Sample records for 4-diphenylacetoxy-n-methylpiperidine methiodide 4-damp

  1. Pharmacologic study of muscarinic receptor subtypes and arteriolar dilations: a comparison of conducted and local responses.


    Rivers, R J


    Arteriolar relaxation caused by the application of muscarinic agonists is mediated by multiple factors. One factor causes dilation only at the point of drug microapplication (local response), and a second factor causes responses remote (500 microm away) from the site of application (conducted response). This study was performed to determine if different muscarinic subtypes mediate the two responses. Arterioles of anesthetized hamster cheek pouch were studied with videomicroscopy. Muscarinic antagonists methscopolamine, scopolamine, pirenzepine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), and AFDX-116 [(11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepin-6-one)] were cumulatively applied, and the K(B) for each antagonist was determined for the local and conducted responses caused by methacholine microapplication (10(-4) M, 5 s). The pK(B) (local, conducted) were not significantly different for the two responses when using scopolamine (10.5, 10.4). When the antagonist AFDX-116 (5.6, 6.3), selective for muscarinic receptor (m2) subtype was applied, the K(B) was greater for the conducted response. The pK(B) was greater, however, for the local response when the m1 subtype-selective pirenzepine (7.7, 6.9) or m3 subtype-selective 4-DAMP (10.1, 9.8) was applied. Thus the antagonist pK(B) ratio for on the local and conducted responses depends on the subtype selectivity of the antagonist. These data strongly suggest that different receptors are involved in the two responses.

  2. Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

    USGS Publications Warehouse

    Jett, D.A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.


    The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

  3. Depolarizing Effects of Daikenchuto on Interstitial Cells of Cajal from Mouse Small Intestine

    PubMed Central

    Kim, Hyungwoo; Kim, Hyun Jung; Yang, Dongki; Jung, Myeong Ho; Kim, Byung Joo


    Background: Daikenchuto (DKT; TJ-100, TU-100), a traditional herbal medicineis used in modern medicine to treat gastrointestinal (GI) functional disorders. Interstitial cells of Cajal (ICCs) are the pacemaker cells of the GI tract and play important roles in the regulation of GI motility. Objective: The objective of this study was to investigate the effects of DKT on the pacemaker potentials (PPs) of cultured ICCs from murine small intestine. Materials and Methods: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record ICC PPs (current clamp mode). All experiments were performed at 30-32°C. Results: In current-clamp modeDKT depolarized and concentration-dependently decreased the amplitudes of PPs. Y25130 (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist) did not block DKT-induced PP depolarization, but RS39604 (a 5-HT4 receptor antagonist) did. Methoctramine (a muscarinic M2 receptor antagonist) failed to block DKT-induced PP depolarization, but pretreating 4-diphenylacetoxy-N-methylpiperidine methiodide (a muscarinic M3 receptor antagonist) facilitated blockade of DKT-induced PP depolarization. Pretreatment with an external Ca2+-free solution or thapsigargin abolished PPsand under these conditions, DKT did not induce PP depolarization. Furthermore Ginseng radix and Zingiberis rhizomes depolarized PPs, whereas Zanthoxyli fructus fruit (the third component of DKT) hyperpolarized PPs. Conclusion: These results suggest that DKT depolarizes ICC PPs in an internal or external Ca2+-dependent manner by stimulating 5-HT4 and M3 receptors. Furthermore, the authors suspect that the component in DKT largely responsible for depolarization is probably also a component of Ginseng radix and Zingiberis rhizomes. SUMMARY Daikenchuto (DKT) depolarized and concentration-dependently decreased the amplitudes of

  4. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    SciTech Connect

    Bencherif, M.; Lukas, R.J. )


    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

  5. Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.

    PubMed Central

    Yang, C. M.; Yo, Y. L.; Wang, Y. Y.


    1. The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 microM, respectively. 2. Atropine (10 microM) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3. AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 119 and high affinity for 4-DAMP in antagonizing this response. 4. The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mM EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mM) in the continued presence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8298822

  6. Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists.


    Kyle, Barry D; Bradley, Eamonn; Large, Roddy; Sergeant, Gerard P; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A


    We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca(2+) was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd(2+) (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca(2+) release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca(2+) release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of

  7. Muscarinic suppression of the M-current in the rat sympathetic ganglion is mediated by receptors of the M1-subtype.

    PubMed Central

    Marrion, N. V.; Smart, T. G.; Marsh, S. J.; Brown, D. A.


    1. Under voltage-clamp dissociated adult and foetal rat superior cervical ganglion (s.c.g.) cells exhibited a non-inactivating voltage- and time-dependent component of K+ current termed the M-current (IM). IM was detected and measured from the current decay during hyperpolarizing voltage steps applied from potentials where IM was pre-activated. 2. Neither the resting membrane current nor the amplitude of these current decay relaxations were reduced by omitting Ca from the bathing fluid, showing that the M-current was not a 'Ca-activated' K-current dependent on a primary Ca-influx. Concentrations of (+)-tubocurarine sufficient to block the slow Ca-activated K-current IAHP did not inhibit IM or antagonize the effect of muscarinic agonists on IM, showing that IM was not contaminated by IAHP. Tetraethylammonium (1 mM), which blocks the fast Ca-activated K-current IC, produced a small inhibition of IM. This was not due to contamination of IM by IC since muscarinic agonists did not consistently block IC. 3. The muscarinic agonists muscarine, oxotremorine, McN-A-343 and methacholine reversibly suppressed IM, resulting in an inward (depolarizing) current. The rank order of potency was: oxotremorine greater than or equal to muscarine greater than McN-A-343 greater than methacholine. 4. The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5. IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 +/- 0.13 (n = 3) and 6.02 +/- 0.13 (n = 4) respectively. 6. The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 microM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nM. 7. Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 +/- 0.03 (n = 8). 8. These results suggest that the receptors mediating suppression of the M

  8. Muscarinic receptor heterogeneity in follicle-enclosed Xenopus oocytes

    PubMed Central

    Arellano, Rogelio O; Garay, Edith; Miledi, Ricardo


    Ionic current responses elicited by acetylcholine (ACh) in follicle-enclosed Xenopus oocytes (follicles) were studied using the two-electrode voltage-clamp technique. ACh generated a fast chloride current (Fin) and inhibited K+ currents gated by cAMP (IK,cAMP) following receptor activation by adenosine, follicle-stimulating hormone or noradrenaline. These previously described cholinergic responses were confirmed to be of the muscarinic type, and were independently generated among follicles from different frogs.Inhibition of IK,cAMP was about 100 times more sensitive to ACh than Fin activation; the half-maximal effective concentrations (EC50) were 6.6 ± 0.4 and 784 ± 4 nm, respectively.Both responses were blocked by several muscarinic receptor antagonists. Using the respective EC50 concentrations of ACh as standard, the antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the two effects with very different potencies. Fin was blocked with a half-maximal inhibitory concentration (IC50) of 2.4 ± 0.07 nm, whilst the IC50 for IK,cAMP inhibition was 5.9 ± 0.2 μm.Oxotremorine, a muscarinic agonist, preferentially stimulated IK,cAMP inhibition (EC50= 15.8 ± 1.4 μm), whilst Fin was only weakly activated. In contrast, oxotremorine inhibited Fin generated by ACh with an IC50 of 2.3 ± 0.7 μm.Fin elicited via purinergic receptor stimulation was not affected by oxotremorine, indicating that the inhibition produced was specific to the muscarinic receptor, and suggesting that muscarinic actions do not exert a strong effect on follicular cell-oocyte coupling.Using reverse transcription-PCR, transcripts of a previously cloned muscarinic receptor from Xenopus (XlmR) were amplified from the RNA of both the isolated follicular cells and the oocyte. The pharmacological and molecular characteristics suggest that XlmR is involved in IK,cAMP inhibition.In conclusion, follicular cells possess two different muscarinic receptors, one resembling the M2 (or M4) subtype

  9. Muscarinic receptors participation in angiogenic response induced by macrophages from mammary adenocarcinoma-bearing mice

    PubMed Central

    de la Torre, Eulalia; Davel, Lilia; Jasnis, María A; Gotoh, Tomomi; de Lustig, Eugenia Sacerdote; Sales, María E


    Introduction The role of macrophages in tumor progression has generated contradictory evidence. We had previously demonstrated the ability of peritoneal macrophages from LMM3 murine mammary adenocarcinoma-bearing mice (TMps) to increase the angiogenicity of LMM3 tumor cells, mainly through polyamine synthesis. Here we investigate the ability of the parasympathetic nervous system to modulate angiogenesis induced by TMps through the activation of the muscarinic acetylcholine receptor (mAchR). Methods Peritoneal macrophages from female BALB/c mice bearing a 7-day LMM3 tumor were inoculated intradermally (3 × 105 cells per site) into syngeneic mice. Before inoculation, TMps were stimulated with the muscarinic agonist carbachol in the absence or presence of different muscarinic antagonists or enzyme inhibitors. Angiogenesis was evaluated by counting vessels per square millimeter of skin. The expression of mAchR, arginase and cyclo-oxygenase (COX) isoforms was analyzed by Western blotting. Arginase and COX activities were evaluated by urea and prostaglandin E2 (PGE2) production, respectively. Results TMps, which stimulate neovascularization, express functional mAchR, because carbachol-treated TMps potently increased new blood vessels formation. This response was completely blocked by preincubating TMps with pirenzepine and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), M1 and M3 receptor antagonists, and partly by the M2 receptor antagonist methoctramine. M1 receptor activation by carbachol in TMps triggers neovascularization through arginase products because Nω-hydroxy-L-arginine reversed the agonist action. Preincubation of TMps with methoctramine partly prevented carbachol-stimulated urea formation. In addition, COX-derived liberation of PGE2 is responsible for the promotion of TMps angiogenic activity by M3 receptor. We also detected a higher expression of vascular endothelial growth factor (VEGF) in TMps than in macrophages from normal mice. Carbachol

  10. Blood-brain barrier penetration of novel pyridinealdoxime methiodide (PAM)-type oximes examined by brain microdialysis with LC-MS/MS

    SciTech Connect

    Okuno, Sou; Sakurada, Koichi Ohta, Hikoto; Ikegaya, Hiroshi; Kazui, Yuko; Akutsu, Tomoko; Takatori, Takehiko; Iwadate, Kimiharu


    To develop a new reactivator of inhibited acetylcholinesterase (AChE) that can easily penetrate the blood-brain barrier (BBB), BBB penetration of 6 known and novel pyridinealdoxime methiodide (PAM)-type oximes (alkylPAMs) with relatively high reactivation activities was examined by in vivo rat brain microdialysis with liquid chromatography-mass spectrometry (LC-MS/MS). The 50% lethal dose (LD{sub 50}) of alkylPAMs was intravenously determined for Wistar rats, then the limit of detection, quantification range and linearity of the calibration curve of the alkylPAMs in dialysate and blood were determined by LC-MS/MS. Following 10% LD{sub 50} intravenous administration of the alkylPAMs, 4-[(hydroxyimino) methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) and 4-[(hydroxyimino) methyl]-1-octylpyridinium bromide (4-PAO) appeared in the dialysate. Striatal extracellular fluid/blood concentration ratios were 0.039 {+-} 0.018 and 0.301 {+-} 0.183 (mean {+-} SEM), respectively, 1 h after treatment. This is the first report of BBB penetration of 4-PAPE, and the concentration ratio was smaller than that of 2-PAM.The mean BBB penetration of 4-PAO was approximately 30%, indicating that intravenous administration of 4-PAO may be effective for the reactivation of blocked cholinesterase in the brain. However, the toxicity of 4-PAO (LD{sub 50}; 8.89 mg/kg) was greater than that of 2-PAM. Further investigation is required to determine the effects of these alkylPAMs in organophosphate poisoning.

  11. Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels

    SciTech Connect

    Garcia-Villalon, A.L.; Krause, D.N.; Ehlert, F.J.; Duckles, S.P. )


    The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 (11-2-((2-(diethylaminomethyl)- 1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)benzodiazepine-6-one), hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of (3H)quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of (3H)-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.

  12. The Shock and Vibration Bulletin. Part 4. Damping and Machinery Dynamics

    DTIC Science & Technology


    vibrations by means of enamel coatings", J . Aircraft, 12(4), pp 226-230. 1975. 8. G. Graves et al , "On tailoring a family of enamels for high...forced vibration res- ponse is determined by using modal analysis. Free vibration tests on rotating blades were conducted by Carnegie et al . [12 J ...Using Component Modes", J . Mech. Des., Trans. ASMS, Vol. 100, 1978, p. 520. 10. J.S. Rao, et al .«"Unsteady Force-Blade Response

  13. Muscarinic receptor subtypes mediating the mucosal response to neural stimulation of guinea pig ileum

    SciTech Connect

    Carey, H.V.; Tien, X.Y.; Wallace, L.J.; Cooke, H.J.


    Muscarinic receptors involved in the secretory response evoked by electrical stimulation of submucosal neutrons were investigated in muscle-stripped flat sheets of guinea pig ileum set up in flux chambers. Neural stimulation produced a biphasic increase in short-circuit current due to active chloride secretion. Atropine and 4-diphenylacetoxy-N-methylpiperadine methiodide (4-DAMP) (10/sup -7/ M) were more potent inhibitors of the cholinergic phase of the response than was pirenzepine. Dose-dependent increases in base-line short-circuit current were evoked by carbachol and bethanechol; 4-hydroxy-2-butynyl trimethylammonium chloride (McN A343) produced a much smaller effect. Tetrodotoxin abolished the effects of McN A343 but did not alter the responses of carbachol and bethanechol. McN A343 significantly reduced the cholinergic phase of the neurally evoked response and caused a rightward shift of the carbachol dose-response curve. All muscarinic compounds inhibited (/sup 3/H)quinuclidinyl benzilate binding to membranes from muscosal scrapings, with a rank order of potency of 4-DAMP > pirenzepine > McN A343 > carbachol > bethanechol. These results suggest that acetylcholine released from submucosal neurons mediates chloride secretion by interacting with muscarinic cholinergic receptors that display a high binding affinity for 4-DAMP. Activation of neural muscarinic receptors makes a relatively small contribution to the overall secretory response.

  14. Airway smooth muscle relaxant effects of the cocaine pyrolysis product, methylecgonidine.


    el-Fawal, H A; Wood, R W


    Methylecgonidine (anhydroecgonine methylester; MEG) is produced when cocaine base ("crack") is heated. Since crack smoking can produce significant airway toxicity and the role of MEG in this toxicity is unknown, we determined the effects of MEG on guinea pig isolated tracheal rings. Trachea do not contract in response to MEG; rather, MEG (10(-9) to 10(-3) M) dose-dependently relaxed tissue precontracted with 2 x 10(-3) M acetylcholine (ACh). MEG (10(-9) to 10(-6) M) reduced the magnitude of contractions induced by ACh, carbachol, histamine and KCl in a nonsurmountable manner; the maximal response to these agents was not restored after repeated washing. MEG did not affect contractions induced by BaCl2. 4-Diphenyl acetoxymethyl piperidine methiodide (4-DAMP; 10(-7) M), in the presence or absence of MEG (10(-7) M), shifted the dose-effect curve for ACh 30-fold to the right. After washing, sensitivity to ACh was fully recovered in tissues exposed to 4-DAMP alone, but was still reduced to 50% of control in tissues exposed to 4-DAMP and MEG. The effects of MEG were unlike those of cocaine which, at 10(-7) to 10(-5) M, increased the magnitude of contractions induced by ACh (10(-9) to 2 x 10(-3) M); MEG (10(-7) M) abolished this increase. The mechanism by which MEG relaxes tracheal smooth muscle has not been established, but it is likely to be independent of direct interaction with sites that mediate the effects of the bronchoconstrictor agents used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Release of acetylcholine by syringin, an active principle of Eleutherococcus senticosus, to raise insulin secretion in Wistar rats.


    Liu, Ko Yu; Wu, Yang-Chang; Liu, I-Min; Yu, Wen Chen; Cheng, Juei-Tang


    The present study is designed to screen the effect of syringin, an active principle purified from the rhizome and root parts of Eleutherococcus senticosus (Araliaceae), on the plasma glucose and investigate the possible mechanisms. Plasma glucose decreased in a dose-dependent manner 60 min after intravenous injection of syringin into fasting Wistar rats. In parallel to the decrease of plasma glucose, increases of plasma insulin level as well as the plasma C-peptide was also observed in rats receiving same treatment. Both the plasma glucose lowering action and the raised plasma levels of insulin and C-peptide induced by syringin were also inhibited by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP), the antagonist of the muscarinic M3 receptors, but not affected by the ganglionic nicotinic antagonist, pentolinium or hexamethonium. Moreover, disruption of synaptic available acetylcholine (ACh) using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished these actions of syringin. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the actions of syringin. Mediation of ACh release from the nerve terminals to enhance insulin secretion by syringin can thus be considered. The results suggest that syringin has an ability to raise the release of ACh from nerve terminals, which in turn to stimulate muscarinic M3 receptors in pancreatic cells and augment the insulin release to result in plasma glucose lowering action.

  16. Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

    NASA Technical Reports Server (NTRS)

    Johnson, R. F.; Beltz, T. G.; Sharma, R. V.; Xu, Z.; Bhatty, R. A.; Johnson, A. K.


    The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

  17. Prelimbic cortex GABAA receptors are involved in the mediation of restraint stress-evoked cardiovascular responses.


    Fassini, Aline; Resstel, Leonardo B M; Corrêa, Fernando M A


    Stress is a response of the organism to homeostasis-threatening stimuli and is coordinated by two main neural systems: the hypothalamic-pituitary-adrenal and the autonomic nervous system. Acute restraint stress (RS) is a model of unavoidable stress, which is characterized by autonomic responses including an increase in mean arterial pressure (MAP) and heart rate (HR), as well as a drop in tail temperature. The prelimbic cortex (PL) has been implicated in the modulation of functional responses caused by RS. The present study aimed to evaluate the role of PL GABAergic neurotransmission in the modulation of autonomic changes induced by RS. Bilateral microinjection of the GABAA receptor antagonist bicuculline methiodide into the PL reduced pressor and tachycardic responses evoked by RS, in a dose-dependent manner, without affecting the tail temperature drop evoked by RS. In order to investigate which peripheral autonomic effector modulated the reduction in RS-cardiovascular responses caused by the blockade of PL GABAA receptors, rats were intravenously pretreated with either atenolol or homatropine methylbromide. The blockade of the cardiac sympathetic nervous system with atenolol blunted the reducing effect of PL treatment with bicuculline methiodide on RS-evoked pressor and tachycardic responses. The blockade of the parasympathetic nervous system with homatropine methylbromide, regardless of affecting the beginning of the tachycardic response, did not impact on the reduction of RS-evoked tachycardic and pressor responses caused by the PL treatment with bicuculline methiodide. The present results indicate that both cardiac sympathetic and parasympathetic activities are involved in the reduction of RS-evoked cardiovascular responses evidenced after the blockade of PL GABAA receptors by bicuculline methiodide.


    DTIC Science & Technology

    Contracture of the isolated frog Rectus abdominis muscle was used to study pharmacological properties of 2-PAM (2-pyridine aldoxime methiodide) and...example, concentrations of 2-PAM in excess of 4 x 10 to the -5th power M potentiate contractures of the frog rectus muscle elicited by acetylcholine...2-PAM inhibits the response to the depolarizing agents, decamethonium and carbamylcholine, which are not susceptible to hydrolysis by the ChE of frog

  19. Involvement of peripheral mu opioid receptors in scratching behavior in mice.


    Yamamoto, Atsuki; Sugimoto, Yukio


    Pruritus is a common adverse effect of opioid treatment. However, the mechanism by which pruritus is induced by opioid administration is unclear. In this study, we examined the effects of the intradermal injection of loperamide, a peripherally restricted opioid receptor agonist, on the itch sensation. When injected intradermally into the rostral part of the back in mice, loperamide elicited scratching behavior. We also examined the effects of the selective mu opioid receptor agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate (DAMGO), the selective delta opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE), and the selective kappa opioid receptor agonist U-50488H on scratching behavior in mice in order to determine which subtype is involved in opioid-induced pruritus. Following intradermal injection into the rostral part of the back in mice, DAMGO elicited scratching behavior, while DPDPE and U-50488H did not. This suggests that peripheral mu opioid activation elicits the itch sensation. Next, we focused on the treatment of opioid-induced itch sensation without central adverse effects. Naloxone methiodide is a peripherally restricted opioid receptor antagonist. In the present study, naloxone methiodide significantly suppressed scratching behavior induced by loperamide and DAMGO. These findings suggest that mu opioid receptors play a primary role in peripheral pruritus and that naloxone methiodide may represent a possible remedy for opioid-induced itching.

  20. Differences in the morphine-induced inhibition of small and large intestinal transit: Involvement of central and peripheral μ-opioid receptors in mice.


    Matsumoto, Kenjiro; Umemoto, Hiroyuki; Mori, Tomohisa; Akatsu, Ryuya; Saito, Shinichiro; Tashima, Kimihito; Shibasaki, Masahiro; Kato, Shinichi; Suzuki, Tsutomu; Horie, Syunji


    Constipation is the most common side effect of morphine. Morphine acts centrally and on peripheral sites within the enteric nervous system. There are a few comprehensive studies on morphine-induced constipation in the small and large intestine by the activation of central and peripheral μ-opioid receptors. We investigated the differences in the inhibition of the small and large intestinal transit in normal and morphine-tolerant mice. Morphine reduced the geometric center in the fluorescein isothiocyanate-dextran assay and prolonged the bead expulsion time in a dose-dependent manner. The inhibitory effects of morphine were blocked by μ-opioid antagonist β-funaltrexamine, but not by δ- and κ-opioid antagonists. The peripheral opioid receptor antagonist, naloxone methiodide, partially blocked morphine's effect in the small intestine and completely blocked its effect in the large intestine. The intracerebroventricular administration of naloxone significantly reversed the delay of small intestinal transit but did not affect morphine-induced inhibition of large intestinal transit. Naloxone methiodide completely reversed the inhibition of large intestinal transit in normal and morphine-tolerant mice. Naloxone methiodide partially reversed the morphine-induced inhibition of small intestinal transit in normal mice but completely reversed the effects of morphine in tolerant mice. Chronic treatment with morphine results in tolerance to its inhibitory effect on field-stimulated contraction in the isolated small intestine but not in the large intestine. These results suggest that peripheral and central opioid receptors are involved in morphine-induced constipation in the small and large intestine during the early stage of treatment, but the peripheral receptors mainly regulate constipation during long-term morphine treatment.

  1. A novel class of cholinergic agents structurally related to 2-morpholinol.


    Feriani, A; Gaviraghi, G; Toson, G; Valsecchi, B; Caldirola, P; Ravasi, M; Merlini, L; Redaelli, C; Grana, E; Zonta, F


    A series of esters and ethers of N-alkylmorpholin-2-ols, and their methiodides, which can be considered cyclic analogues of acetylcholine, were synthesized. The amines were obtained by acylation or etherification of morpholinols with the appropriate acyl chlorides and alcohols. All compounds were tested for their ability to interact with the muscarinic receptor M2 (guinea-pig atria) or M3 (rat ileum and urinary bladder) subtype. Some compounds, although endowed with relatively low potency, proved interesting for their organ selectivity. Some considerations on the structure-activity relationship are made and the results obtained with reference agonists and antagonists are also shown.

  2. GABA-A and GABA-B receptors in the cuneate nucleus of the rat in vivo.


    Orviz, P; Cecchini, B G; Andrés-Trelles, F


    Electric stimulation of the rat forepaw evokes a negative potential (N-wave) at the ipsilateral cuneate nucleus. The responses of the N-wave to microiontophoretically applied GABA agonists and antagonists have been studied. Applications of GABA-A agonists (3-amino-propanesulfonic acid and muscimol) reduce the amplitude of the N-wave. This effect decreases during prolonged application, suggesting a desensitization of GABA-A receptors. In addition the effect of muscimol is reduced by (-)-bicuculline methiodide. Baclofen (a GABA-B agonist) also depresses the N-wave but its action lasts longer, is less reversible, shows no desensitization and is not blocked by (-)-bicuculline methiodide. The different responses of the N-wave to GABA-A and GABA-B agonists are compatible with the existence of different types of functional receptors for them in the cuneate nucleus of the rat. The receptors activated by muscimol (GABA-A) are clearly not the same as the ones activated by baclofen (conceivably GABA-B).

  3. Effects of a 60 Hz magnetic field on central cholinergic systems of the rat

    SciTech Connect

    Lai, H.; Carino, M.A.; Horita, A.; Guy, A.W. )


    The authors studied the effects of an acute exposure to a 60 Hz magnetic field on sodium-dependent, high-affinity choline uptake in the brain of the rat. Decreases in uptake were observed in the frontal cortex and hippocampus after the animals were exposed to a magnetic field at flux densities [>=] 0.75 mT. These effects of the magnetic field were blocked by pretreating the animals with the narcotic antagonist naltrexone, but not by the peripheral opioid antagonist, naloxone methiodide. These data indicate that the magnetic-field-induced decreases in high-affinity choline uptake in the rat brain were mediated by endogenous opioids in the central nervous systems.

  4. Evidence That GABA Mediates Dopaminergic and Serotonergic Pathways Associated with Locomotor Activity in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Clements, S.; Schreck, C.B.


    The authors examined the control of locomotor activity in juvenile salmon (Oncorhynchus tshawytscha) by manipulating 3 neurotransmitter systems-gamma-amino-n-butyric acid (GABA), dopamine, and serotonin-as well as the neuropeptide corticotropin releasing hormone (CRH). Intracerebroventricular (ICV) injections of CRH and the GABAAagonist muscimol stimulated locomotor activity. The effect of muscimol was attenuated by administration of a dopamine receptor antagonist, haloperidol. Conversely, the administration of a dopamine uptake inhibitor (4???,4??? -difluoro-3-alpha-[diphenylmethoxy] tropane hydrochloride [DUI]) potentiated the effect of muscimol. They found no evidence that CRH-induced hyperactivity is mediated by dopaminergic systems following concurrent injections of haloperidol or DUI with CRH. Administration of muscimol either had no effect or attenuated the locomotor response to concurrent injections of CRH and fluoxetine, whereas the GABAA antagonist bicuculline methiodide potentiated the effect of CRH and fluoxetine.

  5. Modulation of the release of norepinephrine by gamma-aminobutyric acid and morphine in the frontal cerebral cortex of the rat

    SciTech Connect

    Peoples, R.W.


    Agents that enhance gamma-aminobutyric acid, or GABA, neurotransmission modulate certain effects of opioids, such as analgesia. Opioid analgesia is mediated in part by norepinephrine in the forebrain. In this study, the interactions between morphine and GABAergic agents on release of ({sup 3}H) norepinephrine from rat frontal cerebral cortical slices were examined. GABA, 5 {times} 10{sup {minus}5}-10{sup {minus}3} M, enhanced potassium stimulated ({sup 3}H) norepinephrine release and reversed the inhibitory effect of morphine in a noncompetitive manner. GABA did not enhance release of ({sup 3}H) norepinephrine stimulated by the calcium ionophore A23187. The effect of GABA was reduced by the GABA{sub A} receptor antagonists bicuculline methiodide or picrotoxin, and by the selective inhibitor of GABA uptake SKF 89976A, but was blocked completely only when bicuculline methiodide and SKF 89976A were used in combination. The GABA{sub A} agonist muscimol, 10{sup {minus}4} M, mimicked the effect of GABA, but the GABA{sub B} agonist ({plus minus})baclofen, 10{sup {minus}4} M, did not affect the release of ({sup 3}H) norepinephrine in the absence or the presence of morphine. Thus GABA appears to produce this effect by stimulating GABA uptake and GABA{sub A}, but not GABA{sub B}, receptors. In contrast to the results that would be predicted for an event involving GABA{sub A} receptors, however, the effect of GABA did not desensitize, and benzodiazepine agonists did not enhance the effect of GABA at any concentration tested between 10{sup {minus}8} and 10{sup {minus}4} M. Thus these receptors may constitute a subclass of GABA{sub A} receptors. These results support a role of GABA uptake and GABA{sub A} receptors in enhancing the release of norepinephrine and modulating its inhibition by opioids in the frontal cortex of the rat.

  6. Hypoxia-induced hypothermia mediated by GABA in the rostral parapyramidal area of the medulla oblongata.


    Osaka, T


    Hypoxia evokes a regulated decrease in the body core temperature (Tc) in a variety of animals. The neuronal mechanisms of this response include, at least in part, glutamatergic activation in the lateral preoptic area (LPO) of the hypothalamus. As the sympathetic premotor neurons in the medulla oblongata constitute a cardinal relay station in the descending neuronal pathway from the hypothalamus for thermoregulation, their inhibition can also be critically involved in the mechanisms of the hypoxia-induced hypothermia. Here, I examined the hypothesis that hypoxia-induced hypothermia is mediated by glutamate-responsive neurons in the LPO that activate GABAergic transmission in the rostral raphe pallidus (rRPa) and neighboring parapyramidal region (PPy) of the medulla oblongata in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. Unilateral microinjection of GABA (15nmol) into the rRPa and PPy regions elicited a prompt increase in tail skin temperature (Ts) and decreases in Tc, oxygen consumption rate (VO2), and heart rate. Next, when the GABAA receptor blocker bicuculline methiodide (bicuculline methiodide (BMI), 10pmol) alone was microinjected into the rRPa, it elicited unexpected contradictory responses: simultaneous increases in Ts, VO2 and heart rate and a decrease in Tc. Then, when BMI was microinjected bilaterally into the PPy, no direct effect on Ts was seen; and thermogenic and tachycardic responses were slight. However, pretreatment of the PPy with BMI, but not vehicle saline, greatly attenuated the hypothermic responses evoked by hypoxic (10%O2-90%N2, 5min) ventilation or bilateral microinjections of glutamate (5nmol, each side) into the LPO. The results suggest that hypoxia-induced hypothermia was mediated, at least in part, by the activation of GABAA receptors in the PPy.

  7. Intrathecal alpha2 adrenoceptor agonist clonidine inhibits mechanical transmission in mouse spinal cord via activation of muscarinic M1 receptors.


    Honda, Kenji; Koga, Kohei; Moriyama, Tomoko; Koguchi, Masako; Takano, Yukio; Kamiya, Hiro-o


    We examined the role of the spinal muscarinic receptor subtype in the anti-nociceptive effect of intrathecal (i.t.) alpha2 adrenoceptor agonist clonidine in mice. I.t. injection of the muscarinic receptor antagonist atropine completely inhibited i.t. clonidine-induced increase in the mechanical threshold, but did not affect the increase in tail-flick latency induced by i.t. clonidine. The clonidine-induced increase in mechanical threshold was inhibited by i.t. injection of the M1 receptor antagonist pirenzepine in a dose-dependent manner, and by the M3 receptor antagonist 4-DAMP, but not by the M2 receptor antagonist methoctramine. The potency of pirenzepine was greater than that of 4-DAMP. These results suggest that the clonidine-induced increase in mechanical threshold is mediated via the activation of M1 receptors in the spinal cord.

  8. Functional characterization of muscarinic receptors in murine airways.

    PubMed Central

    Garssen, J.; Van Loveren, H.; Gierveld, C. M.; Van der Vliet, H.; Nijkamp, F. P.


    1. The effects of muscarinic receptor antagonists considered to be selective for M1 receptors (pirenzepine; PZ), M2 receptors (AFDX-116), and for M3 receptors (4-diphenyl acetoxy N-methyl-piperidine (4-DAMP)) were used to investigate the existence of muscarinic receptors subtypes in murine airways. Atropine was used as a nonselective antagonist. The effects of these antagonists were studied upon tracheal contractions induced either by EFS (electric field stimulation) or by application of an exogenous cholinoceptor agonist (arecoline). 2. The muscarinic receptor antagonists tested inhibited arecoline-induced tracheal contractions with the following rank order of potency: 4-DAMP = atropine > pirenzepine = AFDX-116. The rank order of potency of the muscarinic antagonists used in inhibiting EFS-induced tracheal contractions was: 4-DAMP = atropine > PZ > AFDX-116. The pA2 values for these antagonists were similar when compared to the pA2 values determined in guinea-pig and bovine airway smooth muscle. 3. In addition to in vitro studies, the effects of inhalation of the different muscarinic antagonists on lung function parameters in vivo were investigated. Inhalation of 4-DAMP induced a decrease in airway resistance and an increase in lung compliance. In contrast, inhalation of AFDX-116 induced an increase in airway resistance and almost no change in lung compliance. Apart from some minor effects of atropine on airway resistance, atropine, PZ, and pilocarpine failed to induce changes in lung mechanics as determined by in vivo lung function measurements. 4. The results provide evidence for the existence of M3 receptors on murine tracheae that are involved in the contraction of tracheal smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 6 Figure 7 PMID:8495246

  9. Acetylcholine acts through M3 muscarinic receptor to activate the EGFR signaling and promotes gastric cancer cell proliferation

    PubMed Central

    Yu, Huangfei; Xia, Hongwei; Tang, Qiulin; Xu, Huanji; Wei, Guoqing; Chen, Ying; Dai, Xinyu; Gong, Qiyong; Bi, Feng


    Acetylcholine (ACh), known as a neurotransmitter, regulates the functions of numerous fundamental central and peripheral nervous system. Recently, emerging evidences indicate that ACh also plays an important role in tumorigenesis. However, little is known about the role of ACh in gastric cancer. Here, we reported that ACh could be auto-synthesized and released from MKN45 and BGC823 gastric cancer cells. Exogenous ACh promoted cell proliferation in a does-dependent manner. The M3R antagonist 4-DAMP, but not M1R antagonist trihexyphenidyl and M2/4 R antagonist AFDX-116, could reverse the ACh-induced cell proliferation. Moreover, ACh, via M3R, activated the EGFR signaling to induce the phosphorylation of ERK1/2 and AKT, and blocking EGFR pathway by specific inhibitor AG1478 suppressed the ACh induced cell proliferation. Furthermore, the M3R antagonist 4-DAMP and darifenacin could markedly inhibit gastric tumor formation in vivo. 4-DAMP could also significantly enhance the cytotoxic activity of 5-Fu against the MKN45 and BGC823 cells, and induce the expression of apoptosis-related proteins such as Bax and Caspase-3. Together, these findings indicated that the autocrine ACh could act through M3R and the EGFR signaling to promote gastric cancer cells proliferation, targeting M3R or EGFR may provide us a potential therapeutic strategy for gastric cancer treatment. PMID:28102288

  10. GABAA receptor-mediated positive inotropism in guinea-pig isolated left atria: evidence for the involvement of capsaicin-sensitive nerves.


    Maggi, C A; Giuliani, S; Manzini, S; Meli, A


    1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow. Both capsaicin (1 microM) and CGRP (1 microM) produced marked tachycardias and increases in coronary flow

  11. Characterization of GABA- and glycine-induced currents of solitary rodent retinal ganglion cells in culture.


    Tauck, D L; Frosch, M P; Lipton, S A


    Ganglion cells were fluorescently labeled, dissociated from 7- to 11-day-old rodent retinas, and placed in tissue culture. Whole-cell recordings with patch electrodes were obtained from solitary cells lacking processes, which permitted a high-quality space clamp. Both GABA (1-200 microM) and glycine (10-300 microM) produced large increases in membrane conductance in virtually every ganglion cell tested, including ganglion cells from different size classes in both rats and mice. Taurine evoked responses similar to those of glycine, but considerably greater concentrations of taurine (150-300 microM) were necessary to observe any effect. Since 20 microM GABA produced approximately the same response as 100 microM glycine, the effects of these two concentrations were compared under various conditions. When recording with chloride distributed equally across the membrane, the reversal potential of the agonist-induced currents was approximately 0 mV. When the internal chloride was reduced by substitution with aspartate, the reversal potential shifted in a negative direction by about 42 mV, indicating that the current was carried mainly by chloride ions. Strychnine (1-5 microM) completely and reversibly blocked the actions of glycine (100 microM) but not those of GABA (20 microM); however, higher concentrations of strychnine (20 microM) nearly totally inhibited the current elicited by GABA (20 microM). The responses to glycine (100 microM) were not affected by bicuculline methiodide (20 microM) or picrotoxinin (20 microM). In contrast, bicuculline methiodide (10 microM) and picrotoxinin (10 microM) reversibly blocked the current evoked by GABA (20 microM); d-tubocurarine (100 microM) only slightly decreased the response to GABA (20 microM). The antagonists were effective over a wide range of holding potentials (-90 mV to +30 mV). The responses to a steady application of both GABA and glycine decayed in a few seconds when recorded under conditions of both symmetric and

  12. Comparison of the action of baclofen with gamma-aminobutyric acid on rat hippocampal pyramidal cells in vitro.

    PubMed Central

    Newberry, N R; Nicoll, R A


    Intracellular recordings from CA1 pyramidal cells in the hippocampal slice preparation were used to compare the action of baclofen, a gamma-aminobutyric acid (GABA) analogue, with GABA. Ionophoretic application of GABA or baclofen into stratum (s.) pyramidale evoked hyperpolarizations associated with reductions in the input resistance of the cell. Baclofen responses were easier to elicit in the dendrites than in the cell body layer. Blockade of synaptic transmission, with tetrodotoxin or cadmium, did not reduce baclofen responses, indicating a direct post-synaptic action. (+)-Bicuculline (10 microM) and bicuculline methiodide (100 microM) had little effect on baclofen responses but strongly antagonized somatic GABA responses of equal amplitude. The bicuculline resistance of the baclofen response was not absolute, as higher concentrations of these compounds did reduce it. Pentobarbitone (100 microM) enhanced somatic GABA responses without affecting baclofen responses. (-)-Baclofen was approximately 200 times more potent than (+)-baclofen. The reversal potentials for the somatic GABA and baclofen responses were -70 mV and -85 mV respectively. When the membrane was depolarized, the baclofen response was reduced. This apparent voltage sensitivity was not seen with somatic GABA responses. Altering the chloride gradient across the cell membrane altered the reversal potential of the somatic GABA response but not that of the baclofen response. It was extrapolated that a tenfold shift in the extracellular potassium concentration would cause a 48 mV shift in the reversal potential of the baclofen response. Barium ions reduced the baclofen response, but not the GABA response. Orthodromic stimulation produced a fast inhibitory post-synaptic potential (i.p.s.p.) and a slow i.p.s.p. The properties of the fast and slow i.p.s.p.s were remarkably similar to those of the somatic GABA and baclofen responses, respectively. Application of GABA to the pyramidal cell dendrites evoked, in

  13. Peripheral antinociceptive effects of the cyclic endomorphin-1 analog c[YpwFG] in a mouse visceral pain model.


    Bedini, Andrea; Baiula, Monica; Gentilucci, Luca; Tolomelli, Alessandra; De Marco, Rossella; Spampinato, Santi


    We previously described a novel cyclic endomorphin-1 analog c[Tyr-D-Pro-D-Trp-Phe-Gly] (c[YpwFG]), acting as a mu-opioid receptor (MOR) agonist. This study reports that c[YpwFG] is more lipophilic and resistant to enzymatic hydrolysis than endomorphin-1 and produces preemptive antinociception in a mouse visceral pain model when injected intraperitoneally (i.p.) or subcutaneously (s.c.) before 0.6% acetic acid, employed to evoke abdominal writhing (i.p. ED(50)=1.24 mg/kg; s.c. ED(50)=2.13 mg/kg). This effect is reversed by the selective MOR antagonist β-funaltrexamine and by a high dose of the mu(1)-opioid receptor-selective antagonist naloxonazine. Conversely, the kappa-opioid receptor antagonist nor-binaltorphimine and the delta-opioid receptor antagonist naltrindole are ineffective. c[YpwFG] produces antinociception when injected i.p. after acetic acid (ED(50)=4.80 mg/kg), and only at a dose of 20mg/kg did it elicit a moderate antinociceptive response in the mouse, evaluated by the tail flick assay. Administration of a lower dose of c[YpwFG] (10mg/kg i.p.) apparently produces a considerable part of antinociception on acetic acid-induced writhes through peripheral opioid receptors as this action is fully prevented by i.p. naloxone methiodide, which does not readily cross the blood-brain barrier; whereas this opioid antagonist injected intracerebroventricularly (i.c.v.) is not effective. Antinociception produced by a higher dose of c[YpwFG] (20mg/kg i.p.) is partially reversed by naloxone methiodide i.c.v. administered. Thus, only at the dose of 20mg/kg c[YpwFG] can produce antinociception through both peripheral and central opioid receptors. In conclusion, c[YpwFG] displays sufficient metabolic stability to be effective after peripheral administration and demonstrates the therapeutic potential of endomorphin derivatives as novel analgesic agents to control visceral pain.

  14. Dietary peptides induce satiety via cholecystokinin-A and peripheral opioid receptors in rats.


    Pupovac, Jelena; Anderson, G Harvey


    We hypothesized that the digestion of proteins gives rise to peptides that initiate several satiety signals from the gut, and that the signals arising will be dependent on the protein source. The role of peripheral opioid and cholecystokinin (CCK)-A receptors was investigated. Casein, soy protein, and casein and soy hydrolysates were administered to rats by gavage (0.5 g protein/4 mL water). Food intake was measured over 2 h. The opioid receptor antagonist, naloxone methiodide (1.0 mg/kg) given intraperitoneally (i.p.), increased food intake when given at the same time as the hydrolysate preloads, 25 min after the casein preloads and 55 min after the soy protein preloads. The CCK-A receptor antagonist, devazepide (which reverses protein-induced food intake suppression), when given at 0.25 mg/kg, i.p., 60 min before preloads of each of three soy hydrolysates, also blocked suppression of food intake, but the strength and duration of the interaction depended on the preparation. When the two receptor antagonists were both administered with soy or casein preloads, their effects were additive. We conclude that peptides arising from digestion contribute to satiety by independent activation of both opioid and CCK-A receptors.

  15. Bicuculline, a GABAA-receptor antagonist, blocked HPA axis activation induced by ghrelin under an acute stress.


    Gastón, M S; Cid, M P; Salvatierra, N A


    Ghrelin is a peptide of 28 amino acids with a homology between species, which acts on the central nervous system to regulate different actions, including the control of growth hormone secretion and metabolic regulation. It has been suggested that central ghrelin is a mediator of behavior linked to stress responses and induces anxiety in rodents and birds. Previously, we observed that the anxiogenic-like behavior induced by ghrelin injected into the intermediate medial mesopallium (IMM) of the forebrain was blocked by bicuculline (a GABAA receptor competitive antagonist) but not by diazepam (a GABAA receptor allosteric agonist) in neonatal meat-type chicks (Cobb). Numerous studies have indicated that hypothalamic-pituitary-adrenal (HPA) axis activation mediates the response to stress in mammals and birds. However, it is still unclear whether this effect of ghrelin is associated with HPA activation. Therefore, we investigated whether anxiety behavior induced by intra-IMM ghrelin and mediated through GABAA receptors could be associated with HPA axis activation in the neonatal chick. In the present study, in an Open Field test, intraperitoneal bicuculline methiodide blocked anxiogenic-like behavior as well as the increase in plasma ACTH and corticosterone levels induced by ghrelin (30pmol) in neonatal chicks. Moreover, we showed for the first time that a competitive antagonist of GABAA receptor suppressed the HPA axis activation induced by an anxiogenic dose of ghrelin. These results show that the anxiogenic ghrelin action involves the activation of the HPA axis, with a complex functional interaction with the GABAA receptor.

  16. Recovery of brain and plasma cholinesterase activities in ducklings exposed to organophosphorus pesticides

    USGS Publications Warehouse

    Fleming, W.J.


    Brain and plasma cholinesterase (ChE) activities were determined for mallard ducklings (Anas platyrhynchos) exposed to dicrotophos and fenthion. Recovery rates of brain ChE did not differ between ducklings administered a single oral dose vs. a 2-week dietary dose of these organophosphates. Exposure to the organophosphates, followed by recovery of brain ChE, did not significantly affect the degree of brain ChE inhibition or the recovery of ChE activity at a subsequent exposure. Recovery of brain ChE activity followed the general model Y = a + b(logX) with rapid recovery to about 50% of normal, followed by a slower rate of recovery until normal ChE activity levels were attained. Fenthion and dicrotophos-inhibited brain ChE were only slightly reactivated in vitro by pyridine-2-aldoxime methiodide, which suggested that spontaneous reactivation was not a primary method of recovery of ChE activity. Recovery of brain ChE activity can be modeled for interpretation of sublethal inhibition of brain ChE activities in wild birds following environmental applications of organophosphates. Plasma ChE activity is inferior to brain ChE activity for environmental monitoring, because of its rapid recovery and large degree of variation among individuals.

  17. Transient and reversible parkinsonism after acute organophosphate poisoning.


    Arima, Hajime; Sobue, Kazuya; So, MinHye; Morishima, Tetsuro; Ando, Hirkoshi; Katsuya, Hirotada


    Parkinsonism is a rare complication in patients with organophosphate poisoning. To date there have been two cases of transient parkinsonism after acute and severe cholinergic crisis, both of which were successfully treated using amantadine, an anti-parkinsonism drug. We report on an 81-year-old woman who was admitted for the treatment of acute severe organophosphate poisoning. Although acute cholinergic crisis was treated successfully with large doses of atropine and 2-pyridine aldoxime methiodide (PAM), extrapyramidal manifestations were noticed on hospital day 6. The neurological symptoms worsened, and the diagnosis of parkinsonism was made by a neurologist on hospital day 9. Immediately, biperiden (5mg), an anti-parkinsonism drug, was administered intravenously, and her symptoms markedly improved. From the following day, biperiden (5 mg/day) was given intramuscularly for eight days. Subsequently, neurological symptoms did not relapse, and no drugs were required. Our patient is the third case of parkinsonism developing after an acute severe cholinergic crisis and the first case successfully treated with biperiden. Patients should be carefully observed for the presence of neurological signs in this kind of poisoning. If present, an anti-parkinsonism drug should be considered.

  18. Interaction of anisatin with rat brain gamma-aminobutyric acidA receptors: allosteric modulation by competitive antagonists.


    Kakemoto, E; Okuyama, E; Nagata, K; Ozoe, Y


    Anisatin, a toxic sesquiterpene isolated from the Japanese star anise (Illicium anisatum L.), competitively inhibited the specific binding of [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB), a non-competitive antagonist of gamma-aminobutyric acid (GABA)A receptors, to rat brain membranes with an IC50 value of 0.43 microM. R 5135, a competitive GABA antagonist, decreased the potency of anisatin in inhibiting [3H]EBOB binding in a negatively cooperative manner. Two other competitive antagonists, SR 95531 (gabazine) and (-)-bicuculline methiodide, had similar effects. On the other hand, R 5135 exerted little influence on the potencies of the other non-competitive antagonists tested: EBOB, picrotoxinin, isopropylbicyclophosphate, and dieldrin. Thus, anisatin was clearly different from the other non-competitive antagonists in responding to the action of competitive antagonists on (GABA)A receptors. These findings suggest that the binding region of anisatin might overlap with that of the other non-competitive antagonists, but that anisatin must interact with other specific region(s).



    Avdiyuk, K V; Varbanets, L D


    The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.

  20. Intraplantar injection of bergamot essential oil induces peripheral antinociception mediated by opioid mechanism.


    Sakurada, Tsukasa; Mizoguchi, Hirokazu; Kuwahata, Hikari; Katsuyama, Soh; Komatsu, Takaaki; Morrone, Luigi Antonio; Corasaniti, Maria Tiziana; Bagetta, Giacinto; Sakurada, Shinobu


    This study investigated the effect of bergamot essential oil (BEO) containing linalool and linalyl acetate as major volatile components in the capsaicin test. The intraplantar injection of capsaicin (1.6 μg) produced a short-lived licking/biting response toward the injected paw. The nociceptive behavioral response evoked by capsaicin was inhibited dose-dependently by intraplantar injection of BEO. Both linalool and linalyl acetate, injected into the hindpaw, showed a significant reduction of nociceptive response, which was much more potent than BEO. Intraperitoneal (i.p.) and intraplantar pretreatment with naloxone hydrochloride, an opioid receptor antagonist, significantly reversed BEO- and linalool-induced antinociception. Pretreatment with naloxone methiodide, a peripherally acting μ-opioid receptor preferring antagonist, resulted in a significant antagonizing effect on antinociception induced by BEO and linalool. Antinociception induced by i.p. or intrathecal morphine was enhanced by the combined injection of BEO or linalool. The enhanced effect of combination of BEO or linalool with morphine was antagonized by pretreatment with naloxone hydrochloride. Our results provide evidence for the involvement of peripheral opioids, in the antinociception induced by BEO and linalool. Combined administration of BEO or linalool acting at the peripheral site, and morphine may be a promising approach in the treatment of clinical pain.

  1. Effect of plantar subcutaneous administration of bergamot essential oil and linalool on formalin-induced nociceptive behavior in mice.


    Katsuyama, Soh; Otowa, Akira; Kamio, Satomi; Sato, Kazuma; Yagi, Tomomi; Kishikawa, Yukinaga; Komatsu, Takaaki; Bagetta, Giacinto; Sakurada, Tsukasa; Nakamura, Hitoshi


    This study investigated the effect of bergamot essential oil (BEO) or linalool, a major volatile component of BEO, on the nociceptive response to formalin. Plantar subcutaneous injection of BEO or linalool into the ipsilateral hindpaw reduced both the first and late phases of the formalin-induced licking and biting responses in mice. Plantar subcutaneous injection of BEO or linalool into the contralateral hindpaw did not yield an antinociceptive effect, suggesting that the antinociceptive effect of BEO or linalool in the formalin test occurred peripherally. Intraperitoneal and plantar subcutaneous injection pretreatment with naloxone hydrochloride, an opioid receptor antagonist, significantly attenuated both BEO- and linalool-induced antinociception. Pretreatment with naloxone methiodide, a peripherally acting opioid receptor antagonists, also significantly antagonized the antinociceptive effects of BEO and linalool. Our results provide evidence for the involvement of peripheral opioids in antinociception induced by BEO and linalool. These results suggest that activation of peripheral opioid receptors may play an important role in reducing formalin-induced nociception.

  2. Local GABA receptor blockade reveals hindlimb responses in the SI forelimb-stump representation of neonatally amputated rats.


    Pluto, Charles P; Lane, Richard D; Rhoades, Robert W


    In adult rats that sustained forelimb amputation on the day of birth, there are numerous multi-unit recording sites in the forelimb-stump representation of primary somatosensory cortex (SI) that also respond to cutaneous stimulation of the hindlimb when cortical receptors for GABA are blocked. These normally suppressed hindlimb inputs originate in the SI hindlimb representation and synapse in the dysgranular cortex before exciting SI forelimb-stump neurons. In our previous studies, GABA (A + B) receptor blockade was achieved by topically applying a bicuculline methiodide/saclofen solution (BMI/SAC) to the cortical surface. This treatment blocks receptors throughout SI and does not allow determination of where along the above circuit the GABA-mediated suppression of hindlimb information occurs. In this study, focal injections of BMI/SAC were delivered to three distinct cortical regions that are involved in the hindlimb-to-forelimb-stump pathway. Blocking GABA receptors in the SI hindlimb representation and in the dysgranular cortex was largely ineffective in revealing hindlimb inputs ( approximately 10% of hindlimb inputs were revealed in both cases). In contrast, when the blockade was targeted at forelimb-stump recording sites, >80% of hindlimb inputs were revealed. Thus GABAergic interneurons within the forelimb-stump representation suppress the expression of reorganized hindlimb inputs to the region. A circuit model incorporating these and previous observations is presented and discussed.

  3. Contributions of peripheral and central opioid receptors to antinociception in rat muscle pain models.


    Sánchez, Eva Ma; Bagües, Ana; Martín, Ma Isabel


    Administration of hypertonic saline (HS) is an accepted model to study muscular pain. HS-induced nociceptive responses were tested in masseter, already described, and in two new pain models of spinally innervated muscles (gastrocnemius and triceps) developed in rats at our laboratory. HS administration in the masseter induced vigorous hindpaw shaking and in the gastrocnemius or triceps, paw withdrawal or flexing. Participation of the central and peripheral opioid receptors in HS-induced pain is compared in these muscles: masseter, innervated by trigeminal nerve, and gastrocnemius and triceps by spinal nerves. Morphine and loperamide were used to reveal peripheral and central components of opioid analgesia. Both agonists reduced HS-induced nociceptive behaviours in the masseter and were antagonised by the opioid antagonist naloxone and by naloxone methiodide, an opioid receptor antagonist that poorly penetrates the blood-brain barrier. Unexpectedly, in the gastrocnemius and triceps, morphine, but not loperamide, decreased the nociceptive behaviour and this effect was only reversed by naloxone. So, peripheral opioid receptors seem to participate in HS-induced masseter pain, whereas only central opioid receptors reduced the nociception in gastrocnemius and triceps. Our results suggest that the use of peripheral opioids can be more advantageous than central opioids for treatment of orofacial muscular pain.

  4. Antitussive activity of Withania somnifera and opioid receptors.


    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana


    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

  5. Destruction and creation of spatial tuning by disinhibition: GABA(A) blockade of prefrontal cortical neurons engaged by working memory.


    Rao, S G; Williams, G V; Goldman-Rakic, P S


    Local circuit neurons in the dorsolateral prefrontal cortex (dPFC) of monkeys have been implicated in the cellular basis of working memory. To further elucidate the role of inhibition in spatial tuning, we iontophoresed bicuculline methiodide (BMI) onto functionally characterized neurons in the dPFC of monkeys performing an oculomotor delayed response task. This GABA(A) blockade revealed that both putative interneurons and pyramidal cells possess significant inhibitory tone in the awake, behaving monkey. In addition, BMI application primarily resulted in the loss of previously extant spatial tuning in both cell types through reduction of both isodirectional and cross-directional inhibition. This tuning loss occurred in both the sensorimotor and mnemonic phases of the task, although the delay activity of prefrontal neurons appeared to be particularly affected. Finally, application of BMI also created significant spatial tuning in a sizable minority of units that were untuned in the control condition. Visual field analysis of such tuning suggests that it is likely caused by the unmasking of normally suppressed spatially tuned excitatory input. These findings provide the first direct evidence of directional inhibitory modulation of pyramidal cell and interneuron firing in both the mnemonic and sensorimotor phases of the working memory process, and they implicate a further role for GABAergic interneurons in the construction of spatial tuning in prefrontal cortex.

  6. Respiratory, metabolic and cardiac functions are altered by disinhibition of subregions of the medial prefrontal cortex

    PubMed Central

    Hassan, Sarah F; Cornish, Jennifer L; Goodchild, Ann K


    The prefrontal cortex (PFC) is referred to as the visceral motor cortex; however, little is known about whether this region influences respiratory or metabolic outflows. The aim of this study was to describe simultaneous changes in respiratory, metabolic and cardiovascular functions evoked by disinhibition of the medial PFC (mPFC) and adjacent lateral septal nucleus (LSN). In urethane-anaesthetized rats, bicuculline methiodide was microinjected (2 mm; GABA-A receptor antagonist) into 90 sites in the mPFC at 0.72–4.00 mm from bregma. Phrenic nerve amplitude and frequency, arterial pressure, heart rate, splanchnic and lumbar sympathetic nerve activities (SNA), expired CO2, and core and brown adipose tissue temperatures were measured. Novel findings included disturbances to respiratory rhythm evoked from all subregions of the mPFC. Injections into the cingulate cortex evoked reductions in central respiratory function exclusively, whereas in ventral sites, particularly the infralimbic region, increases in respiratory drive and frequency, and metabolic and cardiac outflows were evoked. Disinhibition of sites in surrounding regions revealed that the LSN could evoke cardiovascular changes accompanied by distinct oscillations in SNA, as well as increases in respiratory amplitude. We show that activation of neurons within the mPFC and LSN influence respiratory, metabolic and cardiac outflows in a site-dependent manner. This study has implications with respect to the altered PFC neuronal activity seen in stress-related and mental health disorders, and suggests how basic physiological systems may be affected. PMID:24042503

  7. Blockade of GABA, type A, receptors in the rat pontine reticular formation induces rapid eye movement sleep that is dependent upon the cholinergic system.


    Marks, G A; Sachs, O W; Birabil, C G


    The brainstem reticular formation is an area important to the control of rapid eye movement (REM) sleep. The antagonist of GABA-type A (GABA(A)) receptors, bicuculline methiodide (BMI), injected into the rat nucleus pontis oralis (PnO) of the reticular formation resulted in a long-lasting increase in REM sleep. Thus, one factor controlling REM sleep appears to be the number of functional GABA(A) receptors in the PnO. The long-lasting effect produced by BMI may result from secondary influences on other neurotransmitter systems known to have long-lasting effects. To study this question, rats were surgically prepared for chronic sleep recording and additionally implanted with guide cannulas aimed at sites in the PnO. Multiple, 60 nl, unilateral injections were made either singly or in combination. GABA(A) receptor antagonists, BMI and gabazine (GBZ), produced dose-dependent increases in REM sleep with GBZ being approximately 35 times more potent than BMI. GBZ and the cholinergic agonist, carbachol, produced very similar results, both increasing REM sleep for about 8 h, mainly through increased period frequency, with little reduction in REM latency. Pre-injection of the muscarinic antagonist, atropine, completely blocked the REM sleep-increase by GBZ. GABAergic control of REM sleep in the PnO requires the cholinergic system and may be acting through presynaptic modulation of acetylcholine release.

  8. The effect of boric acid on acethylcholine, bethanechol and potasssium-evoked responses on ileum of rat.


    Ince, S; Turkmen, R; Yavuz, H


    1 The aim of this study was to clarify the effect of boric acid on contractions of rat isolated ileum. 2 Contractile responses expressed as Emax and pD2 for acetylcholine (10(-3)-10(-8) m, Ach), bethanechol (10(-3)-10(-8) m) and potassium (10-80 × 10(-3) m, KCl) were determined in the absence and presence of boric acid (10(-3); 5 × 10(-4); 10(-4) m). 3 The contractile response to Ach in the presence of verapamil (10(-6) or 10(-8) m) or in calcium-free Tyrode's solution was also determined in the absence and presence of boric acid. 4 Boric acid did not affect the contractile response to Ach, bethanechol or KCl. Single or cumulative treatment of boric acid did not affect ileum muscle contraction evoked by KCl. The atropine-resistant component of Ach-induced contraction and 4-diphenyl-acetoxy-N-methyl-piperidine methiodide-resistant component of bethanechol-induced contraction were not inhibited by boric acid (10(-3) m). The contractile response to Ach was reduced in calcium-free Tyrode's solution, and the contractile response was not affected by (10(-8) m). The addition of boric acid (10(-3) m) in combination with verapamil (10(-8) m) did not significantly affect the contractile response to Ach. 5 In conclusion, boric acid does not affect contractions induced by Ach, bethanechol or potassium in rat isolated ileum.

  9. Action of certain tropine esters on voltage-clamped lobster axon.


    Blaustein, M P


    Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClphiA), which differs from TPTA only by the substitution of a p-Cl for a p-CH(3) group on the benzene ring, had a negligible effect on axonal excitability.

  10. Biochemical basis for the toxic effects of triethyl lead

    PubMed Central

    Galzigna, L.; Ferraro, M. V.; Manani, G.; Viola, A.


    Galzigna, L., Ferraro, M. V., Manani, G., and Viola, A. (1973).British Journal of Industrial Medicine,30, 129-133. Biochemical basis for the toxic effects of triethyl lead. The effects of triethyl lead (PbEt3) have been studied in vitro on the cholinesterase activity of rat diaphragm and in vivo on serum cholinesterase in the dog. PbEt3 dramatically increases the duration of succinylcholine-induced myoneural block and pyridine-2-aldoxime methiodide (PAM) is able to counteract both cholinesterase inhibition and the effects on neuromuscular transmission. On the other hand, PbEt3 catalyses the rearrangement of catecholamines to aminochromes in vitro and inhibits catecholamine effect on smooth muscle contraction. The toxicity of PbEt3 and particularly its action on the central nervous system can be explained by a combination of effects which might result from an upset of cholinergic and adrenergic central pathways due to the formation of endogenous psychotogenic complexes. Images PMID:4349924

  11. Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

    NASA Technical Reports Server (NTRS)

    Moffitt, Julia A.; Heesch, Cheryl M.; Hasser, Eileen M.


    Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.

  12. GABA-ergic neurons in the leach central nervous system

    SciTech Connect

    Cline, H.T.


    GABA is a candidate for an inhibitory neurotransmitter in the leech central nervous system because of the well-documented inhibitory action of GABA in other invertebrates. To demonstrate that GABA meets the criteria used to identify a substance as a neurotransmitter, the author examined GABA metabolism and synaptic interactions of inhibitory motor neurons in two leech species, Hirudo medicinalis and Haementeria ghilianii. Segmental ganglia of the leech ventral nerve cord and identified inhibitors have the capacity to synthesize GABA when incubated in the presence of the precursor glutamate. Application of GABA to cell bodies of excitatory motor neurons or muscle fibers innervated by the inhibitors hyperpolarizes the membrane potential of the target cell and activates a chloride ion conductance channel, similar to the inhibitory membrane response following intracellular stimulation of the inhibitor. Bicuculline methiodide (5 x 10/sup -5/M), GABA receptor antagonist, blocks reversibly the response to applied GABA and the inhibitory synaptic inputs onto the postsynaptic neurons or muscle fibers without interfering with their excitatory inputs. Furthermore, the inhibitors are included among approximately 25 neurons per segmental ganglion that take up GABA by a high affinity uptake system, as revealed by /sup 3/H-GABA-autoradiography. The development of the capacities to synthesize and to take up GABA were examined in leech embryos. The embryos are able to synthesize GABA at early stages of the development of the nervous system, before any neurons have extended neutrites.

  13. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.


    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna


    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  14. Effect of some blocking drugs on the pressor response to physostigmine in the rat

    PubMed Central

    Gokhale, S. D.; Gulati, O. D.; Joshi, N. Y.


    Bretylium and guanethidine blocked the pressor effect of physostigmine and potentiated the responses to adrenaline and noradrenaline on the blood pressure of the rat. Morphine and atropine in small doses blocked the pressor effect of physostigmine without interfering with the actions of adrenaline and noradrenaline. Chlorpromazine in small doses (0.5 to 2.5 mg/kg) blocked the pressor effect of physostigmine and potentiated the responses to noradrenaline whilst those to adrenaline remained unaltered. 3,6-Di(3-diethylaminopropoxy)pyridazine di(methiodide) (Win 4981) blocked the pressor effect of physostigmine and, in its early stages, this block was partially reversed by choline chloride. N-Diethylaminoethyl-N-isopentyl-N'N'-diisopropylurea (P-286), in a dose that reduced the effect of dimethylphenylpiperazinium, had no effect on the pressor response to physostigmine or on the responses to adrenaline and noradrenaline. Hexamethonium, even in large doses (100 mg/kg), only blocked partially the effect of physostigmine while mecamylamine produced a complete block; the responses to adrenaline and noradrenaline were potentiated in both instances. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6 PMID:14081658

  15. GABAergic influence on temporomandibular joint-responsive spinomedullary neurons depends on estrogen status.


    Tashiro, A; Bereiter, D A; Thompson, R; Nishida, Y


    Sensory input from the temporomandibular joint (TMJ) to neurons in superficial laminae at the spinomedullary (Vc/C1-2) region is strongly influenced by estrogen status. This study determined if GABAergic mechanisms play a role in estrogen modulation of TMJ nociceptive processing in ovariectomized female rats treated with high- (HE) or low-dose (LE) estradiol (E2) for 2days. Superficial laminae neurons were activated by ATP (1mM) injections into the joint space. The selective GABAA receptor antagonist, bicuculline methiodide (BMI, 5 or 50μM, 30μl), applied at the site of recording greatly enhanced the magnitude and duration of ATP-evoked responses in LE rats, but not in units from HE rats. The convergent cutaneous receptive field (RF) area of TMJ neurons was enlarged after BMI in LE but not HE rats, while resting discharge rates were increased after BMI independent of estrogen status. By contrast, the selective GABAA receptor agonist, muscimol (50μM, 30μl), significantly reduced the magnitude and duration of ATP-evoked activity, resting discharge rate, and cutaneous RF area of TMJ neurons in LE and HE rats, whereas lower doses (5μM) affected only units from LE rats. Protein levels of GABAA receptor β3 isoform at the Vc/C1-2 region were similar for HE and LE rats. These results suggest that GABAergic mechanisms contribute significantly to background discharge rates and TMJ-evoked input to superficial laminae neurons at the Vc/C1-2 region. Estrogen status may gate the magnitude of GABAergic influence on TMJ neurons at the earliest stages of nociceptive processing at the spinomedullary region.

  16. Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases.


    Radić, Zoran; Sit, Rakesh K; Kovarik, Zrinka; Berend, Suzana; Garcia, Edzna; Zhang, Limin; Amitai, Gabriel; Green, Carol; Radić, Bozica; Fokin, Valery V; Sharpless, K Barry; Taylor, Palmer


    We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.

  17. Modulation of peripheral μ-opioid analgesia by σ1 receptors.


    Sánchez-Fernández, Cristina; Montilla-García, Ángeles; González-Cano, Rafael; Nieto, Francisco Rafael; Romero, Lucía; Artacho-Cordón, Antonia; Montes, Rosa; Fernández-Pastor, Begoña; Merlos, Manuel; Baeyens, José Manuel; Entrena, José Manuel; Cobos, Enrique José


    We evaluated the effects of σ1-receptor inhibition on μ-opioid-induced mechanical antinociception and constipation. σ1-Knockout mice exhibited marked mechanical antinociception in response to several μ-opioid analgesics (fentanyl, oxycodone, morphine, buprenorphine, and tramadol) at systemic (subcutaneous) doses that were inactive in wild-type mice and even unmasked the antinociceptive effects of the peripheral μ-opioid agonist loperamide. Likewise, systemic (subcutaneous) or local (intraplantar) treatment of wild-type mice with the selective σ1 antagonists BD-1063 [1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride] or S1RA [4-[2-[[5-methyl-1-(2-naphthalenyl)1H-pyrazol-3-yl]oxy]ethyl] morpholine hydrochloride] potentiated μ-opioid antinociception; these effects were fully reversed by the σ1 agonist PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate) hydrochloride], showing the selectivity of the pharmacological approach. The μ-opioid antinociception potentiated by σ1 inhibition (by σ1-receptor knockout or σ1-pharmacological antagonism) was more sensitive to the peripherally restricted opioid antagonist naloxone methiodide than opioid antinociception under normal conditions, indicating a key role for peripheral opioid receptors in the enhanced antinociception. Direct interaction between the opioid drugs and σ1 receptor cannot account for our results, since the former lacked affinity for σ1 receptors (labeled with [(3)H](+)-pentazocine). A peripheral role for σ1 receptors was also supported by their higher density (Western blot results) in peripheral nervous tissue (dorsal root ganglia) than in several central areas involved in opioid antinociception (dorsal spinal cord, basolateral amygdala, periaqueductal gray, and rostroventral medulla). In contrast to its effects on nociception, σ1-receptor inhibition did not alter fentanyl- or loperamide-induced constipation, a peripherally mediated nonanalgesic opioid effect. Therefore

  18. The Sensory Impact of Nicotine on Noradrenergic and Dopaminergic Neurons of the Nicotine Reward - Addiction Neurocircuitry.


    Rose, Jed E; Dehkordi, Ozra; Manaye, Kebreten F; Millis, Richard M; Cianaki, Salman Ameri; Jayam-Trouth, Annapurni


    The sensory experience of smoking is a key component of nicotine addiction known to result, in part, from stimulation of nicotinic acetylcholine receptors (nAChRs) at peripheral sensory nerve endings. Such stimulation of nAChRs is followed by activation of neurons at multiple sites in the mesocorticolimbic reward pathways. However, the neurochemical profiles of CNS cells that mediate the peripheral sensory impact of nicotine remain unknown. In the present study in mice, we first used c-Fos immunohistochemistry to identify CNS cells stimulated by nicotine (NIC, 40 μg/kg, IP) and by a peripherally-acting analog of nicotine, nicotine pyrrolidine methiodide (NIC-PM, 30 μg/kg, IP). Sequential double-labelling was then performed to determine whether noradrenergic and dopaminergic neurons of the nicotine reward-addiction circuitry were primary targets of NIC and NIC-PM. Double-labelling of NIC and/or NIC-PM activated c-Fos immunoreactive cells with tyrosine hydroxylase (TH) showed no apparent c-Fos expression by the dopaminergic cells of the ventral tegmental area (VTA). With the exception of sparse numbers of TH immunoreactive D11 cells, dopamine-containing neurons in other areas of the reward-addiction circuitry, namely periaqueductal gray, and dorsal raphe, were also devoid of c-Fos immunoreactivity. Noradrenergic neurons of locus coeruleus (LC), known to innervate VTA, were activated by both NIC and NIC-PM. These results demonstrate that noradrenergic neurons of LC are among the first structures that are stimulated by single acute IP injection of NIC and NIC-PM. Dopaminergic neurons of VTA and other CNS sites, did not respond to acute IP administration of NIC or NIC-PM by induction of c-Fos.

  19. PIP2 in pancreatic β-cells regulates voltage-gated calcium channels by a voltage-independent pathway.


    de la Cruz, Lizbeth; Puente, Erika I; Reyes-Vaca, Arturo; Arenas, Isabel; Garduño, Julieta; Bravo-Martínez, Jorge; Garcia, David E


    Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (CaV) channels promotes insulin secretion in pancreatic β-cells. However, whether CaV channels are regulated by PIP2, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether CaV channels are regulated by PIP2 depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). CaV channel currents were recorded by the patch-clamp technique. The CaV current amplitude was reduced by activation of the muscarinic receptor 1 (M1R) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced CaV inhibition was diminished by a high concentration of Ca(2+) chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP2 dialysis. Localization of PIP2 in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP2 hydrolysis and CaV channel inhibition. Furthermore, the depletion of PIP2 by a voltage-sensitive phosphatase reduced CaV currents in a way similar to that observed following M1R activation. These results indicate that activation of the M1R pathway inhibits the CaV channel via PIP2 depletion by a Ca(2+)-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

  20. Cardiovascular and behavioral effects produced by administration of liposome-entrapped GABA into the rat central nervous system.


    Vaz, G C; Bahia, A P C O; de Figueiredo Müller-Ribeiro, F C; Xavier, C H; Patel, K P; Santos, R A S; Moreira, F A; Frézard, F; Fontes, M A P


    Liposomes are nanosystems that allow a sustained release of entrapped substances. Gamma-aminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter of the central nervous system (CNS). We developed a liposomal formulation of GABA for application in long-term CNS functional studies. Two days after liposome-entrapped GABA was injected intracerebroventricularly (ICV), Wistar rats were submitted to the following evaluations: (1) changes in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) to ICV injection of bicuculline methiodide (BMI) in anesthetized rats; (2) changes in cardiovascular reactivity to air jet stress in conscious rats; and (3) anxiety-like behavior in conscious rats. GABA and saline-containing pegylated liposomes were prepared with a mean diameter of 200 nm. Rats with implanted cannulas targeted to lateral cerebral ventricle (n = 5-8/group) received either GABA solution (GS), empty liposomes (EL) or GABA-containing liposomes (GL). Following (48 h) central microinjection (2 μL, 0.09 M and 99 g/L) of liposomes, animals were submitted to the different protocols. Animals that received GL demonstrated attenuated response of RSNA to BMI microinjection (GS 48 ± 9, EL 43 ± 9, GL 11 ± 8%; P < 0.05), blunted tachycardia in the stress trial (ΔHR: GS 115 ± 14, EL 117 ± 10, GL 74 ± 9 bpm; P<0.05) and spent more time in the open arms of elevated plus maze (EL 6 ± 2 vs. GL 18 ± 5%; P = 0.028) compared with GS and EL groups. These results indicate that liposome-entrapped GABA can be a potential tool for exploring the chronic effects of GABA in specific regions and pathways of the central nervous system.

  1. Inhibition by levetiracetam of a non-GABAA receptor-associated epileptiform effect of bicuculline in rat hippocampus

    PubMed Central

    Margineanu, Doru Georg; Wülfert, Ernst


    Extracellular recording of field potentials, evoked by commissural stimulation in hippocampal area CA3 of anaesthetized rats, was performed in order to study the mode of action of the novel antiepileptic drug levetiracetam (ucb LO59). The amplitude of orthodromic field population spike (PS2) markedly increased and repetitive population spikes appeared when the recording micropipette contained either bicuculline methiodide (BMI), or the specific GABAA antagonist gabazine (SR-95531). BMI-induced increases in PS2 were reduced in a dose-dependent manner by 1 to 320 μmol kg−1 levetiracetam i.v., with a U-shape dose-response relationship. However, levetiracetam did not reduce the increases in PS2 produced by gabazine. Clonazepam (1 mg kg−1, i.p.), carbamazepine (20 mg kg−1, i.p.) and valproate (200 mg kg−1, i.v.) were ineffective in preventing BMI-induced increases in PS2, while the calcium channel antagonist flunarizine, 50 μmol kg−1, i.p., reduced PS2 increments caused by BMI. The L-type calcium channel blocker nifedipine, 100 μmol kg−1, i.p., was without effect. Similar to levetiracetam, flunarizine did not reduce the increases in PS2 induced by gabazine. These data suggest that the increased excitability of CA3 neurones, caused by BMI administered in situ, involves calcium-dependent processes not associated with blockade of GABAA receptors. The inhibition by levetiracetam of this calcium-dependent effect of BMI might contribute to the antiepileptic effects of the drug. PMID:9401779

  2. Seizure-like activity in the disinhibited CA1 minislice of adult guinea-pigs

    PubMed Central

    Karnup, Sergei; Stelzer, Armin


    Spontaneous activity was monitored during pharmacological blockade of GABAA receptor function in the CA1 minislice (CA3 was cut off). Synaptic inhibition was blocked by competitive GABAA antagonists bicuculline-methiodide (Bic) or GABAZINE (GBZ) and the chloride channel blocker picrotoxin (PTX). Extra- and intracellular recordings using sharp electrodes were carried out in stratum radiatum and pyramidale. At low antagonist concentrations (Bic, GBZ: 1-10 μm; PTX: < 100 μm), synchronized bursts (< 500 ms in duration, interictal activity) were seen as described previously. However, in the presence of high concentrations (Bic, GBZ: 50-100 μm; PTX: 100-200 μm), seizure-like, ictal events (duration 4-17 s) were observed in 67 of 88 slices. No other experimental measures to increase excitability were applied: cation concentrations ([Ca2+]o= 2 mm, [Mg2+]o= 1.7 mm, [K+]o= 3 mm) and recording temperature (30-32 °C) were standard and GABAB-mediated inhibition was intact. In whole-slice recordings prominent interictal activity, but fewer ictal events were observed. A reduced ictal activity was also observed when interictal-like responses were evoked by afferent stimulation. Ictal activity was reversibly blocked by antagonists of excitatory transmission, CNQX (40 μm) or d-AP5 (50 μm). Disinhibition-induced ictal development did not rely on group I mGluR activation as it was not prevented in the presence of group I mGluR antagonists (AIDA or 4CPG). (RS)-3,5-DHPG prevented the induction and reversed the tertiary component of the ictal event through a group I mGluR-independent mechanism. PMID:11313441

  3. Rational Basis for the Use of Bergamot Essential Oil in Complementary Medicine to Treat Chronic Pain.


    Rombolà, L; Amantea, D; Russo, R; Adornetto, A; Berliocchi, L; Tridico, L; Corasaniti, M T; Sakurada, S; Sakurada, T; Bagetta, G; Morrone, L A


    In complementary medicine, aromatherapy uses essential oils to improve agitation and aggression observed in dementia, mood, depression, anxiety and chronic pain. Preclinical research studies have reported that the essential oil obtained from bergamot (BEO) fruit (Citrus bergamia, Risso) modifies normal and pathological synaptic plasticity implicated, for instance, in nociceptive and neuropathic pain. Interestingly, recent results indicated that BEO modulates sensitive perception of pain in different models of nociceptive, inflammatory and neuropathic pain modulating endogenous systems. Thus, local administration of BEO inhibited the nociceptive behavioral effect induced by intraplantar injection of capsaicin or formalin in mice. Similar effects were observed with linalool and linalyl acetate, major volatile components of the phytocomplex, Pharmacological studies showed that the latter effects are reversed by local or systemic pretreatment with the opioid antagonist naloxone hydrochloride alike with naloxone methiodide, high affinity peripheral μ-opioid receptor antagonist. These results and the synergistic effect observed following systemic or intrathecal injection of an inactive dose of morphine with BEO or linalool indicated an activation of peripheral opioid system. Recently, in neuropathic pain models systemic or local administration of BEO or linalool induced antiallodynic effects. In particular, in partial sciatic nerve ligation (PSNL) model, intraplantar injection of the phytocomplex or linalool in the ipsilateral hindpaw, but not in the contralateral, reduced PSNL-induced extracellularsignal- regulated kinase (ERK) activation and mechanical allodynia. In neuropathic pain high doses of morphine are needed to reduce pain. Interestingly, combination of inactive doses of BEO or linalool with a low dose of morphine induced antiallodynic effects in mice. Peripheral cannabinoid and opioid systems appear to be involved in the antinociception produced by

  4. Opioids in the nucleus accumbens stimulate ethanol intake.


    Barson, Jessica R; Carr, Ambrose J; Soun, Jennifer E; Sobhani, Nasim C; Leibowitz, Sarah F; Hoebel, Bartley G


    The nucleus accumbens (NAc) participates in the control of both motivation and addiction. To test the possibility that opioids in the NAc can cause rats to select ethanol in preference to food, Sprague-Dawley rats with ethanol, food, and water available, were injected with two doses each of morphine, the mu-receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin (DAMGO), the delta-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), the k-receptor agonist (+/-)-trans-U-50488 methanesulfonate (U-50,488H), or the opioid antagonist naloxone methiodide (m-naloxone). As an anatomical control for drug reflux, injections were also made 2mm above the NAc. The main result was that morphine in the NAc significantly increased ethanol and food intake, whereas m-naloxone reduced ethanol intake without affecting food or water intake. Of the selective receptor agonists, DALA in the NAc increased ethanol intake in preference to food. This is in contrast to DAMGO, which stimulated food but not ethanol intake, and the k-agonist U-50,488H, which had no effect on intake. When injected in the anatomical control site 2mm dorsal to the NAc, the opioids had no effects on ethanol intake. These results demonstrate that ethanol intake produced by morphine in the NAc is driven in large part by the delta-receptor. In light of other studies showing ethanol intake to increase enkephalin expression in the NAc, the present finding of enkephalin-induced ethanol intake suggests the existence of a positive feedback loop that fosters alcohol abuse. Naltrexone therapy for alcohol abuse may then act, in part, in the NAc by blocking this opioid-triggered cycle of alcohol intake.

  5. GABA-A receptors regulate neocortical neuronal migration in vitro and in vivo.


    Heck, Nicolas; Kilb, Werner; Reiprich, Petra; Kubota, Hisahiko; Furukawa, Tomonori; Fukuda, Atsuo; Luhmann, Heiko J


    The cortical migration process depends on a number of trophic factors and on the activation of different voltage- and ligand-gated channels. We investigated the role of gamma-aminobutyric acid (GABA) type A receptors in the neuronal migration process of the newborn rat parietal cortex in vivo and in vitro. Local in vivo application of the GABA-A antagonist bicuculline methiodide (BMI) or the agonist muscimol via cortical surface Elvax implants induced prominent alterations in the cortical architecture when compared with untreated or sham-operated controls. BMI- and muscimol-treated animals revealed heterotopic cell clusters in the upper layers and a complete loss of the cortical lamination in the region underlying the Elvax implant. Immunocytochemical staining for glial fibrillary acidic protein, N-methyl-D-aspartate receptors, and GABA demonstrated that heterotopia was not provoked by glial proliferation and confirmed the presence of both glutamatergic and GABAergic neurons. In organotypic neocortical slices from embryonic day 18-19 embryos, application of BMI and to a lesser extent also muscimol induced an increase in the migration speed and an accumulation of neurons in the upper cortical layers. Spontaneous intracellular calcium ([Ca2+]i) oscillations in neocortical slices from newborn rats were abolished by BMI (5 and 20 microM) and muscimol (1 and 10 microM), indicating that both compounds interfere with [Ca2+]i signaling required for normal neuronal migration. Electrophysiological recordings from migrating neurons in newborn rat neocortical slices indicate that long-term application of muscimol causes a pronounced reduction (1 microM muscimol) or blockade (10 microM) in the responsiveness of postsynaptic GABA-A receptors due to a pronounced receptor desensitization. Our results indicate that modulation of GABA-A receptors by compounds acting as agonists or antagonists may profoundly influence the neuronal migration process in the developing cerebral cortex.

  6. A novel muscarinic receptor-independent mechanism of KCNQ2/3 potassium channel blockade by Oxotremorine-M.


    Zwart, Ruud; Reed, Hannah; Clarke, Sophie; Sher, Emanuele


    Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels.

  7. Rapid Sensitization of Physiological, Neuronal, and Locomotor Effects of Nicotine: Critical Role of Peripheral Drug Actions

    PubMed Central

    Lenoir, Magalie; Tang, Jeremy S.; Woods, Amina S.


    Repeated exposure to nicotine and other psychostimulant drugs produces persistent increases in their psychomotor and physiological effects (sensitization), a phenomenon related to the drugs' reinforcing properties and abuse potential. Here we examined the role of peripheral actions of nicotine in nicotine-induced sensitization of centrally mediated physiological parameters (brain, muscle, and skin temperatures), cortical and VTA EEG, neck EMG activity, and locomotion in freely moving rats. Repeated injections of intravenous nicotine (30 μg/kg) induced sensitization of the drug's effects on all these measures. In contrast, repeated injections of the peripherally acting analog of nicotine, nicotine pyrrolidine methiodide (nicotinePM, 30 μg/kg, i.v.) resulted in habituation (tolerance) of the same physiological, neuronal, and behavioral measures. However, after repeated nicotine exposure, acute nicotinePM injections induced nicotine-like physiological responses: powerful cortical and VTA EEG desynchronization, EMG activation, a large brain temperature increase, but weaker hyperlocomotion. Additionally, both the acute locomotor response to nicotine and nicotine-induced locomotor sensitization were attenuated by blockade of peripheral nicotinic receptors by hexamethonium (3 mg/kg, i.v.). These data suggest that the peripheral actions of nicotine, which precede its direct central actions, serve as a conditioned interoceptive cue capable of eliciting nicotine-like physiological and neural responses after repeated nicotine exposure. Thus, by providing a neural signal to the CNS that is repeatedly paired with the direct central effects of nicotine, the drug's peripheral actions play a critical role in the development of nicotine-induced physiological, neural, and behavioral sensitization. PMID:23761889

  8. Effects of Prunus mume Siebold & Zucc. in the pacemaking activity of interstitial cells of Cajal in murine small intestine

    PubMed Central

    Lee, Sang Weon; Kim, Sung Jin; Kim, Hyungwoo; Yang, Dongki; Kim, Hyun Jung; Kim, Byung Joo


    Interstitial cells of Cajal (ICCs) function as pacemaker cells in the gastrointestinal (GI) tract and therefore, serve an important role in regulating GI motility. The effects of a species of plum (Prunus mume Siebold & Zucc.) on cultured ICC cluster-induced pacemaker potentials in the mouse small intestine were investigated, and the effects of a methanolic extract of Prunus mume (m-PM) on ICC pacemaker activities were examined using the whole-cell patch-clamp technique. ICC pacemaker membrane potentials were depolarized by m-PM in a concentration dependent manner in current clamp mode. 4-Diphenylacetoxy-N-methyl-piperidine methiodide, which is a muscarinic 3 (M3) receptor antagonist, was able to block m-PM-induced pacemaker potential increases, whereas methoctramine, which is a muscarinic 2 (M2) receptor antagonist, was not. When 1 mM guanosine diphosphate β-5 was present in the pipette solution, m-PM induced slight pacemaker depolarization. Following pretreatment in bath solution of Ca2+-free solution or a Ca2+-ATPase inhibitor in endoplasmic reticulum, the pacemaker currents were inhibited. Furthermore, pretreatment with PD98059, SB203580 or SP600125, which is a c-jun NH2-terminal kinase inhibitor, blocked m-PM-induced ICC potential depolarization. Furthermore, m-PM inhibited transient receptor potential melastatin (TRPM) 7 channels, but did not affect Ca2+-activated Cl− channels. These results suggest that m-PM is able to modulate pacemaker potentials through the muscarinic M3 receptor, via G-protein and external and internal Ca2+, in a mitogen-activated protein kinase and TRPM7-dependent manner. Therefore, m-PM may provide a basis for the development of a novel gastroprokinetic agent. PMID:28123510

  9. High fat diet induced-obesity facilitates anxiety-like behaviors due to GABAergic impairment within the dorsomedial hypothalamus in rats.


    de Noronha, Sylvana Rendeiro; Campos, Glenda Viggiano; Abreu, Aline Rezende; de Souza, Aline Arlindo; Chianca, Deoclécio A; de Menezes, Rodrigo C


    Overweight and obesity are conditions associated with an overall range of clinical health consequences, and they could be involved with the development of neuropsychiatric diseases, such as generalized anxiety disorder (GAD) and panic disorder (PD). A crucial brain nuclei involved on the physiological functions and behavioral responses, especially fear, anxiety and panic, is the dorsomedial hypothalamus (DMH). However, the mechanisms underlying the process whereby the DMH is involved in behavioral changes in obese rats still remains unclear. The current study further investigates the relation between obesity and generalized anxiety, by investigating the GABAA sensitivity to pharmacological manipulation within the DMH in obese rats during anxiety conditions. Male Wistar rats were divided in two experimental groups: the first was fed a control diet (CD; 11% w/w) and second was fed a high fat diet (HFD; 45% w/w). Animals were randomly treated with muscimol, a GABAA agonist and bicuculline methiodide (BMI), a GABAA antagonist. Inhibitory avoidance and escape behaviors were investigated using the Elevated T-Maze (ETM) apparatus. Our results revealed that the obesity facilitated inhibitory avoidance acquisition, suggesting a positive relation between obesity and the development of an anxiety-like state. The injection of muscimol (an anxiolytic drug), within the DMH, increased the inhibitory avoidance latency in obese animals (featuring an anxiogenic state). Besides, muscimol prolonged the escape latency and controlling the possible panic-like behavior in these animals. Injection of BMI into the DMH was ineffective to produce an anxiety-like effect in obese animals opposing the results observed in lean animals. These findings support the hypotheses that obese animals are susceptible to develop anxiety-like behaviors, probably through changes in the GABAergic neurotransmission within the DMH.

  10. Asymmetry in the control of cardiac performance by dorsomedial hypothalamus.


    Xavier, Carlos Henrique; Beig, Mirza Irfan; Ianzer, Danielle; Fontes, Marco Antônio Peliky; Nalivaiko, Eugene


    Dorsomedial hypothalamus (DMH) plays a key role in integrating cardiovascular responses to stress. We have recently reported greater heart rate responses following disinhibition of the right side of the DMH (R-DMH) in anesthetized rats and greater suppression of stress-induced tachycardia following inhibition of the R-DMH in conscious rats [both compared with similar intervention in the left DMH (L-DMH)], suggesting existence of right/left side asymmetry in controlling cardiac chronotropic responses by the DMH. The aim of the present study was to determine whether similar asymmetry is present for controlling cardiac contractility. In anesthetized rats, microinjections of the GABAA antagonist bicuculline methiodide (BMI; 40 pmol/100 nl) into the DMH-evoked increases in heart rate (HR), left ventricular pressure (LVP), myocardial contractility (LVdP/dt), arterial pressure, and respiratory rate. DMH disinhibition also precipitated multiple ventricular and supraventricular ectopic beats. DMH-induced increases in HR, LVP, LVdP/dt, and in the number of ectopic beats dependent on the side of stimulation, with R-DMH provoking larger responses. In contrast, pressor and respiratory responses did not depend on the side of stimulation. Newly described DMH-induced inotropic responses were rate-, preload- and (largely) afterload-independent; they were mediated by sympathetic cardiac pathway, as revealed by their sensitivity to β-adrenergic blockade. We conclude that recruitment of DMH neurons causes sympathetically mediated positive chronotropic and inotropic effects, and that there is an asymmetry, at the level of the DMH, in the potency to elicit these effects, with R-DMH > L-DMH.

  11. Dorsomedial/Perifornical Hypothalamic Stimulation Increases Intraocular Pressure, Intracranial Pressure, and the Translaminar Pressure Gradient

    PubMed Central

    Samuels, Brian C.; Hammes, Nathan M.; Johnson, Philip L.; Shekhar, Anantha; McKinnon, Stuart J.; Allingham, R. Rand


    Purpose. Intraocular pressure (IOP) fluctuation has recently been identified as a risk factor for glaucoma progression. Further, decreases in intracranial pressure (ICP), with postulated increases in the translaminar pressure gradient across the lamina cribrosa, has been reported in glaucoma patients. We hypothesized that circadian fluctuations in IOP and the translaminar pressure gradient are influenced, at least in part, by central autonomic regulatory neurons within the dorsomedial and perifornical hypothalamus (DMH/PeF). This study examined whether site-directed chemical stimulation of DMH/PeF neurons evoked changes in IOP, ICP, and the translaminar pressure gradient. Methods. The GABAA receptor antagonist bicuculline methiodide (BMI) was stereotaxically microinjected into the DMH/PeF region of isoflurane-anesthetized male Sprague-Dawley rats (n = 19). The resulting peripheral cardiovascular (heart rate [HR] and mean arterial pressure [MAP]), IOP, and ICP effects were recorded and alterations in the translaminar pressure gradient calculated. Results. Chemical stimulation of DMH/PeF neurons evoked significant increases in HR (+69.3 ± 8.5 beats per minute); MAP (+22.9 ± 1.6 mm Hg); IOP (+7.1 ± 1.9 mm Hg); and ICP (+3.6 ± 0.7 mm Hg) compared with baseline values. However, the peak IOP increase was significantly delayed compared with ICP (28 vs. 4 minutes postinjection), resulting in a dramatic translaminar pressure gradient fluctuation. Conclusions. Chemical stimulation of DMH/PeF neurons evokes substantial increases in IOP, ICP, and the translaminar pressure gradient in the rat model. Given that the DMH/PeF neurons may be a key effector pathway for circadian regulation of autonomic tone by the suprachiasmatic nucleus, these findings will help elucidate novel mechanisms modulating circadian fluctuations in IOP and the translaminar pressure gradient. PMID:23033392

  12. A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity.


    Freier, D O; Fuchs, B A


    Morphine is a drug of abuse with an ability to down-regulate immune responsiveness that could have potentially serious consequences in both heroin addicts and in the clinical environment. The exact mechanism of action by which morphine induces immunosuppression has yet to be clearly determined. A direct mechanism of action is suggested to operate through lymphocyte opiate receptors, but the nature of such receptors is still in question. The alternative, an indirect mechanism of action is proposed to be mediated by two possible pathways, hypothalamic-pituitary-adrenal axis activation with increased production of adrenal corticosteroids, or activation of the sympathetic nervous system and concomitant catecholamine release. Natural killer (NK) cell activity was used to determine potential indirect mechanisms of action for morphine. NK activity in the B6C3F1 mouse was suppressed between 12 and 48 hr after implantation of 75 mg timed-release morphine pellets. Morphine suppressed NK activity in a dose-responsive manner. The opiate antagonists naloxone and naltrexone completely blocked morphine-induced suppression of NK activity, whereas naloxone methiodide, a congener that crosses the blood-brain barrier much more slowly than naloxone, produced very little blockade. Implantation of the 75-mg morphine pellets produced a significant elevation in serum corticosterone levels. In vitro exposure to corticosterone is known to suppress NK activity directly, whereas in vitro morphine was unable to alter directly NK activity. The glucocorticoid receptor antagonist Roussel-Uclaf 38486 blocked morphine-induced suppression of NK activity in a dose-responsive fashion. Naltrexone (10-mg pellet) antagonized the morphine-induced elevation in serum corticosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Hyperalgesic and hypoalgesic mechanisms evoked by the acute administration of CCL5 in mice.


    González-Rodríguez, Sara; Álvarez, Miguel G; García-Domínguez, Mario; Lastra, Ana; Cernuda-Cernuda, Rafael; Folgueras, Alicia R; Fernández-García, María Teresa; Hidalgo, Agustín; Baamonde, Ana; Menéndez, Luis


    We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3-10ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01-0.1μg) and DAPTA (0.3-3μg), respectively. The prevention of this hyperalgesia by diclofenac (1-10μg), the inhibitors of COX-1 SC-560 (0.1-1μg) or COX-2 celecoxib (1-5μg), the TRPV1 antagonist capsazepine (0.03-0.3μg) or the TRPA1 antagonist HC030031 (10-50μg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17μg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5μg), nor-binaltorphimine (10mg/kg) or an anti-dynorphin A antibody (0.62-2.5ng). The administration of 30ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.

  14. Low-dose morphine elicits ventilatory excitant and depressant responses in conscious rats: Role of peripheral μ-opioid receptors

    PubMed Central

    Henderson, Fraser; May, Walter J.; Gruber, Ryan B.; Young, Alex P.; Palmer, Lisa A.; Gaston, Benjamin; Lewis, Stephen J.


    The systemic administration of morphine affects ventilation via a mixture of central and peripheral actions. The aims of this study were to characterize the ventilatory responses elicited by a low dose of morphine in conscious rats; to determine whether tolerance develops to these responses; and to determine the potential roles of peripheral μ-opioid receptors (μ-ORs) in these responses. Ventilatory parameters were monitored via unrestrained whole-body plethysmography. Conscious male Sprague-Dawley rats received an intravenous injection of vehicle or the peripherally-restricted μ-OR antagonist, naloxone methiodide (NLXmi), and then three successive injections of morphine (1 mg/kg) given 30 min apart. The first injection of morphine in vehicle-treated rats elicited an array of ventilatory excitant (i.e., increases in frequency of breathing, minute volume, respiratory drive, peak inspiratory and expiratory flows, accompanied by decreases in inspiratory time and end inspiratory pause) and inhibitory (i.e., a decrease in tidal volume and an increase in expiratory time) responses. Subsequent injections of morphine elicited progressively and substantially smaller responses. The pattern of ventilatory responses elicited by the first injection of morphine was substantially affected by pretreatment with NLXmi whereas NLXmi minimally affected the development of tolerance to these responses. Low-dose morphine elicits an array of ventilatory excitant and depressant effects in conscious rats that are subject to the development of tolerance. Many of these initial actions of morphine appear to involve activation of peripheral μ-ORs whereas the development of tolerance to these responses does not. PMID:24900948

  15. Gα14 subunit-mediated inhibition of voltage-gated Ca2+ and K+ channels via neurokinin-1 receptors in rat celiac-superior mesenteric ganglion neurons.


    Sugino, Shigekazu; Farrag, Mohamed; Ruiz-Velasco, Victor


    The mechanisms by which G proteins modulate voltage-gated Ca(2+)channel currents (CaV), particularly CaV2.2 and CaV2.3, are voltage dependent (VD) or voltage independent (VI). VD pathways are typically mediated by Gαi/oand GαSsubfamilies. On the other hand, VI inhibition modulation is coupled to the Gαqsubfamily and signaling pathways downstream of phospholipase C stimulation. In most studies, this latter pathway has been shown to be linked to Gαqand/or Gα11protein subunits. However, there are no studies that have examined whether natively expressed Gα14subunits (Gαqsubfamily member) couple G protein-coupled receptors (GPCR) with CaV2.2 channels. We report that Gα14subunits functionally couple the substance P (SP)/neurokinin-1 (NK-1) receptor pathway to CaV2.2 channels in acutely dissociated rat celiac-superior mesenteric ganglion (CSMG) neurons. Exposure of CSMG neurons to SP blocked the CaV2.2 currents in a predominantly VD manner that was pertussis toxin and cholera toxin resistant, as well as Gαq/11independent. However, silencing Gα14subunits significantly attenuated the SP-mediated Ca(2+)current block. In another set of experiments, exposure of CSMG neurons to SP led to the inhibition of KCNQ K(+)M-currents. The SP-mediated M-current block was significantly reduced in neurons transfected with Gα14small-interference RNA. Finally, overexpression of the GTP-bound Gαq/11binding protein RGS2 did not alter the block of M-currents by SP but significantly abolished the oxotremorine methiodide-mediated M-current inhibition. Taken together, these results provide evidence of a new Gα14-coupled signaling pathway that modulates CaV2.2 and M-currents via SP-stimulated NK-1 receptors in CSMG neurons.

  16. In Vivo Voltage-Sensitive Dye Study of Lateral Spreading of Cortical Activity in Mouse Primary Visual Cortex Induced by a Current Impulse

    PubMed Central

    Fehérvári, Tamás Dávid; Sawai, Hajime; Yagi, Tetsuya


    In the mammalian primary visual cortex (V1), lateral spreading of excitatory potentials is believed to be involved in spatial integrative functions, but the underlying cortical mechanism is not well understood. Visually-evoked population-level responses have been shown to propagate beyond the V1 initial activation site in mouse, similar to higher mammals. Visually-evoked responses are, however, affected by neuronal circuits prior to V1 (retina, LGN), making the separate analysis of V1 difficult. Intracortical stimulation eliminates these initial processing steps. We used in vivo RH1691 voltage-sensitive dye (VSD) imaging and intracortical microstimulation in adult C57BL/6 mice to elucidate the spatiotemporal properties of population-level signal spreading in V1 cortical circuits. The evoked response was qualitatively similar to that measured in single-cell electrophysiological experiments in rodents: a fast transient fluorescence peak followed by a fast and a slow decrease or hyperpolarization, similar to EPSP and fast and slow IPSPs in single cells. The early cortical response expanded at speeds commensurate with long horizontal projections (at 5% of the peak maximum, 0.08–0.15 m/s) however, the bulk of the VSD signal propagated slowly (at half-peak maximum, 0.05–0.08 m/s) suggesting an important role of regenerative multisynaptic transmission through short horizontal connections in V1 spatial integrative functions. We also found a tendency for a widespread and fast cortical response suppression in V1, which was eliminated by GABAA-antagonists gabazine and bicuculline methiodide. Our results help understand the neuronal circuitry involved in lateral spreading in V1. PMID:26230520

  17. Nuclear medicine progress report for quarter ending September 30, 1984

    SciTech Connect

    Knapp, F.F. Jr.; Ambrose, K.R.; Goodman, M.M.; Srivastava, P.C.


    The preparation and animal testing of a new radio-iodinated p-iodophenylamine-linked dihydropyridine system is described. The model agent, 1-methyl-3-(N-(..beta..-(4-(/sup 125/I)iodophenyl)ethyl)carbamoyl)-1,4-dihydropyridine, was prepared by coupling 4-(/sup 125/I)iodoaniline with the methiodide salt succinimidyl ester of nicotinic acid followed by dithionite reduction to the lipid soluble product. The dihydropyridine agent showed good brain uptake in rats (5 min, 1.14% injected dose/gm; 60 min, 1.12% dose/gm) and good brain to blood ratios (5 min 3.9:1, 60 min, 3.5:1). In contrast the quaternary ammonium compound, prior to reduction, showed only moderate brain uptake (5 min, 0.63; 60 min, 0.46) and low brain to blood ratios (5 min, 0.05; 60 min, 0.06). Also described is further investigation of the effects of fasting on the relative myocardial retention of straight-chain iodovinyl fatty acids. 18-(/sup 125/)Iodo-17-octadecenoic acid showed good retention in unfasted rats. Studies have now been reported for fasted rats where this agent showed rapid myocardial wash-out. In fasted rats, approx. 70% wash-out at 30 min, and in unfasted rats, approx. 15% wash-out at 30 min was observed. During this period several shipments were made to Medical Cooperative investigators including three samples of /sup 191/Os-potassium osmate (Children's Hospital, Boston, and the University of Liege, Belgium) and 15-(p-(/sup 131/I)iodophenyl)-3-R,S-methylpentadecanoic acid (University of Massachusetts and Brookhaven National Laboratory).

  18. Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85.

    PubMed Central

    Zhu, H; Paradis, F W; Krell, P J; Phillips, J P; Forsberg, C W


    The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles. Images PMID:8021170

  19. New Morphine Analogs Produce Peripheral Antinociception within a Certain Dose Range of Their Systemic Administration.


    Lackó, Erzsébet; Riba, Pál; Giricz, Zoltán; Váradi, András; Cornic, Laura; Balogh, Mihály; Király, Kornél; Csekő, Kata; Mousa, Shaaban A; Hosztafi, Sándor; Schäfer, Michael; Zádori, Zoltán Sándor; Helyes, Zsuzsanna; Ferdinandy, Péter; Fürst, Susanna; Al-Khrasani, Mahmoud


    Growing data support peripheral opioid antinociceptive effects, particularly in inflammatory pain models. Here, we examined the antinociceptive effects of subcutaneously administered, recently synthesized 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU) compared with morphine-6-O-sulfate (M6SU) in a rat model of inflammatory pain induced by an injection of complete Freund's adjuvant and in a mouse model of visceral pain evoked by acetic acid. Subcutaneous doses of 14-O-MeM6SU and M6SU up to 126 and 547 nmol/kg, respectively, produced significant and subcutaneous or intraplantar naloxone methiodide (NAL-M)-reversible antinociception in inflamed paws compared with noninflamed paws. Neither of these doses significantly affected thiobutabarbital-induced sleeping time or rat pulmonary parameters. However, the antinociceptive effects of higher doses were only partially reversed by NAL-M, indicating contribution of the central nervous system. In the mouse writhing test, 14-O-MeM6SU was more potent than M6SU after subcutaneous or intracerebroventricular injections. Both displayed high subcutaneous/intracerebroventricular ED50 ratios. The antinociceptive effects of subcutaneous 14-O-MeM6SU and M6SU up to 136 and 3043 nmol/kg, respectively, were fully antagonized by subcutaneous NAL-M. In addition, the test compounds inhibited mouse gastrointestinal transit in antinociceptive doses. Taken together, these findings suggest that systemic administration of the novel compound 14-O-MeM6SU similar to M6SU in specific dose ranges shows peripheral antinociception in rat and mouse inflammatory pain models without central adverse effects. These findings apply to male animals and must be confirmed in female animals. Therefore, titration of systemic doses of opioid compounds with limited access to the brain might offer peripheral antinociception of clinical importance.

  20. Cholinergic submandibular effects and muscarinic receptor expression in blood vessels of the rat.


    Ryberg, Anders T; Selberg, Hanna; Soukup, Ondrej; Gradin, Kathryn; Tobin, Gunnar


    In order to functionally characterise the muscarinic vasodilator responses, effects of cholinergic agonists were studied on isolated preparations of the rat submandibular artery and vein and carotid and jugular vessels. Tentatively, a cholinergic regulatory mechanism having different effects on the arterial and venous vessels would enhance vascular fluid recruitment for the secretory response. In vitro functional findings were correlated to the expression and cellular location of the different receptors that were assessed by immunohistochemistry. In order to find in vivo correlates to the in vitro findings, the influence of muscarinic receptors on permeability was studied on the vasculature of the submandibular gland in anaesthetised rats. Staining for muscarinic M1 receptors occurred in the endothelium, and muscarinic M5 receptors, and possibly M3 also, were detected in the arterial smooth muscle. In venous endothelium, muscarinic M1 and M4 receptors occurred. In the jugular smooth muscle layer, staining for M1, and possibly also for M3, appeared. Muscarinic agonists caused arteries to relax and veins to contract. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-4)M) markedly reduced the cholinergic-evoked relaxation of pre-contracted carotid arterial preparations. In the presence of 4-DAMP (10(-7)M), the relaxation to cholinergic agonists was inhibited. Pirenzepine (10(-5)M) did not only inhibit the relaxatory effects, but even reversed the effects, while it in the jugular vein abolished the cholinergic effects. The arterial nitric oxide-dependent response to muscarinic receptor stimulation consisted of two parts -- one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only, resulted in cholinergic arterial contraction. Also, the submandibular artery and vein responses to muscarinic receptor stimulation show a resemblance with those of the carotid and jugular vessels, i.e. a pronounced arterial

  1. Different antagonist binding properties of rat pancreatic and cardiac muscarinic receptors

    SciTech Connect

    Waelbroeck, M.; Camus, J.; Winand, J.; Christophe, J.


    The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (/sup 3/H)N-methylscopolamine ((/sup 3/)NMS) (K/sub D/ values of 140 and 280nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-(/(2-((diethylamino)-methyl)-1-piperidinyl/acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on)(AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptros also showed different (/sup 3/H)NMS association and dissociation rates. These results support the concept of M2 receptor subtypes have different binding kinetic properties. 20 references, 3 figures, 1 table.

  2. Muscarinic antagonists microinjected into the subthalamic nucleus decrease muscular rigidity in reserpinized rats.


    Hernández-López, S; Flores, G; Rosales, M G; Sierra, A; Martínez-Fong, D; Aceves, J


    The ability of anticholinergic agents microinjected into the subthalamic nucleus to reduce reserpine-induced muscular rigidity was assessed in rats. The electromyographical activity of the gastrocnemius-soleus muscle was used as a parameter of muscular rigidity. Reserpine (5 mg/kg i.p.) produced the appearance of electromyographical activity. The muscarinic antagonists M3 (1.27 nmol of 4-DAMP) and M1 (2.36 nmol of pirenzepine) markedly reduced the reserpine-induced electromyographical activity, whereas the M2 antagonist AFDX-116 (2.37 nmol) had no effect. These results suggest that a high cholinergic tone in the subthalamic nucleus is associated with the reserpine-induced muscular rigidity. Moreover, the M3 muscarinic antagonist is more effective than the M1 muscarinic antagonist in reducing the muscular rigidity in reserpinized rats, a model of Parkinson's disease, by blocking the high cholinergic tone in the subthalamic nucleus.

  3. Enhanced nerve-stimulated muscarinic and neurokinin contractions of ileum from streptozotocin guinea-pigs.


    Cellini, J; Pommier, R; Porter, R; LePard, K J


    Diabetes mellitus can lead to neuropathy of enteric neurons, resulting in abnormal gut motility. These studies investigated voltage-dependent contributions of muscarinic M₃ receptor activation by acetylcholine and neurokinin NK₁ receptor activation by neurokinins to nerve-stimulated contractions of longitudinal ileal strips from STZ guinea-pigs, a type 1 diabetic model with insulin deficiency, but mild hyperglycaemia. Contractions to bethanechol, substance P methyl ester, and nerve stimulation were greater in diabetic as compared to control ileum. The muscarinic M₃ receptor antagonist 4-DAMP at lower voltages and the neurokinin NK₁ receptor antagonist SR140333 at higher voltages, but not the neurokinin NK₁ receptor antagonist CP-96,345, were more effective at inhibiting nerve-stimulated immediate peak contractions and total areas of contraction of ileum from diabetic as compared to control animals. For diabetic ileum, voltage-dependent increases in the areas of nerve-stimulated contraction were observed in the presence of 4-DAMP and CP-96,345 but not SR140333. At low voltages only, nerve-stimulated release of acetylcholine was greater from diabetic as compared to control ileum. Fluorescence intensity of tachykinin-like immunoreactivity was increased in ileal myenteric ganglia from diabetic as compared to control animals. In diabetic guinea-pigs, stronger ileal nerve-stimulated contractions reflected increased release of acetylcholine at lower voltages and tachykinins at higher voltages, as well as increased sensitivity of smooth muscle M₃ and NK₁ receptors to acetylcholine and tachykinins. Hypoinsulinaemia may be a primary contributor to intestinal motility dysfunction in type 1 diabetes mellitus.

  4. Ventral tegmental area neurons are either excited or inhibited by cocaine’s actions in the peripheral nervous system

    PubMed Central

    Mejías-Aponte, Carlos A.; Kiyatkin, Eugene A.


    Cocaine’s multiple pharmacological substrates are ubiquitously present in the peripheral and central nervous system. Thus, upon its administration, cocaine acts in the periphery before directly acting in the brain. We determined whether cocaine alters ventral tegmental area (VTA) neuronal activity via peripheral actions, and whether this precedes its central actions. In urethane-anesthetized rats, we recorded VTA neurons responses to intravenous injections of two cocaine analogs: cocaine-hydrochloride (HCl, 0.25 mg/kg) that readily cross the blood-brain barrier (BBB) and cocaine-methiodide (MI, 0.33 mg/kg) that does not cross the BBB. Both cocaine analogs produced sustained changes in discharge rates that began 5s after the initiation of a 10s drug infusion. Within the first 90s post-injection the magnitudes of neuronal responsive of both cocaine analogs were comparable, but later in time the effects of cocaine-HCl were stronger and persisted longer than those of cocaine-MI. The proportion of neurons responsive to cocaine-HCl was twice to that of cocaine-MI (74% and 35% respectively). Both analogs also differed in the response onsets. Cocaine-MI rarely evoked responses after 1 min whereas cocaine-HCl continued to evoke responses within 3 min post-injection. VTA neurons were either excited or inhibited by both cocaine analogs. Most units responsive to cocaine-MI, regardless of excitation or inhibition, had electrophysiological characteristics of putative DA neurons. Units inhibited by cocaine-HCl also had characteristic of DA neurons whereas excited neurons had widely varying action potential durations and discharge rates. Cocaine-MI and cocaine-HCl each produced changes in VTA neuron activity under full DA receptor blockade. However, the duration of inhibition was shortened, the number of excitations increased, and they occurred with an earlier onset during DA receptor blockade. These findings indicate that cocaine acts peripherally with a short latency and

  5. GABAA and glutamate receptor involvement in dendrodendritic synaptic interactions from salamander olfactory bulb.


    Wellis, D P; Kauer, J S


    1. Whole-cell patch clamp and optical recording techniques were applied to the same in vitro salamander olfactory bulb preparations to study the postsynaptic responses of single mitral/tufted cells in the context of the surrounding neural activity in which they are embedded. Mitral/tufted cells were identified by intracellular filling with biocytin. 2. Single mitral/tufted cells were under a tonic GABAA receptor-mediated inhibitory influence as revealed by the recording of bicuculline methiodide (BMI)/picrotoxin-sensitive inhibitory postsynaptic currents (IPSCs) in symmetrical chloride conditions at a holding potential of -70 mV. Depolarizing voltage steps (100 ms) applied to single cells or electrical stimulation of the olfactory nerve or medial olfactory tract evoked a prolonged increase in the frequency of GABAergic IPSCs. 3. The frequency of spontaneous and driven IPSCs was reduced with application of the glutamate receptor antagonists 6-cyano-2,3-dihydroxy-7-nitro-quionoxaline (CNQX) or 2-amino-5-phosphonopentanoic acid (AP5) whereas olfactory nerve- or medial olfactory tract-driven IPSC frequency was enhanced with removal of bathing Mg2+, indicating that GABAergic interneurones were driven by mitral/tufted cells at both non-NMDA and NMDA receptors. 4. Olfactory nerve or medial olfactory tract stimulation evoked widely distributed changes in fluorescence in preparations stained with the voltage-sensitive dye RH414. The optical response predominantly consisted of a decrease in fluorescence, indicative of depolarization. The presence of the dye did not obviously affect mitral/tufted cell postsynaptic responses. 5. BMI enhanced the amplitude and duration of optical signals related to depolarization within the bulb and in regions central to the bulb. In the presence of BMI, depolarizing activity appeared to spread hundreds of micrometres into regions of the bulb not activated in control conditions showing explicitly that GABAA receptors in the bulb participate in

  6. [Dmt(1)]DALDA is highly selective and potent at mu opioid receptors, but is not cross-tolerant with systemic morphine.


    Riba, Pal; Ben, Yong; Nguyen, Thi M-D; Furst, Susanna; Schiller, Peter W; Lee, Nancy M


    The clinical effectiveness of morphine is limited by several side effects, including the development of tolerance and dependence. Most of these side effects are believed to be mediated by central opioid receptors; therefore, hydrophilic opioids, which don't cross the blood-brain barrier, may have advantages over morphine in some clinical applications. We recently synthesized several analogues of DALDA (Tyr-D-Arg-Phe-Lys-NH2), a highly hydrophilic peptide derived from the endogenous opioid peptide dermorphin; all of them, particularly [Dmt(1)] DALDA (Dmt - 2',6'-dimethyl tyrosine), had high potency and selectivity at mu receptors, the target of morphine, in activity assays. Here we report the pharmacological characterization of [Dmt(1)] DALDA in the whole animal. [Dmt(1)]DALDA was 40 times more potent than morphine in inducing antinociception in mice when both drugs were given s.c., and 6-14 times more potent than DAMGO, a selective m agonist, when both drugs were given it. However, [Dmt(1)]DALDA showed poor cross-tolerance to morphine; thus chronic morphine treatment of animals increased the antinociceptive AD(50) of systemic [Dmt(1)]DALDA two fold or less, as compared to an 8-9-fold increase for morphine and a 4-5-fold increase for DAMGO. The antinociceptive activity of [Dmt(1)]DALDA (i.t) was blocked by CTAP, a selective mu antagonist, but not by TIPP psi, a selective delta antagonist, nor by nor-BNI, a selective kappa antagonist. [Dmt(1)]DALDA-induced antinociception was also blocked by naloxone methiodide, an antagonist that does not cross the blood-brain barrier, when agonist and antagonist were given i.t. or i.c.v., but not when they were given s.c. We conclude that [Dmt(1)] DALDA is a highly potent analgesic acting at mu receptors. Though it appears to penetrate the blood-brain barrier, it exhibits low cross-tolerance to morphine, suggesting that it may have advantages over the latter in certain clinical applications.

  7. Much caution does no harm! Organophosphate poisoning often causes pancreatitis.


    Yoshida, Shozo; Okada, Hideshi; Nakano, Shiho; Shirai, Kunihiro; Yuhara, Toshiyuki; Kojima, Hiromasa; Doi, Tomoaki; Kato, Hisaaki; Suzuki, Kodai; Morishita, Kentaro; Murakami, Eiji; Ushikoshi, Hiroaki; Toyoda, Izumi; Ogura, Shinji


    Organophosphate poisoning (OP) results in various poisoning symptoms due to its strong inhibitory effect on cholinesterase. One of the occasional complications of OP is pancreatitis. A 62-year-old woman drank alcohol and went home at midnight. After she quarreled with her husband and drank 100 ml of malathion, a parasympathomimetic organophosphate that binds irreversibly to cholinesterase, she was transported to our hospital in an ambulance. On admission, activated charcoal, magnesium citrate, and pralidoxime methiodide (PAM) were used for decontamination after gastric lavage. Abdominal computed tomography detected edema of the small intestine and colon with doubtful bowel ischemia, and acute pancreatitis was suspected. Arterial blood gas analysis revealed severe lactic acidosis. The Ranson score was 6 and the APACHE II (Acute Physiology and Chronic Health Evaluation) score was 14. Based on these findings, severe acute pancreatitis was diagnosed. One day after admission, hemodiafiltration (HDF) was started for the treatment of acute pancreatitis. On the third hospital day, OP symptoms were exacerbated, with muscarinic manifestations including bradycardia and hypersalivation and decreased plasma cholinesterase activity. Atropine was given and the symptoms improved. The patient's general condition including hemodynamic status improved. Pancreatitis was attenuated by 5 days of HDF. Ultimately, it took 14 days for acute pancreatitis to improve, and the patient discharged on hospital day 32. Generally, acute pancreatitis associated with OP is mild. In fact, one previous report showed that the influence of organophosphates on the pancreas disappears in approximately 72 hours, and complicated acute pancreatitis often improves in 4-5 days. However, it was necessary to treat pancreatitis for more than 2 weeks in this case. Therefore, organophosphate-associated pancreatitis due to malathion is more severe. Although OP sometime causes severe necrotic pancreatitis or

  8. Intrinsic and Network Mechanisms Constrain Neural Synchrony in the Moth Antennal Lobe

    PubMed Central

    Lei, Hong; Yu, Yanxue; Zhu, Shuifang; Rangan, Aaditya V.


    Projection-neurons (PNs) within the antennal lobe (AL) of the hawkmoth respond vigorously to odor stimulation, with each vigorous response followed by a ~1 s period of suppression—dubbed the “afterhyperpolarization-phase,” or AHP-phase. Prior evidence indicates that this AHP-phase is important for the processing of odors, but the mechanisms underlying this phase and its function remain unknown. We investigate this issue. Beginning with several physiological experiments, we find that pharmacological manipulation of the AL yields surprising results. Specifically, (a) the application of picrotoxin (PTX) lengthens the AHP-phase and reduces PN activity, whereas (b) the application of Bicuculline-methiodide (BIC) reduces the AHP-phase and increases PN activity. These results are curious, as both PTX and BIC are inhibitory-receptor antagonists. To resolve this conundrum, we speculate that perhaps (a) PTX reduces PN activity through a disinhibitory circuit involving a heterogeneous population of local-neurons, and (b) BIC acts to hamper certain intrinsic currents within the PNs that contribute to the AHP-phase. To probe these hypotheses further we build a computational model of the AL and benchmark our model against our experimental observations. We find that, for parameters which satisfy these benchmarks, our model exhibits a particular kind of synchronous activity: namely, “multiple-firing-events” (MFEs). These MFEs are causally-linked sequences of spikes which emerge stochastically, and turn out to have important dynamical consequences for all the experimentally observed phenomena we used as benchmarks. Taking a step back, we extract a few predictions from our computational model pertaining to the real AL: Some predictions deal with the MFEs we expect to see in the real AL, whereas other predictions involve the runaway synchronization that we expect when BIC-application hampers the AHP-phase. By examining the literature we see support for the former, and we

  9. Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M-current inhibition

    PubMed Central

    del Río, Elena; Bevilacqua, Jorge A; Marsh, Stephen J; Halley, Pamela; Caulfield, Malcolm P


    The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M-current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. Phosphoinositide-specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [3H]-cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependent manner with an EC50 of approximately 3.5 μm. The concentration-response curve for oxo-M was shifted to the right by a factor of about 10 by pirenzepine (100 nm), suggesting a pKB (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pKB∼7.1 ± 0.6). This indicates involvement of the M1 muscarinic receptor in this response. The accumulation of CMP-PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. The ability of oxo-M to release Ca2+ from inositol(1,4,5)trisphosphate (InsP3)-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke intracellular Ca2+ (Cai2+) mobilisation in SCG neurones voltage clamped at −60 mV, but produced a significant Cai2+ rise (67 ± 15 nm, n = 9) in cells voltage clamped at −25 mV. Thapsigargin (0.5–1 μm) caused a 70% inhibition of the oxo-M-induced Cai2+ increase, indicating its intracellular origin, while oxo-M-induced inhibition of M-current in the same cells was unaffected by thapsigargin. Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition. PMID:10517804

  10. Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila-pridinol, diphenidol and sila-diphenidol type.

    PubMed Central

    Waelbroeck, M.; Camus, J.; Tastenoy, M.; Lambrecht, G.; Mutschler, E.; Kropfgans, M.; Sperlich, J.; Wiesenberger, F.; Tacke, R.; Christophe, J.


    1. We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using [3H]-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide. 2. The affinity of the antagonists either increased or decreased with temperature. van't Hoff plots were linear in the 278-310 degrees K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to -29 kJ mol-1) to large positive values (up to +30 kJ mol-1). 3. (R)-configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed. 4. When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes. 5. Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change

  11. Muscarine inhibits high-threshold calcium currents with two distinct modes in rat embryonic hippocampal neurons.

    PubMed Central

    Toselli, M; Taglietti, V


    1. Ca2+ channel modulation by muscarine was investigated in primary cultured embryonic rat hippocampal neurons using the whole-cell variant of the patch-clamp technique. 2. Muscarine produced a reversible and concentration-dependent decrease in the Ba2+ current amplitude. In 65% of neurons sensitive to the agonist, current inhibition was time and voltage dependent, being maximal between -20 and 0 mV and decreasing at depolarizing potentials. In the remaining 35% of neurons, the effects of muscarine were voltage independent, inhibition being constant in a wide potential range between -20 and +80 mV. 3. Different receptors might be involved in the two modes of modulation. Muscarine-induced voltage-dependent inhibition of Ba2+ current was best suppressed by the muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide (81% suppression), while voltage-independent inhibition was best suppressed by AFDX116 (75% suppression). 4. In cells treated with omega-conotoxin (omega-CgTX), the voltage-independent mode of inhibition was strongly prevented, suggesting that the two modulatory mechanisms (voltage dependent and voltage independent) operate on separate classes of high-voltage-activated (HVA) Ca2+ channels. 5. A pertussis toxin-sensitive G-protein is involved in both modes of action of muscarine, since both modes were prevented by pretreatment of the cells with 50 ng ml-1 pertussis toxin. 6. Both modes of modulation were mimicked in different cells by intracellular application of GTP-gamma-S. However, the onset of voltage-independent inhibition was about 5 times slower than that of voltage-dependent inhibition, suggesting involvement of a more complex metabolic pathway for the former mode of channel modulation. 7. Relief of the voltage-dependent inhibition was obtained by depolarizing voltage prepulses and occurred with kinetics that depended on agonist concentration. 8. The voltage-dependent inhibition could be simulated by a kinetic model in which

  12. Postsynaptic membrane shifts during frequency potentiation of the hippocampal EPSP.


    Pitler, T A; Landfield, P W


    , at the soma. 4. Studies in which the membrane was hyperpolarized with injected current to approximately the IPSP reversal potential, or in which bicuculline methiodide was applied to the slices, indicated that depression of the IPSP by repetitive stimulation did not account for frequency potentiation of EPSP amplitude. 5. These data are therefore consistent with the conclusion that the frequency potentiation of composite EPSPs in central neurons depends on presynaptic mechanisms, rather than on generalized postsynaptic changes. However, our findings do not rule out localized postsynaptic changes in receptors or spines as possible contributing factors.(ABSTRACT TRUNCATED AT 400 WORDS)

  13. Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

    PubMed Central

    Zholos, Alexander V; Bolton, Thomas B


    The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4±1.4 nS and a mean −log EC50 of 5.12±0.03 (mean±s.e.mean) (n=114). Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of

  14. Muscarinic responses of gastric parietal cells

    SciTech Connect

    Wilkes, J.M.; Kajimura, M.; Scott, D.R.; Hersey, S.J.; Sachs, G. )


    Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide, with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations, suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the (Ca)i transient.

  15. The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion.


    Chernyavsky, Alex I; Nguyen, Vu Thuong; Arredondo, Juan; Ndoye, Assane; Zia, Shaheen; Wess, Jürgen; Grando, Sergei A


    We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility. To determine muscarinic effects on lateral migration of KC, we used an agarose gel keratinocyte outgrowth system (AGKOS) which provides for measurements of the response of large cell populations (> 10(4) cells). Muscarine produced a dose-dependent stimulatory effect on cell migration (p < 0.05). This activity was abolished by atropine, which decreased migration distance when given alone. To identify the mAChR subtype(s) mediating these muscarinic effects, we substituted atropine with subtype-selective antagonists. Tropicamide (M(4)-selective) was more effective at decreasing the migration distance than pirenzepine and 4-DAMP at nanomolar concentrations. We then compared lateral migration of KC obtained from M(4) mAChR knockout mice with that of wild-type murine KC, using AGKOS. In the absence of M(4) mAChR, the migration distance of KC was significantly (p < 0.05) decreased. These results indicate that the M(4) mAChR plays a central role in mediating cholinergic control of keratinocyte migration by endogenous acetylcholine produced by these cells.

  16. Identification of four areas each enriched in a unique muscarinic receptor subtype

    SciTech Connect

    Hoss, W.; Ellerbrock, B.R.; Goldman, P.S.; Collins, D.A.; Messer, W.S. Jr. )


    The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC{sub 50} values and Hill coefficients for the inhibition of the binding of 0.2 nM ({sup 3}H)-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol >> arecoline > bethanechol > McN A343 = oxotremorine = pilocarpine.

  17. The binding of (3H)AF-DX 384 to rat ileal smooth muscle muscarinic receptors

    SciTech Connect

    Entzeroth, M.; Mayer, N. )


    The tritiated cardioselective muscarinic antagonist AF-DX 384 (5,11-dihydro-11-(2-(-(8-dipropylamino)methyl)-1-piperidinyl-ethyl-amino-carbonyl)-6H-pyrido (2,3-b) (1,4)benzodiazepin-6-one) was used to label muscarinic receptors in the rat ileum. Saturation binding to membrane suspensions revealed a high affinity binding site with a Kd of 9.2 nM. The maximal number of binding sites labeled in this tissue (Bmax) is 237 fmol/mg protein. The association and dissociation kinetics were well represented by single exponential reactions, and the dissociation constant obtained from the ratio of rate constants was in agreement with that derived from saturation experiments. Specific binding was inhibited by muscarinic antagonists with a rank order of potencies of atropine (pKi: 8.80) greater than 4-DAMP (pKi: 8.23) = AF-DX 384 (pKi: 8.20) greater than AF-DX 116 (pKi: 7.09) = hexahydro-sila-difenidol (pKi: 6.97) greater than pirenzepine (pKi: 6.49) and is consistent with the interaction of (3H)AF-DX 384 with muscarinic receptors of the M2 subtype. It can be concluded that (3H)AF-DX 384 can be used to selectively label M2 muscarinic receptors in heterogeneous receptor populations.

  18. Identification of a galaxy responsible for a high-redshift Lyman-α absorption system

    NASA Astrophysics Data System (ADS)

    Djorgovski, S. G.; Pahre, M. A.; Bechtold, J.; Elston, R.


    DAMPEDLyman-α systems are high-column-density intergalactic clouds of hydrogen, the existence of which is inferred from absorption lines appearing in the emission spectra of distant quasars. The galaxies believed to be responsible for these absorption systems have been suggested as possible progenitors of the normal disk galaxies observed in the local Universe1. Indeed, Lyman-α systems appear to contain a substantial fraction of the baryons known to exist in galaxies today2,3. Here we report the optical detection of a galaxy (designated DLA2233 + 131) associated with a known4 damped Lyman-α absorption system at a redshift of z = 3.150. The properties of this galaxy correspond closely to those expected of a young disk galaxy in the early stages of formation, and show no evidence for an active nucleus. This finding gives strong support to the idea that damped Lyman-α systems represent a population of young galaxies at high red-shifts.

  19. Label-Free Kinetics: Exploiting Functional Hemi-Equilibrium to Derive Rate Constants for Muscarinic Receptor Antagonists.


    Riddy, Darren M; Valant, Celine; Rueda, Patricia; Charman, William N; Sexton, Patrick M; Summers, Roger J; Christopoulos, Arthur; Langmead, Christopher J


    Drug receptor kinetics is as a key component in drug discovery, development, and efficacy; however, determining kinetic parameters has historically required direct radiolabeling or competition with a labeled tracer. Here we present a simple approach to determining the kinetics of competitive antagonists of G protein-coupled receptors by exploiting the phenomenon of hemi-equilibrium, the state of partial re-equilibration of agonist, antagonist, and receptor in some functional assays. Using functional [Ca(2+)]i-flux and extracellular kinases 1 and 2 phosphorylation assays that have short incubation times and therefore are prone to hemi-equilibrium "behaviors," we investigated a wide range of structurally and physicochemically distinct muscarinic acetylcholine receptor antagonists. Using a combined operational and hemi-equilibrium model of antagonism to both simulate and analyze data, we derived estimates of association and dissociation rates for the test set of antagonists, identifying both rapidly dissociating (4-DAMP, himbacine) and slowly dissociating (tiotropium, glycopyrrolate) ligands. The results demonstrate the importance of assay incubation time and the degree of receptor reserve in applying the analytical model. There was an excellent correlation between estimates of antagonist pK(B), k(on), and k(off) from functional assays and those determined by competition kinetics using whole-cell [(3)H]N-methylscopolamine binding, validating this approach as a rapid and simple method to functionally profile receptor kinetics of competitive antagonists in the absence of a labeled tracer.

  20. New biological properties of coffee melanoidins.


    Argirova, Mariana D; Stefanova, Iliyana D; Krustev, Athanas D


    Water-soluble melanoidins isolated from roasted coffee induced ex vivo changes in the bioelectric and contractile activity of rat circular gastric smooth muscle tissues. They provoked a depolarization of smooth muscle cellular membranes and an increase in Ca²⁺-influx as evidenced by the increase in the frequency and amplitude of Ca²⁺-generated spike potentials. In the presence of 1 × 10⁻⁶ mol L⁻¹ acetylcholine and 1 × 10⁻⁶ mol L⁻¹ arecoline the melanoidin-evoked contraction was significantly reduced. M-cholinergic receptor blocking agents atropine, ipratropium, pirenzepine, and 4-DAMP also significantly reduced the melanoidin-provoked contraction. Nonspecific N-cholinergic receptor blockers hexamethonium and decamethonium (1 × 10⁻⁵ mol L⁻¹ each) did not influence the melanoidin-induced mechanical reaction. The melanoidins did not affect the strength of contractions evoked by adrenaline and dopamine (1 × 10⁻⁶ mol L⁻¹ each). The results obtained support the assumption that melanoidin-evoked contraction is a result of activation of muscarinic-type cholinergic receptors.

  1. The Neutral Hydrogen Cosmological Mass Density at z = 5

    NASA Astrophysics Data System (ADS)

    Crighton, Neil H. M.; Murphy, Michael T.; Prochaska, J. Xavier; Worseck, Gábor; Rafelski, Marc; Becker, George D.; Ellison, Sara L.; Fumagalli, Michele; Lopez, Sebastian; Meiksin, Avery; O’Meara, John M.


    We present the largest homogeneous survey of redshift > 4.4 damped Lyα systems (DLAs) using the spectra of 163 quasars that comprise the Giant Gemini GMOS (GGG) survey. With this survey we make the most precise high-redshift measurement of the cosmological mass density of neutral hydrogen, ΩHI. After correcting for systematic effects using a combination of mock and higher-resolution spectra, we find ΩHI= 0.98+0.20 -0.18 × 10-3 at = 4.9, assuming a 20% contribution from lower column density systems below the DLA threshold. By comparing to literature measurements at lower redshifts, we show that ΩHI can be described by the functional form ΩHI(z) ~ (1 + z)0.4. This gradual decrease from z = 5 to 0 suggests that in the galaxies which dominate the cosmic star formation rate, Hi is a transitory gas phase fuelling star formation which must be continually replenished by more highly-ionized gas from the intergalactic medium, and from recycled galactic winds.

  2. Ligand-binding properties of an unusual nicotinic acetylcholine receptor subtype on isolated outer hair cells from guinea pig cochlea.


    Lawoko, G; Järlebark, L; Heilbronn, E


    Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [125I]alpha-bungarotoxin revealed a KD of 62 +/- 2 nM, Bmax = 7.2 +/- 1.8 x 10(7) binding sites/OHC, and a slowly reversible dissociation rate constant, kappa-1 = 2.2 +/- 0.01 x 10(-4) min-1. L-[3H]Nicotine bound reversibly (estimated KD approximately 230 nM and Bmax approximately 5 x 10(7)) with kinetic rate constants of association kappa-1 = 6.2 +/- 0.06 x 10(4) min-1 nM-1 and dissociation kappa-1 = 0.23 +/- 0.003 min-1. [3H]Strychnine bound to OHC with a KD of 35 +/- 6 nM and Bmax = 2.6 +/- 0.5 x 10(7), and binding increased 3-4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > nM concentrations, of [3H]3-quinuclidinyl benzilate (KD = 11.5 +/- 5 nM; Bmax = 2.5 +/- 0.6 x 10(6)) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 +/- 0.5 and 6.5 +/- 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.

  3. The neutral hydrogen cosmological mass density at z = 5

    NASA Astrophysics Data System (ADS)

    Crighton, Neil H. M.; Murphy, Michael T.; Prochaska, J. Xavier; Worseck, Gábor; Rafelski, Marc; Becker, George D.; Ellison, Sara L.; Fumagalli, Michele; Lopez, Sebastian; Meiksin, Avery; O'Meara, John M.


    We present the largest homogeneous survey of z > 4.4 damped Lyα systems (DLAs) using the spectra of 163 QSOs that comprise the Giant Gemini GMOS (GGG) survey. With this survey we make the most precise high-redshift measurement of the cosmological mass density of neutral hydrogen, Ω_{H I}. At such high redshift, important systematic uncertainties in the identification of DLAs are produced by strong intergalactic medium absorption and QSO continuum placement. These can cause spurious DLA detections, result in real DLAs being missed or bias the inferred DLA column density distribution. We correct for these effects using a combination of mock and higher resolution spectra, and show that for the GGG DLA sample the uncertainties introduced are smaller than the statistical errors on Ω_{H I}. We find Ω _{H I}=0.98^{+0.20}_{-0.18}× 10^{-3} at = 4.9, assuming a 20 per cent contribution from lower column density systems below the DLA threshold. By comparing to literature measurements at lower redshifts, we show that Ω_{H I} can be described by the functional form Ω _{H I}(z)∝ (1+z)^{0.4}. This gradual decrease from z = 5 to 0 is consistent with the bulk of H I gas being a transitory phase fuelling star formation, which is continually replenished by more highly ionized gas from the intergalactic medium and from recycled galactic winds.

  4. The NASA/industry Design Analysis Methods for Vibrations (DAMVIBS) program : Bell Helicopter Textron accomplishments

    NASA Technical Reports Server (NTRS)

    Cronkhite, James D.


    Accurate vibration prediction for helicopter airframes is needed to 'fly from the drawing board' without costly development testing to solve vibration problems. The principal analytical tool for vibration prediction within the U.S. helicopter industry is the NASTRAN finite element analysis. Under the NASA DAMVIBS research program, Bell conducted NASTRAN modeling, ground vibration testing, and correlations of both metallic (AH-1G) and composite (ACAP) airframes. The objectives of the program were to assess NASTRAN airframe vibration correlations, to investigate contributors to poor agreement, and to improve modeling techniques. In the past, there has been low confidence in higher frequency vibration prediction for helicopters that have multibladed rotors (three or more blades) with predominant excitation frequencies typically above 15 Hz. Bell's findings under the DAMVIBS program, discussed in this paper, included the following: (1) accuracy of finite element models (FEM) for composite and metallic airframes generally were found to be comparable; (2) more detail is needed in the FEM to improve higher frequency prediction; (3) secondary structure not normally included in the FEM can provide significant stiffening; (4) damping can significantly affect phase response at higher frequencies; and (5) future work is needed in the areas of determination of rotor-induced vibratory loads and optimization.

  5. Opioids in the Hypothalamic Paraventricular Nucleus Stimulate Ethanol Intake

    PubMed Central

    Barson, Jessica R.; Carr, Ambrose J.; Soun, Jennifer E.; Sobhani, Nasim C.; Rada, Pedro; Leibowitz, Sarah F.; Hoebel, Bartley G.


    Background Specialized hypothalamic systems that increase food intake might also increase ethanol intake. To test this possibility, morphine and receptor-specific opioid agonists were microinjected in the paraventricular nucleus (PVN) of rats that had learned to drink ethanol. To cross-validate the results, naloxone methiodide (m-naloxone), an opioid antagonist, was microinjected with the expectation that it would have the opposite effect of morphine and the specific opioid agonists. Methods Sprague-Dawley rats were trained, without sugar, to drink 4% or 7% ethanol and were then implanted with chronic brain cannulas aimed at the PVN. After recovery, those drinking 7% ethanol, with food and water available, were injected with two doses each of morphine or m-naloxone. To test for receptor specificity, two doses each of the μ-receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO), δ-receptor agonist D-Ala-Gly-Phe-Met-NH2 (DALA), or k-receptor agonist U-50,488H were injected. DAMGO was also tested in rats drinking 4% ethanol without food or water available. As an anatomical control for drug reflux, injections were made 2 mm dorsal to the PVN. Results A main result was a significant increase in ethanol intake induced by PVN injection of morphine. The opposite effect was produced by m-naloxone. The effects of morphine and m-naloxone were exclusively on intake of ethanol, even though food and water were freely available. In the analysis with specific receptor agonists, PVN injection of the δ-agonist DALA significantly increased 7% ethanol intake without affecting food or water intake. This is in contrast to the k-agonist U-50,488H, which decreased ethanol intake, and the μ-agonist DAMGO, which had no effect on ethanol intake in the presence or absence of food and water. In the anatomical control location 2 mm dorsal to the PVN, no drug caused any significant changes in ethanol, food, or water intake, providing evidence that the active site was close to the

  6. Removal of GABAergic inhibition alters subthreshold input in neurons in forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) after digit stimulation.


    Li, Cheng X; Callaway, Joseph C; Waters, Robert S


    Our objective was to test the hypothesis that suppression of GABAergic inhibition results in an enhancement of responses to stimulation of the surround receptive field. Neurons in the forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) receive short latency suprathreshold input from a principal location on the forepaw and longer latency subthreshold input from surrounding forepaw skin regions. Input from principal and surround receptive field sites was examined before, during, and after administration of the GABA(A) receptor blocker bicuculline methiodide (BMI) (in 165 mM NaCl at pH 3.3-3.5). In vivo extracellular recording was used to first identify the location of the glabrous forepaw digit representation within the FBS. In vivo intracellular recording and labeling techniques were then used to impale single FBS neurons in layer IV as well as neurons in layers III and V, determine the receptive field of the cell, and fill the cell with biocytin for subsequent morphological identification. The intracellular recording electrode was fastened with dental wax to a double-barrel pipette for BMI iontophoresis and current balance. A stimulating probe, placed on the glabrous forepaw skin surface, was used to identify principal and surround components of the receptive field. Once a cell was impaled and a stable recording was obtained, a stimulating probe was placed at a selected site within the surround receptive field. Single-pulse stimulation (1 Hz) was then delivered through the skin probe and the percentage of spikes occurring in 1-min intervals before BMI onset was used as a baseline measure. BMI was then iontophoresed while the periphery was simultaneously stimulated, and spike percentage measured during and after BMI ejection was compared with the pre-BMI baseline. The major findings are: (1) suppression of GABAergic inhibition enhanced evoked responses to firing level from sites in surround receptive fields in 65% of the cells ( n=17); (2) evoked

  7. Serotonergic modulation of neurotransmission in the rat basolateral amygdala.


    Rainnie, D G


    Whole cell patch-clamp recordings were obtained from projection neurons and interneurons of the rat basolateral amygdala (BLA) to understand local network interactions in morphologically identified neurons and their modulation by serotonin. Projection neurons and interneurons were characterized morphologically and electrophysiologically according to their intrinsic membrane properties and synaptic characteristics. Synaptic activity in projection neurons was dominated by spontaneous inhibitory postsynaptic currents (IPSCs) that were multiphasic, reached 181 +/- 38 pA in amplitude, lasted 296 +/- 27 mS, and were blocked by the GABAA receptor antagonist, bicuculline methiodide (30 microM). In interneurons, spontaneous synaptic activity was characterized by a burst-firing discharge patterns (200 +/- 40 Hz) that correlated with the occurrence of 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive, high-amplitude (260 +/- 42 pA), long-duration (139 +/- 19 mS) inward excitatory postsynaptic currents (EPSCs). The interevent interval of 831 +/- 344 mS for compound inhibitory postsynaptic potentials (IPSPs), and 916 +/- 270 mS for EPSC bursts, suggested that spontaneous IPSP/Cs in projection neurons are driven by burst of action potentials in interneurons. Hence, BLA interneurons may regulate the excitability of projection neurons and thus determine the degree of synchrony within ensembles of BLA neurons. In interneurons 5-hydroxytryptamine oxalate (5-HT) evoked a direct, dose-dependent, membrane depolarization mediated by a 45 +/- 6.9 pA inward current, which had a reversal potential of -90 mV. The effect of 5-HT was mimicked by the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT), but not by the 5-HT1A receptor agonist, (+/-) 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), or the 5-HT1B agonist, CGS 12066A. In projection neurons, 5-HT evoked an indirect membrane hyperpolarization ( approximately 2 mV) that was associated with a 75 +/- 42 p

  8. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase

    PubMed Central

    Zhao, Yixiu; Zhang, Xin; Li, Jiannan; Bian, Yu; Sheng, Miaomiao; Liu, Bin; Fu, Zidong; Zhang, Yan; Yang, Baofeng


    Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved

  9. Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.


    Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S


    We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.

  10. Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.


    Hirayama, Michiko; Ogata, Masanori; Kawamata, Tomoyuki; Ishibashi, Hitoshi


    Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

  11. Different muscarinic receptor subtypes modulate proliferation of primary human detrusor smooth muscle cells via Akt/PI3K and map kinases.


    Arrighi, Nicola; Bodei, Serena; Zani, Danilo; Michel, Martin C; Simeone, Claudio; Cosciani Cunico, Sergio; Spano, Pierfranco; Sigala, Sandra


    While acetylcholine (ACh) and muscarinic receptors in the bladder are mainly known for their role in the regulation of smooth muscle contractility, in other tissues they are involved in tissue remodelling and promote cell growth and proliferation. In the present study we have used primary cultures of human detrusor smooth muscle cells (HDSMCs), in order to investigate the role of muscarinic receptors in HDSMC proliferation. Samples were obtained as discarded tissue from men >65 years undergoing radical cystectomy for bladder cancer and cut in pieces that were either immediately frozen or placed in culture medium for the cell culture establishment. HDSMCs were isolated from samples, propagated and maintained in culture. [(3)H]-QNB radioligand binding on biopsies revealed the presence of muscarinic receptors, with a Kd of 0.10±0.02nM and a Bmax of 72.8±0.1fmol/mg protein. The relative expression of muscarinic receptor subtypes, based on Q-RT-PCR, was similar in biopsies and HDSMC with a rank order of M2≥M3>M1>M4>M5. The cholinergic agonist carbachol (CCh, 1-100μM) concentration-dependently increased [(3)H]-thymidine incorporation (up to 46±4%). This was concentration-dependently inhibited by the general muscarinic receptor antagonist atropine and by subtype-preferring antagonists with an order of potency of darifenacin >4-DAMP>AF-DX 116. The CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation. This work shows that M2 and M3 receptors can mediate not only HDSM contraction but also proliferation; they may also contribute bladder remodelling including detrusor hypertrophy.

  12. Jujuboside B Reduces Vascular Tension by Increasing Ca2+ Influx and Activating Endothelial Nitric Oxide Synthase.


    Zhao, Yixiu; Zhang, Xin; Li, Jiannan; Bian, Yu; Sheng, Miaomiao; Liu, Bin; Fu, Zidong; Zhang, Yan; Yang, Baofeng


    Jujuboside B has been reported to have protective effect on many cardiovascular diseases. However, the effects of Jujuboside B on vascular tension and endothelial function are unknown. The present study investigated the effects of Jujuboside B on reducing vascular tension, protecting endothelial function and the potential mechanisms. The tension of isolated rat thoracic aorta ring was measured by Wire myograph system. The concentration of nitric oxide (NO) and the activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells (HAECs) were determined by Griess reagent method and enzyme-linked immune sorbent assay. The protein levels of eNOS and p-eNOS at Serine-1177 were determined by western blot analysis. Intracellular Ca2+ concentration in HAECs was measured by laser confocal imaging microscopy. Results showed that Jujuboside B reduced the tension of rat thoracic aorta rings with intact endothelium in a dose-dependent manner. L-NAME, KN93, EGTA, SKF96365, iberiotoxin and glibenclamide significantly attenuated Jujuboside B-induced vasodilation in endothelium-intact tissues. In contrast, indometacin and 4-DAMP had no such effects. Jujuboside B also promoted NO generation and increased eNOS activity, which were attenuated by L-NAME, EGTA and SKF96365. Moreover, Jujuboside B increased intracellular Ca2+ concentration dose-dependently, which was inhibited by EGTA and SKF96365. Besides, Jujuboside B induced a rapid Ca2+ influx instantaneously after depleting intracellular Ca2+ store, which was significantly inhibited by SKF96365. In conclusion, this study preliminarily confirmed that Jujuboside B reduced vascular tension endothelium-dependently. The underlying mechanisms involved that Jujuboside B increased extracellular Ca2+ influx through endothelial transient receptor potential cation (TRPC) channels, phosphorylated eNOS and promoted NO generation in vascular endothelial cells. In addition, Jujuboside B-induced vasodilation involved

  13. Effects of olanzapine on muscarinic M3 receptor binding density in the brain relates to weight gain, plasma insulin and metabolic hormone levels.


    Weston-Green, Katrina; Huang, Xu-Feng; Lian, Jiamei; Deng, Chao


    The second generation antipsychotic drug (SGA) olanzapine has an efficacy to treat schizophrenia, but can cause obesity and type II diabetes mellitus. Cholinergic muscarinic M3 receptors (M3R) are expressed on pancreatic β-cells and in the brain where they influence insulin secretion and may regulate other metabolic hormones via vagal innervation of the gastrointestinal tract. Olanzapine's M3R antagonism is an important risk factor for its diabetogenic liability. However, the effects of olanzapine on central M3Rs are unknown. Rats were treated with 0.25, 0.5, 1.0 or 2.0 mg olanzapine/kg or vehicle (3×/day, 14-days). M3R binding densities in the hypothalamic arcuate (Arc) and ventromedial nuclei (VMH), and dorsal vagal complex (DVC) of the brainstem were investigated using [3H]4-DAMP plus pirenzepine and AF-DX116. M3R binding correlations to body weight, food intake, insulin, ghrelin and cholecystokinin (CCK) were analyzed. Olanzapine increased M3R binding density in the Arc, VMH and DVC, body weight, food intake, circulating plasma ghrelin and CCK levels, and decreased plasma insulin and glucose. M3R negatively correlated to insulin, and positively correlated to ghrelin, CCK, food intake and body weight. Increased M3R density is a compensatory up-regulation in response to olanzapine's M3R antagonism. Olanzapine acts on M3R in regions of the brain that control food intake and insulin secretion. Olanzapine's M3R blockade in the brain may inhibit the acetylcholine pathway for insulin secretion. These findings support a role for M3Rs in the modulation of insulin, ghrelin and CCK via the vagus nerve and provide a mechanism for olanzapine's diabetogenic and weight gain liability.

  14. Autoantibodies against muscarinic type 3 receptor in Sjögren's syndrome inhibit aquaporin 5 trafficking.


    Lee, Byung Ha; Gauna, Adrienne E; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M; Kawai, Toshihisa; Cha, Seunghee


    Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.

  15. Nicotinic receptor activation on primary sensory afferents modulates autorhythmicity in the mouse renal pelvis

    PubMed Central

    Nguyen, M J; Angkawaijawa, S; Hashitani, H; Lang, R J


    BACKGROUND AND PURPOSE The modulation of the spontaneous electrical and Ca2+ signals underlying pyeloureteric peristalsis upon nicotinic receptor activation located on primary sensory afferents (PSAs) was investigated in the mouse renal pelvis. EXPERIMENTAL APPROACH Contractile activity was followed using video microscopy, electrical and Ca2+ signals in typical and atypical smooth muscle cells (TSMCs and ASMCs) within the renal pelvis were recorded separately using intracellular microelectrodes and Fluo-4 Ca2+ imaging. KEY RESULTS Nicotine and carbachol (CCh; 1–100 μM) transiently reduced the frequency and increased the amplitude of spontaneous phasic contractions in a manner unaffected by muscarininc antagonists, 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) and pirenzipine (10 nM) or L-NAME (L-Nω-nitroarginine methyl ester; 200 μM), inhibitor of NO synthesis, but blocked by the nicotinic antagonist, hexamethonium or capsaicin, depletor of PSA neuropeptides. These negative chronotropic and delayed positive inotropic effects of CCh on TSMC contractions, action potentials and Ca2+ transients were inhibited by glibenclamide (Glib; 1 μM), blocker of ATP-dependent K (KATP) channels. Nicotinic receptor-evoked inhibition of the spontaneous Ca2+ transients in ASMCs was prevented by capsaicin but not Glib. In contrast, the negative inotropic and chronotropic effects of the non-selective COX inhibitor indomethacin were not prevented by Glib. CONCLUSIONS AND IMPLICATIONS The negative chronotropic effect of nicotinic receptor activation results from the release of calcitonin gene-related peptide (CGRP) from PSAs, which suppresses Ca2+ signalling in ASMCs. PSA-released CGRP also evokes a transient hyperpolarization in TSMCs upon the opening of KATP channels, which reduces contraction propagation but promotes the recruitment of TSMC Ca2+ channels that underlie the delayed positive inotropic effects of CCh. PMID:24004375

  16. Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.


    Dittrich, Nikolaus P; Kummer, Wolfgang; Clauss, Wolfgang G; Fronius, Martin


    Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 μM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 μM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 μM) and ACh responses were not affected by nicotine or mecamylamine (25 μM). Luminal application of IBMX (I, 100 μM) and forskolin (F, 10 μM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 μM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

  17. Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy.


    Inazu, Masato; Yamada, Tomoko; Kubota, Nobuo; Yamanaka, Tsuyoshi


    Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H

  18. [Developmental mechanism of low myopia and therapeutic possibilities. A review].


    Tokoro, T


    are recognized; they are named M1 to M5. The effects of M1, M2, and M3 on the contractile response to transmural electrical stimulation of the bovine ciliary muscle were studied. The contractions produced by transmural electrical stimulation were greatly attenuated by 4-DAMP as an M3 antagonist. 3. We measured autofluorescence of the lens by fluorophotometry. A statistically significant relation was found between autofluorescence of the lens and refraction. 4. Possibility of axial elongation: The anterior and posterior suprachoroidal spaces are different anatomically and physiologically. When the choroid is stretched forward by accommodation, the intraocular pressure may exert more influence on the posterior part of the sclera, than on the anterior part. Using a fluorophotometer, fluorescence leakage at a site 3 mm in front of the retina was examined. Intensity of fluorescence 3 mm in front of the retina was strong in late-onset myopia. This may indicate disturbance of the barrier of the retinal pigment epithelium. When cultured fibroblasts of the sclera of the chick embryo was stretched by a stretching apparatus, proliferation of the cultured cells was inhibited. Therefore, some influence may involve the posterior part of the eyeball. III. Possibility of drug treatment of low myopia: From these results, muscarinic receptor antagonists (especially M3), ET receptor agtagonists, and NO donors are possible drugs for low myopia treatment. As there are many causative factors of low myopia, there are several treatment methods to be evaluated.

  19. The Quantum World of Ultra-Cold Atoms and Light - Book 1: Foundations of Quantum Optics

    NASA Astrophysics Data System (ADS)

    Gardiner, Crispin; Zoller, Peter


    Quantized Radiation Field * 12.2. Decay of an Excited Atom * 12.3. The Two-Level Atom in a Strong Classical Driving Field * 12.4. Interaction of a Two-Level Atom with a Single Mode * IV - QUANTUM STOCHASTIC PROCESSES * 13. Quantum Markov Processes * 13.1. Two-Level Atom in a Finite-Temperature Electromagnetic Field * 13.2. Derivation of theMaster Equation * 13.3. More General Heat Baths * 13.4. Quantum Correlation Functions and Spectra * 14. Applications of the Master Equation * 14.1. A Two-Level Atom Interacting with a Thermal Heat Bath * 14.2. The Two-Level Atom Driven by a Coherent Light Field * 14.3. Master Equations for Harmonic Oscillator Systems * 14.4. A Simple Model of Laser Cooling * V - PHASE SPACE METHODS * 15. Phase Space Representations for Bosons * 15.1. The Quantum Characteristic Function * 15.2. Phase Space Representations of the Density Operator * 16. Wigner Function Methods * 16.1. Operator Correspondences and Equations of Motion * 16.2. Damped and Driven Systems * 16.3. The Wigner Distribution Function f (x, p) * 16.4. Quantum Fluctuations in Equations of Motion * 17. P-Function Methods * 17.1. Introduction * 17.2. Artificial Neural Networks * 17.3. Clinical Example * VI - QUANTUM MEASUREMENT THEORY * 18. Foundations and Formalism of Quantum Measurement * 18.1. Formulations of Quantum Mechanics * 18.2. Modelling a Measurement-Tracks in a Cloud Chamber * 18.3. Formal Quantum Measurement Theory * 18.4. Multitime Measurements * 19. Continuous Measurements * 19.1. Photon Counting * 19.2. Wavefunction Interpretation of Continuous Measurement * 19.3. Application to Matter Wave Interference * 19.4. Damping of Quantum Coherence * 19.5. The Emergence of the oscopic World * 20. The Quantum Zeno Effect * 20.1. Theoretical Basis for the Quantum Zeno Effect * 20.2. A Quantum Model of Trapped Atoms * 20.3. Quantum Zeno Effect for a Bose-Einstein Condensate * References * Author Index * Subject Index