNA is ultimately produced. In isolated nuclei, both polyadenylation sites appear to be utilized; however, the proximal (calcitonin) site is preferentially used in nuclei from tissues producing calcitonin mRNA. These data suggest that the mechanism dictating production of each mRNA involves the selective use of alternative polyadenylation sites. Images PMID:6334229

  1. Inotropic and lusitropic effects of calcitonin gene-related peptide in the heart

    PubMed Central

    Al-Rubaiee, Mustafa; Gangula, Pandu R.; Millis, Richard M.; Walker, Robin K.; Umoh, Nsini A.; Cousins, Valerie M.; Jeffress, Miara A.

    2013-01-01

    Previous studies have demonstrated positive-inotropic effects of calcitonin gene-related peptide (CGRP), but the mechanisms remain unclear. Therefore, two experiments were performed to determine the physiological correlates of the positive-inotropic effects of CGRP. Treatments designed to antagonize the effects of physiologically active CGRP1–37 included posttreatment with CGRP8–37 and pretreatment with LY-294002 (LY, an inhibitor of phosphatidylinositol 3-kinase), 17β-estradiol (E), and progesterone (P) were also used to modulate the effects of CGRP1–37. Experiment 1 was in vitro studies on sarcomeres and cells of isolated adult rat cardiac myocytes. CGRP1–37, alone and in combination with E and P, decreased sarcomere shortening velocities and increased shortening percentages, effects that were antagonized by CGRP8–37, but not by LY. CGRP1–37 increased resting intracellular calcium ion concentrations and Ca2+ influxes, effects that were also antagonized by both CGRP8–37 and LY. Experiment 2 was in vivo studies on left ventricular pressure-volume (PV) loops. CGRP1–37 increased end-systolic pressure, ejection fraction, and velocities of contraction and relaxation while decreasing stroke volume, cardiac output, stroke work, PV area, and compliance. After partial occlusion of the vena cava, CGRP1–37 increased the slope of the end-systolic PV relationship. CGRP8–37 and LY attenuated most of the CGRP-induced changes. These findings suggest that CGRP-induced positive-inotropic effects may be increased by treatments with estradiol and progesterone and inhibited by LY. The physiological correlates of CGRP-induced positive inotropy observed in rat sarcomeres, cells, and intact hearts are likely to reveal novel mechanisms of heart failure in humans. PMID:23585136

  2. Skeletal muscle microcirculatory response to rat alpha-calcitonin gene-related peptide.

    PubMed

    Arden, W A; Fiscus, R R; Beihn, L D; Derbin, M; Oremus, R; Gross, D R

    1994-07-01

    We used in vivo video microscopy to determine the effect of increasing doses of rat alpha-calcitonin gene-related peptide (rCGRP) on rat cremaster muscle arterioles in the presence or absence of the nitric oxide synthase inhibitor N-omega-nitro-L-arginine (L-NNA). Male Sprague-Dawley rats (118-148 g) were anaesthetized with pentobarbital, and neurovascularly intact cremaster muscles were imaged. Changes in the diameter, erythrocyte velocity and volume flow in second-(A2), third-(A3), and fourth-(A4) order arterioles were determined. To produce uniform arteriolar tone, the cremaster preparation was challenged with norepinephrine (NE: 10(-7) M). L-NNA (10(-4) M), which was shown to inhibit acetylcholine-(ACh: 10(-6) M) induced arteriolar dilations, was added to 16 of the preparations. Preparations were then challenged by adding cumulative log concentrations of rCGRP (10(-12)-10-7) M; n = 16) or an equivalent volume of vehicle (n = 19) to the bath. Following rCGRP challenge, arterioles were maximally dilated with 10(-5) M nitroprusside (NP). rCGRP caused significant dose-dependent increases in erythrocyte velocity and volume flow in A2 arterioles, and in diameter, velocity, and volume flow in A3 and A4 arterioles, by 10(-8) M, when compared with vehicle-treated controls. L-NNA had no significant effect on rCGRP-induced responses. These data indicate that rCGRP causes dose-dependent dilation of skeletal muscle resistance arterioles at a concentration similar to that observed in larger vessels. This dilation does not appear to be dependent on the vascular production of nitric oxide from L-arginine. PMID:7526261

  3. Endosomal proteolysis regulates calcitonin gene-related peptide responses in mesenteric arteries

    PubMed Central

    McNeish, AJ; Roux, BT; Aylett, S-B; Van Den Brink, AM; Cottrell, GS

    2012-01-01

    Background and Purpose Calcitonin gene-related peptide (CGRP) is a potent vasodilator, implicated in the pathogenesis of migraine. CGRP activates a receptor complex comprising, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). In vitro studies indicate recycling of CLR•RAMP1 is regulated by degradation of CGRP in early endosomes by endothelin-converting enzyme-1 (ECE-1). However, it is not known if ECE-1 regulates the resensitization of CGRP-induced responses in functional arterial tissue. Experimental Approach CLR, ECE-1a-d and RAMP1 expression in rat mesenteric artery smooth muscle cells (RMA-SMCs) and mesenteric arteries was analysed by RT-PCR and by immunofluorescence and confocal microscopy. CGRP-induced signalling in cells was examined by measuring cAMP production and ERK activation. CGRP-induced relaxation of arteries was measured by isometric wire myography. ECE-1 was inhibited using the specific inhibitor, SM-19712. Key Results RMA-SMCs and arteries contained mRNA for CLR, ECE-1a-d and RAMP1. ECE-1 was present in early endosomes of RMA-SMCs and in the smooth muscle layer of arteries. CGRP induced endothelium-independent relaxation of arteries. ECE-1 inhibition had no effect on initial CGRP-induced responses but reduced cAMP generation in RMA-SMCs and vasodilation in mesenteric arteries responses to subsequent CGRP challenges. Conclusions And Implications ECE-1 regulated the resensitization of responses to CGRP in RMA-SMCs and mesenteric arteries. CGRP-induced relaxation did not involve endothelium-derived pathways. This is the first report of ECE-1 regulating CGRP responses in SMCs and arteries. ECE-1 inhibitors may attenuate an important vasodilatory pathway, implicated in primary headaches and may represent a new therapeutic approach for the treatment of migraine. PMID:22881710

  4. Phospholipase C gamma mediates endogenous brain-derived neurotrophic factor-regulated calcitonin gene-related peptide expression in colitis-induced visceral pain

    PubMed Central

    Hashmi, Fiza; Liu, Miao; Shen, Shanwei

    2016-01-01

    Background Visceral hypersensitivity is a complex pathophysiological paradigm with unclear mechanisms. Primary afferent neuronal plasticity marked by alterations in neuroactive compounds such as calcitonin gene-related peptide is suggested to underlie the heightened sensory responses. Signal transduction that leads to calcitonin gene-related peptide expression thereby sensory neuroplasticity during colitis remains to be elucidated. Results In a rat model with colitis induced by 2,4,6-trinitrobenzene sulfonic acid, we found that endogenously elevated brain-derived neurotrophic factor elicited an up-regulation of calcitonin gene-related peptide in the lumbar L1 dorsal root ganglia. At seven days of colitis, neutralization of brain-derived neurotrophic factor with a specific brain-derived neurotrophic factor antibody reversed calcitonin gene-related peptide up-regulation in the dorsal root ganglia. Colitis-induced calcitonin gene-related peptide transcription was also inhibited by brain-derived neurotrophic factor antibody treatment. Signal transduction studies with dorsal root ganglia explants showed that brain-derived neurotrophic factor-induced calcitonin gene-related peptide expression was mediated by the phospholipase C gamma, but not the phosphatidylinositol 3-kinase/Akt or the mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. Application of PLC inhibitor U73122 in vivo confirmed that colitis-induced and brain-derived neurotrophic factor-mediated calcitonin gene-related peptide up-regulation in the dorsal root ganglia was regulated by the phospholipase C gamma pathway. In contrast, suppression of the phosphatidylinositol 3-kinase activity in vivo had no effect on colitis-induced calcitonin gene-related peptide expression. During colitis, calcitonin gene-related peptide also co-expressed with phospholipase C gamma but not with p-Akt. Calcitonin gene-related peptide up-regulation during colitis correlated to the activation

  5. Calcitonin Gene-Related Peptide Enhances Release of Native Brain-Derived Neurotrophic Factor from Trigeminal Ganglion Neurons

    PubMed Central

    Buldyrev, Ilya; Tanner, Nathan M.; Hsieh, Hui-ya; Dodd, Emily G.; Nguyen, Loi T.; Balkowiec, Agnieszka

    2008-01-01

    Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is co-expressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1–1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose–dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8–37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity. PMID:17064360

  6. Calcitonin gene-related peptide enhances release of native brain-derived neurotrophic factor from trigeminal ganglion neurons.

    PubMed

    Buldyrev, Ilya; Tanner, Nathan M; Hsieh, Hui-ya; Dodd, Emily G; Nguyen, Loi T; Balkowiec, Agnieszka

    2006-12-01

    Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is coexpressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1-1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose-dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8-37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity. PMID:17064360

  7. [The pharmacological mechanism of gastrodin on calcitonin gene-related peptide of cultured rat trigeminal ganglion].

    PubMed

    Luo, Guo-Gang; Fan, Wen-Jing; Yuan, Xing-Yun; Yuan, Bo-Bo; Lü, She-Min; Cao, Yong-Xiao; Xu, Cang-Bao

    2011-12-01

    The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1

  8. Calcitonin gene-related peptide activated ATP-sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.

    PubMed Central

    Quayle, J M; Bonev, A D; Brayden, J E; Nelson, M T

    1994-01-01

    1. Whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole-cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide-sensitive currents. CGRP increased the glibenclamide-sensitive currents by 3- to 6-fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide-sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current-voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp-cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide-sensitive K+ currents 2.1-, 3.3- and 8.2-fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide-sensitive K+ currents. 5. Dialysis of the cell's interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H-8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP-sensitive K+ channels. PMID:8189394

  9. Demonstration of the neurotransmitter role of calcitonin gene-related peptides (CGRP) by immunoblockade with anti-CGRP monoclonal antibodies.

    PubMed Central

    Tan, K. K.; Brown, M. J.; Longmore, J.; Plumpton, C.; Hill, R. G.

    1994-01-01

    1. Monoclonal antibodies (MAbs) against rat alpha-calcitonin gene-related peptide (alpha CGRP) were produced. Those which bound CGRP in a radioimmunoassay and inhibited the binding of 2-[125I]-iodohistidyl10-CGRP in a receptor binding assay were selected for immunoblockade experiments. 2. The effect of MAbs on CGRP inhibition of electrically stimulated contractions of the rat isolated vas deferens was characterized. Four out of 11 MAbs tested shifted the concentration-response curve of CGRP to the right compared with vehicle or irrelevant MAb control. MAb C4.19 produced equipotent blockade of rat alpha CGRP and rat beta CGRP and was chosen for further studies. MAb C4.19 had no pharmacologically significant effect on the concentration-response relationship of isoprenaline, rat beta-endorphin or somatostatin. 3. We demonstrated that the pharmacological response to CGRP in the presence of MAb C4.19 could be predicted when the dissociation constant and concentration of binding sites of the antibody were known. Comparison of experimental and computer simulated data showed good agreement for EC50 and maximum effect of CGRP in the presence of MAb C4.19. 4. Capsaicin at 1 microM inhibited the electrically stimulated contractions by 60.8% (95% confidence interval 51.8% to 69.9%). This effect was significantly attenuated by MAb C4.19 to 26.0% (95% confidence interval 15.2% to 36.8%; P < 0.003). 5. The immunoblockade of exogenous and endogenous CGRP described here, together with complementary evidence from other studies, strongly suggest that CGRP has a major neurotransmitter role at the neuroeffector junction of the rat vas deferens. PMID:7912623

  10. Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

    PubMed

    Machado, Juliano; Manfredi, Leandro H; Silveira, Wilian A; Gonçalves, Dawit A P; Lustrino, Danilo; Zanon, Neusa M; Kettelhut, Isis C; Navegantes, Luiz C

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 μg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation. PMID:26718975

  11. Pharmacologic Characterization of AMG 334, a Potent and Selective Human Monoclonal Antibody against the Calcitonin Gene-Related Peptide Receptor.

    PubMed

    Shi, Licheng; Lehto, Sonya G; Zhu, Dawn X D; Sun, Hong; Zhang, Jianhua; Smith, Brian P; Immke, David C; Wild, Kenneth D; Xu, Cen

    2016-01-01

    Therapeutic agents that block the calcitonin gene-related peptide (CGRP) signaling pathway are a highly anticipated and promising new drug class for migraine therapy, especially after reports that small-molecule CGRP-receptor antagonists are efficacious for both acute migraine treatment and migraine prevention. Using XenoMouse technology, we successfully generated AMG 334, a fully human monoclonal antibody against the CGRP receptor. Here we show that AMG 334 competes with [(125)I]-CGRP binding to the human CGRP receptor, with a Ki of 0.02 nM. AMG 334 fully inhibited CGRP-stimulated cAMP production with an IC50 of 2.3 nM in cell-based functional assays (human CGRP receptor) and was 5000-fold more selective for the CGRP receptor than other human calcitonin family receptors, including adrenomedullin, calcitonin, and amylin receptors. The potency of AMG 334 at the cynomolgus monkey (cyno) CGRP receptor was similar to that at the human receptor, with an IC50 of 5.7 nM, but its potency at dog, rabbit, and rat receptors was significantly reduced (>5000-fold). Therefore, in vivo target coverage of AMG 334 was assessed in cynos using the capsaicin-induced increase in dermal blood flow model. AMG 334 dose-dependently prevented capsaicin-induced increases in dermal blood flow on days 2 and 4 postdosing. These results indicate AMG 334 is a potent, selective, full antagonist of the CGRP receptor and show in vivo dose-dependent target coverage in cynos. AMG 334 is currently in clinical development for the prevention of migraine. PMID:26559125

  12. Effects of Voluntary Locomotion and Calcitonin Gene-Related Peptide on the Dynamics of Single Dural Vessels in Awake Mice

    PubMed Central

    Gao, Yu-Rong

    2016-01-01

    The dura mater is a vascularized membrane surrounding the brain and is heavily innervated by sensory nerves. Our knowledge of the dural vasculature has been limited to pathological conditions, such as headaches, but little is known about the dural blood flow regulation during behavior. To better understand the dynamics of dural vessels during behavior, we used two-photon laser scanning microscopy (2PLSM) to measure the diameter changes of single dural and pial vessels in the awake mouse during voluntary locomotion. Surprisingly, we found that voluntary locomotion drove the constriction of dural vessels, and the dynamics of these constrictions could be captured with a linear convolution model. Dural vessel constrictions did not mirror the large increases in intracranial pressure (ICP) during locomotion, indicating that dural vessel constriction was not caused passively by compression. To study how behaviorally driven dynamics of dural vessels might be altered in pathological states, we injected the vasodilator calcitonin gene-related peptide (CGRP), which induces headache in humans. CGRP dilated dural, but not pial, vessels and significantly reduced spontaneous locomotion but did not block locomotion-induced constrictions in dural vessels. Sumatriptan, a drug commonly used to treat headaches, blocked the vascular and behavioral the effects of CGRP. These findings suggest that, in the awake animal, the diameters of dural vessels are regulated dynamically during behavior and during drug-induced pathological states. SIGNIFICANT STATEMENT The vasculature of the dura has been implicated in the pathophysiology of headaches, but how individual dural vessels respond during behavior, both under normal conditions and after treatment with the headache-inducing peptide calcitonin gene-related peptide (CGRP), is poorly understood. To address these issues, we imaged individual dural vessels in awake mice and found that dural vessels constricted during voluntary locomotion, and

  13. Interaction of histamine and calcitonin gene-related peptide in the formalin induced pain perception in rats.

    PubMed

    Mobarakeh, Jalal Izadi; Torkaman-Boutorabi, Anahita; Rahimi, Amir Abbas; Ghasri, Shahrooz; Nezhad, Reza Mohammad Ali; Hamzely, Arash; Khoshkholgh Sima, Baharak; Takahashi, Kazuhiro; Nunoki, Kazuo; Yanai, Kazuhiko

    2011-06-01

    Histamine and calcitonin gene-related peptide (CGRP) contribute to the pain perception. The aim of the present study is to clarify the interaction of histamine and CGRP in the perception of inflammatory pain. The effects of a histamine H1 receptor antagonist (pyrilamine, i.p.), an H2 receptor antagonist (ranitidine, i.p.) and a CGRP antagonist (CGRP 8-37, i.t.) on the formalininduced pain was studied in rats. Pyrilamine and ranitidine produced a dose-dependent antinociceptive response in the first and the second phases of the formalin test. A single administration of pyrilamine (1 mg/kg, i.p.), ranitidine (10 mg/kg, i.p.) or CGRP 8-37 (10 µg/µL, i.t.) had no significant effects on the pain perception in the second phase. A combination of CGRP 8-37 and pyrilamine or ranitidine at these sub-effective doses, however, showed nociceptive response in the second phase. Moreover, a histamine (i.t.)-induced hyperalgesia was completely prevented by treatment with GGRP 8-37 at this dose. Our findings have raised the possibility that the CGRP system has interaction with histamine in the perception of inflammatory pain. PMID:21673449

  14. Studies on rat and human thymus to demonstrate immunoreactivity of calcitonin gene-related peptide, tyrosine hydroxylase and neuropeptide Y

    PubMed Central

    KRANZ, ANDREA; KENDALL, MARION D.; VON GAUDECKER, BRITA

    1997-01-01

    The peptidergic and noradrenergic innervation of rat and human thymus was investigated by immunohistochemistry at the light and electron microscopical level (avidin-biotin-complex, sucrose-phosphate-glyoxylic-acid, and immunogold techniques). The distribution of noradrenergic neural profiles, and positive immunoreactivity for calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY) is described in female rats during ageing, and in human children. In the neonatal rat thymus, the arteries and septa are well supplied by fine varicose nerves. In older animals (2 wk–1 y) the number of septa and blood vessels increase and consequently also the innervation. No nerves were found in the cortex. Apart from the innervation of the septal areas, immunoreactivity for CGRP and TH was present in thymic cells. Except for the young rats (neonatal–14 d), all rats showed CGRP positivity in subcapsular/perivascular epithelial cells (type 1 cells). All rat thymuses also contained a few TH positive cells in the medulla, which could only be confirmed as epithelial cells (type 6 cells) in children. Type 1 cells in the human thymus were not CGRP positive, but as in the rat, there were similar TH positive cells in the medulla. It was concluded that in addition to nerves containing CGRP, noradrenaline or dopamine, epithelial cells also contain these transmitters. They could therefore act on different cells (compared with neural targets) in a paracrine manner. PMID:9419001

  15. Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

    PubMed Central

    Yeakley, J M; Hedjran, F; Morfin, J P; Merillat, N; Rosenfeld, M G; Emeson, R B

    1993-01-01

    The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery. Images PMID:8413203

  16. Role of calcitonin gene-related peptide in cerebral vasospasm, and as a therapeutic approach to subarachnoid hemorrhage

    PubMed Central

    Kokkoris, Stelios; Andrews, Peter; Webb, David J.

    2012-01-01

    Calcitonin gene-related peptide (CGRP) is one of the most potent microvascular vasodilators identified to date. Vascular relaxation and vasodilation is mediated via activation of the CGRP receptor. This atypical receptor is made up of a G protein-coupled receptor called calcitonin receptor-like receptor (CLR), a single transmembrane protein called receptor activity-modifying protein (RAMP), and an additional protein that is required for Gas coupling, known as receptor component protein (RCP). Several mechanisms involved in CGRP-mediated relaxation have been identified. These include nitric oxide (NO)-dependent endothelium-dependent mechanisms or cAMP-mediated endothelium-independent pathways; the latter being more common. Subarachnoid hemorrhage (SAH) is associated with cerebral vasoconstriction that occurs several days after the hemorrhage and is often fatal. The vasospasm occurs in 30–40% of patients and is the major cause of death from this condition. The vasoconstriction is associated with a decrease in CGRP levels in nerves and an increase in CGRP levels in draining blood, suggesting that CGRP is released from nerves to oppose the vasoconstriction. This evidence has led to the concept that exogenous CGRP may be beneficial in a condition that has proven hard to treat. The present article reviews: (a) the pathophysiology of delayed ischemic neurologic deficit after SAH (b) the basics of the CGRP receptor structure, signal transduction, and vasodilatation mechanisms and (c) the studies that have been conducted so far using CGRP in both animals and humans with SAH. PMID:23162536

  17. Cells showing immunoreactivity for calcitonin or calcitonin gene-related peptide (CGRP) in the central nervous system of some invertebrates.

    PubMed

    Sasayama, Y; Katoh, A; Oguro, C; Kambegawa, A; Yoshizawa, H

    1991-09-01

    In the central nervous system of some species of several invertebrate phyla, including land planarians (Platyhelminthes), ribbon worms (Nemertina), slugs (Mollusca), polychaetes, earthworms and leeches (Annelida), pill bugs (Arthropoda), and beard worms (Pogonophora), salmon calcitonin-immunoreactive cells and rat calcitonin gene-related peptide (CGRP)-immunoreactive cells were found by immunohistochemistry. These immunoreactive cells were located in the region surrounding the neuropile, although the sizes of the cells varied according to species. Some of them were round or polygonal and regarded as apolar nerve cells because of their lack of cytoplasmic processes, whereas others were spindle-shaped or elongated, being comparable with unipolar nerve cells because of extension of their cytoplasmic processes in the direction of the neuropile. In some cases, it was noted that the cytoplasmic processes had complicated branches or formed loop-like structures at their ends. These observations suggest that a calcitonin-like or CGRP-like substance is extensively present in invertebrates as well as vertebrates. PMID:1936921

  18. Reductions in calcitonin gene-related peptide may be associated with the impairment of the contralateral testis in unilateral cryptorchidism

    PubMed Central

    ZHU, BAOPING; LIU, QING; LIN, LI; ZHENG, XINMIN

    2015-01-01

    The aim of the present study was to investigate the mechanism underlying the impairment of the contralateral testis in unilateral cryptorchidism in experimental rats using a molecular neurophysiological approach. Thirty-six male rats (21 days old) were divided into a cryptorchidism group, a cryptorchidism with division of the genitofemoral nerve (GFN) group and a control group (n=12/group). The distribution of the calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers in the testes was studied using an immunohistochemistry technique. Germ cell apoptosis was detected using the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling method. The concentration of malondialdehyde (MDA) in the testis tissue was evaluated using a spectrophotometric determination method, and the ultrastructure of Sertoli cells was observed using transmission electron microscopy. It was found that, 100 days after the surgery, the concentration of CGRP in the cryptorchidism group was decreased significantly, whereas the levels of MDA and the number of apoptotic germ cells were increased significantly compared with the control group (P<0.01). Following the division of the GFN, the damaging effects were decreased (P<0.01). The impairment mechanism may therefore be associated with a reduction in the level of CGRP in the contralateral testis. The reflex decrease in CGRP may be caused by germ cell apoptosis, decreased blood flow and oxygen levels, and the increase in reactive oxygen free radicals and lipid peroxidation. PMID:26136895

  19. Calcitonin Gene-Related Peptide Modulates Heat Nociception in the Human Brain - An fMRI Study in Healthy Volunteers

    PubMed Central

    Asghar, Mohammad Sohail; Becerra, Lino; Larsson, Henrik B. W.; Borsook, David; Ashina, Messoud

    2016-01-01

    Background Intravenous infusion of calcitonin-gene-related-peptide (CGRP) provokes headache and migraine in humans. Mechanisms underlying CGRP-induced headache are not fully clarified and it is unknown to what extent CGRP modulates nociceptive processing in the brain. To elucidate this we recorded blood-oxygenation-level-dependent (BOLD) signals in the brain by functional MRI after infusion of CGRP in a double-blind placebo-controlled crossover study of 27 healthy volunteers. BOLD-signals were recorded in response to noxious heat stimuli in the V1-area of the trigeminal nerve. In addition, we measured BOLD-signals after injection of sumatriptan (5-HT1B/1D antagonist). Results Brain activation to noxious heat stimuli following CGRP infusion compared to baseline resulted in increased BOLD-signal in insula and brainstem, and decreased BOLD-signal in the caudate nuclei, thalamus and cingulate cortex. Sumatriptan injection reversed these changes. Conclusion The changes in BOLD-signals in the brain after CGRP infusion suggests that systemic CGRP modulates nociceptive transmission in the trigeminal pain pathways in response to noxious heat stimuli. PMID:26990646

  20. Immunohistochemical localization of the calcitonin gene-related peptide binding site in the primate trigeminovascular system using functional antagonist antibodies.

    PubMed

    Miller, Silke; Liu, Hantao; Warfvinge, Karin; Shi, Licheng; Dovlatyan, Mary; Xu, Cen; Edvinsson, Lars

    2016-07-22

    Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology. PMID:27155150

  1. Nitric oxide synthase inhibitors can antagonize neurogenic and calcitonin gene-related peptide induced dilation of dural meningeal vessels

    PubMed Central

    Akerman, S; Williamson, D J; Kaube, H; Goadsby, P J

    2002-01-01

    The detailed pathophysiology of migraine is beginning to be understood and is likely to involve activation of trigeminovascular afferents. Clinically effective anti-migraine compounds are believed to have actions that include peripheral inhibition of calcitonin gene-related peptide (CGRP) release from trigeminal neurones, or preventing dural vessel dilation, or both. CGRP antagonists can block both neurogenic and CGRP-induced dural vessel dilation. Nitric oxide (NO) can induce headache in migraine patients and often triggers a delayed migraine. The initial headache is thought to be caused via a direct action of the NO–cGMP pathway that causes vasodilation by vascular smooth muscle relaxation, while the delayed headache is likely to be a result of triggering trigeminovascular activation. Nitric oxide synthase (NOS) inhibitors are effective in the treatment of acute migraine. The present studies used intravital microscopy to examine the effects of specific NOS inhibitors on neurogenic dural vasodilation (NDV) and CGRP-induced dilation. The non-specific and neuronal NOS (nNOS) inhibitors were able to partially inhibit NDV, while the non-specific and endothelial NOS (eNOS) inhibitors were able to partially inhibit the CGRP induced dilation. There was no effect of the inducible NOS (iNOS) inhibitor. The data suggest that the delayed headache response triggered by NO donors in humans may be due, in part, to increased nNOS activity in the trigeminal system that causes CGRP release and dural vessel dilation. Further, eNOS activity in the endothelium causes NO production and smooth muscle relaxation by direct activation of the NO–cGMP pathway, and may be involved in the initial headache response. PMID:12183331

  2. Flupirtine inhibits calcitonin-gene related peptide release from rat brainstem in vitro.

    PubMed

    Tringali, Giuseppe; Greco, Maria Cristina; Capuano, Alessandro; Guerriero, Giuseppe; Currò, Diego; Navarra, Pierluigi

    2012-01-11

    We have previously shown that the nonopioid analgesic flupirtine possesses analgesic activity in the orofacial formalin test in vivo in the rat. However, this paradigm does not allow to distinguish between central and peripheral site of action of the drug. In this study we used a recently characterized in vitro model, consisting in acute rat brainstem explants, to investigate whether flupirtine analgesia may be, at least in part, attributed to interference with neurotransmission between the first and the second order neurons of the trigeminal system, occurring within the brainstem. We used acute rat brainstem explants; CGRP released into the incubation medium was taken as a marker of CGRP release from central terminals of trigeminal ganglion afferent neurons within the brainstem. CGRP levels were measured by radioimmunoassay under basal conditions or in the presence of flupirtine, alone or with putative antagonist XE-991. We found that flupirtine inhibits in a concentration-dependent manner both basal and capsaicin-stimulated CGRP release from rat brainstem. This effect is mimicked by the flupirtine analogue retigabine, and is counteracted by the Kv7 blocker XE-991. These findings provide in vitro evidence that the analgesic activity of flupirtine may be related to interference with pain neurotransmission at the brainstem level. Pharmacological data suggests that such effect is related to opening of Kv7 channels on first-order neuronal nerve ending, and the subsequent inhibition of neurotransmitter release, since the effect is mimicked by the Kv7 opener retigabine and is counteracted by the Kv7 blocker XE-991. PMID:22155095

  3. Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

    PubMed

    Yang, Hai-Yu; Wu, Zhi-Yuan; Bian, Jin-Song

    2014-09-01

    Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways. PMID:24824948

  4. Involvement of calcitonin gene-related peptide and CCL2 production in CD40-mediated behavioral hypersensitivity in a model of neuropathic pain

    PubMed Central

    MALON, JENNIFER T.; MADDULA, SWATHI; BELL, HARMONY; CAO, LING

    2014-01-01

    The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a pro-nociceptive role after peripheral nerve injury upon its release from primary afferent neurons in preclinical models of neuropathic pain. We previously demonstrated a critical role for spinal cord microglial CD40 in the development of spinal nerve L5 transection (L5Tx)-induced mechanical hypersensitivity. Herein, we investigated whether CGRP is involved in the CD40-mediated behavioral hypersensitivity. First, L5Tx was found to significantly induce CGRP expression in wild-type (WT) mice up to 14 days post-L5Tx. This increase in CGRP expression was reduced in CD40 knockout (KO) mice at day 14 post-L5Tx. Intrathecal injection of the CGRP antagonist CGRP8–37 significantly blocked L5Tx-induced mechanical hypersensitivity. In vitro, CGRP induced glial IL-6 and CCL2 production, and CD40 stimulation added to the effects of CGRP in neonatal glia. Further, there was decreased CCL2 production in CD40 KO mice compared to WT mice 21 days post-L5Tx. However, CGRP8–37 did not significantly affect spinal cord CCL2 production following L5Tx in WT mice. Altogether, these data suggest that CD40 contributes to the maintenance of behavioral hypersensitivity following peripheral nerve injury in part through two distinct pathways, the enhancement of CGRP expression and spinal cord CCL2 production. PMID:22377050

  5. Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

    PubMed Central

    Banerjee, Sugato; Evanson, Janel; Harris, Erik; Lowe, Stephen L; Thomasson, Kathryn A; Porter, James E

    2006-01-01

    Background Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP. Results Two leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant

  6. Effect of vasoactive peptides in Tetrahymena: chemotactic activities of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP).

    PubMed

    Kőhidai, László; Tóth, Katalin; Samotik, Paul; Ranganathan, Kiran; Láng, Orsolya; Tóth, Miklós; Ruskoaho, Heikki

    2016-01-01

    Adrenomedullin (AMD), proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) were studied for chemotaxis, chemotactic selection and G-actin/F-actin transition in Tetrahymena. The aim of the experiments was to study the effects of two different peptides encoded by the same gene compared to a peptide related to one of the two, but encoded by a different gene, at a low level of phylogeny. The positive, chemotactic effect of ADM and the strong negative, chemorepellent effect of PAMP suggest that in Tetrahymena, the two peptides elicit their chemotactic effects via different signalling mechanisms. The complexity of swimming behaviour modulated by the three peptides underlines that chemotaxis, chemokinesis and some characteristics of migratory behaviour (velocity, tortuosity) are working as a sub-population level complex functional unit. Chemotactic responsiveness to ADM and CGRP is short-term, in contrast to PAMP, which as a chemorepellent ligand, has the ability to select sub-populations with negative chemotactic responsiveness. The different effects of ADM and PAMP on the polymerization of actin networks show that the microtubular structure of cilia is more essential to chemotactic response than are transitions of the actin network. The results draw attention to the characteristic effects of vasoactive peptides at this low level of phylogeny. PMID:26481478

  7. Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells

    PubMed Central

    ZHOU, RI; YUAN, ZHI; LIU, JIERONG; LIU, JIAN

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β-catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β-catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β-catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β-catenin pathway, including the mRNA of c-myc, cyclin D1, Lef1, Tcf7 and β-catenin as well as β-catenin protein. However, the upregulation of these genes and β-catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled-related protein, an antagonist of the Wnt/β-catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP- and LiCl-promoted osteoblastic differentiation of BMSCs. PMID:27082317

  8. Calcitonin gene-related peptide promotes the expression of osteoblastic genes and activates the WNT signal transduction pathway in bone marrow stromal stem cells.

    PubMed

    Zhou, Ri; Yuan, Zhi; Liu, Jierong; Liu, Jian

    2016-06-01

    Calcitonin gene-related peptide (CGRP) is known to induce osteoblastic differentiation and alkaline phosphatase activity in bone marrow stromal stem cells (BMSCs). However, it has remained elusive whether this effect is mediated by CGRP receptors directly or whether other signaling pathways are involved. The present study assessed the possible involvement of the Wnt/β‑catenin signaling pathway in the activation of CGRP signaling during the differentiation of BMSCs. First, the differentiation of BMSCs was induced in vitro and the expression of CGRP receptors was examined by western blot analysis. The effects of exogenous CGRP and LiCl, a stimulator of the Wnt/β‑catenin signaling pathway, on the osteoblastic differentiation of BMSCs were assessed; furthermore, the expression of mRNA and proteins involved in the Wnt/β‑catenin signaling pathway was assessed using quantitative PCR and western blot analyses. The results revealed that CGRP receptors were expressed throughout the differentiation of BMSCs, at days 7 and 14. Incubation with CGRP and LiCl led to the upregulation of the expression of osteoblastic genes associated with the Wnt/β‑catenin pathway, including the mRNA of c‑myc, cyclin D1, Lef1, Tcf7 and β‑catenin as well as β‑catenin protein. However, the upregulation of these genes and β‑catenin protein was inhibited by CGRP receptor antagonist or secreted frizzled‑related protein, an antagonist of the Wnt/β‑catenin pathway. The results of the present study therefore suggested that the Wnt/β-catenin signaling pathway may be involved in CGRP‑ and LiCl-promoted osteoblastic differentiation of BMSCs. PMID:27082317

  9. Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function.

    PubMed

    Qi, Tao; Christopoulos, George; Bailey, Richard J; Christopoulos, Arthur; Sexton, Patrick M; Hay, Debbie L

    2008-10-01

    Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to alphaCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology. PMID:18593822

  10. Impact of Food Components on in vitro Calcitonin Gene-Related Peptide Secretion—A Potential Mechanism for Dietary Influence on Migraine

    PubMed Central

    Slavin, Margaret; Bourguignon, Julia; Jackson, Kyle; Orciga, Michael-Angelo

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a pivotal messenger in the inflammatory process in migraine. Limited evidence indicates that diet impacts circulating levels of CGRP, suggesting that certain elements in the diet may influence migraine outcomes. Interruption of calcium signaling, a mechanism which can trigger CGRP release, has been suggested as one potential route by which exogenous food substances may impact CGRP secretion. The objective of this study was to investigate the effects of foods and a dietary supplement on two migraine-related mechanisms in vitro: CGRP secretion from neuroendocrine CA77 cells, and calcium uptake by differentiated PC12 cells. Ginger and grape pomace extracts were selected for their anecdotal connections to reducing or promoting migraine. S-petasin was selected as a suspected active constituent of butterbur extract, the migraine prophylactic dietary supplement. Results showed a statistically significant decrease in stimulated CGRP secretion from CA77 cells following treatment with ginger (0.2 mg dry ginger equivalent/mL) and two doses of grape pomace (0.25 and 1.0 mg dry pomace equivalent/mL) extracts. Relative to vehicle control, CGRP secretion decreased by 22%, 43%, and 87%, respectively. S-petasin at 1.0 μM also decreased CGRP secretion by 24%. Meanwhile, S-petasin and ginger extract showed inhibition of calcium influx, whereas grape pomace had no effect on calcium. These results suggest that grape pomace and ginger extracts, and S-petasin may have anti-inflammatory propensity by preventing CGRP release in migraine, although potentially by different mechanisms, which future studies may elucidate further. PMID:27376323

  11. Impact of Food Components on in vitro Calcitonin Gene-Related Peptide Secretion-A Potential Mechanism for Dietary Influence on Migraine.

    PubMed

    Slavin, Margaret; Bourguignon, Julia; Jackson, Kyle; Orciga, Michael-Angelo

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a pivotal messenger in the inflammatory process in migraine. Limited evidence indicates that diet impacts circulating levels of CGRP, suggesting that certain elements in the diet may influence migraine outcomes. Interruption of calcium signaling, a mechanism which can trigger CGRP release, has been suggested as one potential route by which exogenous food substances may impact CGRP secretion. The objective of this study was to investigate the effects of foods and a dietary supplement on two migraine-related mechanisms in vitro: CGRP secretion from neuroendocrine CA77 cells, and calcium uptake by differentiated PC12 cells. Ginger and grape pomace extracts were selected for their anecdotal connections to reducing or promoting migraine. S-petasin was selected as a suspected active constituent of butterbur extract, the migraine prophylactic dietary supplement. Results showed a statistically significant decrease in stimulated CGRP secretion from CA77 cells following treatment with ginger (0.2 mg dry ginger equivalent/mL) and two doses of grape pomace (0.25 and 1.0 mg dry pomace equivalent/mL) extracts. Relative to vehicle control, CGRP secretion decreased by 22%, 43%, and 87%, respectively. S-petasin at 1.0 μM also decreased CGRP secretion by 24%. Meanwhile, S-petasin and ginger extract showed inhibition of calcium influx, whereas grape pomace had no effect on calcium. These results suggest that grape pomace and ginger extracts, and S-petasin may have anti-inflammatory propensity by preventing CGRP release in migraine, although potentially by different mechanisms, which future studies may elucidate further. PMID:27376323

  12. Expression of calcitonin gene-related peptide, adenosine A2a receptor and adenosine A1 receptor in experiment rat migraine models

    PubMed Central

    LU, WENXIAN; LI, BIN; CHEN, JINBO; SU, YIPENG; DONG, XIAOMENG; SU, XINYANG; GAO, LIXIANG

    2016-01-01

    A migraine is a disabling neurovascular disorder characterized by a unilateral throbbing headache that lasts from 4 to 72 h. The headache is often accompanied by nausea, vomiting, phonophobia and photophobia, and may be worsened by physical exercise. The trigeminovascular system (TVS) is speculated to have an important role in migraines, although the pathophysiology of this disorder remains to be elucidated. Trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (TNC) are important components of the TVS. Several clinical cases have provided evidence for the involvement of the brainstem in migraine initiation. Electrical stimulation of the trigeminal ganglion (ESTG) in rats can activate TVS during a migraine attack. Calcitonin gene-related peptide (CGRP) is an important vasoactive compound produced following TVS activation. Numerous studies have revealed that adenosine and its receptors have an important role in pain transmission and regulation process. However, only a few studies have examined whether adenosine A2a receptor (A2aR) and adenosine A1 receptor (A1R) are involved in migraine and nociceptive pathways. In the present study, CGRP, A2aR and A1R expression levels were detected in the TG and TNC of ESTG models through reverse transcription-quantitative polymerase chain reaction and western blot analysis. Tianshu capsule (TSC), a type of Chinese medicine, was also used in the ESTG rat models to examine its influence on the three proteins. Results demonstrated that CGRP, A2aR and A1R mediated pain transmission and the regulation process during migraine and the expression of the three proteins was regulated by TSC. PMID:26998280

  13. Calcitonin gene-related peptide (CGRP), peptide YY (PYY) gastrin releasing peptide (GRP) and others in hamster lung and plasma

    SciTech Connect

    Ekman, R.; Keith, I.M.

    1986-03-05

    Rabbit antisera to CGRP, PYY, neuropeptide Y (NPY) and GRP were used for immunocytochemical localization of these peptides in lungs of neonate hamsters at birth and 6 d of age and young (70 gm) and adult (107 gm) hamsters. The peroxidase-antiperoxidase method was applied to paraffin sections of tissue fixed in Bouin's or Zamboni's solution. Furthermore, radioimmunoassay (RIA) was used to quantify these peptides in lung tissue and plasma from the young hamsters (n=13). Distinct CGRP-like immunoreactivity (IR) was noted in grouped (NEB) and individual (NEC) neuroendocrine cells at all ages including all airways from trachea (NECs only) to alveoli. In some NEBs this IR coexisted with 5-HT-like IR. PYY- and NPY-like Ir was mainly noted in NEBs and NECs at the level of bronchioles and alveoli, and weak GRP-like IR was present in neuroendocrine-like cells of small airways. Measurable quantities of all peptides were recorded by RIA. Females had higher lung and plasma levels of CGRP and plasma levels of PYY than males and tended to have higher lung levels of GRP. The neuropeptides CGRP, PYY and the analog NPY are putative regulators of local pulmonary blood flow by vasodilation (CGRP) and constriction (PYY, NPY), and GRP is known to regulate peptide release.

  14. Diverse Physiological Roles of Calcitonin Gene-Related Peptide in Migraine Pathology: Modulation of Neuronal-Glial-Immune Cells to Promote Peripheral and Central Sensitization.

    PubMed

    Durham, Paul L

    2016-08-01

    The neuropeptide calcitonin gene-related peptide (CGRP) is implicated in the underlying pathology of migraine by promoting the development of a sensitized state of primary and secondary nociceptive neurons. The ability of CGRP to initiate and maintain peripheral and central sensitization is mediated by modulation of neuronal, glial, and immune cells in the trigeminal nociceptive signaling pathway. There is accumulating evidence to support a key role of CGRP in promoting cross excitation within the trigeminal ganglion that may help to explain the high co-morbidity of migraine with rhinosinusitis and temporomandibular joint disorder. In addition, there is emerging evidence that CGRP facilitates and sustains a hyperresponsive neuronal state in migraineurs mediated by reported risk factors such as stress and anxiety. In this review, the significant role of CGRP as a modulator of the trigeminal system will be discussed to provide a better understanding of the underlying pathology associated with the migraine phenotype. PMID:27334137

  15. Calcitonin gene-related peptide contributes to peripheral nerve injury-induced mechanical hypersensitivity through CCL5 and p38 pathways.

    PubMed

    Malon, Jennifer T; Cao, Ling

    2016-08-15

    The role of calcitonin gene related peptide (CGRP) in neuropathic pain was investigated in a mouse model of neuropathic pain, spinal nerve L5 transection (L5Tx). Intrathecal injection (i.t.) of CGRP8-37, a CGRP antagonist, significantly reduced L5Tx-induced mechanical hypersensitivity and lumbar spinal cord CCL5 expression. i.t. injection of a CCL5 neutralizing antibody significantly inhibited L5Tx-induced mechanical hypersensitivity. Further, pre-treatment with a p38-inhibitor, SB203580, was able to reduce CGRP-induced mechanical hypersensitivity, but not CGRP-induced CCL5 production. Our data indicate that CGRP can play its pro-nociceptive role through both a spinal cord CCL5-dependent, p38-independent pathway, and a p38-depenented, CCL5-independent pathway. PMID:27397078

  16. Effects of rizatriptan on the expression of calcitonin gene-related peptide and cholecystokinin in the periaqueductal gray of a rat migraine model.

    PubMed

    Yao, Gang; Han, Ximei; Hao, Tingting; Huang, Qian; Yu, Tingmin

    2015-02-01

    Triptans are serotonin 5-hydroxytryptamine receptor 1B/D agonists that are highly effective in the treatment of migraine. We previously found that rizatriptan can reduce the expression of proenkephalin and P substance in the rat midbrain, suggesting that rizatriptan may exert its analgesic effects by influencing the endogenous pain modulatory system. Calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK) are mainly responsible for antagonizing the analgesic effects of opioid peptides in the endogenous pain modulatory system. In this study, we investigated the effects of rizatriptan on the expression of CGRP and CCK in the periaqueductal gray (PAG), a key structure of the endogenous pain modulatory system, in a rat migraine model induced by nitroglycerin. We found that the mRNA and protein levels of CGRP and CCK in the PAG of migraine rats were significantly increased compared to those in control rats, and these levels were significantly reduced upon treatment with rizatriptan in migraine rats (P<0.05). Our results suggest that the expression of CGRP and CCK in the endogenous pain modulatory system may be increased during migraine attacks, which further antagonizes the analgesic effects of endogenous opioid peptides and induces sustained migraine. Rizatriptan, however, significantly reduces the levels of CGRP and CCK to enhance the inhibition of pain signals via the endogenous pain modulatory system, resulting in effective treatment of migraine. PMID:25524408

  17. Calcitonin gene-related peptide erases the fear memory and facilitates long-term potentiation in the central nucleus of the amygdala in rats.

    PubMed

    Wu, Xin; Zhang, Jie-Ting; Liu, Jue; Yang, Si; Chen, Tao; Chen, Jian-Guo; Wang, Fang

    2015-11-01

    Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide, which plays a critical role in the central nervous system. CGRP binds to G protein-coupled receptors, including CGRP1, which couples positively to adenylyl cyclase (AC) and protein kinase A (PKA) activation. CGRP and CGRP1 receptors are enriched in central nucleus of the amygdala (CeA), the main part of the amygdala, which regulates conditioned fear memories. Here, we reported the importance of CGRP and CGRP1 receptor for synaptic plasticity in the CeA and the extinction of fear memory in rats. Our electrophysiological and behavioral in vitro and in vivo results showed exogenous application of CGRP induced an immediate and lasting long-term potentiation in the basolateral nucleus of amygdala-CeA pathway, but not in the lateral nucleus of amygdala-CeA pathway, while bilateral intra-CeA infusion CGRP (0, 5, 13 and 21 μM/side) dose dependently enhanced fear memory extinction. The effects were blocked by CGRP1 receptor antagonist (CGRP8-37 ), N-methyl-d-aspartate receptors antagonist MK801 and PKA inhibitor H89. These results demonstrate that CGRP can lead to long-term potentiation of basolateral nucleus of amygdala-CeA pathway through a PKA-dependent postsynaptic mechanism that involved N-methyl-d-aspartate receptors and enhance the extinction of fear memory in rats. Together, the results strongly support a pivotal role of CGRP in the synaptic plasticity of CeA and extinction of fear memory. Calcitonin gene-related peptide (CGRP) plays an essential role in synaptic plasticity in the amygdala and fear memory. We found that CGRP-induced chemical long-term potentiation (LTP) in a dose-dependent way in the BLA-CeA (basolateral and central nucleus of amygdala, respectively) pathway and enhanced fear memory extinction in rats through a protein kinase A (PKA)-dependent postsynaptic mechanism that involved NMDA receptors. These results support a pivotal role of CGRP in amygdala. PMID:26179152

  18. High arterial compliance in cirrhosis is related to low adrenaline and elevated circulating calcitonin gene related peptide but not to activated vasoconstrictor systems

    PubMed Central

    Henriksen, J; Moller, S; Schifter, S; Abrahamsen, J; Becker, U

    2001-01-01

    BACKGROUND AND AIMS—Static and dynamic functions of the wall of large arteries are largely unknown in cirrhosis in vivo. The present study was undertaken to determine arterial compliance (COMPart) in relation to vasodilator and vasoconstrictor systems in patients with cirrhosis. In addition, vasoactivity was manipulated by inhalation of oxygen.
STUDY POPULATION AND METHODS—In 20 patients with alcoholic cirrhosis and 12 controls we determined COMPart (stroke volume relative to pulse pressure), cardiac output, plasma volume, systemic vascular resistance, central circulation time, plasma catecholamines, renin activity, endothelin-1, and calcitonin gene related peptide (CGRP) at baseline and during oxygen inhalation.
RESULTS—COMPart was significantly increased in cirrhotic patients compared with controls (1.32 v 1.06 ml/mm Hg; p< 0.05) and inversely related to plasma adrenaline levels (r=−0.53; p<0.02) but positively related to circulating levels of CGRP (r=0.58; p<0.01). No significant relation was found for plasma noradrenaline, renin activity, or endothelin-1. COMPart was positively related to plasma volume (r=0.50; p<0.02) and inversely to systemic vascular resistance (r=−0.69; p<0.001) and central circulation time (r=−0.49; p<0.02). During oxygen inhalation, COMPart decreased (−13%; p<0.005) and systemic vascular resistance increased (+10%; p<0.001) towards normal values without significant changes in mean arterial pressure. Plasma adrenaline (−16%; p<0.01) decreased and the relation to COMPart disappeared. The relation of COMPart to CGRP and circulatory variables remained unchanged.
CONCLUSION—Elevated arterial compliance in cirrhosis is related to low adrenaline, high CGRP, and systemic hyperdynamics but not to indicators of the activated vasoconstrictor systems (noradrenaline, renin, endothelin-1). Thus the altered static and dynamic characteristics of the wall of large arteries are intimately associated with circulatory and

  19. Calcitonin gene-related peptide is a key factor in the homing of transplanted human MSCs to sites of spinal cord injury.

    PubMed

    Zhang, Yu; Yang, Jinhua; Zhang, Peng; Liu, Tao; Xu, Jianwei; Fan, Zhihai; Shen, Yixin; Li, Wenjie; Zhang, Huanxiang

    2016-01-01

    Mesenchymal stem cells (MSCs) can be used to treat many diseases, including spinal cord injury (SCI). Treatment relies mostly on the precise navigation of cells to the injury site for rebuilding the damaged spinal cord. However, the key factors guiding MSCs to the epicenter of SCI remain unknown. Here, we demonstrated that calcitonin gene-related peptide (CGRP), a neural peptide synthesized in spinal cord, can dramatically aid the homing of human umbilical cord mesenchymal stem cells (HUMSCs) in spinal cord-transected SCI rats. First, HUMSCs exhibited chemotactic responses in vitro to CGRP. By time-lapse video analysis, increased chemotactic index (CMI), forward migration index (FMI) and speed contributed to this observed migration. Then, through enzyme immunoassay, higher CGRP concentrations at the lesion site were observed after injury. The release of CGRP directed HUMSCs to the injury site, which was suppressed by CGRP 8-37, a CGRP antagonist. We also verified that the PI3K/Akt and p38MAPK signaling pathways played a critical role in the CGRP-induced chemotactic migration of HUMSCs. Collectively, our data reveal that CGRP is a key chemokine that helps HUMSCs migrate to the lesion site and thereby can be used as a model molecule to study MSCs homing after SCI. PMID:27296555

  20. Calcitonin gene-related peptide is a key factor in the homing of transplanted human MSCs to sites of spinal cord injury

    PubMed Central

    Zhang, Yu; Yang, Jinhua; Zhang, Peng; Liu, Tao; Xu, Jianwei; Fan, Zhihai; Shen, Yixin; Li, Wenjie; Zhang, Huanxiang

    2016-01-01

    Mesenchymal stem cells (MSCs) can be used to treat many diseases, including spinal cord injury (SCI). Treatment relies mostly on the precise navigation of cells to the injury site for rebuilding the damaged spinal cord. However, the key factors guiding MSCs to the epicenter of SCI remain unknown. Here, we demonstrated that calcitonin gene-related peptide (CGRP), a neural peptide synthesized in spinal cord, can dramatically aid the homing of human umbilical cord mesenchymal stem cells (HUMSCs) in spinal cord-transected SCI rats. First, HUMSCs exhibited chemotactic responses in vitro to CGRP. By time-lapse video analysis, increased chemotactic index (CMI), forward migration index (FMI) and speed contributed to this observed migration. Then, through enzyme immunoassay, higher CGRP concentrations at the lesion site were observed after injury. The release of CGRP directed HUMSCs to the injury site, which was suppressed by CGRP 8–37, a CGRP antagonist. We also verified that the PI3K/Akt and p38MAPK signaling pathways played a critical role in the CGRP-induced chemotactic migration of HUMSCs. Collectively, our data reveal that CGRP is a key chemokine that helps HUMSCs migrate to the lesion site and thereby can be used as a model molecule to study MSCs homing after SCI. PMID:27296555

  1. Lafutidine, a novel histamine H2-receptor antagonist, increases serum calcitonin gene-related peptide in rats after water immersion-restraint stress.

    PubMed

    Sato, Hiroshi; Kawashima, Kousaku; Yuki, Mika; Kazumori, Hideaki; Rumi, Mohammad Azharul Karim; Ortega-Cava, Cesar Francisco; Ishihara, Shunji; Kinoshita, Yoshikazu

    2003-02-01

    Lafutidine is a novel histamine H(2)-receptor antagonist with a potent and long-lasting anti-acid secretory effect that has also been found to have a potent gastroprotective effect. We investigated the effect of lafutidine on gastric mucosal injury induced in rats with the use of water-immersion restraint stress (WRS) by examining serum calcitonin gene-related peptide (CGRP) concentrations, which we measured with the use of an enzyme immunometric assay. WRS-induced mucosal erosive injury in the stomach was reduced significantly by both lafutidine and famotidine pretreatment (from 7.79 +/- 2.02 mm(2) to 3.09 +/- 0.74 mm(2) and 4.05 +/- 1.18 mm(2), respectively). A single administration of lafutidine or famotidine did not change the serum CGRP concentration from the control value when these drugs were administered without WRS. Lafutidine pretreatment before WRS caused a significant increase in serum CGRP concentration compared with famotidine (lafutidine, 86.64 +/- 9.52 pg/mL; famotidine, 47.55 +/- 4.35 pg/mL; control, 58.43 +/- 6.07 pg/mL). Our results suggest that lafutidine augments CGRP release from the rat stomach when administered before the induction of WRS. PMID:12577045

  2. [Suppressing effect of the serotonin 5HT1B/D receptor agonist rizatriptan on calcitonin gene-related peptide (CGRP) concentration in migraine attacks].

    PubMed

    Stepień, Adam; Jagustyn, Piotr; Trafny, Elzbieta Anna; Widerkiewicz, Krzysztof

    2003-01-01

    Calcitonin gene-related peptide (CGRP) is one of the neuropeptides most abundant in the nervous tissue. Recent studies indicate that local cranial release of CGRP from the trigeminal nerve perivascular endings within arachnoidea plays an important role in the pathophysiology of migraine attacks and cluster headaches. Elevated CGRP levels in cranial venous blood (in the jugular vein) during an acute spontaneous migraine attack have been reported in rather few studies so far. Sumatriptan--a selective serotonin 5HT1B/D receptor agonist, highly effective in terminating migraine attacks, decreases the elevated CGRP level back to normal. The aim of our study was to determine the effect of rizatriptan (a drug from a new generation of triptans) on CGRP release in migraine attacks. In 45 patients suffering from migraine attacks with and without aura, plasma CGRP levels were assessed during an attack twice: before treatment and two hours after rizatriptan administration. In the group under study the plasma CGRP level before treatment was significantly higher than that measured two hours after rizatriptan administration. The decrease in CGRP levels was associated with subsidence of the migraine attack. There was no difference between migraine patients with and without aura. These results suggest that triptans as serotonin 5HT1B/D receptor agonists decrease CGRP plasma concentration in migraine attacks. PMID:15174248

  3. Calcitonin gene-related peptide protects rats from cerebral ischemia/reperfusion injury via a mechanism of action in the MAPK pathway

    PubMed Central

    YANG, SI; YUAN, YONGJIE; JIAO, SHAN; LUO, QI; YU, JINLU

    2016-01-01

    The aim of the present study was to investigate the protective function and underlying mechanism of calcitonin gene-related peptide (CGRP) on cerebral ischemia/reperfusion damage in rats. Adult male Wistar rats were selected for the establishment of an ischemia/reperfusion injury model through the application of a middle cerebral artery occlusion. Animals were randomly divided into 6 groups of 24 animals. Drugs were administered according to the design of each group; animals were administered CGRP, CGRP8–37, PD98059 and SB20358. The neurobehavioral scores of the rat cerebral ischemia model in each group were calculated. The infarction range of the rat brain tissues was observed by 2,3,5-triphenyltetrazolium chloride staining. The expression levels of three proteins, phosphorylated c-Jun N-terminal kinase (JNK)/JNK, phosphorylated extracellular signal-regulated protein kinase (ERK)/ERK and p-p38/p38, in the mitogen-activated protein kinase (MAPK) pathway in the brain tissues was detected by western blotting. The results showed that CGRP could improve the neurobehavioral function of the ischemic rats and reduce the infarction range. Western blotting results confirmed that the function of the CGRP was mediated mainly through the reduction of the JNK and p38 phosphorylation and the promotion of ERK phosphorylation. Therefore, the present study confirmed that an increase in the exogenous CRGP could effectively improve ischemia/reperfusion injury of the brain tissue. The mechanisms of action were achieved through a reduction in JNK and p38 phosphorylation and an increase in ERL phosphorylation in the MAPK pathway. These mechanisms were interdependent. PMID:27284409

  4. Calcitonin gene-related peptide cooperates with substance P to inhibit melanogenesis and induces apoptosis of B16F10 cells.

    PubMed

    Zhou, Jia; Feng, Jun-Yi; Wang, Qian; Shang, Jing

    2015-07-01

    Skin is the largest organ in human body and works as biologically active barrier to provide critical preservation of body homeostasis. The skin is highly innervated by a plenitude of nerve fiber subpopulations, each carrying one or more neuronal mediators. Melanocyte itself also intimately contact with nerve fibers to form 'synaptic-like structure' and its functions may be directly regulated by the mediators contained in terminals of intra-epidermal nerve fibers. Clinical and biochemical studies have suggested that calcitonin gene-related peptide (CGRP) is involved in vitiligo skin. The present study was designed to investigate the effect of CGRP on epidermal melanocytes. After treatment with CGRP ranging from 0 to 500 ng/mL for 48 h, tyrosinase activity and melanogenesis were with little changes compared to treatment with medium only in B16F10 cells. Treatment with 500 ng/mL of CGRP cooperates with substance P (SP) (0.1-10 nM) to decrease tyrosinase activity and decrease melanin biosynthesis in B16F10 cells in a concentration-dependent manner. Furthermore, CGRP (8-37) antagonizes the synergistic effect of CGRP. The effect of CGRP on the cell apoptosis was examined. Treatments with 0-500 ng/mL of CGRP for 24 h, the expression levels of cleaved caspase-3, total caspase-3, cleaved caspase-9 and total caspase-9 were increased in a concentration-dependent manner. And 500 ng/mL of CGRP induced B16F10 cell apoptosis showed by TUNEL assay. In addition, Bax expression was up-regulated and Bcl-2 down-regulated in response to CGRP treatment. Hence, the Bax/Bcl-2 ratio was significantly increased. These in vitro observations indicate the pro-apoptotic impact of CGRP on B16F10 cell. PMID:25982845

  5. Exogenous asymmetric dimethylarginine (ADMA) in pathogenesis of ischemia-reperfusion-induced gastric lesions: interaction with protective nitric oxide (NO) and calcitonin gene-related peptide (CGRP).

    PubMed

    Magierowski, Marcin; Jasnos, Katarzyna; Sliwowski, Zbigniew; Surmiak, Marcin; Krzysiek-Maczka, Gracjana; Ptak-Belowska, Agata; Kwiecien, Slawomir; Brzozowski, Tomasz

    2014-01-01

    Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide (NO) synthesis inhibitor and pro-inflammatory factor. We investigated the role of ADMA in rat gastric mucosa compromised through 30 min of gastric ischemia (I) and 3 h of reperfusion (R). These I/R animals were pretreated with ADMA with or without the combination of L-arginine, calcitonin gene-related peptide (CGRP) or a small dose of capsaicin, all of which are known to afford protection against gastric lesions, or with a farnesoid X receptor (FXR) agonist, GW 4064, to increase the metabolism of ADMA. In the second series, ADMA was administered to capsaicin-denervated rats. The area of gastric damage was measured with planimetry, gastric blood flow (GBF) was determined by H2-gas clearance, and plasma ADMA and CGRP levels were determined using ELISA and RIA. ADMA significantly increased I/R-induced gastric injury while significantly decreasing GBF, the luminal NO content, and the plasma level of CGRP. This effect of ADMA was significantly attenuated by pretreatment with CGRP, L-arginine, capsaicin, or a PGE2 analogue. In GW4064 pretreated animals, the I/R injury was significantly reduced and this effect was abolished by co-treatment with ADMA. I/R damage potentiated by ADMA was exacerbated in capsaicin-denervated animals with a further reduction of CGRP. Plasma levels of IL-10 were significantly decreased while malonylodialdehyde (MDA) and plasma TNF-α contents were significantly increased by ADMA. In conclusion, ADMA aggravates I/R-induced gastric lesions due to a decrease of GBF, which is mediated by a fall in NO and CGRP release, and the enhancement of lipid peroxidation and its pro-inflammatory properties. PMID:24658439

  6. Rapid nitric oxide- and prostaglandin-dependent release of calcitonin gene-related peptide (CGRP) triggered by endotoxin in rat mesenteric arterial bed.

    PubMed Central

    Wang, X.; Wu, Z.; Tang, Y.; Fiscus, R. R.; Han, C.

    1996-01-01

    1. Our objective was to determine whether endotoxin (ETX) could directly trigger the release of calcitonin gene-related peptide (CGRP) from perivascular sensory nerves in the isolated mesenteric arterial bed (MAB) of the rat and to determine whether nitric oxide (NO) and prostaglandins (PGs) are involved. 2. ETX caused time- and concentration-dependent release of CGRP, and as much as a 17 fold increase in CGRP levels in the perfusate at 10-15 min after the administration of ETX (50 micrograms ml-1). 3. CGRP-like immunoreactivity in the perfusate was shown to co-elute with synthetic rat CGRP by reverse-phase h.p.l.c. 4. Pretreatment of MAB with capsaicin or ruthenium red inhibited ETX-induced CGRP release by 90% and 71%, respectively. ETX-evoked CGRP release was decreased by 84% during Ca2(+)-free perfusion. 5. The release of CGRP evoked by ETX was enhanced by L-arginine by 43% and inhibited by N omega-nitro-L-arginine (L-NOARG) and methylene blue by 37% and 38%, respectively. L-Arginine reversed the effect of L-NOARG. 6. Indomethacin and ibuprofen also inhibited the ETX-induced CGRP release by 34% and 44%, respectively. No additive inhibition could be found when L-NOARG and indomethacin were concomitantly incubated. 7. The data suggest that ETX triggers the release of CGRP from capsaicin-sensitive sensory nerves innervating blood vessels. The ETX-induced CGRP release is dependent on extracellular Ca2+ influx and involves a ruthenium red-sensitive mechanism. Both NO and PGs appear to be involved in the ETX-induced release of CGRP in the rat mesenteric arterial bed. PMID:8864557

  7. Structure–activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation

    PubMed Central

    Hay, Debbie L; Harris, Paul WR; Kowalczyk, Renata; Brimble, Margaret A; Rathbone, Dan L; Barwell, James; Conner, Alex C; Poyner, David R

    2014-01-01

    Background and Purpose: The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1–7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3–6 and 8–9, excluding Cys-2 and Cys-7. Experimental Approach: CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key Results: Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and Implications: Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1–7 ring also contribute to agonist activity. PMID:24125506

  8. Expression and function of calcitonin gene-related peptide (CGRP) receptors in trigeminal ganglia of R192Q Cacna1a knock-in mice.

    PubMed

    Vilotti, Sandra; Vana, Natascha; Van den Maagdenberg, Arn M J M; Nistri, Andrea

    2016-05-01

    Migraine is a neurovascular brain disorder suggested to be due to dysfunction of the trigeminovascular system with sensitization of trigeminal ganglion (TG) nociceptors. Since the neuropeptide calcitonin gene-related peptide (CGRP) has been established as a key player in the pathogenesis of migraine, CGRP receptor antagonists have been considered useful compounds to block headache originating from hyperactivation of such TG neurons. Whereas there is some information on the expression of CGRP receptors in postmortem human tissue, data are lacking for migraineurs suffering from common or genetic migraine. To help to clarify these issues it is very useful to study a transgenic knock-in (KI) mouse model of hemiplegic migraine expressing a R192Q missense mutation in the α1 subunit of CaV2.1 calcium channels previously found in patients with familial hemiplegic migraine type-1 (FHM-1). The aim of the present study, therefore, was to compare CGRP receptor expression and function in wildtype (WT) versus KI mouse TG. The principal components of the CGRP receptor, namely the CLR and RAMP-1 proteins, were similarly expressed in WT and KI TG neurons (in situ or in culture) and responded to exogenous CGRP with a strong rise in cAMP concentration. Hence, the previously reported phenotype of sensitization of KI TG neurons is not due to up-regulation of CGRP receptors but is likely caused by a constitutively larger release of CGRP. This observation implies that, in FHM-1 TG, normal TG sensory neuron signaling can be restored once the extracellular concentration of CGRP returns to control level with targeted treatment. PMID:27021026

  9. Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

    PubMed Central

    Vohra, Shabana; Taddese, Bruck; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.; Barwell, James; Reeves, Philip J.; Upton, Graham J. G.; Reynolds, Christopher A.

    2013-01-01

    Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg2.39, His2.43 and Glu3.46, which makes a polar lock with T6.37. These alignments and models provide useful tools for understanding class B GPCR function. PMID:23235263

  10. Calcitonin Gene-Related Peptide-Exposed Endothelial Cells Bias Antigen Presentation to CD4+ T Cells toward a Th17 Response.

    PubMed

    Ding, Wanhong; Stohl, Lori L; Xu, Linghui; Zhou, Xi K; Manni, Michela; Wagner, John A; Granstein, Richard D

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with well-established immunomodulatory functions. CGRP-containing nerves innervate dermal blood vessels and lymph nodes. We examined whether CGRP regulates the outcome of Ag presentation by Langerhans cells (LCs) to T cells through actions on microvascular endothelial cells (ECs). Exposure of primary murine dermal microvascular ECs (pDMECs) to CGRP followed by coculture with LCs, responsive CD4(+) T cells and Ag resulted in increased production of IL-6 and IL-17A accompanied by inhibition of IFN-γ, IL-4, and IL-22 compared with wells containing pDMECs treated with medium alone. Physical contact between ECs and LCs or T cells was not required for this effect and, except for IL-4, we demonstrated that IL-6 production by CGRP-treated pDMECs was involved in these effects. CD4(+) cells expressing cytoplasmic IL-17A were increased, whereas cells expressing cytoplasmic IFN-γ or IL-4 were decreased by the presence of CGRP-treated pDMECs. In addition, the level of retinoic acid receptor-related orphan receptor γt mRNA was significantly increased, whereas T-bet and GATA3 expression was inhibited. Immunization at the site of intradermally administered CGRP led to a similar bias in CD4(+) T cells from draining lymph node cells toward IL-17A and away from IFN-γ. Actions of nerve-derived CGRP on ECs may have important regulatory effects on the outcome of Ag presentation with consequences for the expression of inflammatory skin disorders involving Th17 cells. PMID:26829986

  11. Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats

    PubMed Central

    Mimaki, Yuichi; Kawasaki, Hiromu; Okazaki, Masatoshi; Nakatsuma, Akira; Araki, Hiroaki; Gomita, Yutaka

    1998-01-01

    The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin gene-related peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats.The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94±0.22 and 43.9±5.2, respectively.This vasodilator response to insulin was unaffected by 100 nM propranolol (β-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 μM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 μM ouabain (Na+-K+ ATPase inhibitor), or 1 μM glibenclamide (ATP sensitive K+-channel inhibitor).In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62±0.21 and 81.0±4.6, respectively, significantly greater than in preparations with intact endothelium.The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8–37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation.These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors. PMID:9605576

  12. Decreased calcitonin gene-related peptide expression in the dorsal root ganglia of TNF-deficient mice in a monoiodoacetate-induced knee osteoarthritis model

    PubMed Central

    Taniguchi, Aya; Ishikawa, Tetsuhiro; Miyagi, Masayuki; Kamoda, Hiroto; Sakuma, Yoshihiro; Oikawa, Yasuhiro; Kubota, Go; Inage, Kazuhide; Sainoh, Takeshi; Nakamura, Junichi; Aoki, Yasuchika; Toyone, Tomoaki; Inoue, Gen; Suzuki, Miyako; Yamauchi, Kazuyo; Suzuki, Takane; Takahashi, Kazuhisa; Ohtori, Seiji; Orita, Sumihisa

    2015-01-01

    Background: The detailed mechanisms of knee osteoarthritis (OA) pain have not been clarified, but involvement of inflammatory cytokines such as tumor necrosis factor-alpha (TNF) has been suggested. The present study aimed to investigate the more detailed neurological involvement of TNF in joint pain using a TNF-knockout mouse OA model. Methods: The right knees of twelve-week-old C57BL/6J wild and TNF-deficient knockout (TNF-ko) mice (n=15, each group) were given a single intra-articular injection of 10 µg monoiodoacetate in 10 mL sterile saline. The left knees were only punctured as the control. Evaluations were performed immediately after the injection (baseline) and at 7, 14, and 28 days after the injection with a subsequent intra-articular injection of neurotracer into both knees. The animals were evaluated for immunofluorescence of the lumbar dorsal root ganglia (DRG) innervating the knee joints. The injected knees were observed macroscopically and mouse pain-related behaviors were scored. Results: Macroscopic observation showed similar knee OA development in both wild and TNF-ko mice. Calcitonin gene-related peptide (CGRP, a neuropeptide identified as a inflammatory pain-related biomarker) was significantly increased in DRG neurons innervating OA-induced knee joints with significantly less CGRP expression in TNF-ko animals. Pain-related behavior scoring showed a significant increase in pain in OA-induced joints, but there was no significant difference in pain observed between the wild and TNF-ko mice. Conclusions: The result of the present study indicates the possible association of TNF-alpha in OA pain but not OA development. PMID:26722492

  13. Calcitonin gene-related peptide does not mediate the abnormal vascular reactivity observed in a rat model of acute Pseudomonas pneumonia.

    PubMed

    Fox, G A; Paterson, N A; McCormack, D G

    1996-06-01

    Abnormal systemic and pulmonary vascular reactivity has been demonstrated in numerous models of sepsis and pneumonia. Furthermore, the attenuated hypoxic pulmonary pressor response observed in these animals probably is responsible for the ventilation/perfusion (V/Q) mismatching and consequent arterial hypoxemia. We hypothesized that excess release of endogenous vasodilators such as calcitonin gene-related peptide (CGRP) in pneumonia was responsible for the diminished hypoxic pressor response. Using the CGRP receptor antagonist CGRP (8-37), we examined the role of CGRP in the attenuated hypoxic pulmonary response in a rat model of acute Pseudomonas pneumonia. Sixteen Sprague-Dawley rats were instrumented for chronic hemodynamic monitoring and subsequently randomized to either Pneumonia (n = 8), induced by the instillation of 0.2 ml broth containing 2 x 10(8) colony-forming units (CFU)/ml Pseudomonas aeruginosa into the right lower lobe, or Sham (n = 8) procedure. Hemodynamic measurements and the hypoxic (FiO2 = 0.08) pulmonary pressor response were recorded at baseline, 48 h after the pneumonia or sham procedure and after the administration of 250 micrograms CGRP (8-37) (post-CGRP(8-37)). The regional distribution of pulmonary blood flow was determined by the injection of radioactive microspheres. Forty-eight hours after the instillation of Pseudomonas, Pneumonia animals had significantly increased cardiac output (CO) as compared with Sham (193 +/- 7 vs. 154 +/- 7 ml/min, p < 0.05), slightly decreased mean arterial pressure (MAP 109 +/- 4 vs. 118 +/- 3 mm Hg, p = NS), and reduced total systemic vascular resistance (TSVR 0.57 +/- 0.03 vs. 0.78 +/- 0.05 mm Hg.min.ml-1, p < 0.05). Pneumonia animals were further characterized by increased mean pulmonary artery pressure (MPAP) as compared with Sham (24 +/- 2 vs. 20 +/- 1 mm Hg, p < 0.05) animals, and an increased alveolar-arterial (A-a) oxygen gradient (31 +/- 3 vs. 20 +/- 4 mm Hg, p < 0.05). The administration of CGRP

  14. The effect of 17β-estradiol on gene expression of calcitonin gene-related peptide and some pro-inflammatory mediators in peripheral blood mononuclear cells from patients with pure menstrual migraine

    PubMed Central

    Karkhaneh, Azam; Ansari, Mohammad; Emamgholipour, Solaleh; Rafiee, Mohammad Hessam

    2015-01-01

    Objective(s): The neuropeptide calcitonin gene-related peptide (CGRP) has long been postulated to play an integral role in the pathophysiology of migraine. Earlier studies showed that CGRP can stimulate the synthesis and release of nitric oxide (NO) and cytokines from trigeminal ganglion glial cells. The purpose of this study was to determine the effect of 17β-estradiol in regulation of CGRP expression, inducible nitric oxide synthase (iNOS) activity, and NO and interleukin-1beta (IL-1β) release in cultured peripheral blood mononuclear cells (PBMCs) from patients with pure menstrual migraine and healthy individuals. Materials and Methods: This study was performed on twelve patients with pure menstrual migraine and twelve age-and sex-matched healthy individuals. PBMCs treated with 17β-estradiol for 24 hr at physiological and pharmacological doses. Gene expression was evaluated by real time-PCR. CGRP and IL-1β proteins in culture supernatant were determined by ELISA method. Activity of iNOS in PBMCs and total nitrite in the culture supernatant were measured by colorimetric assays. Results: Treatment with 17β-estradiol had a biphasic effect on expression of CGRP. We found that 17β-estradiol treatment at pharmacological dose significantly increases mRNA expression of CGRP in both groups (P<0.001), whereas at physiological dose it could significantly decrease CGRP mRNA expression (P<0.001), CGRP protein levels, IL-1β release, NO production and iNOS activity only in patient groups (P<0.05). Conclusion: Collectively, it appears that 17β-estradiol can exert protective effect on decrease of inflammation in migraine via decrease in levels of CGRP, IL-1β and iNOS activity; however, more studies are necessary in this regard. PMID:26526225

  15. Comparison of the effects of salmon calcitonin (sCT) and calcitonin gene-related peptide (CGRP) in a number of in vivo and in vitro tests

    SciTech Connect

    Welch, S.P.; Brase, D.; Cooper, C.; Dewey, W.L.

    1986-03-05

    sCT and CGRP have been shown previously to have multiple activities in the central nervous system (CNS). Recent work has shown that CGRP (15 ..mu..g) intraventricularly (IVT) produces a naloxone reversible 37% inhibition in the p-phenylquinone test (PPQ) accompanied by severe diarrhea. The ED50 of sCT in the PPQ test is 362 ng and this effect is not reversed totally by naloxone. The onset of CGRP is more rapid than that of sCT. sCT and CGRP (10/sup -6/M) both produce naloxone reversible inhibition of the electrically stimulated guinea pig ileum (GPI) (25% and 50% respectively). Both sCT and CGRP (10/sup -6/ M) produce contracture (15% and 40% respectively) of the non-stimulated GPI that is not blocked by atropine. Both sCT and CGRP block the naloxone-induced contracture of the morphine (MS04) dependent ilea (29% and 68% respectively). Both sCT and CGRP produce biphasic shifts in the MS04 acetylcholine dose-effect curves in the stimulated and nonstimulated GPI, respectively. Neither sCT nor CGRP (10/sup -9/ to 10/sup -4/ M) displaces /sup 3/H-naloxone binding to mouse brain membranes. Both sCT and CGRP may produce their effects by modulation of CA/sup +2/ fluxes in the CNS and GPI.

  16. Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats.

    PubMed

    Mimaki, Y; Kawasaki, H; Okazaki, M; Nakatsuma, A; Araki, H; Gomita, Y

    1998-04-01

    1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors. PMID:9605576

  17. Correlation between algogenic effects of calcitonin-gene-related peptide (CGRP) and activation of trigeminal vascular system, in an in vivo experimental model of nitroglycerin-induced sensitization.

    PubMed

    Capuano, Alessandro; Greco, Maria Cristina; Navarra, Pierluigi; Tringali, Giuseppe

    2014-10-01

    The neural mechanism(s) underlying migraine remain poorly defined at present; preclinical and clinical studies show an involvement of CGRP in this disorder. However current evidence pointed out that CGRP does not exert an algogenic action per se, but it is able to mediate migraine pain only if the trigeminal-vascular system is sensitized. The present study was addressed to investigate CGRP-evoked behavior in nitric oxide (NO) sensitized rats, using an experimental model of nitroglycerin induced sensitization of trigeminal system, looking at neuropeptide release from different cerebral areas after the intra-peritoneal (i.p.) administration of NO-donors. CGRP injected into the rat whisker pad did not induce significant changes in face rubbing behavior compared to controls. On the contrary, CGRP injected in animals pre-treated with 10mg/kg nitroglycerin significantly increased the time spent in face rubbing. Nitroglycerin pre-treated animals did not show any rubbing behavior after locally injected saline. Furthermore, the i.p. treatment with nitroglycerin produced an increase of CGRP levels in brainstem and trigeminal ganglia, but not in the hypothalamus and hippocampus. The absolute amounts of CGRP produced in the brainstem were lower compared to those in the trigeminal ganglion; however, after nitroglycerin stimulation the percentage increase was higher in the brainstem. In conclusion, findings presented in this study suggest that CGRP induces a painful behavior in rats only after sensitization of trigeminal system; thus supporting the concept that a genetic as well as acquired predisposition to trigemino- vascular activation represents the neurobiological basis of CGRP nociceptive effects in migraineurs. PMID:24998872

  18. Discovery of (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-yl 4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxylate (BMS-927711): an oral calcitonin gene-related peptide (CGRP) antagonist in clinical trials for treating migraine.

    PubMed

    Luo, Guanglin; Chen, Ling; Conway, Charles M; Denton, Rex; Keavy, Deborah; Signor, Laura; Kostich, Walter; Lentz, Kimberley A; Santone, Kenneth S; Schartman, Richard; Browning, Marc; Tong, Gary; Houston, John G; Dubowchik, Gene M; Macor, John E

    2012-12-13

    Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated clinical efficacy in the treatment of acute migraine. Herein, we describe the design, synthesis, and preclinical characterization of a highly potent, oral CGRP receptor antagonist BMS-927711 (8). Compound 8 has good oral bioavailability in rat and cynomolgus monkey, attractive overall preclinical properties, and shows dose-dependent activity in a primate model of CGRP-induced facial blood flow. Compound 8 is presently in phase II clinical trials. PMID:23153230

  19. Negative pressure wound therapy-associated tissue trauma and pain: a controlled in vivo study comparing foam and gauze dressing removal by immunohistochemistry for substance P and calcitonin gene-related peptide in the wound edge.

    PubMed

    Malmsjö, Malin; Gustafsson, Lotta; Lindstedt, Sandra; Ingemansson, Richard

    2011-12-01

    Pain upon negative pressure wound therapy (NPWT) dressing removal has been reported and is believed to be associated with the observation that granulation tissue grows into foam. Wound tissue damage upon removal of the foam may cause the reported pain. Calcitonin gene-related peptide (CGRP) and substance P are neuropeptides that cause inflammation and signal pain and are known to be released when tissue trauma occurs. The aim of this controlled in vivo study was to compare the expression of CGRP and substance P in the wound bed in control wounds and following NPWT and foam or gauze dressing removal. Eight pigs with two wounds each were treated with open-pore structure polyurethane foam or AMD gauze and NPWT of 0 (control) or -80 mm Hg for 72 hours. Following removal of the wound filler, the expression of CGRP and substance P was measured, using arbitrary units, in sections of biopsies from the wound bed using immunofluorescence techniques. Substance P and CGRP were more abundant in the wound edge following the removal of foam than of gauze dressings and least abundant in control wounds. The immunofluorescence staining of the wound edge for CGRP was 52 ± 3 au after the removal of gauze and 97 ± 5 au after the removal of foam (P <0.001). For substance P, the staining was 55 ± 3 au after gauze removal and 95 ± 4 au after foam removal (P <0.001). CGRP and substance P staining was primarily located to nerves and leukocytes. The increase in CGRP and substance P immunofluorescence was especially prominent in the dermis but also was seen in subcutaneous and muscle tissue. Using gauze may be one way of reducing NPWT dressing change-related pain. New wound fillers designed to optimize granulation tissue formation and minimize pain issues presumably will be developed in the near future. PMID:22156176

  20. Structure of the mouse calcitonin/calcitonin gene-related peptide alpha and beta genes.

    PubMed

    Thomas, P M; Nasonkin, I; Zhang, H; Gagel, R F; Cote, G J

    2001-01-01

    We report the cloning, genomic organization and sequence of the mouse alpha-CALC and beta-CALC genes. The two genes share extensive sequence homology. The transcription units of both genes contain 6 exons. Transcripts of the alpha-CALC gene were found to alternatively include exon 4 or exons 5 and 6. For the beta-CALC gene exon 4 was not detected in transcripts derived from this gene. The predicted mouse alpha-CGRP was found to be identical to rat alpha-CGRP, however, beta-CGRP predicted amino acid sequences revealed three amino acid differences suggesting these residues are not critical to CGRP function. PMID:11761712

  1. The potential for prazosin and calcitonin gene-related peptide (CGRP) in causing hypoxia in tumours.

    PubMed Central

    Burney, I. A.; Maxwell, R. J.; Griffiths, J. R.; Field, S. B.

    1991-01-01

    Using 31P NMR spectroscopy, changes in tumour metabolic status were studied in a transplanted rat fibrosarcoma following the administration of vasodilators. Mean Arterial Blood Pressure (MABP) was monitored simultaneously. Two vasodilators were studied, prazosin and CGRP, which altered the NMR parameters Pi/sigma P, beta NTP,Pi, PCr/Pi and PME/Pi in a dose dependent manner. There was a good correlation between the various NMR parameters; for analysis, Pi/sigma P was used for convenience. With increasing doses of vasodilator, Pi/sigma P increased and the MABP decreased. Reduction in pHNMR showed a correlation with decreasing MABP following the administration of prazosin but not after CGRP. Both prazosin and CGRP produced changes in 31P NMR spectra consistent with a reduction in tumour blood flow. The results for prazosin and CGRP were comparable and showed a 15-20% increase in Pi/sigma P for a 20% reduction in MABP. These results were compared with those from hydralazine. With hydralazine an acceptable reduction in blood pressure (up to approximately 25%) has little effect and may even alter NMR parameters consistent with an increase in blood flow, a reduction of approximately 40% is required for a significant decrease in flow. Both prazosin and CGRP are shown to be far more effective than hydralazine in causing tumour hypoxia at a clinically acceptable reduction in blood pressure. CGRP may be the more suitable for clinical use because of its short half life, its capability to achieve controlled hypotension and the relatively few side effects associated with its use. PMID:1911217

  2. Indole-3-carbinol protects against cisplatin-induced acute nephrotoxicity: role of calcitonin gene-related peptide and insulin-like growth factor-1

    PubMed Central

    El-Naga, Reem N.; Mahran, Yasmen F.

    2016-01-01

    Nephrotoxicity associated with the clinical use of the anticancer drug cisplatin is a limiting problem. Thus, searching for new protective measures is required. Indole-3-carbinol is a powerful anti-oxidant, anti-inflammatory and anti-tumor agent. The present study aimed to investigate the potential protective effect of indole-3-carbinol against cisplatin-induced acute nephrotoxicity in rats. Rats were pre-treated with 20 mg/kg indole-3-carbinol orally before giving cisplatin (7 mg/kg). Cisplatin-induced acute nephrotoxicity was demonstrated where relative kidney weight, BUN and serum creatinine were significantly increased. Increased oxidative stress was evident in cisplatin group where GSH and SOD tissue levels were significantly depleted. Also, lipid peroxidation and NOX-1 were increased as compared to the control. Additionally, renal expression of pro-inflammatory mediators was induced by cisplatin. Cisplatin-induced cell death was shown by increased caspase-3 and decreased expression of EGF, IGF-1 and IGF-1 receptor. Nephrotoxicity, oxidative stress, inflammation and apoptotic effects induced by cisplatin were significantly ameliorated by indole-3-carbinol pre-treatment. Besides, the role of CGRP in cisplatin-induced nephrotoxicity was explored. Furthermore, cisplatin cytotoxic activity was significantly enhanced by indole-3-carbinol pre-treatment in vitro. In conclusion, indole-3-carbinol provides protection against cisplatin-induced nephrotoxicity. Also, reduced expression of CGRP may play a role in the pathogenesis of cisplatin-induced renal injury. PMID:27417335

  3. Indole-3-carbinol protects against cisplatin-induced acute nephrotoxicity: role of calcitonin gene-related peptide and insulin-like growth factor-1.

    PubMed

    El-Naga, Reem N; Mahran, Yasmen F

    2016-01-01

    Nephrotoxicity associated with the clinical use of the anticancer drug cisplatin is a limiting problem. Thus, searching for new protective measures is required. Indole-3-carbinol is a powerful anti-oxidant, anti-inflammatory and anti-tumor agent. The present study aimed to investigate the potential protective effect of indole-3-carbinol against cisplatin-induced acute nephrotoxicity in rats. Rats were pre-treated with 20 mg/kg indole-3-carbinol orally before giving cisplatin (7 mg/kg). Cisplatin-induced acute nephrotoxicity was demonstrated where relative kidney weight, BUN and serum creatinine were significantly increased. Increased oxidative stress was evident in cisplatin group where GSH and SOD tissue levels were significantly depleted. Also, lipid peroxidation and NOX-1 were increased as compared to the control. Additionally, renal expression of pro-inflammatory mediators was induced by cisplatin. Cisplatin-induced cell death was shown by increased caspase-3 and decreased expression of EGF, IGF-1 and IGF-1 receptor. Nephrotoxicity, oxidative stress, inflammation and apoptotic effects induced by cisplatin were significantly ameliorated by indole-3-carbinol pre-treatment. Besides, the role of CGRP in cisplatin-induced nephrotoxicity was explored. Furthermore, cisplatin cytotoxic activity was significantly enhanced by indole-3-carbinol pre-treatment in vitro. In conclusion, indole-3-carbinol provides protection against cisplatin-induced nephrotoxicity. Also, reduced expression of CGRP may play a role in the pathogenesis of cisplatin-induced renal injury. PMID:27417335

  4. Protective role of ellagitannins from Eucalyptus citriodora against ethanol-induced gastric ulcer in rats: impact on oxidative stress, inflammation and calcitonin-gene related peptide.

    PubMed

    Al-Sayed, Eman; El-Naga, Reem N

    2015-01-15

    The gastroprotective activity of an ellagitannin-rich fraction obtained from Eucalyptus citriodora (ECF) was investigated against ethanol-induced gastric ulceration in rats. The rats were pretreated with ECF (25, 50 and 100mg/kg) 1h before the administration of absolute ethanol to induce acute gastric ulceration. The gastric lesions were significantly reduced by all doses of ECF. Notably, pre-treatment with ECF (100mg/kg) conferred 99.6% gastroprotection, which is significantly higher than that produced by omeprazole. Moreover, ECF administration markedly increased the mucin content in a dose-dependent manner. The potent gastroprotective effect of ECF could be partly mediated by attenuating ethanol-induced oxidative stress. ECF-pre-treatment markedly increased the depleted GSH and SOD levels in a dose-dependent manner. Moreover, ECF significantly decreased the elevated MDA tissue levels induced by ethanol administration. The results demonstrated that ECF administration exerted a powerful anti-inflammatory activity as evidenced by the reduction in the pro-inflammatory markers; IL-1β, TNF-α, 5-LO and COX-2. Additionally, the caspase-3 tissue levels were significantly reduced in the groups pre-treated with ECF. These results suggest that ECF could exert a beneficial gastroprotective effect through their antioxidant, anti-inflammatory and anti-apoptotic properties. Furthermore, ECF pre-treatment significantly attenuated the ethanol-induced decrease in CGRP expression, which has a protective role against gastric ulceration. Histopathological examination revealed intact mucosal layer, absence of hemorrhage and necrosis in groups treated with ECF. Ellagitannins were identified as the major active constituents responsible for the marked antioxidant and gastroprotective properties of ECF. The HPLC-PDA-ESI/MS/MS technique was employed to identify the ellagitannins of E. citriodora. PMID:25636864

  5. Transcutaneous Electrical Nerve Stimulation (TENS) Improves the Diabetic Cytopathy (DCP) via Up-Regulation of CGRP and cAMP

    PubMed Central

    Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n = 15), DM group (n = 15) and control group (n = 15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG. PMID:23468996

  6. Transcutaneous electrical nerve stimulation (TENS) improves the diabetic cytopathy (DCP) via up-regulation of CGRP and cAMP.

    PubMed

    Ding, Liucheng; Song, Tao; Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n=15), DM group (n=15) and control group (n=15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG. PMID:23468996

  7. Inclusion of Cocoa as a Dietary Supplement Represses Expression of Inflammatory Proteins in Spinal Trigeminal Nucleus in Response to Chronic Trigeminal Nerve Stimulation

    PubMed Central

    Cady, Ryan J.; Denson, Jennifer E.; Durham, Paul L.

    2013-01-01

    Scope Central sensitization is implicated in the pathology of temporomandibular joint disorder (TMD) and other types of orofacial pain. We investigated the effects of dietary cocoa on expression of proteins involved in the development of central sensitization in the spinal trigeminal nucleus (STN) in response to inflammatory stimulation of trigeminal nerves. Methods and results Male Sprague Dawley rats were fed either a control diet or an isocaloric diet consisting of 10% cocoa powder 14 days prior to bilateral injection of complete Freund’s adjuvant (CFA) into the temporomandibular joint to promote prolonged activation of trigeminal ganglion neurons and glia. While dietary cocoa stimulated basal expression of GLAST and MKP-1 when compared to animals on a normal diet, cocoa suppressed basal calcitonin gene-related peptide levels in the STN. CFA-stimulated levels of protein kinase A, P2X3, P-p38, GFAP, and OX-42, whose elevated levels in the STN are implicated in central sensitization, were repressed to near control levels in animals on a cocoa enriched diet. Similarly, dietary cocoa repressed CFA-stimulated inflammatory cytokine expression. Conclusion Based on our findings, we speculate that cocoa enriched diets could be beneficial as a natural therapeutic option for TMD and other chronic orofacial pain conditions. PMID:23576361

  8. Local cooling alters neural mechanisms producing changes in peripheral blood flow by spinal cord stimulation.

    PubMed

    Tanaka, Satoshi; Barron, Kirk W; Chandler, Margaret J; Linderoth, Bengt; Foreman, Robert D

    2003-03-28

    This study was performed to investigate the respective role of sensory afferent and sympathetic fibers in peripheral vasodilatation induced by spinal cord stimulation at different hindpaw skin temperatures. Cooling the skin was used as a strategy to enhance sympathetic activity [Am. J. Physiol.: Heart Circ. Physiol. 263 (1992) H1197]. Cutaneous blood flow in the footpad of anesthetized rats was recorded using laser Doppler flowmetry. Local cooling (<25 degrees C) or moderate local cooling (25-28 degrees C) of the hindpaw was produced with a cooling copper coil. Spinal cord stimulation delivered at clinically relevant parameters and with 30%, 60%, and 90% of motor threshold induced the early phase of vasodilatation in the cooled and the moderately cooled hindpaw. In addition, spinal cord stimulation at 90% of motor threshold produced the late phase of vasodilatation only in the cooled hindpaw, which was possible to block by the autonomic ganglion-blocking agent, hexamethonium. The early responses to spinal cord stimulation in the moderately cooled hindpaw were not affected by hexamethonium. In contrast, both the early and the late phase responses were eliminated by CGRP (8-37), an antagonist of the calcitonin gene-related peptide receptor. After dorsal rhizotomy, spinal cord stimulation at 90% of motor threshold elicited hexamethonium-sensitive vasodilatation in the cooled hindpaw (late phase). These results suggest that spinal cord stimulation-induced vasodilatation in the cooled hindpaw (<25 degrees C) is mediated via both the sensory afferent (early phase of vasodilatation) and via suppression of the sympathetic efferent activity (late phase) although the threshold for vasodilatation via the sympathetic efferent fibers is higher than that via sensory nerves. In contrast, vasodilatation via sensory afferent fibers may predominate with moderate temperatures (25-28 degrees C). Thus, two complementary mechanisms for spinal cord stimulation-induced vasodilatation may

  9. The adrenal contribution to the neuroendocrine responses to splanchnic nerve stimulation in conscious calves.

    PubMed Central

    Bloom, S R; Edwards, A V; Jones, C T

    1988-01-01

    1. The extent to which the adrenal gland contributes to neuroendocrine responses to electrical stimulation of the peripheral end of the splanchnic nerve has been investigated in conscious calves in which the right nerve was stimulated either at 4 Hz continuously for 10 min or at 40 Hz in 1 s bursts at 10 s intervals for the same period. 2. It was confirmed that the release of neuropeptide Y (NPY) and of gastrin-releasing peptide (GRP) is potentiated by stimulation in bursts at a relatively high frequency and shown that the adrenal gland made a negligible contribution to these responses. 3. There was no detectable change in the concentration of vasoactive intestinal peptide (VIP) in the arterial plasma but the existence of a very small but highly significant rise in the output of VIP from the adrenal provided evidence that it was released within the gland in response to splanchnic nerve stimulation. 4. The concentration of calcitonin gene-related peptide (CGRP) in the arterial and adrenal venous effluent plasma was consistently below the level of detection of the assay. 5. Splanchnic nerve stimulation resulted in an abrupt rise in the output of both free and total met5-enkephalin-like immunoreactivity from the adrenal gland which was substantially potentiated by stimulating in bursts. This pattern of stimulation also increased the proportion released in a high-molecular-weight form. 6. Stimulation in bursts significantly enhanced the output of both adrenaline and noradrenaline from the adrenal and resulted in the release of proportionately more noradrenaline. Small amounts of dopamine and DOPAC were also released during splanchnic nerve stimulation and the output of dopamine was significantly increased by stimulating in bursts. 7. Both patterns of stimulation elicited an abrupt rise in mean plasma adrenocorticotrophic hormone (ACTH) concentration, which was associated with an increase in mean adrenal cortisol output and the former effect was significantly enhanced

  10. Excitation-induced cell damage and beta2-adrenoceptor agonist stimulated force recovery in rat skeletal muscle.

    PubMed

    Mikkelsen, Ulla Ramer; Gissel, Hanne; Fredsted, Anne; Clausen, Torben

    2006-02-01

    Intensive exercise leads to a loss of force, which may be long lasting and associated with muscle cell damage. To simulate this impairment and to develop means of compensating the loss of force, extensor digitorum longus muscles from 4-wk-old rats were fatigued using intermittent 40-Hz stimulation (10 s on, 30 s off). After stimulation, force recovery, cell membrane leakage, and membrane potential were followed for 240 min. The 30-60 min of stimulation reduced tetanic force to approximately 10% of the prefatigue level, followed by a spontaneous recovery to approximately 20% in 120-240 min. Loss of force was associated with a decrease in K+ content, gain of Na+ and Ca2+ content, leakage of the intracellular enzyme lactic acid dehydrogenase (10-fold increase), and depolarization (13 mV). Stimulation of the Na+-K+ pump with either the beta2-adrenoceptor agonist salbutamol, epinephrine, rat calcitonin gene-related peptide (rCGRP), or dibutyryl cAMP improved force recovery by 40-90%. The beta-blocker propranolol abolished the effect of epinephrine on force recovery but not that of CGRP. Both spontaneous and salbutamol-induced force recovery were prevented by ouabain. The salbutamol-induced force recovery was associated with repolarization of the membrane potential (12 mV) to the level measured in unfatigued muscles. In conclusion, in muscles exposed to fatiguing stimulation leading to a considerable loss of force, cell leakage, and depolarization, stimulation of the Na+-K+ pump induces repolarization and improves force recovery, possibly due to the electrogenic action of the Na+-K+ pump. This mechanism may be important for the restoration of muscle function after intense exercise. PMID:16210418

  11. Inefficient constitutive inhibition of P2X3 receptors by brain natriuretic peptide system contributes to sensitization of trigeminal sensory neurons in a genetic mouse model of familial hemiplegic migraine

    PubMed Central

    Marchenkova, Anna; Vilotti, Sandra; Ntamati, Niels; van den Maagdenberg, Arn MJM

    2016-01-01

    Background On trigeminal ganglion neurons, pain-sensing P2X3 receptors are constitutively inhibited by brain natriuretic peptide via its natriuretic peptide receptor-A. This inhibition is associated with increased P2X3 serine phosphorylation and receptor redistribution to non-lipid raft membrane compartments. The natriuretic peptide receptor-A antagonist anantin reverses these effects. We studied whether P2X3 inhibition is dysfunctional in a genetic familial hemiplegic migraine type-1 model produced by introduction of the human pathogenic R192Q missense mutation into the mouse CACNA1A gene (knock-in phenotype). This model faithfully replicates several properties of familial hemiplegic migraine type-1, with gain-of-function of CaV2.1 Ca2+ channels, raised levels of the algogenic peptide calcitonin gene-related peptide, and enhanced activity of P2X3 receptors in trigeminal ganglia. Results In knock-in neurons, anantin did not affect P2X3 receptor activity, membrane distribution, or serine phosphorylation level, implying ineffective inhibition by the constitutive brain natriuretic peptide/natriuretic peptide receptor-A pathway. However, expression and functional properties of this pathway remained intact together with its ability to downregulate TRPV1 channels. Reversing the familial hemiplegic migraine type-1 phenotype with the CaV2.1-specific antagonist, ω-agatoxin IVA restored P2X3 activity to wild-type level and enabled the potentiating effects of anantin again. After blocking calcitonin gene-related peptide receptors, P2X3 receptors exhibited wild-type properties and were again potentiated by anantin. Conclusions P2X3 receptors on mouse trigeminal ganglion neurons are subjected to contrasting modulation by inhibitory brain natriuretic peptide and facilitatory calcitonin gene-related peptide that both operate via complex intracellular signaling. In the familial hemiplegic migraine type-1 migraine model, the action of calcitonin gene-related peptide appears to

  12. GLP-1 receptor stimulation of the lateral parabrachial nucleus reduces food intake: neuroanatomical, electrophysiological, and behavioral evidence.

    PubMed

    Richard, Jennifer E; Farkas, Imre; Anesten, Fredrik; Anderberg, Rozita H; Dickson, Suzanne L; Gribble, Fiona M; Reimann, Frank; Jansson, John-Olov; Liposits, Zsolt; Skibicka, Karolina P

    2014-11-01

    The parabrachial nucleus (PBN) is a key nucleus for the regulation of feeding behavior. Inhibitory inputs from the hypothalamus to the PBN play a crucial role in the normal maintenance of feeding behavior, because their loss leads to starvation. Viscerosensory stimuli result in neuronal activation of the PBN. However, the origin and neurochemical identity of the excitatory neuronal input to the PBN remain largely unexplored. Here, we hypothesize that hindbrain glucagon-like peptide 1 (GLP-1) neurons provide excitatory inputs to the PBN, activation of which may lead to a reduction in feeding behavior. Our data, obtained from mice expressing the yellow fluorescent protein in GLP-1-producing neurons, revealed that hindbrain GLP-1-producing neurons project to the lateral PBN (lPBN). Stimulation of lPBN GLP-1 receptors (GLP-1Rs) reduced the intake of chow and palatable food and decreased body weight in rats. It also activated lPBN neurons, reflected by an increase in the number of c-Fos-positive cells in this region. Further support for an excitatory role of GLP-1 in the PBN is provided by electrophysiological studies showing a remarkable increase in firing of lPBN neurons after Exendin-4 application. We show that within the PBN, GLP-1R activation increased gene expression of 2 energy balance regulating peptides, calcitonin gene-related peptide (CGRP) and IL-6. Moreover, nearly 70% of the lPBN GLP-1 fibers innervated lPBN CGRP neurons. Direct intra-lPBN CGRP application resulted in anorexia. Collectively, our molecular, anatomical, electrophysiological, pharmacological, and behavioral data provide evidence for a functional role of the GLP-1R for feeding control in the PBN. PMID:25116706

  13. Peptide portions may hold key to amplifying bone against porosis

    SciTech Connect

    Cotton, P.

    1990-02-02

    Pieces of peptides that are encoded in the calcitonin gene may explain enigmas in treatment of bone disease. Amplification of bone formation by two peptides with similar amino acid sequences was reported at the Third International Conference on the Fundamentals of Bone Growth at the University of California, Los Angeles (UCLA), schools of medicine and dentistry. One treatment enigma is that calcitonin regulates normal bone resorption but does not work as well when administered for the treatment of osteoporosis. While hormone therapy does work, it has wide-ranging effects like the potential for an increased risk of breast cancer. Bone-growth promotion by a better-known peptide, calcitonin gene-related peptide (CGRP), was described. The CGRP is usually processed in the nervous system and has a wide range of activity.

  14. Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

    PubMed

    Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

    2015-08-01

    In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. PMID:26129676

  15. Helical synthetic peptides that stimulate cellular cholesterol efflux

    SciTech Connect

    Bielicki, John K.; Natarajan, Pradeep

    2010-04-06

    The present invention provides peptides comprising at least one amphipathic alpha helix and having an cholesterol mediating activity and a ABCA stabilization activity. The invention further provides methods of using such peptides.

  16. Starfish gonadotropic hormone: Relaxin-like gonad-stimulating peptides.

    PubMed

    Mita, Masatoshi

    2016-05-01

    Relaxin-like gonad-stimulating peptide (RGP) of starfish Patiria (= Asterina) pectinifera is the first identified invertebrate gonadotropin to trigger final gamete maturation. Recently, chemical structures of RGP were identified in several species of starfish. Three kinds of RGP molecules are found in the class Asteroidea. The chemical structure of P. pectinifera RGP (PpeRGP) is conserved among starfish of the order Valvatida beyond species. In contrast, the chemical structures of RGP identified in Asterias amurensis and Aphelasterias japonica of the order Forcipulatida are quite different from that of PpeRGP. The chemical structure of RGP in A. amurensis (AamRGP) is exactly the same as that in Asterias rubens (the order Forcipulatida), Astropecten scoparius (the order Paxillosida), Astropecten polyacanthus (the order Paxillosida), and Echinaster luzonicus (the order Spinulosida). The chemical structure of Coscinasterias acutispina RGP (the order Forcipulatida) is consistent with that of A. japonica RGP (AjaRGP). In cross-experiments using P. pectinifera, A. amurensis, and A. japonica ovaries, AamRGP and AjaRGP can induce each species of ovaries. Neither AamRGP nor AjaRGP induce oocyte maturation and ovulation in the ovary of P. pectinifera, although the PpeRGP is active in ovaries of A. amurensis and A. japonica. This suggests that the AamRGP and AjaRGP partly act species specificity. PMID:27102940

  17. Sensory nerves contribute to cutaneous vasodilator response to cathodal stimulation in healthy rats.

    PubMed

    Gohin, Stéphanie; Decorps, Johanna; Sigaudo-Roussel, Dominique; Fromy, Bérengère

    2015-09-01

    Cutaneous current-induced vasodilation (CIV) in response to galvanic current application is an integrative model of neurovascular interaction that relies on capsaicin-sensitive fiber activation. The upstream and downstream mechanisms related to the activation of the capsaicin-sensitive fibers involved in CIV are not elucidated. In particular, the activation of cutaneous transient receptor potential vanilloid type-1 (TRPV1) channels and/or acid-sensing ion channels (ASIC) (activators mechanisms) and the release of calcitonin gene-related peptide (CGRP) and substance P (SP) (effector mechanisms) have been tested. To assess cathodal CIV, we measured cutaneous blood flow using laser Doppler flowmetry for 20min following cathodal current application (240s, 100μA) on the skin of the thigh in anesthetized healthy rats for 20min. CIV was studied in rats treated with capsazepine and amiloride to inhibit TRPV1 and ASIC channels, respectively; CGRP8-37 and SR140333 to antagonize CGRP and neurokinin-1 (NK1) receptors, respectively; compared to their respective controls. Cathodal CIV was attenuated by capsazepine (12±2% vs 54±6%, P<0.001), amiloride (19±8% vs 61±6%, P<0.01), CGRP8-37 (15±6% vs 61±6%, P<0.001) and SR140333 (9±5% vs 54±6%, P<0.001) without changing local acidification. This is the first integrative study performed in healthy rats showing that cutaneous vasodilation in response to cathodal stimulation is initiated by activation of cutaneous TRPV1 and ASIC channels likely through local acidification. The involvement of CGRP and NK1 receptors suggests that cathodal CIV is the result of CGRP and SP released through activated capsaicin-sensitive fibers. Therefore cathodal CIV could be a valuable method to assess sensory neurovascular function in the skin, which would be particularly relevant to evaluate the presence of small nerve fiber disorders and the effectiveness of treatments. PMID:26205659

  18. CGRP may regulate bone metabolism through stimulating osteoblast differentiation and inhibiting osteoclast formation.

    PubMed

    He, Haitao; Chai, Jianshen; Zhang, Shengfu; Ding, Linlin; Yan, Peng; Du, Wenjun; Yang, Zhenzhou

    2016-05-01

    Calcitonin-gene-related peptide (CGRP) is a neuropeptide, which is widely distributed throughout the central and peripheral nervous systems. Numerous mechanisms underlying the action of CGRP in osteoblast-associated cells have been suggested for bone growth and metabolism. The present study was designed to closely investigate the osteoblast‑ and osteoclast-associated mechanisms of the effect of CGRP administration on bone metabolism in primary osteoblasts. Primary osteoblasts were obtained from newborn rabbit calvaria and incubated with different concentrations of human CGRP (hCGRP), hCGRP and hCGRP (8‑37), or without treatment as a control. Intracellular calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) were detected following treatment, as well as the expression levels of osteoblast differentiation markers, including activating transcription factor‑4 (ATF4) and osteocalcin (OC), and receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG). The isolated primary osteoblasts were found to stain positively for ALP. hCGRP treatment had no significant effect on transient intracellular Ca2+ in the osteoblasts. Treatment of the osteoblasts with hCGRP led to elevations in the expression levels of cAMP, ATF4 and OPG, and downregulation in the expression of RANKL, in a dose‑dependent manner. These effects were markedly reversed by the addition of hCGRP (8‑37). The results of the present study demonstrated that CGRP administration not only stimulated osteoblast differentiation, as demonstrated by upregulated expression levels of ATF4 and OC in the hCGRP‑treated osteoblasts, but also inhibited OPG/RANKL‑regulated osteoclastogenesis. CGRP may act as a modulator of bone metabolism through osteoblast and osteoclast-associated mechanisms, which result in osteoblast formation with subsequent activation of bone formation. PMID:27035229

  19. SPARC is a source of copper-binding peptides that stimulate angiogenesis.

    PubMed

    Lane, T F; Iruela-Arispe, M L; Johnson, R S; Sage, E H

    1994-05-01

    SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113-130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo. PMID:7514608

  20. SPARC is a source of copper-binding peptides that stimulate angiogenesis

    PubMed Central

    1994-01-01

    SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113- 130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo. PMID:7514608

  1. TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout.

    PubMed

    Trevisan, Gabriela; Hoffmeister, Carin; Rossato, Mateus Fortes; Oliveira, Sara Marchesan; Silva, Mariane Arnoldi; Silva, Cássia Regina; Fusi, Camilla; Tonello, Raquel; Minocci, Daiana; Guerra, Gustavo Petri; Materazzi, Serena; Nassini, Romina; Geppetti, Pierangelo; Ferreira, Juliano

    2014-07-01

    Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H2O2), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1(+/+)) or TRPA1-deficient (Trpa1(-/-)) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H2O2 generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H2O2 in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H2O2-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H2O2 mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H2O2 production as potential targets for treatment of acute gout attacks. PMID:24780252

  2. Atrial natriuretic peptide stimulates salt secretion by shark rectal gland by releasing VIP

    SciTech Connect

    Silva, P.; Stoff, J.S.; Solomon, R.J.; Lear, S.; Kniaz, D.; Greger, R.; Epstein, F.H.

    1987-01-01

    Salt secretion by the isolated perfused rectal gland of the spiny dogfish shark, Squalus acanthias, is stimulated by synthetic rat atrial natriuretic peptide (ANP II) as well as extracts of shark heart, but not by 8-bromo-cyclic guanosine 5'-monophosphate. Cardiac peptides have no effect on isolated rectal gland cells or perfused tubules, suggesting that stimulation requires an intact gland. The stimulation of secretion by ANP II is eliminated by maneuvers that block neurotransmitter release. Cardiac peptides stimulate the release of vasoactive intestinal peptide (VIP), known to be present in rectal glands nerves, into the venous effluent of perfused glands in parallel with their stimulation of salt secretion, but the release of VIP induced by ANP II is prevented by perfusion with procaine. VIP was measured by radioimmunoassay. Cardiac peptides thus appear to regulate rectal gland secretion by releasing VIP from neural stores within the gland. It is possible that other physiological effects of these hormones might be explained by an action to enhanced local release of neurotransmitters.

  3. Purification and identification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein.

    PubMed

    Tsou, May-June; Kao, Fuh-Juin; Lu, Hsi-Chi; Kao, Hao-Chun; Chiang, Wen-Dee

    2013-06-01

    The aim of this study was to purify and identify lipolysis-stimulating peptides derived from Flavourzyme®-soy protein isolate (SPI) hydrolysate (F-SPIH). Glycerol release was employed as a marker for lipolysis in 3T3-L1 adipocytes. A higher glycerol release represents a better lipolysis-stimulating activity. The peptide fraction with highest glycerol release obtained from F-SPIH fractionated by sequential ultrafiltration membranes was further purified using gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography. The peptides were identified using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Three lipolysis-stimulating peptides were obtained, and the amino acid sequences were ILL, LLL and VHVV, respectively. The in vitro effect of gastrointestinal proteases on lipolysis-stimulating activity of synthetic ILL, LLL and VHVV, respectively, was also investigated. The result suggested that the gastrointestinal protease did not affect lipolysis-stimulating activity of the three novel peptides, which reveals their potential to act as anti-obesity ingredients. PMID:23411267

  4. Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides1

    PubMed Central

    Cascieri, T.; Mallette, M. F.

    1973-01-01

    A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect. PMID:4588201

  5. Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.

    PubMed

    Shao, Hongwei; Lin, Yanmei; Wang, Teng; Ou, Yusheng; Shen, Han; Tao, Changli; Wu, Fenglin; Zhang, Wenfeng; Bo, Huaben; Wang, Hui; Huang, Shulin

    2015-07-10

    Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells. PMID:25890221

  6. Preferred recycling pathway by internalized PGE2 EP4 receptor following agonist stimulation in cultured dorsal root ganglion neurons contributes to enhanced EP4 receptor sensitivity.

    PubMed

    St-Jacques, Bruno; Ma, Weiya

    2016-06-21

    Prostaglandin E2 (PGE2), a well-known pain mediator abundantly produced in injured tissues, sensitizes nociceptive dorsal root ganglion (DRG) neurons (nociceptors) through its four EP receptors (EP1-4). Our prior study showed that PGE2 or EP4 agonist stimulates EP4 externalization and this event was not only suppressed by the inhibitor of anterograde export, but also by the recycling inhibitor (St-Jacques and Ma, 2013). These data suggest that EP4 recycling also contributes to agonist-enhanced EP4 surface abundance. In the current study, we tested this hypothesis using antibody-feeding-based internalization assay, recycling assay and FITC-PGE2 binding assay. We observed that selective EP4 agonist 1-hydroxy-PGE1 (1-OH-PGE1) or CAY10850 time- and concentration-dependently increased EP4 internalization in cultured DRG neuron. Internalized EP4 was predominantly localized in the early endosomes and recycling endosomes, but rarely in the late endosomes and lysosomes. These observations were confirmed by FITC-PGE2 binding assay. We further revealed that 1-OH-PGE1 or CAY10850 time- and concentration-dependently increased EP4 recycling. Double exposures to 1-OH-PGE1 induced a greater increase in calcitonin gene-related peptide (CGRP) release than a single exposure or vehicle exposure, an event blocked by pre-treatment with the recycling inhibitor monensin. Our data suggest that EP4 recycling contributes to agonist-induced cell surface abundance and consequently enhanced receptor sensitivity. Facilitating EP4 externalization and recycling is a novel mechanism underlying PGE2-induced nociceptor sensitization. PMID:27060485

  7. Peptides having reduced toxicity that stimulate cholesterol efflux

    DOEpatents

    Bielicki, John K.; Johansson, Jan; Danho, Waleed

    2016-08-16

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  8. Peptide YY antagonizes beta-adrenergic-stimulated release of insulin in dogs

    SciTech Connect

    Greeley, G.H. Jr.; Lluis, F.; Gomex, G.; Ishizuka, J.; Holland, B.; Thompson, J.C. )

    1988-04-01

    Peptide YY (PYY) and neuropeptide Y (NPY) are peptides of 36 amino acids that share structural homologies with pancreatic polypeptide (PP). PP is predominantly found in the endocrine pancreas. PYY is primarily found in mucosal endocrine cells of the distal ileum, colon, and rectum, whereas NPY is found in both the peripheral and central nervous system. Previous studies indicate that these peptides can interact with the autonomic nervous system. The objective of the present experiments was to study the effect of PYY on neurally stimulated insulin release in conscious dogs. Intravenous administration of PYY (100, 200, and 400 pmol{center dot}kg{sup {minus}1} {center dot}h{sup {minus}1}) reduced 2-DG-stimulated insulin release in a dose-dependent manner (P <0.05) without affecting plasma glucose levels. Administration of NPY, but not PP, reduced 2-DG-stimulated release of insulin. The inhibitory action of PYY on 2-DG-stimulated insulin release persisted in the presence of atropine or phentolamine treatment; however, hexamethonium alone or phentolamine plus propranolol treatment blocked the inhibitory action of PYY. Release of insulin stimulated by the {beta}-agonist isoproterenol was also inhibited by PYY. These results indicate that PYY can inhibit autonomic neurotransmission by a mechanism that may involve ganglionic or postganglionic inhibition of {beta}-adrenergic stimulation. The findings suggest a role for PYY and NPY in the autonomic regulation of insulin release.

  9. Vasoactive intestinal peptide stimulates protein phosphorylation in a colonic epithelial cell line

    SciTech Connect

    Cohn, J.A.

    1987-09-01

    The T/sub 84/ colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T/sub 84/ cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using /sup 32/P/sub i/. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in /sup 32/P incorporation into four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasocactive intestinal peptide and suggests that T/sub 84/ cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.

  10. Transient expression of somatostatin messenger RNA and peptide in the hypoglossal nucleus of the neonatal rat.

    PubMed

    Seroogy, K B; Bayliss, D A; Szymeczek, C L; Hökfelt, T; Millhorn, D E

    1991-06-21

    The postnatal developmental expression of somatostatin mRNA and peptide in the rat hypoglossal nucleus was analyzed using immunocytochemical and in situ hybridization techniques. Both the neuropeptide and its cognate mRNA were found to be transiently present within a subpopulation of hypoglossal motoneurons during the neonatal period. At the day of birth, a large population of perikarya situated in caudal, ventral regions of the hypoglossal nucleus expressed somatostatin. By postnatal day 7, the number of hypoglossal somata which expressed somatostatin had diminished considerably, and by 2 weeks postnatal, only few such cell bodies were found. By 3-4 weeks postnatal, somatostatin peptide- and mRNA-containing hypoglossal motoneurons were rarely observed, and in the adult, they were never detected, despite the use of colchicine. A double-labeling co-localization technique was used to demonstrate that somatostatin, when present perinatally, always coexisted with calcitonin gene-related peptide in hypoglossal motoneurons. The latter peptide, in contrast to somatostatin, was expressed in large numbers of somata throughout the entire hypoglossal nucleus and persisted within the motoneurons throughout development into adulthood. These results demonstrate that somatostatin is transiently expressed in motoneurons of the caudal, ventral tier of the hypoglossal nucleus in the neonatal rat. The developmental disappearance of somatostatin is most likely not due to cell death; hypoglossal somata continue to express calcitonin gene-related peptide, with which somatostatin coexisted perinatally, a high levels throughout development. Thus, it appears that the regulation of somatostatin expression in hypoglossal neurons occurs at the level of gene transcription or mRNA stability/degradation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1680035

  11. Expression of messenger RNAs for peptides and tyrosine hydroxylase in primary sensory neurons that innervate arterial baroreceptors and chemoreceptors.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Lawson, E E; Millhorn, D E

    1991-08-01

    Retrograde fiber tracing and in situ hybridization were used to determine expression of mRNAs for preprotachykinin A (ppTA), calcitonin gene related peptide (CGRP), preproenkephalin A (ENK), neuropeptide tyrosine (NPY) and somatostatin (SOM) as well as tyrosine hydroxylase (TH) in the petrosal ganglia primary sensory neurons which innervate carotid sinus baroreceptors and carotid body chemoreceptors. Perfusion of the carotid sinus with the retrogradely transported dye (Fluoro-Gold) labeled primary sensory neurons in petrosal ganglion. Numerous somata in the petrosal ganglion labeled with dye contained mRNAs for all the above peptides, except SOM. Moreover, TH mRNA was found in a substantial number of retrogradely labeled cells in the petrosal ganglion. This study provides information concerning which of the numerous peptides identified in sensory neurons of petrosal ganglion may be involved in modulation of the arterial baroreceptor and chemoreceptor reflexes. PMID:1681484

  12. β-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

    PubMed Central

    Oliveira, Maria José; Van Damme, Jozef; Lauwaet, Tineke; De Corte, Veerle; De Bruyne, Georges; Verschraegen, Gerda; Vaneechoutte, Mario; Goethals, Marc; Ahmadian, Mohammad Reza; Müller, Oliver; Vandekerckhove, Joël; Mareel, Marc; Leroy, Ancy

    2003-01-01

    In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer β-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a β-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (δOR) is a candidate receptor for the 13mer peptide since naloxone, an δOR antagonist, blocks both δOR serine phosphorylation and 13mer peptide-mediated invasion. PMID:14609961

  13. ZP-binding peptides identified via phage display stimulate production of sperm antibodies in dogs.

    PubMed

    Samoylova, Tatiana I; Cox, Nancy R; Cochran, Anna M; Samoylov, Alexandre M; Griffin, Brenda; Baker, Henry J

    2010-07-01

    Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs. PMID:20434854

  14. A new relaxin-like gonad-stimulating peptide identified in the starfish Asterias amurensis.

    PubMed

    Mita, Masatoshi; Daiya, Misaki; Haraguchi, Shogo; Tsutsui, Kazuyoshi; Nagahama, Yoshitaka

    2015-10-01

    Relaxin-like gonad-stimulating peptide (RGP) of starfish Asterina pectinifera was the first invertebrate gonadotropin to have its chemical structure identified. However, it is unclear whether gonadotropic hormones in other species starfish are relaxin-like peptides. Thus, this study tried to identify the molecular structure of gonadotropic hormone in Asterias amurensis. As a result, we identified A. amurensis gonadotropic hormone as the RGP (AamRGP). The DNA sequence encoding AamRGP consisted of 330 base pairs with an open reading frame encoding a peptide of 109 amino acids (aa), including a signal peptide (26 aa), B-chain (20 aa), C-peptide (38 aa) and A-chain (25 aa). Comparing with A. pectinifera RGP (ApeRGP), the amino acid identity levels between AmaRGP and ApeRGP were 58% for the A-chain and 73% for the B-chain. Furthermore, chemical synthetic AamRGP induced gamete spawning and oocyte maturation in ovarian fragments of A. amurensis. In contrast, the ovary of A. pectinifera failed to respond to the AamRGP. This suggested that AamRGP is a new relaxin-like peptide. PMID:26163025

  15. Alpha-Melanocyte Stimulating Hormone: An Emerging Anti-Inflammatory Antimicrobial Peptide

    PubMed Central

    Singh, Madhuri; Mukhopadhyay, Kasturi

    2014-01-01

    The alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide belonging to the melanocortin family. It is well known for its anti-inflammatory and antipyretic effects and shares several characteristics with antimicrobial peptides (AMPs). There have been some recent reports about the direct antimicrobial activity of α-MSH against various microbes belonging to both fungal and bacterial pathogens. Similar to α-MSH's anti-inflammatory properties, its C-terminal residues also exhibit antimicrobial activity parallel to that of the entire peptide. This review is focused on the current findings regarding the direct antimicrobial potential and immunomodulatory mechanism of α-MSH and its C-terminal fragments, with particular emphasis on the prospects of α-MSH based peptides as a strong anti-infective agent. PMID:25140322

  16. Replacement of the Disulfide Bridge in a KLK3-Stimulating Peptide Using Orthogonally Protected Building Blocks

    PubMed Central

    2013-01-01

    Peptide “B-2”, which is one of the most potent kallikrein-related peptidase 3 (KLK3)-stimulating compounds, consists of 12 amino acids and is cyclized by a disulfide bridge between the N- and C-terminal cysteines. Orthogonally protected building blocks were used in the peptide synthesis to introduce a disulfide bridge mimetic consisting of four carbon atoms. The resulting pseudopeptides with alkane and E-alkene linkers doubled the proteolytic activity of KLK3 at a concentration of 14 μM. They were almost as potent as the parent “B-2” peptide, which gives a 3.6-fold increase in the proteolytic activity of KLK3 at the same concentration. PMID:24900791

  17. A relaxin-like gonad-stimulating peptide from the starfish Aphelasterias japonica.

    PubMed

    Mita, Masatoshi; Katayama, Hidekazu

    2016-04-01

    Relaxin-like gonad-stimulating peptide (RGP) in starfish is the first identified invertebrate gonadotropin responsible for final gamete maturation. In this study, a new ortholog RGP was identified from Aphelasterias japonica. The DNA sequence encoding A. japonica RGP (AjaRGP) consists of 342 base pairs with an open reading frame encoding a peptide of 113 amino acids (aa), including a signal peptide (26aa), B-chain (20aa), C-peptide (42aa), and A-chain (25aa). AjaRGP is a heterodimeric peptide with disulfide cross-linkages. Comparing with Asterias amurensis RGP (AamRGP) and Patiria (=Asterina) pectinifera RGP (PpeRGP), the amino acid identity levels of AjaRGP with respect to AamRGP and PpeRGP are 84% and 58% for the A-chain and 90% and 68% for the B-chain, respectively. This suggests that AjaRGP is closer to AmaRGP rather than PpeRGP. Although chemical synthetic AjaRGP can induce gamete spawning and oocyte maturation in ovarian fragments of A. japonica, the ovary of P. pectinifera fails to respond to AjaRGP. This suggests that AjaRGP acts species-specifically. PMID:26944483

  18. Targeted Melanoma Imaging and Therapy with Radiolabeled Alpha-Melanocyte Stimulating Hormone Peptide Analogues

    PubMed Central

    Quinn, Thomas; Zhang, Xiuli; Miao, Yubin

    2010-01-01

    Radiolabeled alpha-melanocyte stimulating hormone (α-MSH) analogues have been used to define the expression, affinity and function of the melanocortin-1 receptor (MC1-R). The MC1-R is one of a family of five G-protein linker receptors, which is primarily involved in regulation of skin pigmentation. Over-expression of the MC1-R on melanoma tumor cells has made it an attractive target for the development of α-MSH peptide based imaging and therapeutic agents. Initially, the native α-MSH peptide was radiolabeled directly, but it suffered from low specific activity and poor stability. The addition of non-natural amino acids yielded α-MSH analogues with greater MC-1R affinity and stability. Furthermore, peptide cyclization via disulfide and lactam bond formation as well as site-specific metal coordination resulted in additional gains in receptor affinity and peptide stability in vitro and in vivo. Radiochemical stability of the α-MSH analogues was improved through the conjugation of metal chelators to the peptide’s N-terminus or lysine residues for radionuclide coordination. In vitro cell binding studies demonstrated that the radiolabeled α-MSH analogues had low to subnanomolar affinities for the MC1-R. Biodistribution and imaging studies in the B16 mouse melanoma modeled showed rapid tumor uptake of the radiolabeled peptides, with the cyclic peptides demonstrating prolonged tumor retention. Cyclic α-MSH analogues labeled with beta and alpha emitting radionuclides demonstrated melanoma therapeutic efficacy in the B16 melanoma mouse model. Strong pre-clinical imaging and therapy data highlight the clinical potential use of radiolabeled α-MSH peptides for melanoma imaging and treatment of disseminated disease. PMID:20467398

  19. Natriuretic peptides stimulate the cardiac sodium pump via NPR-C-coupled NOS activation.

    PubMed

    William, M; Hamilton, E J; Garcia, A; Bundgaard, H; Chia, K K M; Figtree, G A; Rasmussen, H H

    2008-04-01

    Natriuretic peptides (NPs) and their receptors (NPRs) are expressed in the heart, but their effects on myocyte function are poorly understood. Because NPRs are coupled to synthesis of cGMP, an activator of the sarcolemmal Na(+)-K(+) pump, we examined whether atrial natriuretic peptide (ANP) regulates the pump. We voltage clamped rabbit ventricular myocytes and identified electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange and normalized for membrane capacitance) as the shift in membrane current induced by 100 micromol/l ouabain. Ten nanomoles per liter ANP stimulated the Na(+)-K(+) pump when the intracellular compartment was perfused with pipette solutions containing 10 mmol/l Na(+) but had no effect when the pump was at near maximal activation with 80 mmol/l Na(+) in the pipette solution. Stimulation was abolished by inhibition of cGMP-activated protein kinase with KT-5823, nitric oxide (NO)-activated guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), or NO synthase with N(G)-nitro-L-arginine methyl ester (L-NAME). Since synthesis of cGMP by NPR-A and NPR-B is not NO dependent or ODQ sensitive, we exposed myocytes to AP-811, a highly selective ligand for the NPR-C "clearance" receptor. It abolished ANP-induced pump stimulation. Conversely, the selective NPR-C agonist ANP(4-23) reproduced stimulation. The stimulation was blocked by l-NAME. To examine NO production in response to ANP(4-23), we loaded myocytes with the NO-sensitive fluorescent dye diacetylated diaminofluorescein-2 and examined them by confocal microscopy. ANP(4-23) induced a significant increase in fluorescence, which was abolished by L-NAME. We conclude that NPs stimulate the Na(+)-K(+) pump via an NPR-C and NO-dependent pathway. PMID:18272821

  20. Antimicrobial peptides trigger a division block in Escherichia coli through stimulation of a signalling system.

    PubMed

    Yadavalli, Srujana S; Carey, Jeffrey N; Leibman, Rachel S; Chen, Annie I; Stern, Andrew M; Roggiani, Manuela; Lippa, Andrew M; Goulian, Mark

    2016-01-01

    Antimicrobial peptides are an important component of the molecular arsenal employed by hosts against bacteria. Many bacteria in turn possess pathways that provide protection against these compounds. In Escherichia coli and related bacteria, the PhoQ/PhoP signalling system is a key regulator of this antimicrobial peptide defence. Here we show that treating E. coli with sublethal concentrations of antimicrobial peptides causes cells to filament, and that this division block is controlled by the PhoQ/PhoP system. The filamentation results from increased expression of QueE, an enzyme that is part of a tRNA modification pathway but that, as we show here, also affects cell division. We also find that a functional YFP-QueE fusion localizes to the division septum in filamentous cells, suggesting QueE blocks septation through interaction with the divisome. Regulation of septation by PhoQ/PhoP may protect cells from antimicrobial peptide-induced stress or other conditions associated with high-level stimulation of this signalling system. PMID:27471053

  1. Antimicrobial peptides trigger a division block in Escherichia coli through stimulation of a signalling system

    PubMed Central

    Yadavalli, Srujana S.; Carey, Jeffrey N.; Leibman, Rachel S.; Chen, Annie I.; Stern, Andrew M.; Roggiani, Manuela; Lippa, Andrew M.; Goulian, Mark

    2016-01-01

    Antimicrobial peptides are an important component of the molecular arsenal employed by hosts against bacteria. Many bacteria in turn possess pathways that provide protection against these compounds. In Escherichia coli and related bacteria, the PhoQ/PhoP signalling system is a key regulator of this antimicrobial peptide defence. Here we show that treating E. coli with sublethal concentrations of antimicrobial peptides causes cells to filament, and that this division block is controlled by the PhoQ/PhoP system. The filamentation results from increased expression of QueE, an enzyme that is part of a tRNA modification pathway but that, as we show here, also affects cell division. We also find that a functional YFP–QueE fusion localizes to the division septum in filamentous cells, suggesting QueE blocks septation through interaction with the divisome. Regulation of septation by PhoQ/PhoP may protect cells from antimicrobial peptide-induced stress or other conditions associated with high-level stimulation of this signalling system. PMID:27471053

  2. Antibacterial Activity of a Competence-Stimulating Peptide in Experimental Sepsis Caused by Streptococcus pneumoniae

    PubMed Central

    Oggioni, Marco R.; Iannelli, Francesco; Ricci, Susanna; Chiavolini, Damiana; Parigi, Riccardo; Trappetti, Claudia; Claverys, Jean-Pierre; Pozzi, Gianni

    2004-01-01

    Streptococcus pneumoniae, a major cause of human disease, produces a 17-mer autoinducer peptide pheromone (competence-stimulating peptide [CSP]) for the control of competence for genetic transformation. Due to previous work linking CSP to stress phenotypes, we set up an in vivo sepsis model to assay its effect on virulence. Our data demonstrate a significant increase in the rates of survival of mice, reductions of blood S. pneumoniae counts, and prolonged times to death for mice treated with CSP. In vitro the dose of CSP used in the animal model produced a transitory inhibition of growth. When a mutant with a mutation in the CSP sensor histidine kinase was assayed, no bacteriostatic phenotype was detected in vitro and no change in disease outcome was observed in vivo. The data demonstrate that CSP, which induces in vitro a temporary growth arrest through stimulation of its cognate histidine kinase receptor, is able to block systemic disease in mice. This therapeutic effect is novel, in that the drug-like effect is obtained by stimulation, rather than inhibition, of a bacterial drug target. PMID:15561850

  3. Protective effect of atrial natriuretic peptide on electrical-field-stimulated rat ventricular strips during hypoxia.

    PubMed

    Ljusegren, M E; Andersson, R G

    1994-12-01

    We have previously shown that atrial natriuretic peptide reduces lactate accumulation in non-beating rat ventricular myocardium exposed to hypoxic conditions, and that hypoxia induces release of atrial natriuretic peptide from isolated rat atrial tissue. In these studies we suggested that atrial natriuretic peptide may be physiologically important for protection of the myocardium during periods of oxygen deficit. In the present study, we used isolated strips of rat right ventricle, contracted by electrical-field-stimulation, as a model of a beating myocardium. After contraction stabilization, hypoxic conditions were introduced through aeration with 20% O2, held for 20 or 30 min., and then interrupted by reoxygenation with 95% O2. The contractile force was recorded and the percentage regain of the contractions after reoxygenation was considered as an indication of the amount of cell damage induced during the period of hypoxia. The results show that after 30 min. of hypoxia and subsequent reoxygenation, ventricular strips treated with atrial natriuretic peptide (0.1 microM) recovered 67.9 +/- 2.8% of the prehypoxic force of contraction; control strips from the same ventricle regained 44.9 +/- 4.4% (P = 0.015) of their initial contractile activity. After 20 min. of hypoxia followed by reoxygenation, a ventricular strip incubated together with an atrium regained 78.6 +/- 2.4% of the prehypoxic force of contraction as compared to a 60.2 +/- 2.7% regain (P = 0.002) for the control strip. We conclude that atrial natriuretic peptide protects the working ventricular myocardium during hypoxia, which further supports our previously reported suggestion that the effect on myocardial metabolism is physiologically relevant during situations of oxygen deficit in heart muscle. PMID:7899254

  4. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

    PubMed Central

    Bab, I; Gazit, D; Chorev, M; Muhlrad, A; Shteyer, A; Greenberg, Z; Namdar, M; Kahn, A

    1992-01-01

    It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo. Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP-OGP binding protein complex. A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post-ablation marrow regeneration and associated systemic osteogenic response. Authentic OGP is identical to the C-terminus of histone H4 and shares a five residue motif with a T-cell receptor beta-chain V-region and the Bacillus subtilis outB locus. Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides. Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation. Images PMID:1582415

  5. Activation of EP4 receptors contributes to prostaglandin E2-mediated stimulation of renal sensory nerves.

    PubMed

    Kopp, Ulla C; Cicha, Michael Z; Nakamura, Kazuhiro; Nüsing, Rolf M; Smith, Lori A; Hökfelt, Tomas

    2004-12-01

    Induction of cyclooxygenase-2 (COX-2) in the renal pelvic wall increases prostaglandin E(2) (PGE(2)) leading to stimulation of cAMP production, which results in substance P (SP) release and activation of renal mechanosensory nerves. The subtype of PGE receptors involved, EP2 and/or EP4, was studied by immunohistochemistry and renal pelvic administration of agonists and antagonists of EP2 and EP4 receptors. EP4 receptor-like immunoreactivity (LI) was colocalized with calcitonin gene-related peptide (CGRP)-LI in dorsal root ganglia (DRGs) at Th(9)-L(1) and in nerve terminals in the renal pelvic wall. Th(9)-L(1) DRG neurons also contained EP3 receptor-LI and COX-2-LI, each of which was colocalized with CGRP-LI in some neurons. No renal pelvic nerves contained EP3 receptor-LI and only very few nerves COX-2-LI. The EP1/EP2 receptor antagonist AH-6809 (20 microM) had no effect on SP release produced by PGE(2) (0.14 microM) from an isolated rat renal pelvic wall preparation. However, the EP4 receptor antagonist L-161,982 (10 microM) blocked the SP release produced by the EP2/EP4 receptor agonist butaprost (10 microM) 12 +/- 2 vs. 2 +/- 1 and PGE(2), 9 +/- 1 vs. 1 +/- 0 pg/min. The SP release by butaprost and PGE(2) was similarly blocked by the EP4 receptor antagonist AH-23848 (30 microM). In anesthetized rats, the afferent renal nerve activity (ARNA) responses to butaprost 700 +/- 100 and PGE(2).780 +/- 100%.s (area under the curve of ARNA vs. time) were unaffected by renal pelvic perfusion with AH-6809. However, 1 microM L-161,982 and 10 microM AH-23848 blocked the ARNA responses to butaprost by 94 +/- 5 and 78 +/- 10%, respectively, and to PGE(2) by 74 +/- 16 and 74 +/- 11%, respectively. L-161,982 also blocked the ARNA response to increasing renal pelvic pressure 10 mmHg, 85 +/- 5%. In conclusion, PGE(2) increases renal pelvic release of SP and ARNA by activating EP4 receptors on renal sensory nerve fibers. PMID:15292051

  6. Self-Assembly and Collagen-Stimulating Activity of a Peptide Amphiphile Incorporating a Peptide Sequence from Lumican.

    PubMed

    Hamley, Ian W; Dehsorkhi, Ashkan; Castelletto, Valeria; Walter, Merlin N M; Connon, Che J; Reza, Mehedi; Ruokolainen, Janne

    2015-04-21

    The self-assembly and bioactivity of a peptide amphiphile (PA) incorporating a 13-residue sequence derived from the last 13 amino acids of the C-terminus of lumican, C16-YEALRVANEVTLN, attached to a hexadecyl (C16) lipid chain have been examined. Lumican is a proteoglycan found in many types of tissue and is involved in collagen fibril organization. A critical aggregation concentration (cac) for the PA was determined through pyrene fluorescence measurements. The structure of the aggregates was imaged using electron microscopy, and twisted and curved nanotapes were observed. In situ small-angle X-ray scattering and fiber X-ray diffraction reveal that these tapes contain interdigitated bilayers of the PA molecules. FTIR and circular dichroism spectroscopy and fiber X-ray diffraction indicate that the lumican sequence in the PA adopts a β-sheet secondary structure. Cell assays using human dermal fibroblasts show that below the cac the PA displays good biocompatibility and also stimulates collagen production over a period of 3 weeks, exceeding a 2-fold enhancement for several concentrations. Thus, this PA has promise in future biological applications, in particular, in tissue engineering. PMID:25835126

  7. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein kinase C.

    PubMed Central

    De la Fuente, M; Bernaez, I; Del Rio, M; Hernanz, A

    1993-01-01

    The peptides neuropeptide Y (NPY) and peptide YY (PYY) at concentrations from 10(-12) M to 10(-8) M have been shown in this study to stimulate significantly, in vitro, several functions of resting peritoneal macrophages from BALB/c mice: adherence to substrate, chemotaxis, ingestion of inert particles (latex beads) and foreign cells (Candida albicans), and production of superoxide anion measured by nitroblue tetrazolium reduction. A dose-response relationship was observed, with a maximal stimulation of the macrophage functions studied at 10(-10) M. These effects seem to be produced by specific receptors for the neuropeptides studied in peritoneal macrophages. Whereas the two peptides induced no change of intracellular cyclic AMP, they caused a significant stimulation of protein kinase C (PKC) in murine macrophages. These results suggest that NPY and PYY produce their effects on macrophage function through PKC activation. PMID:8262554

  8. Flavonoids stimulate cholecystokinin peptide secretion from the enteroendocrine STC-1 cells.

    PubMed

    Al Shukor, Nadin; Ravallec, Rozenn; Van Camp, John; Raes, Katleen; Smagghe, Guy

    2016-09-01

    Animal experiments showed that flavonoids might have the potential for an anti-obesity effect by reducing weight and food intake. However, the exact mechanisms that could be involved in these proposed effects are still under investigation. The complex process of food intake is partially regulated by gastrointestinal hormones. Cholecystokinin (CCK) is the best known gastrointestinal hormone to induce satiety signal that plays a key role in food intake regulation. It is released from the endocrine cells (I cell) in response to the ingestion of nutrients into the small intestine. In this study, we investigated the possible effects of flavonoids (quercetin, kaempferol, apigenin, rutin and baicalein) on stimulation of CCK release in vitro using enteroendocrine STC-1 cells. In comparison with the control, quercetin, kaempferol and apigenin resulted in a significant increase in CCK secretion with quercetin showing the highest activity. On the other hand, no significant effect was seen by rutin and baicalein. To our knowledge, this is the first report to study the stimulation of CCK peptide hormone secretion from STC-1 cells by quercetin and kaempferol, rutin, apigenin and baicalein. Based on the cell-based results in this work, it can be suggested that the reported activity of flavonoids against food intake and weight could be mediated by stimulation of CCK signal which in turn is responsible for food intake reduction, but future animal and human studies are needed to confirm this conclusion at organism level. PMID:27496247

  9. Peptide 19-2.5 inhibits heparan sulfate-triggered inflammation in murine cardiomyocytes stimulated with human sepsis serum.

    PubMed

    Martin, Lukas; Schmitz, Susanne; De Santis, Rebecca; Doemming, Sabine; Haase, Hajo; Hoeger, Janine; Heinbockel, Lena; Brandenburg, Klaus; Marx, Gernot; Schuerholz, Tobias

    2015-01-01

    Myocardial dysfunction in sepsis has been linked to inflammation caused by pathogen-associated molecular patterns (PAMPs) as well as by host danger-associated molecular patterns (DAMPs). These include soluble heparan sulfate (HS), which triggers the devastating consequences of the pro-inflammatory cascades in severe sepsis and septic shock. Thus, there is increasing interest in the development of anti-infective agents, with effectiveness against both PAMPs and DAMPs. We hypothesized that a synthetic antimicrobial peptide (peptide 19-2.5) inhibits inflammatory response in murine cardiomyocytes (HL-1 cells) stimulated with PAMPs, DAMPs or serum from patients with septic shock by reduction and/or neutralization of soluble HS. In the current study, our data indicate that the treatment with peptide 19-2.5 decreases the inflammatory response in HL-1 cells stimulated with either PAMPs or DAMPs. Furthermore, our work shows that soluble HS in serum from patients with Gram-negative or Gram-positive septic shock induces a strong pro-inflammatory response in HL-1 cells, which can be effectively blocked by peptide 19-2.5. Based on these findings, peptide 19-2.5 is a novel anti-inflammatory agent interacting with both PAMPs and DAMPs, suggesting peptide 19-2.5 may have the potential for further development as a broad-spectrum anti-inflammatory agent in sepsis-induced myocardial inflammation and dysfunction. PMID:26024383

  10. Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling.

    PubMed

    Sugamori, Yasutaka; Mise-Omata, Setsuko; Maeda, Chizuko; Aoki, Shigeki; Tabata, Yasuhiko; Murali, Ramachandran; Yasuda, Hisataka; Udagawa, Nobuyuki; Suzuki, Hiroshi; Honma, Masashi; Aoki, Kazuhiro

    2016-08-01

    Both W9 and OP3-4 were known to bind the receptor activator of NF-κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide-induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3-4 accelerated BMP-2-induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL-binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP-2-induced bone regeneration by the RANKL-binding peptides. PMID:27345003

  11. Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans.

    PubMed Central

    Schalling, M; Stieg, P E; Lindquist, C; Goldstein, M; Hökfelt, T

    1989-01-01

    Thoracic ganglia in humans were studied after electrical, preganglionic stimulation using in situ hybridization with synthetic oligonucleotide probes against the catecholamine-synthesizing enzymes tyrosine hydroxylase (EC 1.14.16.2) and dopamine beta-hydroxylase (EC 1.14.17.1) and neuropeptide tyrosine. Immunohistochemical analysis was also performed. Following short peroperative stimulation a severalfold increase in all three mRNAs was found in principal ganglion cells, whereas no definite changes could be detected in enzyme or peptide levels with immunohistochemistry. The results suggest a very rapid and sensitive regulation of genes involved in signal transmission in the sympathetic nervous system of humans. Moreover, they indicate that electrical stimulation of neurons and/or pathways combined with in situ hybridization may be used as a method to define neuronal projections by visualizing increases in mRNAs for transmitter enzymes and/or peptide in target cells. Images PMID:2567003

  12. PD-L1 peptide co-stimulation increases immunogenicity of a dendritic cell-based cancer vaccine.

    PubMed

    Munir Ahmad, Shamaila; Martinenaite, Evelina; Hansen, Morten; Junker, Niels; Borch, Troels Holz; Met, Özcan; Donia, Marco; Svane, Inge Marie; Andersen, Mads Hald

    2016-08-01

    We recently described naturally occurring PD-L1-specific T cells that recognize PD-L1-expressing immune cells as well as malignant cells. In the present study, we investigated whether the immunogenicity of a dendritic cell (DC)-based vaccine could be influenced by co-stimulation with a known PD-L1-derived epitope. We incubated a PD-L1-derived peptide epitope (19 amino acids long) or a control peptide (an irrelevant HIV epitope) with peripheral blood mononuclear cells from patients with malignant melanoma who had received a DC-based vaccine. We observed a significantly higher number of T cells that reacted to the vaccine in cultures that had been co-stimulated with the PD-L1 peptide epitope compared to cultures incubated with control peptide. Next, we characterized a novel PD-L1-derived epitope (23 amino acids long) and found that co-stimulation with both PD-L1 epitopes boosted the immune response elicited by the DC vaccine even further. Consequently, we observed a significant increase in the number of vaccine-reacting T cells in vitro. In conclusion, activation of PD-L1-specific T cells may directly modulate immunogenicity of DC vaccines. Addition of PD-L1 epitopes may thus be an easily applicable and attractive option to augment the effectiveness of cancer vaccines and other immunotherapeutic agents. PMID:27622072

  13. Analysis of the proteolysis of bioactive peptides using a peptidomics approach

    PubMed Central

    Kim, Yun-Gon; Lone, Anna Mari; Saghatelian, Alan

    2014-01-01

    Identifying the peptidases that inactivate bioactive peptides (e.g. peptide hormones and neuropeptides) in mammals is an important unmet challenge. This protocol describes a recent approach that combines liquid chromatography-mass spectrometry peptidomics to identify endogenous cleavage sites of a bioactive peptide, the subsequent biochemical purification of a candidate peptidase based on these cleavage sites, and validation of the candidate peptidase’s role in the physiological regulation of the bioactive peptide by examining a peptidase knockout mouse. We highlight successful application of this protocol to discover that insulin-degrading enzyme (IDE) regulates physiological calcitonin gene-related peptide (CGRP) levels and detail the key stages and steps in this approach. This protocol requires 7 days of work; however, the total time for this protocol is highly variable because of its dependence on the availability of biological reagents, namely purified enzymes and knockout mice. The protocol is valuable because it expedites the characterization of mammalian peptidases, such as IDE, which in certain instances can be used to develop novel therapeutics. PMID:23949379

  14. Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion.

    PubMed

    Mizokami, Akiko; Yasutake, Yu; Higashi, Sen; Kawakubo-Yasukochi, Tomoyo; Chishaki, Sakura; Takahashi, Ichiro; Takeuchi, Hiroshi; Hirata, Masato

    2014-12-01

    Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion and pancreatic β-cell proliferation. We previously showed that the effect of GluOC on insulin secretion is mediated largely by glucagon-like peptide-1 (GLP-1) secreted from the intestine in response to GluOC exposure. We have now examined the effect of oral administration of GluOC on glucose utilization as well as the fate of such administered GluOC in mice. Long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level and improved glucose tolerance in mice without affecting insulin sensitivity. It also increased the fasting serum insulin concentration as well as the β-cell area in the pancreas. A small proportion of orally administered GluOC reached the small intestine and remained there for at least 24h. GluOC also entered the general circulation, and the serum GLP-1 concentration was increased in association with the presence of GluOC in the intestine and systemic circulation. The putative GluOC receptor, GPRC6A was detected in intestinal cells, and was colocalized with GLP-1 in some of these cells. Our results suggest that orally administered GluOC improved glucose handling likely by acting from both the intestinal lumen and the general circulation, with this effect being mediated in part by stimulation of GLP-1 secretion. Oral administration of GluOC warrants further study as a safe and convenient option for the treatment or prevention of metabolic disorders. PMID:25230237

  15. Angiotensin II-stimulated secretion of arginine vasopressin is inhibited by atrial natriuretic peptide in humans.

    PubMed

    Matsukawa, Toshiyoshi; Miyamoto, Takenori

    2011-03-01

    We investigated the effect of the intravenous infusion of atrial natriuretic peptide (ANP) on the response of plasma arginine vasopressin (AVP) levels to intravenous infusion of angiotensin II (ANG II) in healthy individuals. Intravenous infusion of ANP (10 ng·kg(-1)·min(-1)) slightly but significantly decreased plasma AVP levels, while intravenous infusion of ANG II (10 ng·kg(-1)·min(-1)) resulted in slightly increased plasma AVP levels. ANG II infused significant elevations in arterial blood pressure and central venous pressure (CVP). Because the elevation in blood pressure could have potentially inhibited AVP secretion via baroreceptor reflexes, the effect of ANG II on blood pressure was attenuated by the simultaneous infusion of nitroprusside. ANG II alone produced a remarkable increase in plasma AVP levels when infused with nitroprusside, whereas the simultaneous ANP intravenous infusion (10 ng·kg(-1)·min(-1)) abolished the increase in plasma AVP levels induced by ANG II when blood pressure elevation was attenuated by nitroprusside. Thus, ANG II increased AVP secretion and ANP inhibited not only basal AVP secretion but also ANG II-stimulated AVP secretion in humans. These findings support the hypothesis that circulating ANP modulates AVP secretion, in part, by antagonizing the action of circulating ANG II. PMID:21123762

  16. 83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

    PubMed Central

    Nadeau, K.; Sullivan, M. A.; Bradley, M.; Engman, D. M.; Walsh, C. T.

    1992-01-01

    A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids. PMID:1304385

  17. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

    PubMed

    Booe, Jason M; Walker, Christopher S; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A; Warner, Margaret L; Bill, Roslyn M; Harris, Paul W; Brimble, Margaret A; Poyner, David R; Hay, Debbie L; Pioszak, Augen A

    2015-06-18

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  18. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  19. Sumatriptan inhibits the release of CGRP and substance P from the rat spinal cord.

    PubMed

    Arvieu, L; Mauborgne, A; Bourgoin, S; Oliver, C; Feltz, P; Hamon, M; Cesselin, F

    1996-08-12

    The possible presynaptic action of the anti-migraine drug sumatriptan on primary afferent fibres containing substance P and/or calcitonin gene-related peptide was investigated on superfused rat horizontal spinal cord slices with attached dorsal roots. Electrical stimulation of dorsal roots triggered a significant overflow of both peptides; this could be reduced by sumatriptan in a concentration-dependent manner. As expected from the involvement of 5-HT1B/1.D beta receptors, methiothepin, (-)tertatolol and GR 127,935, but not WAY 100,635, prevented the inhibitors effect of sumatriptan. These data support the idea that the anti-migraine action of sumatriptan may involve, at least in part, a presynaptic inhibitory control of nociceptive (trigeminovascular) substance P- and/or calcitonin gene-related peptide-containing sensory fibres. PMID:8905706

  20. Participation of the ascending serotonergic system in the stimulation of atrial natriuretic peptide release.

    PubMed Central

    Reis, L C; Ramalho, M J; Favaretto, A L; Gutkowska, J; McCann, S M; Antunes-Rodrigues, J

    1994-01-01

    Results obtained in our laboratories have provided evidence for the participation of the hypothalamic atrial natriuretic peptide (ANP) neuronal system in the regulation of water and electrolyte homeostasis. The anterior ventral third ventricular (AV3V) region, a site of the perikarya of the ANP neurons, receives important afferent input from ascending serotoninergic axons. We hypothesized that the ascending serotoninergic tract might be involved in control of the liberation of ANP. Therefore, electrolytic lesions were produced in the mesencephalic dorsal raphé nucleus (DRN), the site of perikarya of serotonin (5-HT) neurons whose axons project to the AV3V region. Rats with sham lesions constituted the control group. In a second group of animals, the serotoninergic system was depleted of 5-HT by lateral ventricular administration of p-chlorophenylalanine (PCPA), an amino acid that causes depletion of 5-HT from the serotoninergic neurons. Control animals were injected with an equal amount of isotonic saline. The DRN lesions induced an increase of water intake and urine output beginning on the first day that lasted for 1 week after lesions were produced. There was a concomitant sodium retention that lasted for the same period of time. When water-loaded, DRN-lesioned and PCPA-injected animals showed diminished excretion of sodium, accompanied by a decrease in basal plasma ANP concentrations, and blockade of the increase in plasma ANP, which followed blood volume expansion by intraatrial injection of hypertonic saline. The results are interpreted to mean that ascending stimulatory serotoninergic input into the ANP neuronal system in the AV3V region produces a tonic stimulation of ANP release, which augments sodium excretion and inhibits water intake. Therefore, in the absence of this serotoninergic input following destruction of the serotoninergic neurons by DRN lesions or intraventricular injection of PCPA, an antinatriuretic effect is obtained that is associated with

  1. Molecular and functional characterization of amylin, a peptide associated with type 2 diabetes mellitus

    SciTech Connect

    Roberts, A.N.; Leighton, B.; Todd, J.A.; Schofield, P.N.; Sutton, R.; Day, A.J.; Foot, E.A.; Willis, A.C.; Reid, K.B.M.; Cooper, H.J.S. ); Holt, S.; Boyd, Y. Medical Research Council Radiobiology Unit, Chilton )

    1989-12-01

    The 37-amino acid peptide called amylin is a major component of the islet amyloid deposited in the pancreases of persons with type 2 diabetes mellitus. The authors report the isolation of a partial cDNA clone and a phage {lambda} genomic clone of the coding region of the amylin gene. The DNA sequence encodes a protein sequences identical to that of amylin isolated from the amyloid found in the diabetic pancreas and shows that amylin is likely to be synthesized as a precursor peptide, now named proamylin. They have demonstrated that the amylin gene is present on chromosome 12 and that it is probably transcribed in the islets of Langerhans. The sequences of the genes for amyli and the calcitonin gene-related peptides (CGRPs) show strong similarity, especially over their 5{prime} coding regions, where both peptides have a conserved intramolecular disulfide bridge, and also over their 3{prime} coding regions, where the presence of a glycine codon strongly suggests that the carboxylterminal residue of amylin, like that of CGRP, is amidated. To examine the functional relevance of these posttranslational modifications, the biological activity of amylin synthesized with or without the disulfide bridge and/or amidation was measured. It was found that both features are necessary for full biological activity, thereby confirming the functional importance of those regions of the molecule whose sequences are conserved at both protein and genetic levels.

  2. Comparison of the effects of pantethine and fursultiamine on plasma gastrointestinal peptide levels in healthy volunteers.

    PubMed

    Suzuki, Yosuke; Itoh, Hiroki; Abe, Tomohide; Nishimura, Fumihiro; Sato, Yuhki; Takeyama, Masaharu

    2011-01-01

    Pantethine and fursultiamine have been evaluated for their clinical usefulness in the treatment and prevention of uncomplicated postoperative adhesive intestinal obstruction. In recent years, the actions of drugs used to treat gastrointestinal diseases have been elucidated pharmacologically from the viewpoints of gastrointestinal peptide levels. We examined the effects of pantethine and fursultiamine on plasma levels of calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)-, motilin- and substance P (SP)-like immunoreactive substances (IS) in healthy subjects. An open-labeled study was conducted on five healthy volunteers. Each subject was administered a single oral dose of pantethine, fursultiamine and placebo at intervals of one month. Venous blood samples were collected before and at 20, 40, 60, 90, 120, 180 and 240 min after each administration. Plasma peptide levels were measured using a highly sensitive enzyme immunoassay. A single oral dose of pantethine resulted in significant increases of plasma CGRP- and VIP-IS levels compared to placebo. Furthermore, areas under the plasma concentration-time curves (AUC(0-240)) of CGRP- and VIP-IS were significantly higher after pantethine administration compared with placebo. On the other hand, fursultiamine had no effect on plasma levels and AUC(0-240) of CGRP-, VIP-, motilin- and SP-IS. This study demonstrated the different effects of pantethine and fursultiamine from the viewpoint of plasma gastrointestinal peptide changes. The pharmacological effects of pantethine may be closely related to the changes in plasma CGRP- and VIP-IS levels. PMID:21963510

  3. Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

    2014-02-01

    Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side

  4. Subpopulation-Specific Transcriptome Analysis of Competence-Stimulating-Peptide-Induced Streptococcus mutans▿†

    PubMed Central

    Lemme, André; Gröbe, Lothar; Reck, Michael; Tomasch, Jürgen; Wagner-Döbler, Irene

    2011-01-01

    Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ∼3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity. PMID:21317319

  5. Molecular mechanisms underlying bile acid-stimulated glucagon-like peptide-1 secretion

    PubMed Central

    Parker, HE; Wallis, K; le Roux, CW; Wong, KY; Reimann, F; Gribble, FM

    2012-01-01

    BACKGROUND AND PURPOSE The glucagon-like peptides GLP-1 and GLP-2 are secreted from enteroendocrine L-cells following nutrient ingestion. Drugs that increase activity of the GLP-1 axis are highly successful therapies for type 2 diabetes, and boosting L-cell secretion is a potential strategy for future diabetes treatment. The aim of the present study was to further our understanding of the bile acid receptor GPBA (TGR5), an L-cell target currently under therapeutic exploration. EXPERIMENTAL APPROACH GLUTag cells and mixed primary murine intestinal cultures were exposed to bile acids and a specific agonist, GPBAR-A. Secretion was measured using hormone assays and intracellular calcium and cAMP responses were monitored using real-time imaging techniques. KEY RESULTS Bile acid-triggered GLP-1 secretion from GLUTag cells was GPBA-dependent, as demonstrated by its abolition following tgr5 siRNA transfection. Bile acids and GPBAR-A increased GLP-1 secretion from intestinal cultures, with evidence for synergy between the effects of glucose and GPBA activation. Elevation of cAMP was observed following GPBA activation in individual GLUTag cells. Direct calcium responses to GPBAR-A were small, but in the presence of the agonist, a subpopulation of cells that was previously poorly glucose-responsive exhibited robust glucose responses. In vivo, increased delivery of bile to more distal regions of the ileum augmented L-cell stimulation. CONCLUSIONS AND IMPLICATIONS GPBA signalling in L-cells involves rapid elevation of cAMP, and enhanced calcium and secretory responses to glucose. Modulation of this receptor therapeutically may be an attractive strategy to enhance GLP-1 secretion and achieve better glycaemic control in diabetic patients. PMID:21718300

  6. Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

    PubMed

    Szabados, Hajnalka; Uray, Katalin; Majer, Zsuzsa; Silló, Pálma; Kárpáti, Sarolta; Hudecz, Ferenc; Bősze, Szilvia

    2015-09-01

    Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors. PMID:26250896

  7. An Arg-Gly-Asp peptide stimulates Ca2+ efflux from osteoclast precursors through a novel mechanism

    NASA Technical Reports Server (NTRS)

    Yamakawa, K.; Duncan, R.; Hruska, K. A.

    1994-01-01

    We examined the effect of a peptide containing the Arg-Gly-Asp (RGD) sequence on 45Ca2+ efflux from osteoclast precursors. 45Ca(2+)-loaded osteoclast precursors were treated with GRGDSP (170 microM) for 10 min after 30 min of basal perfusion with a bicarbonate-containing buffer. GRGDSP significantly increased fractional efflux of Ca2+ from treated cells compared with vehicle-treated cells (P < 0.01) or cells treated with up to 200 micrograms/ml of a control peptide containing GRGESP. The effect of RGD was sustained for 15 min after the peptide was removed from the perfusate, but control levels of Ca2+ efflux returned by 1 h. The Ca2+ efflux effect of GRGDSP was most likely due to activation of the plasma membrane Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase) pump, as indicated by its inhibition with vanadate and a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and the absence of an effect of Na+/Ca2+ exchange inhibition. An inhibitor of cyclic nucleotide-dependent protein kinases, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (0.1 mM), failed to inhibit GRGDSP-stimulated Ca2+ efflux. However, genistein and herbimycin A, inhibitors of protein-tyrosine kinases, blocked Ca2+ efflux stimulated by GRGDSP. The results indicate that RGD sequences of matrix proteins may stimulate Ca2+ efflux from osteoclasts through activation of protein-tyrosine kinases and suggest that GRGDSP-stimulated Ca2+ efflux is mediated via the plasma membrane Ca(2+)-ATPase.

  8. Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic

    SciTech Connect

    Avdic, Vanja; Zhang, Pamela; Lanouette, Sylvain; Voronova, Anastassia; Skerjanc, Ilona; Couture, Jean-Francois

    2011-08-24

    The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an N{alpha} acetylated form of histone H3 peptide (N{alpha}H3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of N{alpha}H3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.-Avdic, V., Zhang, P., Lanouette, S., Voronova, A., Skerjanc, I., Couture, J.-F. Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic.

  9. Helodermin-like peptides in thyroid C cells: stimulation of thyroid hormone secretion and suppression of calcium incorporation into bone.

    PubMed Central

    Grunditz, T; Persson, P; Håkanson, R; Absood, A; Böttcher, G; Rerup, C; Sundler, F

    1989-01-01

    Helodermin is a vasoactive intestinal peptide-like peptide in the salivary gland venom of the lizard Heloderma suspectum. Helodermin-like immunofluorescence was observed in the parafollicular (C) cells in several mammals and in the C cell homologues of the chicken ultimobranchial gland. Thus, helodermin-like peptides coexist with calcitonin. The results of radioimmunoassay agreed with the immunocytochemical findings. HPLC of rat thyroid extracts revealed one major peak of helodermin-like immunoreactivity, which eluted in a position close to that of lizard helodermin. Helodermin stimulated basal thyroid hormone secretion and colloid droplet formation in conscious mice. The effect of large doses of helodermin was quite long-lasting and the maximal response occurred after 2-6 hr. In addition, helodermin suppressed the incorporation of calcium into bone in conscious rats. The findings suggest that helodermin-like peptides in C cells may be involved in the local regulation of thyroid hormone secretion and in the maintenance of calcium homeostasis. Images PMID:2645580

  10. Melanoma Therapy with Rhenium-Cyclized Alpha Melanocyte Stimulating Hormone Peptide Analogs

    SciTech Connect

    Thomas P Quinn

    2005-11-22

    Malignant melanoma is the 6th most commonly diagnosed cancer with increasing incidence in the United States. It is estimated that 54,200 cases of malignant melanoma will be newly diagnosed and 7,600 cases of death will occur in the United States in the year 2003 (1). At the present time, more than 1.3% of Americans will develop malignant melanoma during their lifetime (2). The average survival for patients with metastatic melanoma is about 6-9 months (3). Moreover, metastatic melanoma deposits are resistant to conventional chemotherapy and external beam radiation therapy (3). Systematic chemotherapy is the primary therapeutic approach to treat patients with metastatic melanoma. Dacarbazine is the only single chemotherapy agent approved by FDA for metastatic melanoma treatment (5). However, the response rate to Dacarbazine is only approximately 20% (6). Therefore, there is a great need to develop novel treatment approaches for metastatic melanoma. The global goal of this research program is the rational design, characterization and validation of melanoma imaging and therapeutic radiopharmaceuticals. Significant progress has been made in the design and characterization of metal-cyclized radiolabeled alpha-melanocyte stimulating hormone peptides. Therapy studies with {sup 188}Re-CCMSH demonstrated the therapeutic efficacy of the receptor-targeted treatment in murine and human melanoma bearing mice (previous progress report). Dosimetry calculations, based on biodistribution data, indicated that a significant dose was delivered to the tumor. However, {sup 188}Re is a very energetic beta-particle emitter. The longer-range beta-particles theoretically would be better for larger tumors. In the treatment of melanoma, the larger primary tumor is usually surgically removed leaving metastatic disease as the focus of targeted radiotherapy. Isotopes with lower beta-energies and/or shorter particle lengths should be better suited for targeting metastases. The {sup 177}Lu

  11. Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen-induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts.

    PubMed

    Shiratsuchi, Eri; Ura, Megumi; Nakaba, Misako; Maeda, Iori; Okamoto, Kouji

    2010-11-01

    We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5- to 4.5-fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10(-1) µg/ml, while maximal response was at 10(2) µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. PMID:20853312

  12. Nucleobindin-1 encodes a nesfatin-1-like peptide that stimulates insulin secretion.

    PubMed

    Ramesh, Naresh; Mohan, Haneesha; Unniappan, Suraj

    2015-05-15

    Nesfatin-1 (82 amino acid) is an anorexigenic and insulinotropic peptide encoded in a secreted precursor, nucleobindin-2 (NUCB2). Nucleobindin-1 (NUCB1) is a protein with very high sequence similarity to NUCB2. We hypothesized that a nesfatin-1 like peptide (NLP) is encoded in NUCB1, and this peptide is biologically active. In silico analysis found a signal peptide cleavage site at position 25 (Arginine) and 26 (Valine) preceding the NLP region in NUCB1 sequence, and potential proprotein convertase cleavage sites at Lys-Arg (KR), forming a 77 amino acid NLP. RT-PCR studies found NUCB1 mRNA in both pancreas and MIN6 cells. NUCB1-like immunoreactivity was detected in mouse insulinoma (MIN6) cells, and pancreatic islet beta cells of mice. In order to determine the biological activity of NLP, MIN6 cells were incubated with synthetic rat NLP. NLP (10nM and 100nM) upregulated preproinsulin mRNA expression and insulin secretion at 1h post-incubation. In identical experiments using MIN6 cells, a scrambled peptide based on the NLP sequence did not elicit any effects on preproinsulin mRNA expression or insulin secretion. From this result, it is clear that an intact NLP sequence is required for its biological activity. NLP appears as another endogenous insulinotropic peptide encoded in NUCB1. PMID:25907657

  13. Ingramon, a Peptide Inhibitor of MCP-1 Chemokine, Reduces Migration of Blood Monocytes Stimulated by Glioma-Conditioned Medium.

    PubMed

    Krasnikova, T L; Arefieva, T I; Pylaeva, E A; Sidorova, M V

    2016-02-01

    Malignant gliomas are most common and fatal primary brain tumors. In addition to neoplastic cells, the tumor tissue contains microglial cells and monocyte-derived macrophages. It is an established fact that monocyte recruiting promotes the tumor growth and dissemination. Monocyte chemotactic protein-1 (MCP-1) is the major attractant for monocytes. We have previously synthesized an MCP-1 antagonist ingramon, a synthetic peptide fragment (65-76) of this chemokine. In the present study, we demonstrated that glioma-conditioned medium contains MCP-1 and stimulates migration of blood monocytes. Ingramon inhibited the effect of glioma-conditioned medium on monocyte migration. PMID:26906197

  14. Proteolytic degradation and deactivation of amphibian skin peptides obtained by electrical stimulation of their dorsal glands.

    PubMed

    Samgina, Tatiana Yu; Tolpina, Miriam I; Hakalehto, Elias; Artemenko, Konstantin A; Bergquist, Jonas; Lebedev, Albert T

    2016-05-01

    Amphibians are among the oldest creatures on our planet. Their only defensive weapon efficient against microorganisms and predators involves their skin secretion. The wide range of biological activities of the peptides in the skin secretion of amphibians makes these compounds rather interesting for generation of prospective pharmaceuticals. The first step in studying these molecules requires their structures to be established. Mass spectrometry is the most powerful tool for this purpose. The sampling and sample preparation stages preceding mass spectrometry experiments appear to be rather crucial. The results obtained here demonstrate that these preparation procedures might lead to partial or complete loss of the bioactive peptides in the secretion. Five minutes in water was enough to completely destroy all of the bioactive peptides in the skin secretion of the marsh frog (Rana ridibunda); even immediate addition of methanol to the water solution of the peptides did not prevent partial destruction. Concerted effort should be directed towards development of the most efficient procedure to keep the secreted peptides intact. Graphical Abstract ᅟ. PMID:26975184

  15. Brain Natriuretic Peptide Stimulates Lipid Metabolism through Its Receptor NPR1 and the Glycerolipid Metabolism Pathway in Chicken Adipocytes.

    PubMed

    Huang, H Y; Zhao, G P; Liu, R R; Li, Q H; Zheng, M Q; Li, S F; Liang, Z; Zhao, Z H; Wen, J

    2015-11-01

    Brain natriuretic peptide (BNP) is related to lipid metabolism in mammals, but its effect and the molecular mechanisms underlying it in chickens are incompletely understood. We found that the level of natriuretic peptide precursor B (NPPB, which encodes BNP) mRNA expression in high-abdominal-fat chicken groups was significantly higher than that of low-abdominal-fat groups. Partial correlations indicated that changes in the weight of abdominal fat were positively correlated with NPPB mRNA expression level. In vitro, compared with the control group, preadipocytes with NPPB interference showed reduced levels of proliferation, differentiation, and glycerin in media. Treatments of cells with BNP led to enhanced proliferation and differentiation of cells and glycerin concentration, and mRNA expression of its receptor natriuretic peptide receptor 1 (NPR1) was upregulated significantly. In cells exposed to BNP, 482 differentially expressed genes were identified compared with controls without BNP. Four genes known to be related to lipid metabolism (diacylglycerol kinase; lipase, endothelial; 1-acylglycerol-3-phosphate O-acyltransferase 1; and 1-acylglycerol-3-phosphate O-acyltransferase 2) were enriched in the glycerolipid metabolism pathway and expressed differentially. In conclusion, BNP stimulates the proliferation, differentiation, and lipolysis of preadipocytes through upregulation of the levels of expression of its receptor NPR1 and key genes enriched in the glycerolipid metabolic pathway. PMID:26463554

  16. Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts

    PubMed Central

    Chou, Ming-Lun; Chu, Chiung-Chih; Chen, Lih-Jen; Akita, Mitsuru; Li, Hsou-min

    2006-01-01

    Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing. The Tic40 C-terminal domain, which is homologous to the C terminus of cochaperones Sti1p/Hop and Hip but with no known function, stimulates adenosine triphosphate hydrolysis by Hsp93. Hsp93 dissociates from Tic40 in the presence of adenosine diphosphate, suggesting that Tic40 functions as an adenosine triphosphatase activation protein for Hsp93. Our data suggest that chloroplasts have evolved the Tic40 cochaperone to increase the efficiency of precursor processing and translocation. PMID:17158958

  17. Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti.

    PubMed

    Castillo, Julio; Brown, Mark R; Strand, Michael R

    2011-10-01

    All vector mosquito species must feed on the blood of a vertebrate host to produce eggs. Multiple cycles of blood feeding also promote frequent contacts with hosts, which enhance the risk of exposure to infectious agents and disease transmission. Blood feeding triggers the release of insulin-like peptides (ILPs) from the brain of the mosquito Aedes aegypti, which regulate blood meal digestion and egg formation. In turn, hemocytes serve as the most important constitutive defense in mosquitoes against pathogens that enter the hemocoel. Prior studies indicated that blood feeding stimulates hemocytes to increase in abundance, but how this increase in abundance is regulated is unknown. Here, we determined that phagocytic granulocytes and oenocytoids express the A. aegypti insulin receptor (AaMIR). We then showed that: 1) decapitation of mosquitoes after blood feeding inhibited hemocyte proliferation, 2) a single dose of insulin-like peptide 3 (ILP3) sufficient to stimulate egg production rescued proliferation, and 3) knockdown of the AaMIR inhibited ILP3 rescue activity. Infection studies indicated that increased hemocyte abundance enhanced clearance of the bacterium Escherichia coli at lower levels of infection. Surprisingly, however, non-blood fed females better survived intermediate and high levels of E. coli infection than blood fed females. Taken together, our results reveal a previously unrecognized role for the insulin signaling pathway in regulating hemocyte proliferation. Our results also indicate that blood feeding enhances resistance to E. coli at lower levels of infection but reduces tolerance at higher levels of infection. PMID:21998579

  18. Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

    PubMed Central

    Held, Katharina; Kichko, Tatjana; De Clercq, Katrien; Klaassen, Hugo; Van Bree, Rieta; Vanherck, Jean-Christophe; Marchand, Arnaud; Reeh, Peter W.; Chaltin, Patrick; Voets, Thomas; Vriens, Joris

    2015-01-01

    Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation. PMID:25733887

  19. Structure and function of a peptide pheromone family that stimulate the vomeronasal sensory system in mice.

    PubMed

    Abe, Takayuki; Touhara, Kazushige

    2014-08-01

    Mammals use pheromones to communicate with other animals of the same species. In mice, the VNO (vomeronasal organ) has a pivotal role in pheromone detection. We discovered a 7 kDa peptide, ESP1 (exocrine-gland-secreting peptide 1), in tear fluids from male mice that enhances the sexual behaviour of female mice via the VNO. NMR studies demonstrate that ESP1 adopts a compact structure with a helical fold stabilized by an intramolecular disulfide bridge. Functional analysis in combination with docking simulation indicates that ESP1 is recognized by a specific G-protein-coupled vomeronasal receptor, V2Rp5, via charge-charge interactions in the large extracellular region of the receptor. ESP1 is a member of the ESP family, which comprises 38 homologous genes in mice, and some of these genes are expressed in a sex- or age-dependent manner. Most recently, ESP22 was found to be released specifically in juvenile tear fluids and to inhibit the sexual behaviour of adult male mice. These studies demonstrate that peptide pheromones are used for chemical communication in mice, and they indicate a structural basis for the narrowly tuned perception of mammalian peptide pheromones by vomeronasal receptors. PMID:25109971

  20. Hypergravity differentially modulates cGMP efflux in human melanocytic cells stimulated by nitric oxide and natriuretic peptides

    NASA Astrophysics Data System (ADS)

    Ivanova, K.; Stieber, C.; Lambers, B.; Block, I.; Krieg, R.; Wellmann, A.; Gerzer, R.

    Nitric oxide NO plays a key role in many patho physiologic processes including inflammation and skin cancer The diverse cellular effects of NO are mainly mediated by activation of the soluble guanylyl cyclase sGC isoform that leads to increases in intracellular cGMP levels whereas the membrane-bound isoforms serve as receptors for natriuretic peptides e g ANP In human skin epidermal melanocytes represent the principal cells for skin pigmentation by synthesizing the pigment melanin Melanin acts as a scavenger for free radicals that may arise during metabolic stress as a result of potentially harmful effects of the environment In previous studies we found that long-term exposure to hypergravity stimulated cGMP efflux in normal human melanocytes NHMs and non-metastatic melanoma cells at least partly by an enhanced expression of the multidrug resistance proteins MRP and cGMP transporters MRP4 5 The present study investigated whether hypergravity generated by centrifugal acceleration may modulate the cGMP efflux in NO-stimulated NHMs and melanoma cells MCs with different metastatic potential The NONOates PAPA-NO and DETA-NO were used as direct NO donors for cell stimulation In the presence of 0 1 mM DETA-NO t 1 2 sim 20 h long-term application of hypergravity up to 5 g for 24 h reduced intracellular cGMP levels by stimulating cGMP efflux in NHMs and non-metastatic MCs in comparison to 1 g whereas exposure to 5 g for 6 h in the presence of 0 1 mM PAPA-NO t 1 2 sim 30 min was not effective The hypergravity-stimulated

  1. Peptides in the gastrointestinal tract in human immunodeficiency virus infection. The GI/HIV Study Group of the University of Calgary.

    PubMed

    Sharkey, K A; Sutherland, L R; Davison, J S; Zwiers, H; Gill, M J; Church, D L

    1992-07-01

    The presence of immunoreactivity to the neuronal phosphoprotein B-50 and the peptides bombesin, calcitonin gene-related peptide, galanin, neurotensin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal polypeptide was examined in biopsy specimens from the duodenum and rectum of human immunodeficiency virus (HIV)-seropositive and HIV-seronegative male homosexual patients. The distribution of B-50 and the peptides was correlated with HIV serology, number of CD4+ lymphocytes, and the presence of HIV in biopsy culture. There was a very low incidence of enteric pathogens in both groups of patients. It was found that HIV-seropositive patients had a greater incidence of abnormal patterns of immunoreactivity (reduced intensity and/or density of innervation) in enteric nerves and enteroendocrine cells than HIV-seronegative patients. A reduction of substance P immunoreactivity was significantly correlated with reduced CD4+ lymphocyte count and HIV status; a similar trend was also seen for somatostatin and vasoactive intestinal polypeptide. Using B-50 as a marker, it was found that both groups of patients had altered patterns of immunoreactivity in rectal nerves. The findings of this study suggest that some of the clinical symptoms associated with HIV infection may be caused by a specific HIV enteropathy that influences enteric nerve and/or enteroendocrine cell function by altering the density of peptide immunoreactivity. PMID:1535325

  2. Extracellular Life Cycle of ComS, the Competence-Stimulating Peptide of Streptococcus thermophilus

    PubMed Central

    Besset, Colette; Gitton, Christophe; Guillot, Alain; Fontaine, Laetitia; Hols, Pascal; Monnet, Véronique

    2013-01-01

    In streptococci, ComX is the alternative sigma factor controlling the transcription of the genes encoding the genetic transformation machinery. In Streptococcus thermophilus, comX transcription is controlled by a complex consisting of a transcriptional regulator of the Rgg family, ComR, and a signaling peptide, ComS, which controls ComR activity. Following its initial production, ComS is processed, secreted, and imported back into the cell by the Ami oligopeptide transporter. We characterized these steps and the partners interacting with ComS during its extracellular circuit in more detail. We identified the mature form of ComS and demonstrated the involvement of the membrane protease Eep in ComS processing. We found that ComS was secreted but probably not released into the extracellular medium. Natural competence was first discovered in a chemically defined medium without peptides. We show here that the presence of a high concentration of nutritional peptides in the medium prevents the triggering of competence. In milk, the ecological niche of S. thermophilus, competence was found to be functional, suggesting that the concentration of nutritional peptides was too low to interfere with ComR activation. The kinetics of expression of the comS, comR, and comX genes and of a late competence gene, dprA, in cultures inoculated at different initial densities revealed that the activation mechanism of ComR by ComS is more a timing device than a quorum-sensing mechanism sensu stricto. We concluded that the ComS extracellular circuit facilitates tight control over the triggering of competence in S. thermophilus. PMID:23396911

  3. A dimeric peptide with erythropoiesis-stimulating activity uniquely affects erythropoietin receptor ligation and cell surface expression.

    PubMed

    Verma, Rakesh; Green, Jennifer M; Schatz, Peter J; Wojchowski, Don M

    2016-08-01

    Erythropoiesis-stimulating agents (ESAs) that exert long-acting antianemia effects have been developed recently, but their mechanisms are poorly understood. Analyses reveal unique erythropoietin receptor (EPOR)-binding properties for one such ESA, the synthetic EPOR agonist peginesatide. Compared with recombinant human EPO and darbepoietin, peginesatide exhibited a slow on rate, but sustained EPOR residency and resistant displacement. In EPO-dependent human erythroid progenitor UT7epo cells, culture in peginesatide unexpectedly upmodulated endogenous cell surface EPOR levels with parallel increases in full-length EPOR-68K levels. These unique properties are suggested to contribute to the durable activity of this (and perhaps additional) dimeric peptide hematopoietic growth factor receptor agonist. PMID:27174804

  4. Nesfatin-1 stimulates cholecystokinin and suppresses peptide YY expression and secretion in mice.

    PubMed

    Ramesh, Naresh; Mortazavi, Sima; Unniappan, Suraj

    2016-03-25

    Nesfatin-1 is an 82 amino acid secreted peptide encoded in the precursor, nucleobindin-2 (NUCB2). It is an insulinotropic anorexigen abundantly expressed in the stomach and hypothalamus. Post-prandial insulin secretion is predominantly regulated by incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Nesfatin-1 was previously reported to modulate GLP-1 and GIP secretion in vitro in an enteroendocrine (STC-1) cell line. Intestine is a source of additional hormones including cholecystokinin (CCK) and peptide YY (PYY) that regulate metabolism. We hypothesized that nesfatin-1 modulates CCK and PYY secretion. Immunofluorescence histochemistry showed NUCB2/nesfatin-1 co-localizing CCK and PYY in the intestinal mucosa of mice. Static incubation of STC-1 cells with nesfatin-1 upregulated both CCK mRNA expression (1 and 10 nM) and secretion (0.1, 1 and 10 nM) at 1 h post-incubation. In contrast, nesfatin-1 treatment for 1 h downregulated PYY mRNA expression (all doses tested) and secretion (0.01 and 0.1 nM) in STC-1 cells. Continuous infusion of nesfatin-1 using osmotic mini-pumps for 12 h upregulated CCK mRNA expression in large intestine, and downregulated PYY mRNA expression in both large and small intestines of male C57BL/6J mice. In these tissues, Western blot analysis found a corresponding increase in CCK and a decrease in PYY content. Collectively, we provide new information on the cell specific localization of NUCB2/nesfatin-1 in the intestinal mucosa, and a novel function for nesfatin-1 in modulating intestinal CCK and PYY expression and secretion in mice. PMID:26920055

  5. Reducing renal uptake of 90Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues

    SciTech Connect

    Miao, Yubin; Fisher, Darrell R.; Quinn, Thomas P.

    2006-06-15

    The purpose of this study was to improve the tumor-to-kidney uptake ratios of 90Y- and 177Lu-[1,2,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Re-Cys,D-Phe,Arg]alpha-melanocyte stimulating hormone (DOTA-RE(Arg)CCMSH), through coupling a negatively charged glutamic acid (Glu) to the peptide sequence. A new peptide of DOTA-Re(Glu,Arg)CCMSH was designed, synthesized and labeled with 90Y and 177Lu. Pharmacokinetics of 90Y- and 177Lu-DOTA-RE(Glu,Arg)CCNSH were determined in B16/F1 murine melanoma-bearing C57 mice. Both exhibited significantly less renal uptake than 90Y- and 177Lu-DOTA-Re(Arg)CCMSH at 30 min and at 2, 3, and 24 h after dose administration. The renal uptake values of 90Y- and 177Lu-DOTA-Re(Glu,Arg)CCMSH were 28.16% and 28.81% of those of 90Y- and 177Lu-DOTA-RE(Arg)CCMSH, respectively, at 4 hr post-injection. We also showed higher tumor-to-kidney uptake ratios 2.28 and 1.69 times that of 90Y- and 177Lu-DOTA-Re(Arg)CCMSH, respectively, at 4 h post-injection. The90Y- and 177Lu-DOTA-Re(Glu,Arg)CCMSH activity accumulation was low in normal organs except for kidneys. Coupling a negatively charged amino acid (Glu) to the CCMSH peptide sequence dramatically reduced the renal uptake values and increased the tumor-to-kidney uptake ratios of 90Y- and 177Lu-DOTA-Re(Glu,Arg)CCMSH, facilitating their potential applications as radiopharmaceuticals for targeted radionuclide therapy of melanoma.

  6. Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors.

    PubMed

    Thibault, G; Doubell, A F; Garcia, R; Larivière, R; Schiffrin, E L

    1994-03-01

    Endothelin (ET), a potent vasoconstrictor peptide, is known to enhance the secretion of atrial natriuretic factor (ANF) by the heart. In the present study, we investigated the potency of ET isopeptides to stimulate ANF and brain natriuretic peptide (BNP) secretion in primary cultures of neonatal atrial myocytes, and we characterized the receptor mediating these effects. All ET isopeptides caused a twofold increase of ANF and BNP secretion with the following order of potency: ET-1 approximately ET-2 > sarafotoxin 6b > ET-3. Secretion of the natriuretic peptides was blocked by BQ-123, an ETA-receptor antagonist, but was not affected by either IRL-1620 or [Ala1,3,11,15]ET-1, two ETB-receptor agonists. ET receptors were localized by autoradiography on the surface of atrial myocytes, indicating that contaminating cells were not responsible for 125I-ET-1 binding. Competition binding analyses were then used to assess the ET-receptor subtype on atrial myocyte membrane preparations. A high-affinity (100 pmol/L) binding site with high density (approximately 1500 fmol/mg) was found to preferentially bind the ET isopeptides in the following order: ET-1 > or = ET-2 > or = sarafotoxin 6b > ET-3. Binding was totally displaced by BQ-123 but not by IRL-1620. The ET binding site therefore had the characteristics of an ETA-like receptor. Analysis by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that it possessed a molecular mass of approximately 50 kD. Northern blot analysis of both ETA- and ETB-receptor mRNAs allowed only the detection of the former, indicating that the ETB receptor may be expressed in very small amounts. These results demonstrate that ANF and BNP secretion by atrial myocytes is enhanced by ET via binding to an ETA-like receptor. PMID:8118954

  7. GH-releasing peptide-2 does not stimulate arginine vasopressin secretion in healthy men.

    PubMed

    Kamoi, Kyuzi; Minagawa, Shinichi; Kimura, Keita; Ishizawa, Masahiro; Ohara, Nobumasa; Uemura, Yasuyuki; Tsuchiya, Junpei

    2010-01-01

    Ghrelin has a stimulating effect on arginine vasopressin (AVP). However, it is not known whether GHRP-2, a synthetic ghrelin receptor agonist, also has a stimulating effect on AVP release in men. To determine whether the GHRP-2 test is useful for assessing AVP secretion, blood ACTH, GH, FSH, LH, PRL, TSH and AVP levels, as well as glucose, osmolality, sodium and hematocrit, were measured before and 15, 30, 45 and 60 min after an intravenous bolus of 100 microg GHRP-2 in 10 healthy men with and without fasting. Blood pressure was measured at 15-min intervals. AVP secretion was not stimulated by the GHRP-2 test with and without fasting. There were no significant differences in hematocrit, blood pressure and plasma osmolality before and after GFRP-2 injection, although significant (p<0.001) peak blood GH, and ACTH and PRL levels were observed 30 and 15 min after GHRP-2 injection with and without fasting, respectively, and the maximal peaks were significantly (p<0.05) higher with fasting than without fasting. These results suggest that AVP secretion is not stimulated by the GHRP-2 test both with and without fasting, though GH, ACTH and PRL levels were higher with than without fasting. PMID:19907099

  8. The selective non-peptidic delta opioid agonist SNC80 does not facilitate intracranial self-stimulation in rats

    PubMed Central

    Carmo, Gail Pereira Do; Folk, John E.; Rice, Kenner C.; Chartoff, Elena; Carlezon, William A.; Negus, S. Stevens

    2009-01-01

    Delta opioid receptor agonists are under development for a variety of clinical applications, and some findings in rats raise the possibility that agents with this mechanism have abuse liability. The present study assessed the effects of the non-peptidic delta opioid agonist SNC80 in an assay of intracranial self-stimulation (ICSS) in rats. ICSS was examined at multiple stimulation frequencies to permit generation of frequency-response rate curves and evaluation of curve shifts produced by experimental manipulations. Drug-induced leftward shifts in ICSS frequency-rate curves are often interpreted as evidence of abuse liability. However, SNC80 (1.0-10 mg/kg s.c.; 10-56 mg/kg i.p.) failed to alter ICSS frequency-rate curves at doses up to those that produced convulsions in the present study or other effects (e.g. antidepressant effects) in previous studies. For comparison, the monoamine releaser d-amphetamine (0.1-1.0 mg/kg, i.p.) and the kappa agonist U69,593 (0.1-0.56 mg/kg, i.p.) produced dose-dependent leftward and rightward shifts, respectively, in ICSS frequency-rate curves, confirming the sensitivity of the procedure to drug effects. ICSS frequency-rate curves were also shifted by two non-pharmacological manipulations (reductions in stimulus intensity and increases in response requirement). Thus, SNC80 failed to facilitate or attenuate ICSS-maintained responding under conditions in which other pharmacological and non-pharmacological manipulations were effective. These results suggest that non-peptidic delta opioid receptor agonists have negligible abuse-related effects in rats. PMID:19133255

  9. A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA*

    PubMed Central

    Yordanova, Martina M.; Wu, Cheng; Andreev, Dmitry E.; Sachs, Matthew S.; Atkins, John F.

    2015-01-01

    The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3′ end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5′ and 3′ of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5′ of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5′ part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3′ part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3′ of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting. PMID:25998126

  10. Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides.

    PubMed

    Roberts, Andrew G; Johnston, Eric V; Shieh, Jae-Hung; Sondey, Joseph P; Hendrickson, Ronald C; Moore, Malcolm A S; Danishefsky, Samuel J

    2015-10-14

    Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation-desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone. PMID:26401918

  11. Short-term glucagon stimulation test of C-peptide effect on glucose utilization in patients with type 1 diabetes mellitus.

    PubMed

    Mojto, Viliam; Rausova, Zuzana; Chrenova, Jana; Dedik, Ladislav

    2015-12-01

    This work aimed to evaluate the use of a four-point glucagon stimulation test of C-peptide effect on glucose utilization in type 1 diabetic patients using a new mathematical model. A group of 32 type 1 diabetic patients and a group of 10 healthy control subjects underwent a four-point glucagon stimulation test with blood sampling at 0, 6, 15 and 30 min after 1 mg glucagon bolus intravenous administration. Pharmacokinetic and pharmacokinetic/pharmacodynamic models of C-peptide effect on glucose utilization versus area under curve (AUC) were used. A two-sample t test and ANOVA with Bonferroni correction were used to test the significance of differences between parameters. A significant difference between control and patient groups regarding the coefficient of whole-body glucose utilization and AUC C-peptide/AUC glucose ratio (p ≪ 0.001 and p = 0.002, respectively) was observed. The high correlation (r = 0.97) between modeled coefficient of whole-body glucose utilization and numerically calculated AUC C-peptide/AUC glucose ratio related to entire cohort indicated the stability of used method. The short-term four-point glucagon stimulation test allows the numerically calculated AUC C-peptide/AUC glucose ratio and/or the coefficient of whole-body glucose utilization calculated from model to be used to diagnostically identify type 1 diabetic patients. PMID:26607818

  12. Biphasic Peptide Amphiphile Nanomatrix Embedded with Hydroxyapatite Nanoparticles for Stimulated Osteoinductive Response

    PubMed Central

    Anderson, Joel M.; Patterson, Jessica L.; Vines, Jeremy B.; Javed, Amjad; Gilbert, Shawn R.; Jun, Ho-Wook

    2013-01-01

    Formation of the native bone extracellular matrix (ECM) provides an attractive template for bone tissue engineering. The structural support and biological complexity of bone ECM are provided within a composite microenvironment that consists of an organic fibrous network reinforced by inorganic hydroxyapatite (HA) nanoparticles. Recreating this biphasic assembly, a bone ECM analogous scaffold comprised of self-assembling peptide amphiphile (PA) nanofibers and interspersed HA nanoparticles was investigated. PAs were endowed with biomolecular ligand signaling using a synthetically inscribed peptide sequence (i.e. RGDS) and integrated with HA nanoparticles to form a biphasic nanomatrix hydrogel. It was hypothesized the biphasic hydrogel would induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) and improve bone healing as mediated by RGDS ligand signaling within PA nanofibers and embedded HA mineralization source. Viscoelastic stability of the biphasic PA hydrogels was evaluated with different weight concentrations of HA for improved gelation. After demonstrating initial viability, long-term cellularity and osteoinduction of encapsulated hMSCs in different PA hydrogels were studied in vitro. Temporal progression of osteogenic maturation was assessed by gene expression of key markers. A preliminary animal study demonstrated bone healing capacity of the biphasic PA nanomatrix under physiological conditions using a critical size femoral defect rat model. The combination of RGDS ligand signaling and HA nanoparticles within the biphasic PA nanomatrix hydrogel demonstrated the most effective osteoinduction and comparative bone healing response. Therefore, the biphasic PA nanomatrix establishes a well-organized scaffold with increased similarity to natural bone ECM with the prospect for improved bone tissue regeneration. PMID:22077993

  13. Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa.

    PubMed

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  14. Human Host Defense Peptide LL-37 Stimulates Virulence Factor Production and Adaptive Resistance in Pseudomonas aeruginosa

    PubMed Central

    Strempel, Nikola; Neidig, Anke; Nusser, Michael; Geffers, Robert; Vieillard, Julien; Lesouhaitier, Olivier; Brenner-Weiss, Gerald; Overhage, Joerg

    2013-01-01

    A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor. PMID:24349231

  15. Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Seroogy, K B; Millhorn, D E

    1991-01-01

    In situ hybridization was used to determine whether genes for neuropeptides [substance P/neurokinin A (SP/NKA), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide tyrosine (NPY) and cholecystokinin (CCK)] are expressed in inferior ganglia of the vagus (nodose) and glossopharyngeal (petrosal) nerves. Synthetic oligodeoxyribonucleotides, complementary to the cognate, mRNAs were labeled with [32P] or [35S], and hybridized to 10 microns thick sections of unperfused tissue which were then processed for film and emulsion autoradiography. We found numerous, clustered neuronal perikarya throughout the nodose and petrosal ganglia that expressed preprotachykinin A (SP/NKA) and CGRP mRNAs to varying degrees. Neurons expressing preproSOM mRNA were less abundant and more scattered throughout both ganglia. Notably, we found mRNA for NPY in cells (usually 5-10 per section) in both ganglia. To our knowledge, this is first evidence for NPY in these sensory ganglia. In contrast to previous immunohistochemical findings, we found no evidence for expression of preproCCK in either the nodose or petrosal ganglia. The present findings demonstrate that cells of the nodose and petrosal ganglia express the genes for a number of neuropeptides that are presumably involved with transmission of visceral sensory afferent information to higher order neurons of the central nervous system. PMID:1708726

  16. alpha-Lactalbumin hydrolysate stimulates glucagon-like peptide-2 secretion and small intestinal growth in suckling rats.

    PubMed

    Izumi, Hirohisa; Ishizuka, Satoshi; Inafune, Ayako; Hira, Tohru; Ozawa, Kazuhiro; Shimizu, Takashi; Takase, Mitsunori; Hara, Hiroshi

    2009-07-01

    We investigated whether bovine milk constituents influenced glucagon-like peptide (GLP)-2 secretion and intestinal growth in suckling rats. Male Sprague-Dawley rats (14 d old) received i.g. infusions of a milk protein fraction, a lactose solution, or the cream fraction of milk. The serum concentration of GLP-2, but not GLP-1, markedly increased in rats administered milk protein compared with those given the lactose solution or the cream fraction from 60 to 120 min after administration. In another experiment, both casein (CN) and whey protein isolate stimulated GLP-2 secretion at 120 min after administration, but soy protein and ovalbumin did not. Stimulation of GLP-2 secretion by several milk proteins was similar, including alpha-CN, alpha-lactalbumin (alpha-La), and beta-lactoglobulin, in a separate experiment. A hydrolysate of alpha-La obtained by incubation with protease A extracted from Aspergillus oryzae (LaHPA) caused almost twice the GLP-2 release due to intact alpha-La and other alpha-La hydrolysates. Free amino acid concentrations and molecular size distributions did not differ among alpha-La hydrolysates, including LaHPA. In rat pups reared with milk formulae containing alpha-La or LaHPA, LaHPA significantly promoted small intestinal elongation and increased the number of crypt epithelial cells compared with a formula containing intact alpha-La. LaHPA administration also increased the maltase:lactase activity ratio, a marker of maturation of the intestinal mucosa. In conclusion, milk proteins stimulate GLP-2 secretion and contribute to growth and maturation of the small intestine in suckling rats. PMID:19494023

  17. Acute effects of morphine and opioid peptides on the motility and responses of rat colon to electrical stimulation.

    PubMed Central

    Gillan, M. G.; Pollock, D.

    1980-01-01

    1 Morphine and leucine- and methionine-enkephalins inhibited the contractile response of the pithed rat colon to electrical stimulation of the spinal motor outflows and inhibited motor responses of the isolated colon to field stimulation. 2 Morphine and the opioid peptides also had an excitatory action in the colon. In the pithed rat, opiates caused regular fluctuations in intracolonic pressure and in the isolated colon, caused regular waves of contraction. This excitatory response was produced by low concentrations of the enkephalins (2 X 10(-8) M, 2 X 10(-9) M), was stereospecific and was antagonized by naloxone. 3 Opiate-induced contractions in the isolated colon were inhibited by catecholamines, adenine nucleotides and by phosphodiesterase inhibitors. These contractions were unaffected by ergotamine and tolazoline, or by propranolol. 4 The excitatory action of opiates in the isolated colon was not antagonized and usually was potentiated by atropine, (+)-tubocurarine and hexamethonium. In the absence of opiates, these drugs also produced similar waves of contraction, which were unaffected by naloxone. 5 Opiate-induced contractions occurred in colon rendered unresponsive to 5-hydroxytryptamine (5-HT) and these contractions were potentiated by the 5-HT antagonist, lysergic acid diethylamide, which, when administered alone, caused similar contractions. The 5-HT antagonist, cyproheptadine, inhibited opiate-induced contractions but was non-specific, since it also inhibited responses of the colon to carbachol and KC1. 6 Opiate-induced contractions were unaffected by procaine and were potentiated by tetrodotoxin. Both of these drugs, when administered alone, produced waves of contractions, which were similar to those produced by opiates but were unaffected by naloxone. 7 Contractions produced in the isolated colon either by opiates, atropine or (+)-tubocurarine, or any combination of these drugs, were inhibited by field stimulation applied at the peak of a wave of

  18. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C

    SciTech Connect

    Zurier, B.B.; Kozma, M.; Sinnett-Smith, J.; Rozengurt, E. )

    1988-05-01

    Vasoactive intestinal peptide synergistically stimulated initiation of DNA synthesis in Swiss 3T3 cells. The peptide stimulated ({sup 3}H)thymidine incorporation in the presence of insulin and either forskolin or an inhibitor of cAMP phosphodiesterase in a concentration-dependent manner. Half-maximal effect was obtained at 1 nM. At mitogenic concentrations, VIP stimulated a marked accumulation (eightfold) of cAMP. In contrast to other growth-promoting neuropeptides, VIP did not induce an increase in cytosolic free Ca{sup 2+} or the activation of protein kinase C. The authors conclude that neuropeptides can modulate long-term cell proliferation through multiple signaling pathways.

  19. Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism.

    PubMed

    García-Rincón, Juan; Darszon, Alberto; Beltrán, Carmen

    2016-04-01

    Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca(2+) concentration ([Ca(2+)]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca(2+)]i in an external Ca(2+) concentration ([Ca(2+)]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca(2+)]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca(2+) and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca(2+)]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it. PMID:26772728

  20. Tc-99m-labeled RGD-conjugated alpha-melanocyte stimulating hormone hybrid peptides with reduced renal uptake

    PubMed Central

    Yang, Jianquan; Hu, Chien-An

    2015-01-01

    The purpose of this study was to examine whether the replacement of the positively-charged Lys or Arg linker with a neutral linker could reduce the renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide. The RGD motif {cyclic(Arg-Gly-Asp-dTyr-Asp)} was coupled to [Cys3,4,10, d-Phe7, Arg11]α-MSH3–13 {(Arg11)CCMSH} through the neutral βAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH hybrid peptides. The receptor binding affinity and cytotoxicity of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of 99mTc-RGD-βAla-(Arg11)CCMSH and 99mTc-RGD-Ahx-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the βAla or Ahx linker retained nanomolar receptor binding affinities and remarkable cytotoxicity of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH. The receptor binding affinities of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH were 0.8 and 1.3 nM. Three-hour incubation with 0.1 µM of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH decreased the survival percentages of B16/F1 cells by 71 and 67% as compared to the untreated control cells five days post the treatment. The replacement of the Arg linker with the βAla or Ahx linker reduced the non-specific renal uptake of 99mTc-RGD-βAla-(Arg11)CCMSH and 99mTc-RGD-Ahx-(Arg11)CCMSH by 62% and 61% at 2 h post-injection. 99mTc-RGD-βAla-(Arg11)CCMSH displayed higher melanoma uptake than 99mTc-RGD-Ahx-(Arg11)CCMSH at 0.5, 2, 4 and 24 h post-injection. Enhanced tumor to kidney uptake ratio of 99mTc-RGD-βAla-(Arg11)CCMSH warranted the further evaluation of 188Re-labeled RGD-βAla-(Arg11)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future. PMID:25557051

  1. Amyloid β peptide stimulates platelet activation through RhoA-dependent modulation of actomyosin organization.

    PubMed

    Sonkar, Vijay K; Kulkarni, Paresh P; Dash, Debabrata

    2014-04-01

    Platelets contribute to 95% of circulating amyloid precursor protein in the body and have widely been employed as a "peripheral" model of neurons in Alzheimer's disease. We sought to analyze the effects of amyloid β (Aβ) on platelets and to understand the underlying molecular mechanism. The Aβ active fragment containing amino acid sequence 25-35 (Aβ(25-35); 10-20 μM) was found to induce strong aggregation of human platelets, granule release, and integrin activation, similar to that elicited by physiological agonists. Platelets exposed to Aβ(25-35) retracted fibrin clot and displayed augmented adhesion to collagen under arterial shear, reflective of a switch to prothrombotic phenotype. Exposure of platelets to Aβ peptide (20 μM) resulted in a 4.2- and 2.3-fold increase in phosphorylation of myosin light chain (MLC) and MLC phosphatase, respectively, which was reversed by Y27632, an inhibitor of Rho-associated coiled-coil protein kinase (ROCK). Aβ(25-35)-induced platelet aggregation and clot retraction were also significantly attenuated by Y27632. Consistent with these findings, Aβ(25-35) elicited a significant rise in the level of RhoA-GTP in platelets. Platelets pretreated with reverse-sequenced Aβ fragment (Aβ(35-25)) and untreated resting platelets served as controls. We conclude that Aβ induces cellular activation through RhoA-dependent modulation of actomyosin, and hence, RhoA could be a potential therapeutic target in Alzheimer's disease and cerebral amyloid angiopathy. PMID:24421399

  2. Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1

    NASA Technical Reports Server (NTRS)

    Ding, Ke-Hong; Zhong, Qing; Isales, Carlos M.; Iscules, C. M. (Principal Investigator)

    2003-01-01

    We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

  3. Conformations of peptide fragments comprising the complete sequence of component III of Chi t I and their relationship to T-cell stimulation.

    PubMed

    Czisch, M; Liebers, V; Bernstein, R; Chen, Z; Baur, X; Holak, T A

    1994-08-16

    Conformational preferences of synthetic peptides that span the complete sequence of Chironomus thummi hemoglobin (Chi t I) component III were studied by nuclear magnetic resonance (NMR) and CD spectroscopies. The peptides, 19-21 amino acids in length, were studied in water, except for the C-terminal peptide, which was investigated in DMSO-d6. NMR showed that all investigated peptides lacked uniquely folded conformations in water at 4 degrees C and pH 3.0 or at 10 degrees C and pD 6.6 in DMSO. However, some preferential helix-like conformations for the peptides corresponding to the helices of the folded protein could be seen in solution. These peptides showed characteristic interactions for conformations in both the beta- and alpha-regions of phi-psi space, based on strong C alpha H(i)-NH(i + 1) interactions, and on NH-NH, C alpha H(i)-NH-(i + 2), C alpha H(i)-NH(i + 3), and C alpha H(i)-C beta H(i + 3) interactions, respectively. Helical motifs seem not to be the most important factors in determining MHC-binding and/or T-cell recognition. However, there is a tendency that more stabilized secondary structures show higher T-cell stimulation. PMID:8068617

  4. The vasorelaxant effect of adrenomedullin, proadrenomedullin N-terminal 20 peptide and amylin in human skin.

    PubMed

    Hasbak, Philip; Eskesen, Karen; Lind, Henrik; Holst, Jan; Edvinsson, Lars

    2006-08-01

    In this study we aimed to assess in vivo, the vasodilator effects of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and amylin in human skin vasculature and compare the responses to the effects mediated by the endogenous neuropeptides calcitonin gene-related peptide (CGRP) and substance P and to examine the mRNA expression of calcitonin receptor-like receptor (CL-R) and receptor-activity modifying proteins, RAMP1, RAMP 2 and RAMP3 in human subcutaneous arteries. Changes in skin blood flow of the forearm were measured using a Laser Doppler Imager after intradermal injection of the peptides. The mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction (real-time PCR). CGRP, adrenomedullin and amylin induced concentration-dependent, long-lasting increases in skin blood flow. The response to PAMP was shorter in duration appearing similar to the transient response induced by substance P. PAMP (10(-6)-10(-5) M) caused distinct itch sensation and local erythema. This effect could be abolished when combining the histamine H1-receptor antagonist mepyramin and PAMP. Real-time PCR data showed a higher level of mRNA for RAMP2 than CL-R, RAMP1 and RAMP3 in the tissue. Though the PCR data demonstrated the presence of mRNA for both CGRP1 and adrenomedullin receptors the rank order of potency (CGRP>adrenomedullin>amylin) for the blood flow increase indicated vasodilatation for these peptides was induced by activation of CGRP1 receptors. Intradermal injection of CGRP, adrenomedullin and amylin induces long lasting dilatation of human skin vasculature by activation of CGRP1 receptors. PAMP induces transient vasodilatation. PAMP but not CGRP, adrenomedullin and amylin causes itch sensation and local erythema. The transient effect on vasodilatation as response to PAMP is discussed. PMID:16918718

  5. Comparative Effects of Prolonged and Intermittent Stimulation of the Glucagon-Like Peptide 1 Receptor on Gastric Emptying and Glycemia

    PubMed Central

    Umapathysivam, Mahesh M.; Lee, Michael Y.; Jones, Karen L.; Annink, Christopher E.; Cousins, Caroline E.; Trahair, Laurence G.; Rayner, Chris K.; Chapman, Marianne J.; Nauck, Michael A.; Horowitz, Michael; Deane, Adam M.

    2014-01-01

    Acute administration of glucagon-like peptide 1 (GLP-1) and its agonists slows gastric emptying, which represents the major mechanism underlying their attenuation of postprandial glycemic excursions. However, this effect may diminish during prolonged use. We compared the effects of prolonged and intermittent stimulation of the GLP-1 receptor on gastric emptying and glycemia. Ten healthy men received intravenous saline (placebo) or GLP-1 (0.8 pmol/kg ⋅ min), as a continuous 24-h infusion (“prolonged”), two 4.5-h infusions separated by 20 h (“intermittent”), and a 4.5-h infusion (“acute”) in a randomized, double-blind, crossover fashion. Gastric emptying of a radiolabeled mashed potato meal was measured using scintigraphy. Acute GLP-1 markedly slowed gastric emptying. The magnitude of the slowing was attenuated with prolonged but maintained with intermittent infusions. GLP-1 potently diminished postprandial glycemia during acute and intermittent regimens. These observations suggest that short-acting GLP-1 agonists may be superior to long-acting agonists when aiming specifically to reduce postprandial glycemic excursions in the treatment of type 2 diabetes. PMID:24089511

  6. Vasoactive intestinal peptide enhanced aromatase activity in the neonatal rat ovary before development of primary follicles or responsiveness to follicle-stimulating hormone

    SciTech Connect

    George, F.W.; Ojeda, S.R.

    1987-08-01

    The authors have investigated the factors that regulate aromatase activity in fetal-neonatal rat ovaries. Ovarian aromatase activity (assessed by measuring the amount of /sup 3/H/sub 2/O formed from (1..beta..-/sup 3/H)testosterone) is low prior to birth and increases to values greater than 30 pmol/hr per mg of protein between days 8 and 12 after birth. The appearance of ovarian aromatase coincides with the development of primordial follicles. Fetal-neonatal ovaries maintained in serum-free organ culture do not develop aromatase activity at the expected time. Ovine follicle-stimulating hormone, ovine luteinizing hormone, or their combination failed to induce the enzyme activity in cultured fetal ovaries, whereas follicle-stimulating hormone is effective in preventing the decline in aromatase activity when postnatal day 8 ovaries are placed in culture. In contrast to follicle-stimulating hormone, dibutyryl-cAMP markedly enhances ovarian aromatase in cultured fetal ovaries. Likewise, enhancement of endogenouse cAMP formation with forskolin or cholera toxin caused an increase in enzyme activity within 24 hr. Vasoactive intestinal peptide, a peptide known to occur in ovarian nerves, caused a dose-dependent increase in aromatase activity in fetal ovaries prior to folliculogenesis. Of related peptides tested, only the peptide having N-terminal histidine and C-terminal isoleucine amide was capable of inducing aromatase activity in fetal ovaries. The fact that VIP can induce aromatase activity in fetal rat ovaries prior to follicle formation and prior to responsiveness to follicle-stimulating hormone suggests that this neuropeptide may play a critical role in ovarian differentiation.

  7. Selective CGRP and adrenomedullin peptide binding by tethered RAMP-calcitonin receptor-like receptor extracellular domain fusion proteins

    PubMed Central

    Moad, Heather E; Pioszak, Augen A

    2013-01-01

    Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries. PMID:24115156

  8. Asthma as an axon reflex.

    PubMed

    Barnes, P J

    1986-02-01

    In asthma, damage to airway epithelium, possibly caused by eosinophil products, exposes C-fibre afferent nerve endings. Stimulation of these endings by inflammatory mediators such as bradykinin may result in an axon (local) reflex with antidromic conduction down afferent nerve collaterals and release of sensory neuropeptides such as substance P, neurokinin A, and calcitonin gene-related peptide. These peptides are potent inducers of airway smooth muscle contraction, bronchial oedema, extravasation of plasma, mucus hypersecretion, and possibly inflammatory cell infiltration and secretion. Thus, axon reflexes could account for at least some of the pathophysiology of asthma and this concept might lead to new strategies for treatment. PMID:2418322

  9. Atrial Natriuretic Peptide Stimulates Dopamine Tubular Transport by Organic Cation Transporters: A Novel Mechanism to Enhance Renal Sodium Excretion

    PubMed Central

    Kouyoumdzian, Nicolás M.; Rukavina Mikusic, Natalia L.; Kravetz, María C.; Lee, Brenda M.; Carranza, Andrea; Del Mauro, Julieta S.; Pandolfo, Marcela; Gironacci, Mariela M.; Gorzalczany, Susana; Toblli, Jorge E.; Fernández, Belisario E.

    2016-01-01

    The aim of this study was to demonstrate the effects of atrial natriuretic peptide (ANP) on organic cation transporters (OCTs) expression and activity, and its consequences on dopamine urinary levels, Na+, K+-ATPase activity and renal function. Male Sprague Dawley rats were infused with isotonic saline solution during 120 minutes and randomized in nine different groups: control, pargyline plus tolcapone (P+T), ANP, dopamine (DA), D-22, DA+D-22, ANP+D-22, ANP+DA and ANP+DA+D-22. Renal functional parameters were determined and urinary dopamine concentration was quantified by HPLC. Expression of OCTs and D1-receptor in membrane preparations from renal cortex tissues were determined by western blot and Na+, K+-ATPase activity was determined using in vitro enzyme assay. 3H-DA renal uptake was determined in vitro. Compared to P+T group, ANP and dopamine infusion increased diuresis, urinary sodium and dopamine excretion significantly. These effects were more pronounced in ANP+DA group and reversed by OCTs blockade by D-22, demonstrating that OCTs are implied in ANP stimulated-DA uptake and transport in renal tissues. The activity of Na+, K+-ATPase exhibited a similar fashion when it was measured in the same experimental groups. Although OCTs and D1-receptor protein expression were not modified by ANP, OCTs-dependent-dopamine tubular uptake was increased by ANP through activation of NPR-A receptor and protein kinase G as signaling pathway. This effect was reflected by an increase in urinary dopamine excretion, natriuresis, diuresis and decreased Na+, K+-ATPase activity. OCTs represent a novel target that links the activity of ANP and dopamine together in a common mechanism to enhance their natriuretic and diuretic effects. PMID:27392042

  10. Increased Litter Size and Suckling Intensity Stimulate mRNA of RFamide-related Peptide in Rats

    PubMed Central

    Noroozi, Atefeh; Jafarzadeh Shirazi, Mohammad Reza; Tamadon, Amin; Moghadam, Ali; Niazi, Ali

    2015-01-01

    Background RFamide-related peptide-3 (RFRP-3) inhibits gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in rats. This study evaluates the effects of litter size and suckling intensity on RFRP mRNA expression in the dorsomedial hypothalamic nucleus (DMH) of rats. Materials and Methods A total of 32 pregnant and 4 non-lactating ovariectomized (control group) Sprague-Dawley rats were used in this experimental study. Lactating rats were allotted to 8 equal groups. In 3 groups, the litter size was adjusted to 5, 10, or 15 pups upon parturition. Dams were allowed to suckle their pups continuously until 8 days postpartum. In the other 3 groups, the litter size was adjusted to 5 pups following birth. These pups were separated from the dams for 6 hours on day 8 postpartum, after which the pups were allowed to suckle for 2.5, 5, or 7.5 minutes prior to killing the dams. In 2 groups, lactating rats with 10 and 15 pups were separated from their pups for 6 hours on day 8 postpartum. In these groups, the pups were allowed to suckle their dams for 5 minutes before the dams were killed. All rats were killed on day 8 postpartum and the DMH was removed from each rat. We evaluated RFRP mRNA expression using realtime polymerase chain reaction (PCR). Results The expression of RFRP mRNA in the DMH increased with increased litter size and suckling intensity compared to the controls. The effect of suckling intensity on the expression of RFRP mRNA was more pronounced compared to the litter size. Conclusion Increased litter size and suckling intensity stimulated RFRP mRNA expression in the DMH which might contribute to lactation anestrus in rats. PMID:26644862

  11. The novel anti-migraine agent rizatriptan inhibits neurogenic dural vasodilation and extravasation.

    PubMed

    Williamson, D J; Shepheard, S L; Hill, R G; Hargreaves, R J

    1997-06-01

    These studies in anaesthetised rats showed, using intravital microscopy, that the novel anti-migraine agent, rizatriptan, significantly reduced electrically stimulated dural vasodilation but had no effect on increases in dural vessel diameter produced by exogenous substance P or calcitonin gene-related peptide (CGRP). Rizatriptan also significantly inhibited dural plasma protein extravasation produced by high intensity electrical stimulation of the trigeminal ganglion. We suggest that rizatriptan inhibits the release of sensory neuropeptides from perivascular trigeminal nerves to prevent neurogenic vasodilation and extravasation in the dura mater. These prejunctional inhibitory effects may be involved in the anti-migraine action of rizatriptan. PMID:9203569

  12. Vasoactive intestinal peptide: A potent stimulator of adenosine 3′:5′-cyclic monophosphate accumulation in gut carcinoma cell lines in culture*

    PubMed Central

    Laburthe, M.; Rousset, M.; Boissard, C.; Chevalier, G.; Zweibaum, A.; Rosselin, G.

    1978-01-01

    Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3′:5′-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3×10-12 M. Maximum VIP-induced cAMP levels were observed with 10-9 M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3×10-10 M VIP. 125I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10-10 and 10-7 M. Half-maximum inhibition of binding was observed with 2×10-9 M VIP. Secretin also stimulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10-7 M, such as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, octapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin E1 and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at 10-8 M; half-maximum stimulation was observed at about 10-9 M. These results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (HT 29 and HRT 18) in culture and provide a model for studying the action of VIP on cell proliferation. PMID:208077

  13. 203Pb-Labeled Alpha-Melanocyte-Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection

    SciTech Connect

    Yubin, Miao; Figueroa, Said D.; Fisher, Darrell R.; Moore, Herbert A.; Testa, Richard F.; Hoffman, Timothy J.; Quinn, Thomas P.

    2008-05-01

    Abbreviations: a-MSH; alpha melanocyte stimulating hormone, DOTA; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Re(Arg11)CCMSH; DOTA-[Cys3,4,10, D-Phe7, Arg11]a-MSH3-13, NDP; [Nle4,d-Phe7] a-MSH3-13. Abstract Peptide-targeted alpha therapy with 200 mCi of 212Pb-DOTA-Re(Arg11)CCMSH cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-day study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient specific dosimetry and to monitor the tumor response to 212Pb-DOTA-Re(Arg11)CCMSH therapy. The purpose of this study was to evaluate the potential of 203Pb-DOTA-Re(Arg11)CCMSH as a matched-pair SPECT imaging agent for 212Pb-DOTA-Re(Arg11)CCMSH. Method: DOTA-Re(Arg11)CCMSH was labeled with 203Pb in 0.5 M NH4OAc buffer at pH 5.4. The internalization and efflux of 203Pb-DOTA-Re(Arg11)CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of 203Pb-DOTA-Re(Arg11)CCMSH were examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT imaging study was performed with 203Pb-DOTA-Re(Arg11)CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection. Results: 203Pb-DOTA-Re(Arg11)CCMSH was easily prepared in NH4OAc buffer and completely separated from the excess non-radiolabeled peptide by RP-HPLC. 203Pb-DOTA-Re(Arg11)CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of 203Pb-DOTA-Re(Arg11)CCMSH activity internalized after a 20-min incubation at 25C. After incubating the cells in culture media for 20 min, 78% of internalized activity remained in the cells. 203Pb-DOTA-Re(Arg11)CCMSH exhibited similar biodistribution pattern with 212Pb-DOTA-Re(Arg11)CCMSH in B16/F1 melanoma-bearing mice. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor uptake of 12.00 +/- 3.20 %ID/g at 1 h post-injection. The tumor uptake gradually decreased to 3.43 +/- 1.12 %ID/g at 48 h post-injection. 203Pb-DOTA-Re(Arg11)CCMSH exhibited the peak tumor to kidney

  14. Melanoma-targeting properties of (99m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues.

    PubMed

    Chen, J; Cheng, Z; Hoffman, T J; Jurisson, S S; Quinn, T P

    2000-10-15

    Preliminary reports have demonstrated that (99m)technetium (Tc)-labeled cyclic [Cys(3,4,10), D-Phe7]alpha-MSH(3-13) (CCMSH) exhibits high tumor uptake and retention values in a murine melanoma mouse model. In this report, the tumor targeting mechanism of 99mTc-CCMSH was studied and compared with four other radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) peptide analogues: 125I-(Tyr2)-[Nle4, D-Phe7]alpha-MSH [125I-(Tyr2)-NDP]; 99mTc-CGCG-NDP; 99mTc-Gly11-CCMSH; and 99mTc-Nle11-CCMSH. In vitro receptor binding, internalization, and cellular retention of radiolabeled alpha-MSH analogues in B16/F1 murine cell line demonstrated that >70% of the receptor-bound radiolabeled analogues were internalized together with the receptor. Ninety % of the internalized 125I-(Tyr2)-NDP, whereas only 36% of internalized 99mTc-CCMSH, was released from the cells into the medium during a 4-h incubation at 37 degrees C. Two mouse models, C57 mice and severe combined immunodeficient (Scid) mice, inoculated s.c. with B16/F1 murine and TXM-13 human melanoma cells were used for the in vivo studies. Tumor uptake values of 11.32 and 2.39 [% injected dose (ID)/g] for 99mTc-CCMSH at 4 h after injection, resulted in an uptake ratio of tumor:blood of 39.0 and 11.5 in murine melanoma-C57 and human melanoma-Scid mouse models, respectively. Two strategies for decreasing the nonspecific kidney uptake of 99mTc-CCMSH, substitution of Lys11 in CCMSH with Gly11 or Nle11, and lysine coinjection, were evaluated. The biodistribution data for the modified peptides showed that Lys11 replacement dramatically decreased the kidney uptake, whereas the tumor uptakes of 99mTc-Nle11- and 99mTc-Gly11-CCMSH were significantly lower than that of 99mTc-CCMSH. Lysine coinjection significantly decreased the kidney uptake (e.g., from 14.6% ID/g to 4.5% ID/g at 4 h after injection in murine melanoma-C57 mice) without significantly changing the value of tumor uptake of 99mTc-CCMSH. In conclusion, the compact

  15. Synthetic peptides based upon a three-dimensional model for the receptor recognition site of follicle-stimulating hormone exhibit antagonistic or agonistic activity at low concentrations.

    PubMed Central

    Hage-van Noort, M; Puijk, W C; Plasman, H H; Kuperus, D; Schaaper, W M; Beekman, N J; Grootegoed, J A; Meloen, R H

    1992-01-01

    Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations

  16. Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

    PubMed

    Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A

    2016-04-15

    Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. PMID:26895962

  17. Regeneration of putative sensory and sympathetic cutaneous nerve endings in the rat foot after sciatic nerve injury.

    PubMed

    Stankovic, N; Johansson, O; Hildebrand, C

    1996-01-01

    The present study examines the occurrence of calcitonin gene-related peptide-, substance P- and tyrosine hydroxylase-like immunoreactive profiles in glabrous and hairy foot skin from normal and nerve-injured rats. After neurotomy/suture, glabrous skin samples contain few calcitonin gene-related peptide-, substance P- and tyrosine hydroxylase-like immunoreactive profies. The number of calcitonin gene-related peptide- and substance P-like immunoreacive profiles in the epidermis is significantly subnormal. Hairy skin from these rats does also contain few calcitonin gene-related peptide-, substance P- and tyrosine hydroxylase-like immunoreactive profiles. In addition, the presence of epidermal calcitonin gene-related peptide-like imunoreactive profiles in glabrous skin is subnormal on the contralateral side. After nerve crush injury, the occurrence of calcitonin gene-related peptide-like, but not substance P-like, immunoreactive profiles in th epidermis of the glabrous skin is significantly subnormal. The occurrence of tyrosine hylase-like immnunoreactive fibres in relation to the digital artery is also subnormal. The occurrence in hairy skin of calcitonin gene-related peptide-like immunoreactive, substance P-like immunoreactive and tyrosine hydroxylase-like immunoreactive profiles is subnormal. In both skin types, the contralateral occurrence of such profiles is subjectively normal. These results show that the occurrence of calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-like immunoreactive profiles in glabrous and hairy foot skin is clearly subnormal after neurotomy and suture and less abnormal after nerve crush. After neurotomy and suture the contralateral side is also affected. PMID:10970110

  18. Pregnancy Increases Relaxation in Human Omental Arteries to the CGRP Family of Peptides.

    PubMed

    Dong, Yuanlin; Betancourt, Ancizar; Chauhan, Madhu; Balakrishnan, Meena; Lugo, Fernando; Anderson, Matthew L; Espinoza, Jimmy; Fox, Karin; Belfort, Michael; Yallampalli, Chandrasekhar

    2015-12-01

    Calcitonin gene-related peptide (CALCB) and its family members adrenomedullin (ADM) and intermedin (ADM2) play important roles in maintaining vascular adaptations during pregnancy in animal models. The present study was designed to evaluate the responses of omental arteries to CALCB, ADM, and ADM2 in pregnant and nonpregnant women, and to determine the mechanisms involved. By using resistance omental arteries collected from nonpregnant women (n = 15) during laparotomy and from term pregnant women (n = 15) at cesarean delivery, this study shows that the receptor components--calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1, 2 and 3--are localized to endothelial and smooth muscle cells in omental arteries, with increased expressions of both mRNA and protein in pregnant compared with nonpregnant women. The myography study demonstrated that CALCB, ADM, and ADM2 (0.1-100 nM) dose dependently relax U46619 (1 muM) precontracted omental artery segments, and the maximum possible effects to CALCB and ADM2, but not to ADM, are significantly enhanced in pregnant compared with nonpregnant women. Further, the vasodilatory responses to CALCB, ADM, and ADM2 are reduced by inhibitors of nitric oxide (NO) synthase (L-NAME), adenylyl cyclase (SQ22536), voltage-activated potassium channels (4-aminopyrodin and tetrabutylammonium), Ca(2+)-activated potassium channel (charybdotoxin), and cyclooxygenase (indomethacin). In conclusion, the CALCB family of peptides, CALCB and ADM2, increase human omental artery relaxation during pregnancy through diverse mechanisms, including NO, endothelium-derived hyperpolarizing factors (EDHFs) and prostaglandins, and thus could contribute to the vascular adaptations during pregnancy in the human. PMID:26510864

  19. LPXRFamide peptide stimulates growth hormone and prolactin gene expression during the spawning period in the grass puffer, a semi-lunar synchronized spawner.

    PubMed

    Shahjahan, Md; Doi, Hiroyuki; Ando, Hironori

    2016-02-01

    Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes. PMID:26385315

  20. Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

    PubMed

    Coffelt, Seth B; Tomchuck, Suzanne L; Zwezdaryk, Kevin J; Danka, Elizabeth S; Scandurro, Aline B

    2009-06-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  1. Leucine Leucine-37 Uses Formyl Peptide Receptor–Like 1 to Activate Signal Transduction Pathways, Stimulate Oncogenic Gene Expression, and Enhance the Invasiveness of Ovarian Cancer Cells

    PubMed Central

    Coffelt, Seth B.; Tomchuck, Suzanne L.; Zwezdaryk, Kevin J.; Danka, Elizabeth S.; Scandurro, Aline B.

    2009-01-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor–like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein–coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37–induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37–stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37–treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1. PMID:19491199

  2. Graphene oxide-stimulated myogenic differentiation of C2C12 cells on PLGA/RGD peptide nanofiber matrices

    NASA Astrophysics Data System (ADS)

    Shin, Y. C.; Lee, J. H.; Kim, M. J.; Hong, S. W.; Oh, J.-W.; Kim, C.-S.; Kim, B.; Hyun, J. K.; Kim, Y.-J.; Han, D.-W.

    2015-07-01

    During the last decade, much attention has been paid to graphene-based nanomaterials because they are considered as potential candidates for biomedical applications such as scaffolds for tissue engineering and substrates for the differentiation of stem cells. Until now, electrospun matrices composed of various biodegradable copolymers have been extensively developed for tissue engineering and regeneration; however, their use in combination with graphene oxide (GO) is novel and challenging. In this study, nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 phage with RGD peptide displayed on its surface (RGD peptide-M13 phage) were prepared as extracellular matrix (ECM)-mimicking substrates. RGD peptide is a tripeptide (Arg-Gly-Asp) found on ECM proteins that promotes various cellular behaviors. The physicochemical properties of PLGA and RGD peptide-M13 phage (PLGA/RGD peptide) nanofiber matrices were characterized by atomic force microscopy, Fourier-transform infrared spectroscopy and thermogravimetric analysis. In addition, the growth of C2C12 mouse myoblasts on the PLGA/RGD peptide matrices was examined by measuring the metabolic activity. Moreover, the differentiation of C2C12 mouse myoblasts on the matrices when treated with GO was evaluated. The cellular behaviors, including growth and differentiation of C2C12 mouse myoblasts, were substantially enhanced on the PLGA/RGD peptide nanofiber matrices when treated with GO. Overall, these findings suggest that the PLGA/RGD peptide nanofiber matrices can be used in combination with GO as a novel strategy for skeletal tissue regeneration.

  3. The 'headache tree' via umbellulone and TRPA1 activates the trigeminovascular system.

    PubMed

    Nassini, Romina; Materazzi, Serena; Vriens, Joris; Prenen, Jean; Benemei, Silvia; De Siena, Gaetano; la Marca, Giancarlo; Andrè, Eunice; Preti, Delia; Avonto, Cristina; Sadofsky, Laura; Di Marzo, Vincenzo; De Petrocellis, Luciano; Dussor, Greg; Porreca, Frank; Taglialatela-Scafati, Orazio; Appendino, Giovanni; Nilius, Bernd; Geppetti, Pierangelo

    2012-02-01

    The California bay laurel or Umbellularia californica (Hook. & Arn.) Nutt., is known as the 'headache tree' because the inhalation of its vapours can cause severe headache crises. However, the underlying mechanism of the headache precipitating properties of Umbellularia californica is unknown. The monoterpene ketone umbellulone, the major volatile constituent of the leaves of Umbellularia californica, has irritating properties, and is a reactive molecule that rapidly binds thiols. Thus, we hypothesized that umbellulone stimulates the transient receptor potential ankyrin 1 channel in a subset of peptidergic, nocioceptive neurons, activating the trigeminovascular system via this mechanism. Umbellulone, from µM to sub-mM concentrations, selectively stimulated transient receptor potential ankyrin 1-expressing HEK293 cells and rat trigeminal ganglion neurons, but not untransfected cells or neurons in the presence of the selective transient receptor potential ankyrin 1 antagonist, HC-030031. Umbellulone evoked a calcium-dependent release of calcitonin gene-related peptide from rodent trigeminal nerve terminals in the dura mater. In wild-type mice, umbellulone elicited excitation of trigeminal neurons and released calcitonin gene-related peptide from sensory nerve terminals. These two responses were absent in transient receptor potential ankyrin 1 deficient mice. Umbellulone caused nocioceptive behaviour after stimulation of trigeminal nerve terminals in wild-type, but not transient receptor potential ankyrin 1 deficient mice. Intranasal application or intravenous injection of umbellulone increased rat meningeal blood flow in a dose-dependent manner; a response selectively inhibited by systemic administration of transient receptor potential ankyrin 1 or calcitonin gene-related peptide receptor antagonists. These data indicate that umbellulone activates, through a transient receptor potential ankyrin 1-dependent mechanism, the trigeminovascular system, thereby causing

  4. Selective cleavage of SNAREs in sensory neurons unveils protein complexes mediating peptide exocytosis triggered by different stimuli.

    PubMed

    Meng, Jianghui; Dolly, J Oliver; Wang, Jiafu

    2014-10-01

    Oligomerisation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes is required for synaptic vesicle fusion and neurotransmitter release. How these regulate the release of pain peptides elicited by different stimuli from sensory neurons has not been established. Herein, K(+) depolarization was found to induce multiple sodium dodecyl sulfate (SDS)-resistant SNARE complexes in sensory neurons exposed to botulinum neurotoxins (BoNTs), with molecular weights ranging from 104-288 k (large) to 38-104 k (small). Isoform 1 of vesicle-associated membrane protein 1 (VAMP 1) assembled into stable complexes upon depolarisation and was required for the participation of intact synaptosome-associated protein of relative molecular mass 25 k (SNAP-25) or BoNT/A-truncated form (SNAP-25A) in the large functional and small inactive SDS-resistant SNARE complexes. Cleaving VAMP 1 decreased SNAP-25A in the functional complexes to a much greater extent than the remaining intact SNAP-25. Syntaxin 1 proved essential for the incorporation of intact and SNAP-25A into the large complexes. Truncation of syntaxin 1 by BoNT/C1 caused /A- and/or /C1-truncated SNAP-25 to appear in non-functional complexes and blocked the release of calcitonin gene-related peptide (CGRP) elicited by capsaicin, ionomycin, thapsigargin or K(+) depolarization. Only the latter two were susceptible to /A. Inhibition of CGRP release by BoNT/A was reversed by capsaicin and/or ionomycin, an effect overcome by BoNT/C1. Unlike BoNT/B, BoNT/D cleaved VAMP 1 in addition to 2 and 3 in rat sensory neurons and blocked both CGRP and substance P release. Thus, unlike SNAP-25, syntaxin 1 and VAMP 1 are more suitable targets to abolish functional SNARE complexes and pain peptide release evoked by any stimuli. PMID:24604356

  5. Statins Promote the Degradation of Extracellular Amyloid β-Peptide by Microglia via Stimulation of Exosome-associated Insulin-degrading Enzyme (IDE) Secretion*

    PubMed Central

    Tamboli, Irfan Y.; Barth, Esther; Christian, Leonie; Siepmann, Martin; Kumar, Sathish; Singh, Sandesh; Tolksdorf, Karen; Heneka, Michael T.; Lütjohann, Dieter; Wunderlich, Patrick; Walter, Jochen

    2010-01-01

    Epidemiological studies indicate that intake of statins decrease the risk of developing Alzheimer disease. Cellular and in vivo studies suggested that statins might decrease the generation of the amyloid β-peptide (Aβ) from the β-amyloid precursor protein. Here, we show that statins potently stimulate the degradation of extracellular Aβ by microglia. The statin-dependent clearance of extracellular Aβ is mainly exerted by insulin-degrading enzyme (IDE) that is secreted in a nonconventional pathway in association with exosomes. Stimulated IDE secretion and Aβ degradation were also observed in blood of mice upon peripheral treatment with lovastatin. Importantly, increased IDE secretion upon lovastatin treatment was dependent on protein isoprenylation and up-regulation of exosome secretion by fusion of multivesicular bodies with the plasma membrane. These data demonstrate a novel pathway for the nonconventional secretion of IDE via exosomes. The modulation of this pathway could provide a new strategy to enhance the extracellular clearance of Aβ. PMID:20876579

  6. The Anorexigenic Peptide Neuromedin U (NMU) Attenuates Amphetamine-Induced Locomotor Stimulation, Accumbal Dopamine Release and Expression of Conditioned Place Preference in Mice.

    PubMed

    Vallöf, Daniel; Vestlund, Jesper; Engel, Jörgen A; Jerlhag, Elisabet

    2016-01-01

    Amphetamine dependence, besides its substantial economical consequence, is a serious cause of mortality and morbidity. By investigations of the neurochemical correlates through which addictive drugs, such as amphetamine, activate the mesoaccumbal dopamine system unique targets for treatment of drug addiction can be identified. This reward link consists of a dopamine projection from the ventral tegmental area to the nucleus accumbens (NAc) suggesting that these brain areas are important for reward. The physiological function of gut-brain peptides has expanded beyond food intake modulation and involves regulation of drug reinforcement. A novel candidate for reward regulation is the anorexigenic peptide neuromedin U (NMU). We therefore investigated the effects of intracerebroventricular (icv) administration of NMU on amphetamine's well-documented effects on the mesoaccumbal dopamine system, i.e. locomotor stimulation and accumbal dopamine release in mice. In addition, the effect of accumbal NMU administration on locomotor activity was examined. The effect of NMU, icv or intra-NAc, on the expression of conditioned place preference (CPP) was elucidated. Firstly, we showed that icv administration of NMU attenuate the amphetamine-induced locomotor stimulation, accumbal dopamine release and expression of CPP in mice. Secondly, we found that a lower dose of NMU (icv) reduce the amphetamine-induced locomotor stimulation in mice. Thirdly, we demonstrated that NMU administration into the NAc block the ability of amphetamine to cause a locomotor stimulation in mice. However, accumbal NMU administration did not attenuate the amphetamine-induced expression of CPP in mice. Our novel data suggest that central NMU signalling is involved in development of amphetamine dependence. PMID:27139195

  7. The Anorexigenic Peptide Neuromedin U (NMU) Attenuates Amphetamine-Induced Locomotor Stimulation, Accumbal Dopamine Release and Expression of Conditioned Place Preference in Mice

    PubMed Central

    Vallöf, Daniel; Vestlund, Jesper; Engel, Jörgen A.; Jerlhag, Elisabet

    2016-01-01

    Amphetamine dependence, besides its substantial economical consequence, is a serious cause of mortality and morbidity. By investigations of the neurochemical correlates through which addictive drugs, such as amphetamine, activate the mesoaccumbal dopamine system unique targets for treatment of drug addiction can be identified. This reward link consists of a dopamine projection from the ventral tegmental area to the nucleus accumbens (NAc) suggesting that these brain areas are important for reward. The physiological function of gut-brain peptides has expanded beyond food intake modulation and involves regulation of drug reinforcement. A novel candidate for reward regulation is the anorexigenic peptide neuromedin U (NMU). We therefore investigated the effects of intracerebroventricular (icv) administration of NMU on amphetamine’s well-documented effects on the mesoaccumbal dopamine system, i.e. locomotor stimulation and accumbal dopamine release in mice. In addition, the effect of accumbal NMU administration on locomotor activity was examined. The effect of NMU, icv or intra-NAc, on the expression of conditioned place preference (CPP) was elucidated. Firstly, we showed that icv administration of NMU attenuate the amphetamine-induced locomotor stimulation, accumbal dopamine release and expression of CPP in mice. Secondly, we found that a lower dose of NMU (icv) reduce the amphetamine-induced locomotor stimulation in mice. Thirdly, we demonstrated that NMU administration into the NAc block the ability of amphetamine to cause a locomotor stimulation in mice. However, accumbal NMU administration did not attenuate the amphetamine-induced expression of CPP in mice. Our novel data suggest that central NMU signalling is involved in development of amphetamine dependence. PMID:27139195

  8. Electrophysiological and neurochemical techniques to investigate sensory neurons in analgesia research.

    PubMed

    Babes, Alexandru; Fischer, Michael J M; Reid, Gordon; Sauer, Susanne K; Zimmermann, Katharina; Reeh, Peter W

    2010-01-01

    The primary afferent nociceptive neuron has recently attracted major research interest because of the cloning of very selectively expressed and well-conserved ion channel genes. All parts of the neuron, sensory terminals, axon and cell body, are accessible to validated research techniques in vitro using various isolated tissues or cells taken from laboratory animals. Single-unit recording and measuring stimulated calcitonin gene-related peptide (CGRP) release as well as patch-clamping and calcium imaging of cultured sensory neurons provide different kinds of information, and no model alone answers all questions. In combination, however, consistent results and complementary evidence form a solid basis for translational research to follow. PMID:20336427

  9. The Anthocyanin Delphinidin 3-Rutinoside Stimulates Glucagon-Like Peptide-1 Secretion in Murine GLUTag Cell Line via the Ca2+/Calmodulin-Dependent Kinase II Pathway

    PubMed Central

    Kato, Masaki; Tani, Tsubasa; Terahara, Norihiko; Tsuda, Takanori

    2015-01-01

    Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Although several nutrients induce GLP-1 secretion, there is little evidence to suggest that non-nutritive compounds directly increase GLP-1 secretion. Here, we hypothesized that anthocyanins induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. Delphinidin 3-rutinoside (D3R) was shown to increase GLP-1 secretion in GLUTag L cells. The results suggested that three hydroxyl or two methoxyl moieties on the aromatic ring are essential for the stimulation of GLP-1 secretion. Notably, the rutinose moiety was shown to be a potent enhancer of GLP-1 secretion, but only in conjunction with three hydroxyl moieties on the aromatic ring (D3R). Receptor antagonist studies revealed that D3R-stimulates GLP-1 secretion involving inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca2+ mobilization. Treatment of GLUTag cells with a Ca2+/calmodulin-dependent kinaseII (CaMKII) inhibitor (KN-93) abolished D3R-stimulated GLP-1 secretion. In addition, treatment of GLUTag cells with D3R resulted in activation of CaMKII. Pre-treatment of cells with a G protein-coupled receptor (GPR) 40/120 antagonist (GW1100) also significantly decreased D3R-stimulated GLP-1 secretion. These observations suggest that D3R stimulates GLP-1 secretion in GLUTag cells, and that stimulation of GLP-1 secretion by D3R is mediated via Ca2+-CaMKII pathway, which may possibly be mediated by GPR40/120. These findings provide a possible molecular mechanism of GLP-1 secretion in intestinal L-cells mediated by foods or drugs and demonstrate a novel biological function of anthocyanins in regards to GLP-1 secretion. PMID:25962102

  10. Nesfatin-1 stimulates glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide secretion from STC-1 cells in vitro.

    PubMed

    Ramesh, Naresh; Mortazavi, Sima; Unniappan, Suraj

    2015-06-26

    Nesfatin-1 is an 82 amino acid peptide encoded in a secreted precursor, nucleobindin 2. It is an anorexigenic and insulinotropic peptide found abundantly in the hypothalamus, pancreas and gastric oxyntic mucosa. NUCB2 mRNA expression is 10 fold higher in the gastric mucosa than in brain, suggesting gastrointestinal tract as a main source of nesfatin-1. Meal responsive insulin secretion is regulated by incretins glucagon-like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP). Since both nesfatin-1 and incretins modulate insulin secretion, we hypothesized that nesfatin-1 is present in the enteroendocrine cells, and that it regulates incretin secretion. RT-PCR analysis found NUCB2 mRNA expression, and immunofluorescence microscopy determined nesfatin-1 immunoreactivity in STC-1, an enteroendocrine cell line. NUCB2/nesfatin-1 is co-localized with GLP-1 and GIP in mouse small intestinal cells. Static incubation of STC-1 cells with nesfatin-1 upregulated preproglucagon (GLP-1 precursor) mRNA (0.01, 0.1, 1 and 10 nM) and GLP-1 secretion (0.1, 1 and 10 nM). Nesfatin-1 also enhanced GIP mRNA (0.1, 1 and 10 nM) and GIP secretion (1 and 10 nM). Together, our data support the hypothesis that nesfatin-1 is present in enteroendocrine cells and that it stimulates incretin secretion. Future studies should aim for nesfatin-1 and incretin interactions in vivo. PMID:25930999

  11. Regulatory peptides modulate adhesion of polymorphonuclear leukocytes to bronchial epithelial cells through regulation of interleukins, ICAM-1 and NF-kappaB/IkappaB.

    PubMed

    Zhang, Jian-Song; Tan, Yu-Rong; Xiang, Yang; Luo, Zi-Qiang; Qin, Xiao-Qun

    2006-02-01

    A complex network of regulatory neuropeptides controls airway inflammation reaction, in which airway epithelial cells adhering to and activating leukocytes is a critical step. To study the effect of intrapulmonary regulatory peptides on adhesion of polymorphonuclear leukocytes (PMNs) to bronchial epithelial cells (BECs) and its mechanism, several regulatory peptides including vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), endothelin-1 (ET-1) and calcitonin gene-related peptide (CGRP), were investigated. The results demonstrated that VIP and EGF showed inhibitory effects both on the secretion of IL-1, IL-8 and the adhesion of PMNs to BECs, whereas ET-1 and CGRP had the opposite effect. Anti-intercellular adhesion molecule-1 (ICAM-1) antibody could block the adhesion of PMNs to ozone-stressed BECs. Using immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR), it was shown that VIP and EGF down-regulated the expression of ICAM-1 in BECs, while ET-1 and CGRP up-regulated ICAM-1 expression. NF-kappaB inhibitor MG132 blocked ICAM-1 expression induced by ET-1 and CGRP. Furthermore, in electric mobility shift assay (EMSA), VIP and EGF restrained the binding activity of NF-kappaB to the NF-kappaB binding site within the ICAM-1 promoter in ozone-stressed BECs, while CGRP and ET-1 promoted this binding activity. IkappaB degradation was consistent with NF-kappaB activation. These observations indicate that VIP and EGF inhibit inflammation, while ET-1 and CGRP enhance the inflammation reaction. PMID:16474903

  12. A TLR4-interacting peptide inhibits lipopolysaccharide-stimulated inflammatory responses, migration and invasion of colon cancer SW480 cells

    PubMed Central

    Rakhesh, Madhusoodhanan; Cate, Moriasi; Vijay, Ramani; Shrikant, Anant; Shanjana, Awasthi

    2012-01-01

    Inflammation is a major risk factor for carcinogenesis in patients affected by chronic colitis, yet the molecular mechanisms underlying the progression from chronic inflammation to cancer are not completely understood. Activation of the Toll-like receptor 4 (TLR4)-NFκB signaling axis is associated with inflammation. Thus, we hypothesized that inhibition of TLR4-NFκB signaling might help in limiting inflammatory responses and inflammation-induced oncogenesis. In this work, we studied the effects of a TLR4-interacting surfactant protein A-derived (SPA4) peptide on lipopolysaccharide (LPS)-induced TLR4-NFκB signaling and cancer progression. We first characterized this peptide for its ability to bind the TLR4 ligand-LPS and for physico-chemical characteristics. Inflammation was induced by challenging the colon cancer SW480 cells with Escherichia coli LPS. Cells were then treated with varying amounts of the SPA4 peptide. Changes in the expression of TLR4, interleukin (IL)-1β and IL-6, in intracellular NFκB-related signal transducers (IKBα, p65, phosphorylated IKBα, phosphorylated p65, RelB, COX-2) as well as in the transcriptional activity of NFκB were studied by immunocytochemistry, immunoblotting and NFκB reporter assay, respectively. Simultaneously, the effects on LPS-induced cell migration and invasion were determined. We found that the SPA4 peptide does not bind to LPS. Rather, its binding to TLR4 inhibits the LPS-induced phosphorylation of p65, production of IL-1β and IL-6, activity of NFκB, migration and invasion of SW480 cells. In conclusion, our results suggest that the inhibition of TLR4-NFκB signaling by a TLR4-binding peptide may help for the treatment of chronic inflammation and prevention of inflammation-induced cancer in patients with colitis. PMID:23264896

  13. Hsp90-peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I

    PubMed Central

    Murshid, Ayesha; Gong, Jianlin; Calderwood, Stuart K

    2016-01-01

    Molecular chaperones such as heat shock protein 90 (Hsp90) have been shown to form complexes with tumor antigens and can be used to prepare anticancer vaccines largely due to this property. Earlier studies had suggested that, mice immunized with a molecular chaperone based vaccine derived from tumors became immune to further vaccination and that both CD8+ and CD4+ T cells were activated by the chaperone vaccine in a manner dependent on scavenger receptor SREC-I. Here we have investigated mechanisms whereby SREC-I might facilitate uptake of Hsp90 conjugated peptides by APC into the MHC class II pathway for presentation to CD4+ T cells. Our studies showed that antigenic peptides associated with Hsp90 were taken up into the Class II pathway by a mechanism dependent on SREC-I binding and internalization and presented to CD4+ T cells. In addition our studies showed that SREC-I could associate with MHC class II molecules on the cell surface and in intracellular endosomes, suggesting a mechanism involving facilitated uptake of peptides into the MHC class II pathway. These studies in addition to our earlier findings showed SREC-I to play a primary role in chaperone-associated antigen uptake both through cross priming of MHC class I molecules and entry into the class II pathway. PMID:25155057

  14. Effects of membrane polyunsaturated fatty acids on opiate peptide inhibition of basal and prostaglandin E1-stimulated cyclic AMP formation in intact N1E-115 neuroblastoma cells.

    PubMed

    Murphy, M G; Moak, C M; Rao, B G

    1987-12-01

    The effects of membrane polyunsaturated fatty acids (PUFA) on opiate peptide-mediated inhibition of basal and prostaglandin E1-stimulated cyclic AMP formation were examined in intact N1E-115 neuroblastoma cells. Addition of opiate peptides such as methionine 5-enkephalin (metEnk) to control cultures and to cultures that had been supplemented for 48 hr with 50 microM linoleic acid resulted in dose-dependent decreases in cAMP formation; these decreases were blocked by naloxone. Maximum inhibition of basal cyclase activity was 50-55% in both control and PUFA-enriched cells; however, half-maximal inhibition required ten times more metEnk in supplemented cultures than in controls. This is consistent with our observation that the affinity of binding of [tyrosyl-3',5'-3H(N)](2-D-alanine-5-D-leucine)enkephalin ([3H]DADLE) to intact PUFA-enriched cells was lower than that to control cells. Receptor density was not modified as a result of supplementation. Addition of prostaglandin E1 (PGE1) to the cells produced rapid dose-dependent increases in cAMP formation. Maximum responses were higher in PUFA-enriched than in control cells (1924 and 972 pmol cAMP formed/mg protein respectively). Also, the apparent value for EC50 for PGE1 was consistently lower in supplemented cultures. MetEnk reduced PGE1-stimulated cAMP formation by 45-55% in both control and supplemented cells, and values for IC50 were similar (approximately 30 nM) in both. In the presence of the opiate peptide, values for EC50 for PGE1 were similar in control and PUFA-enriched cultures (0.07 and 0.09 microM respectively). The data from these studies suggest that membrane PUFA increase the efficiency of coupling of receptors that stimulate cAMP formation and decrease the efficiency of those that mediate inhibition. PMID:2825714

  15. Surface-associated material from the bacterium Actinobacillus actinomycetemcomitans contains a peptide which, in contrast to lipopolysaccharide, directly stimulates fibroblast interleukin-6 gene transcription.

    PubMed

    Reddi, K; Nair, S P; White, P A; Hodges, S; Tabona, P; Meghji, S; Poole, S; Wilson, M; Henderson, B

    1996-03-15

    The oral commensal Gram-negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth. Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface-associated material. This material contained potent bone-resorbing activity. We now report that surface-associated material is also a potent stimulator of cytokines, and in particular, interleukin-6 (IL-6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL-6 synthesis by fibroblasts and monocytes. In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, surface-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of IL-6 synthesis). Reverse transcriptase PCR also failed to detect mRNA for IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated cells. Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 receptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesis, but did not inhibit surface-associated-material-induced synthesis. In addition, dexamethasone, which completely suppressed LPS-induced IL-6 synthesis, only inhibited surface-associated-material-induced IL-6 synthesis by 50%. This suggests that the active constituent in the surface-associated material stimulates IL-6 gene transcription by a transcriptional control mechanism distinct to that of E. coli LPS. The IL-6 stimulating activity of the surface-associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous. The activity has been isolated using anion

  16. Prenatal exposure to ethanol stimulates hypothalamic CCR2 chemokine receptor system: Possible relation to increased density of orexigenic peptide neurons and ethanol drinking in adolescent offspring.

    PubMed

    Chang, G-Q; Karatayev, O; Leibowitz, S F

    2015-12-01

    Clinical and animal studies indicate that maternal consumption of ethanol during pregnancy increases alcohol drinking in the offspring. Possible underlying mechanisms may involve orexigenic peptides, which are stimulated by prenatal ethanol exposure and themselves promote drinking. Building on evidence that ethanol stimulates neuroimmune factors such as the chemokine CCL2 that in adult rats is shown to colocalize with the orexigenic peptide, melanin-concentrating hormone (MCH) in the lateral hypothalamus (LH), the present study sought to investigate the possibility that CCL2 or its receptor CCR2 in LH is stimulated by prenatal ethanol exposure, perhaps specifically within MCH neurons. Our paradigm of intraoral administration of ethanol to pregnant rats, at low-to-moderate doses (1 or 3g/kg/day) during peak hypothalamic neurogenesis, caused in adolescent male offspring twofold increase in drinking of and preference for ethanol and reinstatement of ethanol drinking in a two-bottle choice paradigm under an intermittent access schedule. This effect of prenatal ethanol exposure was associated with an increased expression of MCH and density of MCH(+) neurons in LH of preadolescent offspring. Whereas CCL2(+) cells at this age were low in density and unaffected by ethanol, CCR2(+) cells were dense in LH and increased by prenatal ethanol, with a large percentage (83-87%) identified as neurons and found to colocalize MCH. Prenatal ethanol also stimulated the genesis of CCR2(+) and MCH(+) neurons in the embryo, which co-labeled the proliferation marker, BrdU. Ethanol also increased the genesis and density of neurons that co-expressed CCR2 and MCH in LH, with triple-labeled CCR2(+)/MCH(+)/BrdU(+) neurons that were absent in control rats accounting for 35% of newly generated neurons in ethanol-exposed rats. With both the chemokine and MCH systems believed to promote ethanol consumption, this greater density of CCR2(+)/MCH(+) neurons in the LH of preadolescent rats suggests that

  17. Subcomponent Vaccine Based on CTA1-DD Adjuvant with Incorporated UreB Class II Peptides Stimulates Protective Helicobacter pylori Immunity

    PubMed Central

    Nedrud, John G.; Bagheri, Nayer; Schön, Karin; Xin, Wei; Bergroth, Hilda; Eliasson, Dubravka Grdic; Lycke, Nils Y.

    2013-01-01

    A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform. PMID:24391754

  18. Subcomponent vaccine based on CTA1-DD adjuvant with incorporated UreB class II peptides stimulates protective Helicobacter pylori immunity.

    PubMed

    Nedrud, John G; Bagheri, Nayer; Schön, Karin; Xin, Wei; Bergroth, Hilda; Eliasson, Dubravka Grdic; Lycke, Nils Y

    2013-01-01

    A mucosal vaccine against Helicobacter pylori infection could help prevent gastric cancers and peptic ulcers. While previous attempts to develop such a vaccine have largely failed because of the requirement for safe and effective adjuvants or large amounts of well defined antigens, we have taken a unique approach to combining our strong mucosal CTA1-DD adjuvant with selected peptides from urease B (UreB). The protective efficacy of the selected peptides together with cholera toxin (CT) was first confirmed. However, CT is a strong adjuvant that unfortunately is precluded from clinical use because of its toxicity. To circumvent this problem we have developed a derivative of CT, the CTA1-DD adjuvant, that has been found safe in non-human primates and equally effective compared to CT when used intranasally. We genetically fused the selected peptides into the CTA1-DD plasmid and found after intranasal immunizations of Balb/c mice using purified CTA1-DD with 3 copies of an H. pylori urease T cell epitope (CTA1-UreB3T-DD) that significant protection was stimulated against a live challenge infection. Protection was, however, weaker than with the gold standard, bacterial lysate+CT, but considering that we only used a single epitope in nanomolar amounts the results convey optimism. Protection was associated with enhanced Th1 and Th17 immunity, but immunizations in IL-17A-deficient mice revealed that IL-17 may not be essential for protection. Taken together, we have provided evidence for the rational design of an effective mucosal subcomponent vaccine against H. pylori infection based on well selected protective epitopes from relevant antigens incorporated into the CTA1-DD adjuvant platform. PMID:24391754

  19. The non-peptide GLP-1 receptor agonist WB4-24 blocks inflammatory nociception by stimulating β-endorphin release from spinal microglia

    PubMed Central

    Fan, Hui; Gong, Nian; Li, Teng-Fei; Ma, Ai-Niu; Wu, Xiao-Yan; Wang, Ming-Wei; Wang, Yong-Xiang

    2015-01-01

    BACKGROUND AND PURPOSE Two peptide agonists of the glucagon-like peptide-1 (GLP-1) receptor, exenatide and GLP-1 itself, exert anti-hypersensitive effects in neuropathic, cancer and diabetic pain. In this study, we have assessed the anti-allodynic and anti-hyperalgesic effects of the non-peptide agonist WB4-24 in inflammatory nociception and the possible involvement of microglial β-endorphin and pro-inflammatory cytokines. EXPERIMENTAL APPROACH We used rat models of inflammatory nociception induced by formalin, carrageenan or complete Freund's adjuvant (CFA), to test mechanical allodynia and thermal hyperalgesia. Expression of β-endorphin and pro-inflammatory cytokines was measured using real-time quantitative PCR and fluorescent immunoassays. KEY RESULTS WB4-24 displaced the specific binding of exendin (9–39) in microglia. Single intrathecal injection of WB4-24 (0.3, 1, 3, 10, 30 and 100 μg) exerted dose-dependent, specific, anti-hypersensitive effects in acute and chronic inflammatory nociception induced by formalin, carrageenan and CFA, with a maximal inhibition of 60–80%. Spinal WB4-24 was not effective in altering nociceptive pain. Subcutaneous injection of WB4-24 was also antinociceptive in CFA-treated rats. WB4-24 evoked β-endorphin release but did not inhibit expression of pro-inflammatory cytokines in either the spinal cord of CFA-treated rats or cultured microglia stimulated by LPS. WB4-24 anti-allodynia was prevented by a microglial inhibitor, β-endorphin antiserum and a μ-opioid receptor antagonist. CONCLUSIONS AND IMPLICATIONS Our results suggest that WB4-24 inhibits inflammatory nociception by releasing analgesic β-endorphin rather than inhibiting the expression of proalgesic pro-inflammatory cytokines in spinal microglia, and that the spinal GLP-1 receptor is a potential target molecule for the treatment of pain hypersensitivity including inflammatory nociception. PMID:25176008

  20. Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity.

    PubMed

    Walter, Merlin N M; Dehsorkhi, Ashkan; Hamley, Ian W; Connon, Che J

    2016-02-01

    C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes. PMID:26626506

  1. Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I

    PubMed Central

    Lim, Chae Jin; Jeon, Jung Eun; Jeong, Se Kyoo; Yoon, Seok Jeong; Kwon, Seon Deok; Lim, Jina; Park, Keedon; Kim, Dae Yong; Ahn, Jeong Keun; Kim, Bong-Woo

    2015-01-01

    Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition. [BMB Reports 2015; 48(9): 501-506] PMID:25644636

  2. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    PubMed

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  3. Growth hormone-releasing peptide-biotin conjugate stimulates myocytes differentiation through insulin-like growth factor-1 and collagen type I.

    PubMed

    Lim, Chae Jin; Jeon, Jung Eun; Jeong, Se Kyoo; Yoon, Seok Jeong; Kwon, Seon Deok; Lim, Jina; Park, Keedon; Kim, Dae Yong; Ahn, Jeong Keun; Kim, Bong-Woo

    2015-09-01

    Based on the potential beneficial effects of growth hormone releasing peptide (GHRP)-6 on muscle functions, a newly synthesized GHRP-6-biotin conjugate was tested on cultured myoblast cells. Increased expression of myogenic marker proteins was observed in GHRP-6-biotin conjugate-treated cells. Additionally, increased expression levels of insulin-like growth factor-1 and collagen type I were observed. Furthermore, GHRP-6-biotin conjugate-treated cells showed increased metabolic activity, as indicated by increased concentrations of energy metabolites, such as ATP and lactate, and increased enzymatic activity of lactate dehydrogenase and creatine kinase. Finally, binding protein analysis suggested few candidate proteins, including desmin, actin, and zinc finger protein 691 as potential targets for GHRP6-biotin conjugate action. These results suggest that the newly synthesized GHRP-6-biotin conjugate has myogenic stimulating activity through, at least in part, by stimulating collagen type I synthesis and several key proteins. Practical applications of the GHRP-6-biotin conjugate could include improving muscle condition. PMID:25644636

  4. ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex

    PubMed Central

    Park, Kyungho; Ikushiro, Hiroko; Shin, Kyong-Oh; Kim, Young il; Kim, Jong Youl; Lee, Yong-Moon; Yano, Takato; Holleran, Walter M.; Elias, Peter; Uchida, Yoshikazu

    2016-01-01

    We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP. PMID:26903652

  5. CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

    PubMed Central

    2012-01-01

    Background The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822–829 (2011)]. Results and discussion Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. Conclusions Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target. PMID

  6. Solid-Phase Peptide Head-to-Side Chain Cyclodimerization: Discovery of C2-Symmetric Cyclic Lactam Hybrid α-Melanocyte-Stimulating Hormone (MSH)/Agouti-Signaling Protein (ASIP) Analogues with Potent Activities at the Human Melanocortin Receptors

    PubMed Central

    Mayorov, Alexander V.; Cai, Minying; Palmer, Erin S.; Liu, Zhihua; Cain, James P.; Vagner, Josef; Trivedi, Dev; Hruby, Victor J.

    2011-01-01

    A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the Agouti signaling protein (ASIP), an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide lactam cyclodimerization yielded nanomolar range (25-120 nM) hMC1R-selective full and partial agonists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the development of novel GPCR peptide ligands. PMID:20688117

  7. Vasoactive intestinal peptide (VIP) differentially affects inflammatory immune responses in human monocytes infected with viable Salmonella or stimulated with LPS.

    PubMed

    Askar, Basim; Ibrahim, Hiba; Barrow, Paul; Foster, Neil

    2015-09-01

    We compared the effect of VIP on human blood monocytes infected with Salmonella typhimurium 4/74 or stimulated with LPS. VIP (10(-7)M) increased monocyte viability by 24% and 9% when cultured for 24h with 4/74 or Salmonella LPS (100ng/ml), respectively. Significantly increased (P<0.05) numbers of 4/74 were also recovered from monocytes co-cultured with VIP after 6h post-infection (pi) and this remained high after 24h pi. Both 4/74 and LPS increased (P<0.05) the concentration of TNF-α, IL-1β and IL-6 measured in monocyte supernatants. However, LPS induced this effect more rapidly while, with the exception of IL-6, 4/74 induced higher concentrations (P<0.05). VIP significantly decreased (P<0.05) TNF-α and IL-1β production by 4/74-infected monocytes after 6 pi, but only after 24h in LPS-cultured monocytes. This trend was reversed for IL-6 production. However, TNF-α and IL-1β production by 4/74-infected monocytes, cultured with VIP, still remained higher (P<0.05) than concentrations measured in supernatants cultured only with LPS. VIP also increased (P<0.05) production of anti-inflammatory IL-10 in both 4/74 and LPS cultures after 24h. We also show a differential effect of VIP on the expression of TNFα and IL-6 receptors, since VIP was only able to decreased expression in LPS-stimulated monocytes but not in 4/74-infected monocytes. In conclusion, we show a differential effect of VIP on human monocytes infected with virulent Salmonella or stimulated with LPS. Our study suggests that the use of VIP in bacteraemia and/or sepsis may be limited to an adjunctive therapy to antibiotic treatment. PMID:26206287

  8. Vasoactive intestinal peptide stimulates melanogenesis in B16F10 mouse melanoma cells via CREB/MITF/tyrosinase signaling.

    PubMed

    Yuan, Xing-Hua; Yao, Cheng; Oh, Jang-Hee; Park, Chi-Hyun; Tian, Yu-Dan; Han, Mira; Kim, Ji Eun; Chung, Jin Ho; Jin, Zhe-Hu; Lee, Dong Hun

    2016-08-26

    Vasoactive intestinal peptide (VIP), one of the major skin neuropeptides, has been suggested to have active roles in the pathogenesis of inflammatory skin disorders such as atopic dermatitis and psoriasis, which can commonly cause post-inflammatory hyperpigmentation. However, the effect of VIP on melanogenesis remains unknown. In this study, we showed that the melanin contents, tyrosinase activity, and gene expression of tyrosinase and microphthalmia-associated transcription factor (MITF) were significantly increased by treatment with VIP in B16F10 mouse melanoma cells and the stimulatory melanogenic effect was further examined in human epidermal melanocytes (HEMns). In addition, phosphorylated levels of CRE-binding protein (CREB) and protein kinase A (PKA) were markedly increased after VIP treatment, but not p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), or Akt, indicating the possible PKA-CREB signaling pathway involved in VIP-induced melanogenesis. This result was further verified by the fact that VIP induced increased melanin synthesis, and protein levels of phosphorylated CREB, MITF, tyrosinase were significantly attenuated by H89 (a specific PKA inhibitor). These data suggest that VIP-induced upregulation of tyrosinase through the CREB-MITF signaling pathway plays an important role in finding new treatment strategy for skin inflammatory diseases related pigmentation disorders. PMID:27343558

  9. Stimulation of glucagon-like peptide-1 secretion downstream of the ligand-gated ion channel TRPA1

    PubMed Central

    Emery, Edward C.; Diakogiannaki, Eleftheria; Gentry, Clive; Psichas, Arianna; Habib, Abdella M.; Bevan, Stuart; Fischer, Michael J. M.; Reimann, Frank; Gribble, Fiona M.

    2015-01-01

    Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis, and could be targeted for the treatment of type-2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L-cells producing glucagon-like peptide-1 (GLP-1). By microarray and qPCR analysis we identified trpa1 as an L-cell enriched transcript in the small intestine. Calcium imaging of primary L-cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (AITC, mustard oil), carvacrol and polyunsaturated fatty acids, that were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells; an effect that was abolished in cultures from trpa1−/− mice or by pharmacological TRPA1 inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L-cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes. PMID:25325736

  10. Metabolic Syndrome Abolishes Glucagon-Like Peptide 1 Receptor Agonist Stimulation of SERCA in Coronary Smooth Muscle.

    PubMed

    Dineen, Stacey L; McKenney, Mikaela L; Bell, Lauren N; Fullenkamp, Allison M; Schultz, Kyle A; Alloosh, Mouhamad; Chalasani, Naga; Sturek, Michael

    2015-09-01

    Metabolic syndrome (MetS) doubles the risk of adverse cardiovascular events. Glucagon-like peptide 1 (GLP-1) receptor agonists induce weight loss, increase insulin secretion, and improve glucose tolerance. Studies in healthy animals suggest cardioprotective properties of GLP-1 receptor agonists, perhaps partially mediated by improved sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) activity. We examined the acute effect of GLP-1 receptor agonists on coronary smooth muscle cells (CSM) enzymatically isolated from lean, healthy Ossabaw miniature swine. Intracellular Ca(2+) handling was interrogated with fura-2. The GLP-1 receptor agonist exenatide activated SERCA but did not alter other Ca(2+) transporters. Further, we tested the hypothesis that chronic, in vivo treatment with GLP-1 receptor agonist AC3174 would attenuate coronary artery disease (CAD) in swine with MetS. MetS was induced in 20 swine by 6 months' feeding of a hypercaloric, atherogenic diet. Swine were then randomized (n = 10/group) into placebo or AC3174 treatment groups and continued the diet for an additional 6 months. AC3174 treatment attenuated weight gain, increased insulin secretion, and improved glucose tolerance. Intravascular ultrasound and histology showed no effect of AC3174 on CAD. MetS abolished SERCA activation by GLP-1 receptor agonists. We conclude that MetS confers vascular resistance to GLP-1 receptor agonists, partially through impaired cellular signaling steps involving SERCA. PMID:25845661

  11. Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids

    PubMed Central

    Schleifman, Erica B.; Bindra, Ranjit; Leif, Jean; Campo, Jacob del; Rogers, Faye A.; Uchil, Pradeep; Kutsch, Olaf; Shultz, Leonard D.; Kumar, Priti; Greiner, Dale L.; Glazer, Peter M.

    2011-01-01

    SUMMARY Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA and protein levels and was shown to confer resistance to infection with HIV-1. Targeting of CCR5 was achieved in human CD34+ hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months post-transplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function. PMID:21944757

  12. High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons

    PubMed Central

    Qiu, Jian; Nestor, Casey C; Zhang, Chunguang; Padilla, Stephanie L; Palmiter, Richard D

    2016-01-01

    Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1ARH) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1ARH neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1ARH neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1ARH neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1ARH neurons. We propose that Kiss1ARH neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function. DOI: http://dx.doi.org/10.7554/eLife.16246.001 PMID:27549338

  13. High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons.

    PubMed

    Qiu, Jian; Nestor, Casey C; Zhang, Chunguang; Padilla, Stephanie L; Palmiter, Richard D; Kelly, Martin J; Rønnekleiv, Oline K

    2016-01-01

    Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1(ARH)) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1(ARH) neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1(ARH) neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1(ARH) neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1(ARH) neurons. We propose that Kiss1(ARH) neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function. PMID:27549338

  14. Na+ transport across rumen epithelium of hay-fed sheep is acutely stimulated by the peptide IGF-1 in vitro.

    PubMed

    Shen, Zanming; Martens, Holger; Schweigel-Röntgen, Monika

    2012-04-01

    An energy-rich diet leads to enhanced ruminal Na(+) absorption, which is associated with elevated plasma insulin-like growth factor 1 (IGF-1) levels and an increased number of IGF-1 receptors in rumen papillae. This study examined the in vitro effect of IGF-1 on Na(+) transport across the rumen epithelium of hay-fed sheep, in which the IGF-1 concentration in plasma is lower than in concentrate-fed animals. At concentrations ranging from 20 to 100 μg l(-1), serosal LR3-IGF-1, a recombinant analogue of IGF-1, rapidly (within 30 min) stimulated the mucosal-to-serosal Na(+) flux (J(ms)Na) and consequently the net Na(+) flux (J(net)Na). Compared with controls, J(net)Na increased by about 60% (P < 0.05) following the serosal application of LR3-IGF-1 (20 μg l(-1)). The IGF-1-induced increment of J(ms)Na and J(net)Na was inhibited by mucosal amiloride (1 mmol l(-1)). Neither IGF-1 nor amiloride altered tissue conductance or the short-circuit current of the isolated rumen epithelium. These data support the assumption that the stimulating effect of serosally applied IGF-1 on Na(+) transport across the rumen epithelium is mediated by Na(+)-H(+) exchange (NHE). A further study was performed with cultured rumen epithelial cells and a fluorescent probe (BCECF) to estimate the rate of pH(i) recovery after acid loading. The pH(i) of isolated rumen epithelial cells was 6.43 ± 0.15 after butyrate loading and recovered by 0.26 ± 0.02 pH units (15 min)(-1). Application of LR3-IGF-1 (20 μg l(-1)) significantly increased the rate of pH(i) recovery to 0.33 ± 0.02 pH units (15 min)(-1). Amiloride administration reduced the recovery rate in both control and IGF-1-stimulated cells. These results show, for the first time, that an acute effect of IGF-1 on Na(+) absorption across rumen epithelium results from increased NHE activity. Insulin-like growth factor 1 is thus important for the fast functional adaptation of ruminal Na(+) transport via NHE. PMID:22227200

  15. Effect of different intestinal conditions on the intermolecular interaction between insulin and cell-penetrating peptide penetratin and on its contribution to stimulation of permeation through intestinal epithelium.

    PubMed

    Kamei, Noriyasu; Aoyama, Yukina; Khafagy, El-Sayed; Henmi, Mao; Takeda-Morishita, Mariko

    2015-08-01

    Our recent studies have shown that the coadministration of cell-penetrating peptides (CPPs) is a potential strategy for oral delivery of peptide- and protein-based biopharmaceuticals. The intermolecular interaction between drug and CPP is an essential factor in the effective delivery of these drugs, but the characteristics of the interaction under the conditions of the intestinal lumen remain unknown. In this study, therefore, we examined the characteristics of binding of the amphipathic CPP penetratin to insulin and the efficiency of its enhancement of epithelial insulin transport at different pH and in simulated intestinal fluids (SIFs). The binding between insulin and penetratin was pH dependent and particularly decreased at pH 5.0. In addition, we clarified that the sodium taurocholate (NaTC) present in two types of SIF (fasted-state SIF [FaSSIF] and fed-state SIF [FeSSIF]) affected binding efficiency. However, the permeation of insulin through a Caco-2 cell monolayer was significantly facilitated by coincubation with l- or d-penetratin at various pH values. Moreover, the permeation-stimulating effect of l-penetratin was observed in FaSSIF containing NaTC and lecithin, but not in 3mM NaTC solution, suggesting that the presence of lecithin was the key factor in maintaining the ability of penetratin to enhance the intestinal absorption of biopharmaceuticals. This report describes the essential considerations for in vivo use and clinical application of a CPP-based oral delivery strategy. PMID:25960330

  16. Interaction of myenteric neurons and extrinsic nerves in the intestinal inhibitory response induced by mesenteric nerve stimulation.

    PubMed

    Yamasato, T; Nakayama, S

    1991-04-01

    Effects of the mesenteric nerve stimulation (MNS) on the twitch contraction induced by field stimulation were investigated regarding the relationship between myenteric neurons and extrinsic cholinergic nerves in the guinea-pig mesenteric nerve-ileal preparation. The twitch contraction was inhibited after MNS. The inhibition of the twitch contraction after MNS was induced twice, just after MNS (1st inhibition) and 2-3 min later (2nd inhibition) (type I), or once, just after MNS (1st inhibition) (type II), in recovery course of twitch contraction for 6-8 min. The 1st inhibition was slightly decreased by guanethidine and hexamethonium. The inhibitory response (1st inhibition) in both types I and II was recovered to the control level by pretreatment with naloxone (recovered twitch contraction), but the late inhibitory response (2nd inhibition) was markedly observed after 2-3 min in types I and II. Either the 1st or the 2nd inhibition was not altered by capsaicin, desensitization to calcitonin gene-related polypeptide (CGRP), vasoactive intestinal polypeptide (VIP), somatostatin, or galanin. The recovered twitch contraction in types I and II was decreased by CGRP-desensitization, or capsaicin. These results suggest that the first inhibitory response was induced by enteric opioid neurons connected with extrinsic cholinergic nerves, but the 2nd inhibition was induced by unknown substances other than CGRP, VIP, somatostatin, and galanin. The twitch contraction may partly be induced by endogenous neurokinin-like substances. And, some CGRP containing neurons, which connect with extrinsic cholinergic nerves, probably activate the intrinsic excitatory neurons. PMID:1678243

  17. Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells

    PubMed Central

    Cools, Nathalie; Ponsaerts, Peter; Lenjou, Marc; Nijs, Griet; Van Bockstaele, Dirk R; Van Tendeloo, Viggo FI; Berneman, Zwi N

    2006-01-01

    Background Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity. Results In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors. Conclusion We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro. PMID:17067378

  18. Differential gene expression of RAW 264.7 macrophages in response to the RGD peptide lunasin with and without lipopolysaccharide stimulation.

    PubMed

    Dia, Vermont P; de Mejia, Elvira Gonzalez

    2011-10-01

    Lunasin is a novel peptide from soybean with demonstrated chemopreventive property. We compared the effect of lunasin on gene expression of RAW 264.7 macrophages with and without lipopolysaccharide (LPS)-stimulation. Our hypothesis was that lunasin will have a differential effect in RAW 264.7 gene expression in a normal and challenged state. Analysis of the microarray data using False Discovery Rate (FDR) method resulted in the identification of 340 up-regulated and 162 down-regulated genes (FDR p-value <0.05) associated with simultaneous treatment of lunasin and LPS for 24h. Treatment of lunasin with no LPS for 24h resulted in the up-regulation of 855 genes and down-regulation of 397 genes. Pre-treatment of lunasin for 24h resulted in the up-regulation of 35 genes and down-regulation of 65 genes in LPS-stimulated RAW 264.7 macrophages. GeneVenn analysis of these three sets of genes showed that there are 66 genes common among the three groups which are mostly associated with regulation of cell death, ion binding and transcription as datamined by DAVID. Analysis of the 838 genes unique to lunasin alone by functional annotation clustering tool showed that lunasin mostly affected genes associated with RNA processing, apoptosis and protein kinase activity. Further datamining of these genes by ingenuity pathway analysis (IPA) showed that lunasin affected genes involved in cellular growth and proliferation, cellular function and maintenance, and cell to cell signaling and interaction. These findings support the potential chemopreventive and chemotherapeutic use of lunasin against cancer. PMID:21964376

  19. Cytokine production by mononuclear cells following stimulation with a peptide-containing, endotoxin-free Escherichia coli extract.

    PubMed

    Thomsen, A; Loppnow, H

    1995-05-01

    The beneficial effects of the E. coli extract Colibiogen inj. N (Cb) observed in therapy of inflammatory bowel diseases, allergies, or gastrointestinal tumors are possibly mediated by the induction of cytokines in human leukocytes or vascular cells. Thus, the induction of the cytokines interleukin 1 (IL1), IL6 and tumor necrosis factor (TNF) in human mononuclear cells (MNC) and vascular cells was investigated in vitro. Various administration forms of the extract (including Cb-inj. N, Cb-oral, and Cb-infantibus N) induced the release of IL1 and IL6 from MNC. The compounds stimulated TNF production less potently, possibly due to a lower sensitivity of the TNF assay system, as compared to the IL1 and IL6 detection system. The MNC produced the cytokines with a kinetics similar to that observed with other stimuli. Monospecific antibodies abolished the respective cytokine activity in the biological assays. Addition of submaximal amounts of endotoxin potently enhanced the IL1- and IL6-inducing activity of the bacterial extract, indicating synergism of the extract and endotoxin. These results provide evidence that cytokines produced by MNC following administration of the tested bacterial extract may contribute to the regulation of the immune response during therapy of gastrointestinal tumors. At present the in vivo production of cytokines following treatment with the bacterial extract tested is under investigation in a phase III study. PMID:7612070

  20. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    SciTech Connect

    Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

    2011-08-05

    Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  1. Serotonin and CGRP in migraine.

    PubMed

    Aggarwal, Milan; Puri, Veena; Puri, Sanjeev

    2012-04-01

    Migraine is defined as recurrent attack of headache that are commonly unilateral and accompanied by gastrointestinal and visual disorders. Migraine is more prevalent in females than males with a ratio of 3:1. It is primarily a complex neurovascular disorder involving local vasodilation of intracranial, extracerebral blood vessels and simultaneous stimulation of surrounding trigeminal sensory nervous pain pathway that results in headache. The activation of 'trigeminovascular system' causes release of various vasodilators, especially calcitonin gene-related peptide (CGRP) that induces pain response. At the same time, decreased levels of neurotransmitter, serotonin have been observed in migraineurs. Serotonin receptors have been found on the trigeminal nerve and cranial vessels and their agonists especially triptans prove effective in migraine treatment. It has been found that triptans act on trigeminovascular system and bring the elevated serum levels of key molecules like calcitonin gene related peptide (CGRP) to normal. Currently CGRP receptor antagonists, olcegepant and telcagepant are under consideration for antimigraine therapeutics. It has been observed that varying levels of ovarian hormones especially estrogen influence serotonin neurotransmission system and CGRP levels making women more predisposed to migraine attacks. This review provides comprehensive information about the role of serotonin and CGRP in migraine, specifically the menstrual migraine. PMID:25205974

  2. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  3. Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

    PubMed Central

    Tatone, Carla; Carbone, Maria Cristina

    2006-01-01

    Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the

  4. Dual Receptor-Targeting Tc-99m-Labeled Arg-Gly-Asp-Conjugated Alpha-Melanocyte Stimulating Hormone Hybrid Peptides for Human Melanoma Imaging

    PubMed Central

    Xu, Jingli; Yang, Jianquan; Miao, Yubin

    2014-01-01

    Introduction The aim of this study was to examine whether the substitution of the Lys linker with the aminooctanoic acid (Aoc) and polyethylene glycol (PEG) linker could substantially decrease the non-specific renal uptake of 99mTc-labeled Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptides. Methods The RGD motif {Arg-Gly-Asp-DTyr-Asp} was coupled to [Cys3,4,10, D-Phe7, Arg11]α-MSH3–13 via the Aoc or PEG2 linker to generate RGD-Aoc-(Arg11)CCMSH and RGD-PEG-(Arg11)CCMSH. The biodistribution results of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH were examined in M21 human melanoma-xenografted nude mice. Results The substitution of Lys linker with Aoc and PEG2 linker significantly reduced the renal uptake of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH by 58% and 63% at 2 h post-injection. The renal uptake of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH was 27.93 ± 3.98 and 22.01 ± 9.89% ID/g at 2 h post-injection. 99mTc-RGD-Aoc-(Arg11)CCMSH displayed higher tumor uptake than 99mTc-RGD-PEG2-(Arg11)CCMSH (2.35 ± 0.12 vs. 1.71 ± 0.25% ID/g at 2 h post-injection). The M21 human melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RGD-Aoc-(Arg11)CCMSH as an imaging probe. Conclusions The favorable effect of Aoc and PEG2 linker in reducing the renal uptake provided a new insight into the design of novel dual receptor-targeting radiolabeled peptides. PMID:25577037

  5. Administration of insulin-like growth factor-I (IGF-I) peptides for three days stimulates proliferation of the small intestinal epithelium in rats.

    PubMed Central

    Steeb, C B; Trahair, J F; Read, L C

    1995-01-01

    It has previously been shown that longterm administration of insulin-like growth factor-I (IGF-I) or the analogue Long R3 IGF-I (LR3IGF-I) selectively stimulate growth of the gastrointestinal tract in gut resected, dexamethasone treated, and normal rats. In this study, the short-term effects of IGF-I administration on intestinal proliferation have been investigated. Female rats (110 g, five-six/group) were infused for three days with 2.5 mg/kg/day of either IGF-I or LR3IGF-I and compared with vehicle treated or untreated control rats. LR3IGF-I but not IGF-I increased body weight and wet tissue weight of the small and large intestine (+20%), compared with controls. Tissue weight responses were independent of food intake and were reflected in the histology of the tissue. In LR3IGF-I treated animals, duodenal and ileal crypts length were increased by 13 and 22%, respectively, associated with an increase in crypt cell number. No such histological changes were seen in IGF-I treated rats. Tritiated thymidine labelling indices were significantly increased after administration of either IGF-I or LR3IGF-I (up to 14%) in both the duodenum and ileum. In IGF-I treated rats, increased nuclear labelling was not associated with an increase in the crypt compartment. In contrast, LR3IGF-I induced proportional increments in thymidine labelling and crypt size, suggesting that LR3IGF-I is not only more potent than the native peptide but also induced proliferative events more rapidly. In the colon, the thymidine labelling index was low, however, a non-significant increase in the number of cells labelled with thymidine was seen. These results suggest that within a three day treatment period intestinal mitogenesis is more advanced in animals treated with LR3IGF-I. The differences in proliferative response between the two peptides may be accounted for by variations in pharmacokinetics, clearance rates, and interactions with circulating and tissue specific binding proteins. PMID:8549937

  6. Administration of insulin-like growth factor-I (IGF-I) peptides for three days stimulates proliferation of the small intestinal epithelium in rats.

    PubMed

    Steeb, C B; Trahair, J F; Read, L C

    1995-11-01

    It has previously been shown that longterm administration of insulin-like growth factor-I (IGF-I) or the analogue Long R3 IGF-I (LR3IGF-I) selectively stimulate growth of the gastrointestinal tract in gut resected, dexamethasone treated, and normal rats. In this study, the short-term effects of IGF-I administration on intestinal proliferation have been investigated. Female rats (110 g, five-six/group) were infused for three days with 2.5 mg/kg/day of either IGF-I or LR3IGF-I and compared with vehicle treated or untreated control rats. LR3IGF-I but not IGF-I increased body weight and wet tissue weight of the small and large intestine (+20%), compared with controls. Tissue weight responses were independent of food intake and were reflected in the histology of the tissue. In LR3IGF-I treated animals, duodenal and ileal crypts length were increased by 13 and 22%, respectively, associated with an increase in crypt cell number. No such histological changes were seen in IGF-I treated rats. Tritiated thymidine labelling indices were significantly increased after administration of either IGF-I or LR3IGF-I (up to 14%) in both the duodenum and ileum. In IGF-I treated rats, increased nuclear labelling was not associated with an increase in the crypt compartment. In contrast, LR3IGF-I induced proportional increments in thymidine labelling and crypt size, suggesting that LR3IGF-I is not only more potent than the native peptide but also induced proliferative events more rapidly. In the colon, the thymidine labelling index was low, however, a non-significant increase in the number of cells labelled with thymidine was seen. These results suggest that within a three day treatment period intestinal mitogenesis is more advanced in animals treated with LR3IGF-I. The differences in proliferative response between the two peptides may be accounted for by variations in pharmacokinetics, clearance rates, and interactions with circulating and tissue specific binding proteins. PMID:8549937

  7. Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages

    PubMed Central

    Shim, Do-Wan; Heo, Kang-Hyuck; Kim, Young-Kyu; Sim, Eun-Jeong; Kang, Tae-Bong; Choi, Jae-Wan; Sim, Dae-Won; Cheong, Sun-Hee; Lee, Seung-Hong; Bang, Jeong-Kyu; Won, Hyung-Sik; Lee, Kwang-Ho

    2015-01-01

    Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation. PMID:26017270

  8. Chromanol 293B, an inhibitor of KCNQ1 channels, enhances glucose-stimulated insulin secretion and increases glucagon-like peptide-1 level in mice.

    PubMed

    Liu, Lijie; Wang, Fanfan; Lu, Haiying; Ren, Xiaomei; Zou, Jihong

    2014-01-01

    Glucose-stimulated insulin secretion (GSIS) is a highly regulated process involving complex interaction of multiple factors. Potassium voltage-gated channel subfamily KQT member 1 (KCNQ1) is a susceptibility gene for type 2 diabetes (T2D) and the risk alleles of the KCNQ1 gene appear to be associated with impaired insulin secretion. The role of KCNQ1 channel in insulin secretion has been explored by previous work in clonal pancreatic β-cells but has yet to be investigated in the context of primary islets as well as intact animals. Genetic studies suggest that altered incretin glucagon-like peptide-1 (GLP-1) secretion might be a potential link between KCNQ1 variants and impaired insulin secretion, but this hypothesis has not been verified so far. In the current study, we examined KCNQ1 expression in pancreas and intestine from normal mice and then investigated the effects of chromanol 293B, a KCNQ1 channel inhibitor, on insulin secretion in vitro and in vivo. By double-immunofluorescence staining, KCNQ1 was detected in insulin-positive β-cells and GLP-1-positive L-cells. Administration of chromanol 293B enhanced GSIS in cultured islets and intact animals. Along with the potentiated insulin secretion during oral glucose tolerance tests (OGTT), plasma GLP-1 level after gastric glucose load was increased in 293B treated mice. These data not only provided new evidence for the participation of KCNQ1 in GSIS at the level of pancreatic islet and intact animal but also indicated the potential linking role of GLP-1 between KCNQ1 and insulin secretion. PMID:25437377

  9. Novel non-peptide small molecules preventing IKKβ/NEMO association inhibit NF-κB activation in LPS-stimulated J774 macrophages.

    PubMed

    De Falco, Francesca; Di Giovanni, Carmen; Cerchia, Carmen; De Stefano, Daniela; Capuozzo, Antonella; Irace, Carlo; Iuvone, Teresa; Santamaria, Rita; Carnuccio, Rosa; Lavecchia, Antonio

    2016-03-15

    Nuclear Factor-κB (NF-κB) is a transcription factor regulating several genes involved in important physiological and pathological processes. NF-κB has been found constitutively activated in many inflammatory/immune diseases. In addition, a positive correlation between persistent activation of NF-κB and tumor promotion has been demonstrated. Since the IKK (IκB kinase) activation is an indispensable component of all pro-inflammatory signaling pathways leading to NF-κB activation, considerable efforts have been done in order to develop novel anti-inflammatory therapeutics targeting IKK. Association of the IKK complex relies on critical interactions between the C-terminus NBD (NEMO binding domain) of the catalytic subunits IKKα and IKKβ, and the regulatory subunit NEMO (NF-κB Essential Modulator). Thus, this IKK/NEMO interacting region provides an attractive target to prevent the IKK complex formation and NF-κB activation. In this regard, we have identified non-peptide small molecule disruptors of IKKβ/NEMO complex through a structure-based virtual screening (SBVS) of the NCI chemical library. Phenothiazine 22 and its close analogues (22.2, 22.4 and 22.10) were able to reduce nitrite production and iNOS mRNA expression in J774 murine macrophages stimulated with LPS for 24h. These effects were associated with a reduced NF-κB/DNA binding activity as well as a decreased expression of phosphorylated IKKβ, IκBα and NF-κB/p65 in these cells. These observations suggest that compound 22 and its three structural analogues by inhibiting IKKβ/NEMO association mediate the blockage of NF-κB signaling pathway and may prove effective in treatment of diseases in which the IKK/NF-κB pathway is dysregulated. PMID:26776306

  10. Sensory and autonomic innervation of the rat eyelid: neuronal origins and peptide phenotypes.

    PubMed

    Simons, E; Smith, P G

    1994-07-01

    Neuronal origins, peptide phenotypes and target distributions were determined for sensory and autonomic nerves projecting to the eyelid. The retrograde tracer, Fluoro-Ruby, was injected into the superior tarsal muscle and meibomian gland of Sprague-Dawley rats. Labelled neurons were observed within the pterygopalatine (31 +/- 6 of a total of 8238 +/- 1610 ganglion neurons), trigeminal (173 +/- 43 of 62,082 +/- 5869) and superior cervical ganglia (184 +/- 35 of 21,900 +/- 1741). Immunostaining revealed vasoactive intestinal polypeptide immunoreactivity (VIP-ir) in nearly all Fluoro-Ruby-labelled pterygopalatine ganglion neurons (86 +/- 5%) but only rarely in trigeminal (0.3 +/- 0.3%) or superior cervical (1.4 +/- 1.4%) ganglion neurons. Calcitonin gene-related peptide (CGRP)-ir was not observed in pterygopalatine or superior cervical ganglion somata, but was present in 24 +/- 4% of trigeminal neurons. Bright dopamine beta-hydroxylase (DBH) immunofluorescence was observed in the majority of eyelid-projecting neurons within the superior cervical ganglia (65 +/- 5%) and lighter staining was detected in pterygopalatine neurons (63 +/- 3%), but no DBH-ir was observed in trigeminal neurons. Examination of eyelid sections revealed dense VIP-ir innervation of meibomian gland acini and vasculature and modest distribution within tarsal muscle. CGRP-ir fibers surrounded ductal and vascular elements of the meibomian gland and the perimeter of tarsal muscle. DBH-ir fibers were associated with meibomian gland blood vessels and acini, and were more densely distributed within tarsal muscle. This study provides evidence for prominent meibomian gland innervation by parasympathetic pterygopalatine ganglion VIP-ir neurons, with more restricted innervation by sensory trigeminal CGRP-ir and sympathetic neurons. Tarsal muscle receives abundant sympathetic innervation, as well as moderate parasympathetic and sensory CGRP-ir projections. The eyelid contains substantial non-CGRP-ir sensory