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Sample records for gmp-dependent calcium-activated chloride

  1. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.

    PubMed

    Dibattista, Michele; Amjad, Asma; Maurya, Devendra Kumar; Sagheddu, Claudia; Montani, Giorgia; Tirindelli, Roberto; Menini, Anna

    2012-07-01

    The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction. PMID:22732308

  2. International Union of Basic and Clinical Pharmacology. LXXXV: Calcium-Activated Chloride Channels

    PubMed Central

    Huang, Fen; Wong, Xiuming

    2012-01-01

    Calcium-activated chloride channels (CaCCs) are widely expressed in various tissues and implicated in physiological processes such as sensory transduction, epithelial secretion, and smooth muscle contraction. Transmembrane proteins with unknown function 16 (TMEM16A) has recently been identified as a major component of CaCCs. Detailed molecular analysis of TMEM16A will be needed to understand its structure-function relationships. The role this channel plays in physiological systems remains to be established and is currently a subject of intense investigation. PMID:22090471

  3. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    PubMed

    Zhang, Weiping; Schmelzeisen, Steffen; Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

  4. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

    PubMed Central

    Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

  5. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea.

    PubMed

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

  6. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea

    PubMed Central

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl- current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl- currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K+ channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

  7. Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels.

    PubMed

    Reyes, J P; Huanosta-Gutiérrez, A; López-Rodríguez, A; Martínez-Torres, A

    2015-01-01

    We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na(+) permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na(+). Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade. PMID:25853341

  8. Calcium-activated chloride currents prolongs the duration of contractions in pregnant rat myometrial tissue.

    PubMed

    Young, Roger C; Bemis, Adam

    2009-08-01

    We investigated the importance of pharmacologically blocking calcium-activated chloride (I(Cl(Ca))) and L-type calcium currents on isometric contractions of strips of D21 pregnant rat myometrial tissue, while simultaneously measuring the electrical activity of the tissue strips with extracellular contact electrodes. When measured with contact electrodes, the duration of the spiking activity directly reflects the duration of the tissue-level plateau potential. We correlated the number of spikes, durations of spiking activity, and the spiking frequencies with changes of the area under the force curves as a function of exposure to low doses of anthracene-9-carboxylate (9-AC, a non-specific Cl channel blocker), chlorotoxin (a specific I(Cl(Ca)) blocker) and nifedipine (an L-type calcium channel blocker). The area under the force curve was measured only during spiking electrical activity, thereby separating pharmacological effects on tissue relaxation from those that modulate force production. Blocking chloride channels reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes generated in each contraction. This was observed without a change in the frequency of spike production or a reduction of peak force. Nifedipine reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes. In contrast to chloride channel blockade, nifedipine reduced maximum spike frequency and peak force. Taken together, our data suggest that blocking L-type calcium channels reduces impulse directly by reducing peak force, and indirectly by reducing activation of I(Cl(Ca)) , which shortens the duration of the contraction. PMID:19380901

  9. Calcium-activated chloride current expression in axotomized sensory neurons: what for?

    PubMed Central

    Boudes, Mathieu; Scamps, Frédérique

    2012-01-01

    Calcium-activated chloride currents (CaCCs) are activated by an increase in intracellular calcium concentration. Peripheral nerve injury induces the expression of CaCCs in a subset of adult sensory neurons in primary culture including mechano- and proprioceptors, though not nociceptors. Functional screenings of potential candidate genes established that Best1 is a molecular determinant for CaCC expression among axotomized sensory neurons, while Tmem16a is acutely activated by inflammatory mediators in nociceptors. In nociceptors, such CaCCs are preferentially activated under receptor-induced calcium mobilization contributing to cell excitability and pain. In axotomized mechano- and proprioceptors, CaCC activation does not promote electrical activity and prevents firing, a finding consistent with electrical silencing for growth competence of adult sensory neurons. In favor of a role in the process of neurite growth, CaCC expression is temporally correlated to neurons displaying a regenerative mode of growth. This perspective focuses on the molecular identity and role of CaCC in axotomized sensory neurons and the future directions to decipher the cellular mechanisms regulating CaCC during neurite (re)growth. PMID:22461766

  10. Activation and inhibition of TMEM16A calcium-activated chloride channels.

    PubMed

    Ni, Yu-Li; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1. PMID:24489780

  11. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

    PubMed

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-01-01

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. PMID:24980701

  12. Developmental change in calcium-activated chloride current during the differentiation of Xenopus spinal neurons in culture.

    PubMed

    Hussy, N

    1991-09-01

    The duration and ionic dependence of action potentials change during the differentiation of embryonic amphibian spinal neurons both in vivo and in culture. The development of sodium, calcium, and potassium currents has been characterized in these cells and the shortening of the action potential has been shown to depend to a great extent on developmental changes of potassium currents. Previous evidence suggests that a chloride current may also be present in these embryonic neurons. Chloride currents were investigated with intracellular current-clamp and single-electrode and whole-cell voltage-clamp techniques. Most neurons exhibited a calcium-activated chloride current (ICl(Ca] that contributed to the postdepolarization following the action potential recorded in the absence of sodium and potassium currents. This current appeared to decrease in density and its deactivation rate increased during the first day in culture. Its incidence also declined during this period. A much larger Ca(2+)-dependent Cl- current was also observed in a subset of neurons after 24 hr, but was absent at earlier stages of development. The results suggest the presence of two Cl- currents with different developmental fates. The early current probably contributes to the repolarization of long calcium-dependent action potentials at initial stages of neuronal development, when potassium currents are small, and may serve to reduce the extent of repetitive firing. PMID:1715301

  13. Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction

    PubMed Central

    Namkung, Wan; Yao, Zhen; Finkbeiner, Walter E.; Verkman, A. S.

    2011-01-01

    TMEM16A (ANO1) is a calcium-activated chloride channel (CaCC) expressed in secretory epithelia, smooth muscle, and other tissues. Cell-based functional screening of ∼110,000 compounds revealed compounds that activated TMEM16A CaCC conductance without increasing cytoplasmic Ca2+. By patch-clamp, N-aroylaminothiazole “activators” (Eact) strongly increased Cl− current at 0 Ca2+, whereas tetrazolylbenzamide “potentiators” (Fact) were not active at 0 Ca2+ but reduced the EC50 for Ca2+-dependent TMEM16A activation. Of 682 analogs tested, the most potent activator (Eact) and potentiator (Fact) produced large and more sustained CaCC Cl− currents than general agonists of Ca2+ signaling, with EC50 3–6 μM and Cl− conductance comparable to that induced transiently by Ca2+-elevating purinergic agonists. Analogs of activators were identified that fully inhibited TMEM16A Cl− conductance, providing further evidence for direct TMEM16A binding. The TMEM16A activators increased CaCC conductance in human salivary and airway submucosal gland epithelial cells, and IL-4 treated bronchial cells, and stimulated submucosal gland secretion in human bronchi and smooth muscle contraction in mouse intestine. Small-molecule, TMEM16A-targeted activators may be useful for drug therapy of cystic fibrosis, dry mouth, and gastrointestinal hypomotility disorders, and for pharmacological dissection of TMEM16A function.—Namkung, W., Yao, Z., Finkbeiner, W. E., Verkman, A. S. Small-molecule activators of TMEM16A, a calcium-activated chloride channel, stimulate epithelial chloride secretion and intestinal contraction. PMID:21836025

  14. Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

    PubMed Central

    Wang, Yong-Xiao; Kotlikoff, Michael I.

    1997-01-01

    To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation. PMID:9405714

  15. Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule.

    PubMed

    Bidet, M; Tauc, M; Rubera, I; de Renzis, G; Poujeol, C; Bohn, M T; Poujeol, P

    1996-10-01

    Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions. PMID:8898026

  16. Calcium-activated chloride currents in olfactory sensory neurons from mice lacking bestrophin-2

    PubMed Central

    Pifferi, Simone; Dibattista, Michele; Sagheddu, Claudia; Boccaccio, Anna; Al Qteishat, Ahmed; Ghirardi, Filippo; Tirindelli, Roberto; Menini, Anna

    2009-01-01

    Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca2+ into the cilia. Ca2+ activates a Cl− current that produces an efflux of Cl− ions and amplifies the depolarization. The molecular identity of Ca2+-activated Cl− channels is still elusive, although some bestrophins have been shown to function as Ca2+-activated Cl− channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca2+-activated Cl− currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca2+-activated Cl− currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons. PMID:19622610

  17. Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

    PubMed Central

    Nilius, B; Prenen, J; Szücs, G; Wei, L; Tanzi, F; Voets, T; Droogmans, G

    1997-01-01

    1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i

  18. Location of Release Sites and Calcium-Activated Chloride Channels Relative to Calcium Channels at the Photoreceptor Ribbon Synapse

    PubMed Central

    Mercer, A. J.; Rabl, K.; Riccardi, G. E.; Brecha, N. C.; Stella, S. L.

    2011-01-01

    Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca2+ channels, which are in turn regulated by Cl− moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca2+ channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca2+ buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca2+ channels. Comparing Cl(Ca) currents with predicted Ca2+ diffusion profiles suggested that Cl(Ca) and Ca2+ channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca2+ channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca2+]i) elevation through flash photolysis of DM-nitrophen exhibited EC50 values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca2+]i in photoreceptor terminals. Consistent with control of exocytosis by [Ca2+] nanodomains near Ca2+ channels, average submembrane [Ca2+]i remained below the vesicle release threshold (∼400 nM) over much of the physiological voltage range for cones. Positioning Ca2+ channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca2+ influx at one site to influence relatively distant Ca2+ channels. PMID:21084687

  19. Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

    PubMed

    Hendrick, Siobhán M; Mroz, Magdalena S; Greene, Catherine M; Keely, Stephen J; Harvey, Brian J

    2014-09-01

    Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 μM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid. PMID:24993131

  20. Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

    PubMed Central

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

    2012-01-01

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

  1. Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies.

    PubMed

    Bolduc, Véronique; Marlow, Gareth; Boycott, Kym M; Saleki, Khalil; Inoue, Hiroshi; Kroon, Johan; Itakura, Mitsuo; Robitaille, Yves; Parent, Lucie; Baas, Frank; Mizuta, Kuniko; Kamata, Nobuyuki; Richard, Isabelle; Linssen, Wim H J P; Mahjneh, Ibrahim; de Visser, Marianne; Bashir, Rumaisa; Brais, Bernard

    2010-02-12

    The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies. PMID:20096397

  2. Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies

    PubMed Central

    Bolduc, Véronique; Marlow, Gareth; Boycott, Kym M.; Saleki, Khalil; Inoue, Hiroshi; Kroon, Johan; Itakura, Mitsuo; Robitaille, Yves; Parent, Lucie; Baas, Frank; Mizuta, Kuniko; Kamata, Nobuyuki; Richard, Isabelle; Linssen, Wim H.J.P.; Mahjneh, Ibrahim; de Visser, Marianne; Bashir, Rumaisa; Brais, Bernard

    2010-01-01

    The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies. PMID:20096397

  3. Effects of Small Molecule Calcium-Activated Chloride Channel Inhibitors on Structure and Function of Accessory Cholera Enterotoxin (Ace) of Vibrio cholerae

    PubMed Central

    Chatterjee, Tanaya; Sheikh, Irshad Ali; Chakravarty, Devlina; Chakrabarti, Pinak; Sarkar, Paramita; Saha, Tultul; Chakrabarti, Manoj K.; Hoque, Kazi Mirajul

    2015-01-01

    Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders. PMID:26540279

  4. TMEM16A Inhibitors Reveal TMEM16A as a Minor Component of Calcium-activated Chloride Channel Conductance in Airway and Intestinal Epithelial Cells*

    PubMed Central

    Namkung, Wan; Phuan, Puay-Wah; Verkman, A. S.

    2011-01-01

    TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of ∼110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC50 < 10 μm, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16Ainh-A01), had an IC50 of ∼1 μm. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCCinh-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16Ainh-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production. PMID:21084298

  5. Contribution of calcium-activated chloride channel to elevated pulmonary artery pressure in pulmonary arterial hypertension induced by high pulmonary blood flow

    PubMed Central

    Wang, Kai; Chen, Chuansi; Ma, Jianfa; Lao, Jinquan; Pang, Yusheng

    2015-01-01

    The correlation between calcium-activated chloride channel (CaCC) and pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow remains uncertain. In this study, we investigated the possible role and effects of CaCC in this disease. Sixty rats were randomly assigned to normal, sham, and shunt groups. Rats in the shunt group underwent abdominal aorta and inferior vena cava shunt surgery. The pulmonary artery pressure was measured by catheterization. Pathological changes, right ventricle hypertrophy index (RVHI), arterial wall area/vessel area (W/V), and arterial wall thickness/vessel external diameter (T/D) were analyzed by optical microscopy. Electrophysiological characteristics of pulmonary arterial smooth muscle cells (PASMCs) were investigated using patch clamp technology. After 11 weeks of shunting, PAH and pulmonary vascular structural remodeling (PVSR) developed, accompanied by increased pulmonary pressure and pathological interstitial pulmonary changes. Compared with normal and sham groups, pulmonary artery pressure, RVHI, W/V, and T/D of the shunt group rats increased significantly. Electrophysiological results showed primary CaCC characteristics. Compared with normal and sham groups, membrane capacitance and current density of PASMCs in the shunt group increased significantly, which were subsequently attenuated following chloride channel blocker niflumic acid (NFA) treatment. To conclude, CaCC contributed to PAH induced by high pulmonary blood flow and may represent a potential target for treatment of PAH. PMID:25755701

  6. Contribution of calcium-activated chloride channel to elevated pulmonary artery pressure in pulmonary arterial hypertension induced by high pulmonary blood flow.

    PubMed

    Wang, Kai; Chen, Chuansi; Ma, Jianfa; Lao, Jinquan; Pang, Yusheng

    2015-01-01

    The correlation between calcium-activated chloride channel (CaCC) and pulmonary arterial hypertension (PAH) induced by high pulmonary blood flow remains uncertain. In this study, we investigated the possible role and effects of CaCC in this disease. Sixty rats were randomly assigned to normal, sham, and shunt groups. Rats in the shunt group underwent abdominal aorta and inferior vena cava shunt surgery. The pulmonary artery pressure was measured by catheterization. Pathological changes, right ventricle hypertrophy index (RVHI), arterial wall area/vessel area (W/V), and arterial wall thickness/vessel external diameter (T/D) were analyzed by optical microscopy. Electrophysiological characteristics of pulmonary arterial smooth muscle cells (PASMCs) were investigated using patch clamp technology. After 11 weeks of shunting, PAH and pulmonary vascular structural remodeling (PVSR) developed, accompanied by increased pulmonary pressure and pathological interstitial pulmonary changes. Compared with normal and sham groups, pulmonary artery pressure, RVHI, W/V, and T/D of the shunt group rats increased significantly. Electrophysiological results showed primary CaCC characteristics. Compared with normal and sham groups, membrane capacitance and current density of PASMCs in the shunt group increased significantly, which were subsequently attenuated following chloride channel blocker niflumic acid (NFA) treatment. To conclude, CaCC contributed to PAH induced by high pulmonary blood flow and may represent a potential target for treatment of PAH. PMID:25755701

  7. Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells.

    PubMed

    Winpenny, J P; Harris, A; Hollingsworth, M A; Argent, B E; Gray, M A

    1998-05-01

    Using the whole-cell patch-clamp technique, a calcium-activated chloride conductance (CACC) could be elicited in HPAF cells by addition of 1 microM ionomycin to the bath solution (66 +/- 22 pA/pF;Vm + 60 mV) or by addition of 1 microM calcium to the pipette solution (136 +/- 17 pA/pF; Vm + 60 mV). Both conductances had similar biophysical characteristics, including time-dependent inactivation at hyperpolarising potentials and a linear/slightly outwardly rectifying current/voltage (I/V) curve with a reversal potential (Erev) close to the calculated chloride equilibrium potential. The anion permeability sequence obtained from shifts in Erev was I > Br >/= Cl. 4,4'-Diisothiocyanatostilbene disulphonic acid (DIDS, 500 microM) caused a 13% inhibition of the current (Vm + 60 mV) while 100 microM glibenclamide, 30 nM TS-TM-calix[4]arene and 10 microM tamoxifen, all chloride channel blockers, had no marked effects (8%, -6% and -2% inhibition respectively). Niflumic acid (100 microM) caused a voltage-dependent inhibition of the current of 48% and 17% (Vm +/- 60 mV, respectively). In freshly isolated human pancreatic duct cells (PDCs) a CACC was elicited with 1 microM calcium in the pipette solution (260 +/- 62 pA/pF; Vm + 60 mV). The presence of this CACC in human PDCs could provide a possible therapeutic pathway for treatment of pancreatic insufficiency of the human pancreas in cystic fibrosis. PMID:9518508

  8. Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation*

    PubMed Central

    Bill, Anke; Popa, M. Oana; van Diepen, Michiel T.; Gutierrez, Abraham; Lilley, Sarah; Velkova, Maria; Acheson, Kathryn; Choudhury, Hedaythul; Renaud, Nicole A.; Auld, Douglas S.; Gosling, Martin; Groot-Kormelink, Paul J.; Gaither, L. Alex

    2015-01-01

    The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases. PMID:25425649

  9. Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.

    PubMed

    Jia, Linghan; Liu, Wen; Guan, Lizhao; Lu, Min; Wang, KeWei

    2015-01-01

    Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy. PMID:26305547

  10. The Porcine Chloride Channel Calcium-Activated Family Member pCLCA4a Mirrors Lung Expression of the Human hCLCA4

    PubMed Central

    Plog, Stephanie; Grötzsch, Tanja; Klymiuk, Nikolai; Kobalz, Ursula; Gruber, Achim D.

    2012-01-01

    Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4. PMID:22205680

  11. Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer

    PubMed Central

    Jia, Linghan; Liu, Wen; Guan, Lizhao; Lu, Min; Wang, KeWei

    2015-01-01

    Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca2+-activated Cl- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy. PMID:26305547

  12. Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.

    PubMed

    Peters, Christian J; Yu, Haibo; Tien, Jason; Jan, Yuh Nung; Li, Min; Jan, Lily Yeh

    2015-03-17

    TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore. PMID:25733897

  13. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1).

    PubMed

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P

    2015-07-10

    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted. PMID:26004777

  14. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1)*

    PubMed Central

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S.; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P.

    2015-01-01

    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted. PMID:26004777

  15. Noradrenaline activates a calcium-activated chloride conductance and increases the voltage-dependent calcium current in cultured single cells of rat portal vein.

    PubMed

    Pacaud, P; Loirand, G; Mironneau, C; Mironneau, J

    1989-05-01

    1. Membrane responses were recorded by a patch pipette technique in cultured cells isolated from rat portal vein. Using the whole-cell mode, pressure ejections of noradrenaline evoked depolarization (current clamp) and inward current (voltage clamp) at membrane potentials of -60 to -70 mV. The noradrenaline-induced response was reversibly blocked by prazosin indicating that the response was mediated by alpha 1-adrenoceptors. 2. The ionic mechanism of the noradrenaline-induced inward current was investigated in potassium-free caesium-containing solutions. Alteration of the chloride equilibrium potential produced similar changes in the reversal potential of the noradrenaline-induced current, indicating that noradrenaline opened chloride-selective channels. There was no evidence implicating sodium or calcium as the charge-carrying ion. 3. Caffeine applied in the bathing solution also induced a transient increase in chloride conductance but the noradrenaline-induced response was lost after application of caffeine. This is interpreted to mean that the increase in chloride conductance induced by noradrenaline and caffeine can occur as a consequence of a rise in intracellular calcium concentration depending on release of calcium from the same intracellular stores. 4. In the presence of caffeine, noradrenaline increased both the voltage-dependent calcium and chloride membrane conductances during application of repetitive depolarizing pulses. It is concluded that in isolated cells of the rat portal vein the depolarization in response to noradrenaline is mediated by an increase in chloride conductance depending on both the calcium release from intracellular stores and the increase of the voltage-dependent calcium current. PMID:2470458

  16. A Short History of cGMP, Guanylyl Cyclases, and cGMP-Dependent Protein Kinases

    PubMed Central

    Kots, Alexander Y.; Martin, Emil; Sharina, Iraida G.

    2014-01-01

    Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules. PMID:19089322

  17. Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells.

    PubMed

    Linley, J E; Simmons, N L; Gray, M A

    2007-01-01

    We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed. PMID:17021797

  18. cGMP-Dependent Kinase Regulates Response Sensitivity of the Mouse On Bipolar Cell

    PubMed Central

    Snellman, Josefin; Nawy, Scott

    2007-01-01

    The visual system can adjust its sensitivity over a wide range of light intensities. Photoreceptors account for some of this adjustment, but there is evidence that postreceptoral processes also exist. To investigate the latter, we pharmacologically mimicked the effects of light stimulation on mouse On bipolar cells, thus avoiding confounding effects of receptoral mechanisms. Here, we report that cGMP selectively enhances responses to dim, but not bright, stimuli through a purely postsynaptic mechanism. This action of cGMP was completely blocked by inhibitors of cGMP-dependent kinase. We propose that cGMP-dependent kinase decreases coupling of the On bipolar cell glutamate receptor to the downstream cascade, thus amplifying small decreases in photoreceptor transmitter levels that would otherwise go undetected by the visual system. PMID:15269274

  19. Sildenafil Potentiates a cGMP-Dependent Pathway to Promote Melanoma Growth.

    PubMed

    Dhayade, Sandeep; Kaesler, Susanne; Sinnberg, Tobias; Dobrowinski, Hyazinth; Peters, Stefanie; Naumann, Ulrike; Liu, He; Hunger, Robert E; Thunemann, Martin; Biedermann, Tilo; Schittek, Birgit; Simon, Hans-Uwe; Feil, Susanne; Feil, Robert

    2016-03-22

    Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk. PMID:26971999

  20. Nitric oxide signaling modulates synaptic inhibition in the superior paraolivary nucleus (SPN) via cGMP-dependent suppression of KCC2

    PubMed Central

    Yassin, Lina; Radtke-Schuller, Susanne; Asraf, Hila; Grothe, Benedikt; Hershfinkel, Michal; Forsythe, Ian D.; Kopp-Scheinpflug, Cornelia

    2014-01-01

    Glycinergic inhibition plays a central role in the auditory brainstem circuitries involved in sound localization and in the encoding of temporal action potential firing patterns. Modulation of this inhibition has the potential to fine-tune information processing in these networks. Here we show that nitric oxide (NO) signaling in the auditory brainstem (where activity-dependent generation of NO is documented) modulates the strength of inhibition by changing the chloride equilibrium potential. Recent evidence demonstrates that large inhibitory postsynaptic currents (IPSCs) in neurons of the superior paraolivary nucleus (SPN) are enhanced by a very low intracellular chloride concentration, generated by the neuronal potassium chloride co-transporter (KCC2) expressed in the postsynaptic neurons. Our data show that modulation by NO caused a 15 mV depolarizing shift of the IPSC reversal potential, reducing the strength of inhibition in SPN neurons, without changing the threshold for action potential firing. Regulating inhibitory strength, through cGMP-dependent changes in the efficacy of KCC2 in the target neuron provides a postsynaptic mechanism for rapidly controlling the inhibitory drive, without altering the timing or pattern of the afferent spike train. Therefore, this NO-mediated suppression of KCC2 can modulate inhibition in one target nucleus (SPN), without influencing inhibitory strength of other target nuclei (MSO, LSO) even though they are each receiving collaterals from the same afferent nucleus (a projection from the medial nucleus of the trapezoid body, MNTB). PMID:24987336

  1. Activity of cGMP-Dependent Protein Kinase (PKG) Affects Sucrose Responsiveness and Habituation in "Drosophila melanogaster"

    ERIC Educational Resources Information Center

    Scheiner, Ricarda; Sokolowski, Marla B.; Erber, Joachim

    2004-01-01

    The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The "foraging" ("for") gene encodes a PKG in "Drosophila melanogaster." Here, we demonstrate a function for the "for" gene in sensory responsiveness and nonassociative learning. Larvae of the…

  2. Redox regulation of cGMP-dependent protein kinase Iα in the cardiovascular system

    PubMed Central

    Prysyazhna, Oleksandra; Eaton, Philip

    2015-01-01

    Elevated levels of oxidants in biological systems have been historically referred to as “oxidative stress,” a choice of words that perhaps conveys an imbalanced view of reactive oxygen species in cells and tissues. The term stress suggests a harmful role, whereas a contemporary view is that oxidants are also crucial for the maintenance of homeostasis or adaptive signaling that can actually limit injury. This regulatory role for oxidants is achieved in part by them inducing oxidative post-translational modifications of proteins which may alter their function or interactions. Such mechanisms allow changes in cell oxidant levels to be coupled to regulated alterations in enzymatic function (i.e., signal transduction), which enables “redox signaling.” In this review we focus on the role of cGMP-dependent protein kinase (PKG) Ia disulfide dimerisation, an oxidative modification that is induced by oxidants that directly activates the enzyme, discussing how this impacts on the cardiovascular system. Additionally, how this oxidative activation of PKG may coordinate with or differ from classical activation of this kinase by cGMP is also considered. PMID:26236235

  3. Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation.

    PubMed

    Köseoğlu, Volkan K; Heiss, Christian; Azadi, Parastoo; Topchiy, Elena; Güvener, Zehra T; Lehmann, Teresa E; Miller, Kurt W; Gomelsky, Mark

    2015-05-01

    Elevated levels of the second messenger c-di-GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food-borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface-bound. Secreted carbohydrates represent exclusively cell-wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β-1,4-linked N-acetylmannosamine chain decorated with terminal α-1,6-linked galactose. All genes of the pssA-E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS-specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS-mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c-di-GMP-dependent EPS production. PMID:25662512

  4. cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

    PubMed Central

    Wolfertstetter, Stefanie; Huettner, Johannes P.; Schlossmann, Jens

    2013-01-01

    cGMP-dependent protein kinases (PKG) exhibit diverse physiological functions in the mammalian system e.g., in vascular and gastrointestinal smooth muscles, in platelets, in kidney, in bone growth, nociception and in the central nervous system. Furthermore, PKG were found in insects and in the malaria parasite Plasmodium falciparum. Two different genes of PKG exist: a) the PKG-I gene that is expressed as cytosolic PKG-Iα or PKG-Iβ isoform, and b) the PKG-II gene, which expresses the membrane associated PKG-II protein. The enzyme kinetics, the localization and the substrates of these PKG enzymes differ utilizing different physiological functions. Various inhibitors of PKG were developed directed against diverse functional regions of the kinase. These inhibitors of PKG have been used to analyse the specific functions of these enzymes. The review article will summarize these different inhibitors regarding their specificity and their present applications in vitro and in vivo. Furthermore, it will be discussed that the distinct inhibition of the PKG enzymes could be used as a valuable pharmacological target e.g., in the treatment of cardiovascular diseases, diarrhea, cancer or malaria. PMID:24275951

  5. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis

    PubMed Central

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H.; Zhang, Huili

    2015-01-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. PMID:25855081

  6. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

    PubMed

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

    2015-06-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. PMID:25855081

  7. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling.

    PubMed

    Turek, Ilona; Gehring, Chris

    2016-06-01

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3',5'-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. PMID:26945740

  8. Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

    PubMed Central

    Chen, Li-Hong; Köseoğlu, Volkan K.; Güvener, Zehra T.; Myers-Morales, Tanya; Reed, Joseph M.; D'Orazio, Sarah E. F.; Miller, Kurt W.; Gomelsky, Mark

    2014-01-01

    We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence. PMID:25101646

  9. Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium.

    PubMed

    Ann, K S; Nelson, D L

    1995-01-01

    In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7796456

  10. Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse*

    PubMed Central

    Corradini, Eleonora; Vallur, Raghavan; Raaijmakers, Linsey M.; Feil, Susanne; Feil, Robert; Heck, Albert J. R.; Scholten, Arjen

    2014-01-01

    The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca2+ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity. PMID:24925903

  11. cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

    PubMed Central

    Busch, Jennifer L.; Corbin, Jackie D.

    2010-01-01

    To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge. PMID:20716671

  12. A nonsense mutation in cGMP-dependent type II protein kinase (PRKG2) causes dwarfism in American Angus cattle

    PubMed Central

    Koltes, James E.; Mishra, Bishnu P.; Kumar, Dinesh; Kataria, Ranjit S.; Totir, Liviu R.; Fernando, Rohan L.; Cobbold, Rowland; Steffen, David; Coppieters, Wouter; Georges, Michel; Reecy, James M.

    2009-01-01

    Historically, dwarfism was the major genetic defect in U.S. beef cattle. Aggressive culling and sire testing were used to minimize its prevalence; however, neither of these practices can eliminate a recessive genetic defect. We assembled a 4-generation pedigree to identify the mutation underlying dwarfism in American Angus cattle. An adaptation of the Elston-Steward algorithm was used to overcome small pedigree size and missing genotypes. The dwarfism locus was fine-mapped to BTA6 between markers AFR227 and BM4311. Four candidate genes were sequenced, revealing a nonsense mutation in exon 15 of cGMP-dependant type II protein kinase (PRKG2). This C/T transition introduced a stop codon (R678X) that truncated 85 C-terminal amino acids, including a large portion of the kinase domain. Of the 75 mutations discovered in this region, only this mutation was 100% concordant with the recessive pattern of inheritance in affected and carrier individuals (log of odds score = 6.63). Previous research has shown that PRKG2 regulates SRY (sex-determining region Y) box 9 (SOX9)-mediated transcription of collagen 2 (COL2). We evaluated the ability of wild-type (WT) or R678X PRKG2 to regulate COL2 expression in cell culture. Real-time PCR results confirmed that COL2 is overexpressed in cells that overexpressed R678X PRKG2 as compared with WT PRKG2. Furthermore, COL2 and COL10 mRNA expression was increased in dwarf cattle compared with unaffected cattle. These experiments indicate that the R678X mutation is functional, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. Therefore, we present PRKG2 R678X as a causative mutation for dwarfism cattle. PMID:19887637

  13. Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

    PubMed

    Liu, Juan; Liu, Huixia; Li, Yanjing; Xu, Xiaojian; Chen, Zhengju; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng; Dou, Dou

    2014-11-01

    cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation. PMID:24977346

  14. Cardioprotection by H2S engages a cGMP-dependent protein kinase G/phospholamban pathway

    PubMed Central

    Bibli, Sofia-Iris; Andreadou, Ioanna; Chatzianastasiou, Athanasia; Tzimas, Christos; Sanoudou, Despina; Kranias, Evangelia; Brouckaert, Peter; Coletta, Ciro; Szabo, Csaba; Kremastinos, Dimitrios Th.; Iliodromitis, Efstathios K.; Papapetropoulos, Andreas

    2015-01-01

    Aims H2S is known to confer cardioprotection; however, the pathways mediating its effects in vivo remain incompletely understood. The purpose of the present study is to evaluate the contribution of cGMP-regulated pathways in the infarct-limiting effect of H2S in vivo. Methods and results Anaesthetized rabbits were subjected to myocardial ischaemia (I)/reperfusion (R), and infarct size was determined in control or H2S-exposed groups. The H2S donor sodium hydrosulfide (NaHS, an agent that generates H2S) increased cardiac cGMP and reduced the infarct size. The cGMP-dependent protein kinase (PKG)-I inhibitor DT2 abrogated the protective effect of NaHS, whereas the control peptide TAT or l-nitroarginine methyl ester (l-NAME) did not alter the effect of NaHS. Moreover, the KATP channel inhibitor, glibenclamide, partially reversed the effects of NaHS, whereas inhibition of mitochondrial KATP did not modify the NaHS response. NaHS enhanced phosphorylation of phospholamban (PLN), in a PKG-dependent manner. To further investigate the role of PLN in H2S-mediated cardioprotection, wild-type and PLN KO mice underwent I/R. NaHS did not exert cardioprotection in PLN KO mice. Unlike what was observed in rabbits, genetic or pharmacological inhibition of eNOS abolished the infarct-limiting effect of NaHS in mice. Conclusions Our findings demonstrate (i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and (ii) contribution of nitric oxide to the H2S response is species-specific. PMID:25870184

  15. Type II cGMP-dependent protein kinase directly inhibits HER2 activation of gastric cancer cells.

    PubMed

    Zhu, Miaolin; Yao, Xiaoyuan; Wu, Min; Qian, Hai; Wu, Yan; Chen, Yongchang

    2016-02-01

    Our previous study demonstrated that type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced phosphorylation/activation of epidermal growth factor receptor (EGFR). Since human epidermal growth factor receptor 2 (HER2) has a similar molecular structure to EGFR, the present study was designed to investigate whether PKG II also inhibits HER2 activation. The human gastric cancer cell line HGC‑27 was infected with an adenoviral construct encoding cDNA of PKG II (Ad‑PKG II) to increase the expression of PKG II and treated with 8‑(4‑chlorophenylthio)guanosine‑3',5'‑cyclic monophosphate (8‑pCPT‑cGMP) to activate the kinase. Western blotting was performed to detect the tyrosine and serine/threonine phosphorylation of HER2. Co‑immunoprecipitation was performed in order to determine the binding between PKG II and HER2. In addition, a QuikChange Lightning Site‑Directed Mutagenesis kit was used to mutate threonine 686 of HER2 to glutamic acid or alanine. The results demonstrated that EGF treatment increased the tyrosine phosphorylation (activation) of HER2. Increasing the PKG II activity of HGC‑27 cells through infection with Ad‑PKG II and stimulation with 8‑pCPT‑cGMP inhibited the EGF‑induced tyrosine phosphorylation/activation of HER2. PKG II bound directly with HER2 and caused phosphorylation of threonine 686. When threonine 686 of HER2 was mutated to alanine, which could not be phosphorylated by PKG II, the inhibitory effect of PKG II on the activation of HER2 was eradicated. When threonine 686 of HER2 was mutated to glutamic acid, which mimicked the phosphorylation of this site, treatment with EGF had no stimulating effect on tyrosine phosphorylation/activation of the mutant HER2. The results suggested that PKG II inhibits EGF‑induced activation of HER2 through binding with and causing threonine 686 phosphorylation of this oncogenic protein. PMID:26676300

  16. Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs

    PubMed Central

    Hong, Nancy J.; Garvin, Jeffrey L.

    2009-01-01

    We have shown that increased luminal flow induces O2− and nitric oxide (NO) production in thick ascending limbs (TALs). However, the interaction of flow-stimulated NO and O2− in TALs is unclear. We hypothesized that NO inhibits flow-induced O2− production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated O2− production in rat TALs using dihydroethidium in the absence and presence of l-arginine (0.3 mM), the substrate for NO synthase. The addition of l-arginine reduced flow-induced net O2− production from 68 ± 9 to 17 ± 4 AU/s (P < 0.002). The addition of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 5 mM) in the presence of l-arginine stimulated production (l-arginine: 15 ± 4 AU/s vs. l-arginine + l-NAME: 63 ± 7 AU/s; P < 0.002). The guanylate cyclase inhibitor LY-83583 (10 μM) also enhanced flow-induced net O2− production in the presence of l-arginine (l-arginine: 7 ± 4 AU/s vs. l-arginine + LY-83583: 53 ± 7 AU/s; P < 0.01). In the presence of LY-83583, l-arginine only reduced flow-induced net O2− by 36% (LY-83583: 80 ± 7 AU/s vs. LY-83583 + l-arginine: 51 ± 3 AU/s; P < 0.006). The cGMP analog dibutyryl (db)-cGMP reduced flow-induced net O2− from 39 ± 9 to 7 ± 3 AU/s (P < 0.03). The PKG inhibitor KT-5823 (5 μM) partially restored flow-induced net O2− in the presence of l-arginine (l-arginine: 4 ± 4 AU/s vs. l-arginine + KT-5823: 32 ± 9 AU/s; P < 0.03) and db-cGMP (db-cGMP: 9 ± 7 AU/s vs. db-cGMP + KT-5823: 54 ± 5 AU/s; P < 0.01). Phosphodiesterase II inhibition had no effect on arginine-inhibited O2− production. We conclude that 1) NO reduces flow-stimulated O2− production, 2) this occurs primarily via the cGMP/PKG pathway, and 3) O2− scavenging by NO plays a minor role. PMID:19244401

  17. Genetic Ablation of cGMP-Dependent Protein Kinase Type I Causes Liver Inflammation and Fasting Hyperglycemia

    PubMed Central

    Lutz, Stefan Z.; Hennige, Anita M.; Feil, Susanne; Peter, Andreas; Gerling, Andrea; Machann, Jürgen; Kröber, Stefan M.; Rath, Michaela; Schürmann, Annette; Weigert, Cora; Häring, Hans-Ulrich; Feil, Robert

    2011-01-01

    OBJECTIVE The nitric oxide/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway regulates cell functions that play a pivotal role in the pathogenesis of type 2 diabetes. However, the impact of a dysfunction of this pathway for glucose metabolism in vivo is unknown. RESEARCH DESIGN AND METHODS The expression of cGKI in tissues relevant to insulin action was analyzed by immunohistochemistry. The metabolic consequences of a genetic deletion of cGKI were studied in mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice). RESULTS In wild-type mice, cGKI protein was detected in hepatic stellate cells, but not in hepatocytes, skeletal muscle, fat cells, or pancreatic β-cells. Compared with control animals, cGKI-SM mice had higher energy expenditure in the light phase associated with lower body weight and fat mass and increased insulin sensitivity. Mutant mice also showed higher fasting glucose levels, whereas insulin levels and intraperitoneal glucose tolerance test results were similar to those in control animals. Interleukin (IL)-6 signaling was strongly activated in the liver of cGKI-SM mice as demonstrated by increased levels of IL-6, phospho-signal transducer and activator of transcription 3 (Tyr 705), suppressor of cytokine signaling-3, and serum amyloid A2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in the liver was impaired in cGKI-SM mice. The fraction of Mac-2–positive macrophages in the liver was significantly higher in cGKI-SM mice than in control mice. In contrast with cGKI-SM mice, conditional knockout mice lacking cGKI only in the nervous system were normal with respect to body weight, energy expenditure, fasting glucose, IL-6, and insulin action in the liver. CONCLUSIONS Genetic deletion of cGKI in non-neuronal cells results in a complex metabolic phenotype, including liver inflammation and fasting hyperglycemia. Loss of cGKI in hepatic stellate cells may affect liver

  18. Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS.

    PubMed Central

    Butt, E.; Pöhler, D.; Genieser, H. G.; Huggins, J. P.; Bucher, B.

    1995-01-01

    1. The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1, N2-ethenoguanosine 3':5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2. Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 microM. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 microM. 3. In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5, 6-dichloro-1-beta-D-ribofurano-sylbenzimidazole- 3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM). 4. (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cyclic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5. The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-beta-phenyl-1, N2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 microM) shifted the vasoconstriction response to the right without affecting stimulation evoked

  19. Neutron diffraction reveals hydrogen bonds critical for cGMP-selective activation: insights for cGMP-dependent protein kinase agonist design.

    PubMed

    Huang, Gilbert Y; Gerlits, Oksana O; Blakeley, Matthew P; Sankaran, Banumathi; Kovalevsky, Andrey Y; Kim, Choel

    2014-11-01

    High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP. PMID:25271401

  20. Neutron Diffraction Reveals Hydrogen Bonds Critical for cGMP-Selective Activation: Insights for cGMP-Dependent Protein Kinase Agonist Design

    PubMed Central

    2015-01-01

    High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP. PMID:25271401

  1. Structures of cGMP-Dependent Protein Kinase (PKG) Iα Leucine Zippers Reveal an Interchain Disulfide Bond Important for Dimer Stability

    PubMed Central

    Qin, Liying; Reger, Albert S.; Guo, Elaine; Yang, Matthew P.; Zwart, Peter; Casteel, Darren E.; Kim, Choel

    2016-01-01

    cGMP-dependent protein kinase (PKG) Iα is a central regulator of smooth muscle tone and vasorelaxation. The N-terminal leucine zipper (LZ) domain dimerizes and targets PKG Iα by interacting with G-kinase-anchoring proteins. The PKG Iα LZ contains C42 that is known to form a disulfide bond upon oxidation and to activate PKG Iα. To understand the molecular details of the PKG Iα LZ and C42–C42′ disulfide bond, we determined crystal structures of the PKG Iα wild-type (WT) LZ and C42L LZ. Our data demonstrate that the C42–C42′ disulfide bond dramatically stabilizes PKG Iα and that the C42L mutant mimics the oxidized WT LZ structurally. PMID:26132214

  2. The large conductance, voltage-dependent, and calcium-sensitive K+ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo.

    PubMed

    Alioua, A; Tanaka, Y; Wallner, M; Hofmann, F; Ruth, P; Meera, P; Toro, L

    1998-12-01

    Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels. PMID:9830046

  3. cGMP/cGMP-dependent protein kinase pathway modulates nicotine-induced currents through the activation of α-bungarotoxin-insensitive nicotinic acetylcholine receptors from insect neurosecretory cells.

    PubMed

    Mannai, Safa; Bitri, Lofti; Thany, Steeve H

    2016-06-01

    Insect neurosecretory cells, called dorsal unpaired median neurons, are known to express two α-bungarotoxin-insensitive nicotinic acetylcholine receptor (nAChR) subtypes, nAChR1 and nAChR2. It was demonstrated that nAChR1 was sensitive to cAMP/cAMP-dependent protein kinase (PKA) regulation, resulting in a modulation of nicotine currents. In this study, we show that cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway modulates nicotine-induced currents, as increased cGMP affects the second compound of the biphasic current-voltage curve, corresponding to the nAChR2 receptors. Indeed, maintaining the guanosine triphosphate level with 100 μM guanosine triphosphate-γ-S increased nicotine currents through nAChR2. We also demonstrated that inhibition of PKG activity with 0.2 μM (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823), a PKG specific inhibitor, reduced nicotine-induced current amplitudes. KT5823 effect on nicotine currents is associated with calcium (Ca(2+) ) activity because inhibition of Ca(2+) concentration with cadmium chloride (CdCl2 ) abolished KT5823-induced inhibition mediated by nAChR2. However, specific inhibition of nitric oxide-guanylyl cyclase (GC) complex by 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) significantly increased nicotine-induced current amplitudes on both nAChR1 and nAChR2. These results suggest that nicotine-induced currents mediated by both α-bungarotoxin-insensitive nAChR1 and nAChR2 are coupled to the cGMP/PKG pathway. We propose that nicotinic acetylcholine receptor activation induces an increase in intracellular calcium (Ca(2+) ) concentration. Elevation of intracellular Ca(2+) results in the formation of Ca(2+) -calmodulin (CaM) complex, which activates guanylyl cyclase (GC) and/or adenylyl cyclase (AC). Ca(2+) -CaM complex could activate Ca(2+) calmodulin kinase II which

  4. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

    PubMed Central

    Issinger, O G; Beier, H; Speichermann, N; Flokerzi, V; Hofmann, F

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme. Images Fig. 1. Fig. 2. PMID:6246882

  5. Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

    PubMed Central

    Markert, T; Vaandrager, A B; Gambaryan, S; Pöhler, D; Häusler, C; Walter, U; De Jonge, H R; Jarchau, T; Lohmann, S M

    1995-01-01

    Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia. Images PMID:7543493

  6. Rats with minimal hepatic encephalopathy show reduced cGMP-dependent protein kinase activity in hypothalamus correlating with circadian rhythms alterations.

    PubMed

    Felipo, Vicente; Piedrafita, Blanca; Barios, Juan A; Agustí, Ana; Ahabrach, Hanan; Romero-Vives, María; Barrio, Luis C; Rey, Beatriz; Gaztelu, Jose M; Llansola, Marta

    2015-01-01

    Patients with liver cirrhosis show disturbances in sleep and in its circadian rhythms which are an early sign of minimal hepatic encephalopathy (MHE). The mechanisms of these disturbances are poorly understood. Rats with porta-caval shunt (PCS), a model of MHE, show sleep disturbances reproducing those of cirrhotic patients. The aims of this work were to characterize the alterations in circadian rhythms in PCS rats and analyze the underlying mechanisms. To reach these aims, we analyzed in control and PCS rats: (a) daily rhythms of spontaneous and rewarding activity and of temperature, (b) timing of the onset of activity following turning-off the light, (c) synchronization to light after a phase advance and (d) the molecular mechanisms contributing to these alterations in circadian rhythms. PCS rats show altered circadian rhythms of spontaneous and rewarding activities (wheel running). PCS rats show more rest bouts during the active phase, more errors in the onset of motor activity and need less time to re-synchronize after a phase advance than control rats. Circadian rhythm of body temperature is also slightly altered in PCS rats. The internal period length (tau) of circadian rhythm of motor activity is longer in PCS rats. We analyzed some mechanisms by which hypothalamus modulate circadian rhythms. PCS rats show increased content of cGMP in hypothalamus while the activity of cGMP-dependent protein kinase was reduced by 41% compared to control rats. Altered cGMP-PKG pathway in hypothalamus would contribute to altered circadian rhythms and synchronization to light. PMID:26203935

  7. cGMP-Dependent Protein Kinase Type II Knockout Mice Exhibit Working Memory Impairments, Decreased Repetitive Behavior, and Increased Anxiety-like Traits

    PubMed Central

    Wincott, Charlotte M.; Abera, Sinedu; Vunck, Sarah A.; Choi, Yoon; Titcombe, Roseann F.; Antoine, Shannon O.; Tukey, David S.; DeVito, Loren M.; Hofmann, Franz; Hoeffer, Charles A.; Ziff, Edward B.

    2015-01-01

    Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle, Zhang, Ninan, Puzzo, McCarthy, Khatri, Arancio, and Ziff, 2007; Wincott, Kim, Titcombe, Tukey, Girma, Pick, Devito, Hofmann, Hoeffer, and Ziff, 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response. PMID:24752151

  8. Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry*

    PubMed Central

    Pinkse, Martijn W. H.; Rijkers, Dirk T. S.; Dostmann, Wolfgang R.; Heck, Albert J. R.

    2009-01-01

    The inhibitor peptide DT-2 (YGRKKRRQRRRPPLRKKKKKH) is the most potent and selective inhibitor of the cGMP-dependent protein kinase (PKG) known today. DT-2 is a construct of a PKG tight binding sequence (W45, LRKKKKKH, KI = 0.8 μm) and a membrane translocating sequence (DT-6, YGRKKRRQRRRPP, KI = 1.1 μm), that combined strongly inhibits PKG catalyzed phosphorylation (KI = 12.5 nm) with ∼1000-fold selectivity toward PKG over protein kinase A, the closest relative of PKG. However, the molecular mechanism behind this inhibition is not entirely understood. Using a combination of photoaffinity labeling, stable isotope labeling, and mass spectrometry, we have located the binding sites of PKG-specific substrate and inhibitor peptides. Covalent linkage of a PKG-specific substrate analogue was localized in the catalytic core on residues 356–372, also known as the glycine-rich loop, essential for ATP binding. By analogy, the individual inhibitor peptides W45 and DT-6 were also found to cross-link near the glycine-rich loop, suggesting these are both substrate competitive inhibitors. A bifunctional photoreactive analogue of DT-2 was found to generate dimers of PKG. This cross-linking induced covalent PKG dimerization was not observed for an N-terminal deletion mutant of PKG, which lacks the dimerization domain. In addition, non-covalent mass spectrometry was used to determine binding stoichiometry and binding order of the inhibitor peptides. Dimeric PKG binds two W45 and DT-6 peptides, whereas only one DT-2 molecule was observed to bind to the dimeric PKG. Taken together, these findings imply that (i) the two individual components making up DT-2 are both targeted against the substrate-binding site and (ii) binding of a single DT-2 molecule inactivates both PKG monomers simultaneously, which is an indication that (iii) in cGMP-activated PKG the catalytic centers of both subunits may be in each other's proximity. PMID:19369251

  9. Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

    PubMed Central

    Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung

    2011-01-01

    Background Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue. Methods and Findings Single-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions. Conclusion The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in

  10. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    PubMed

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  11. Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

    PubMed

    Xu, Long; Ting-Lou; Lv, Nonghua; Zhu, Xuan; Chen, Youxiang; Yang, Jing

    2009-08-01

    Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway. PMID:19409890

  12. Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

    PubMed

    Cherkashin, Alexander P; Kolesnikova, Alisa S; Tarasov, Michail V; Romanov, Roman A; Rogachevskaja, Olga A; Bystrova, Marina F; Kolesnikov, Stanislav S

    2016-02-01

    Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated. PMID:26530828

  13. Differential effect of calcium-activated potassium and chloride channels on rat basilar artery vasomotion.

    PubMed

    Li, Li; Wang, Rui; Ma, Ke-tao; Li, Xin-zhi; Zhang, Chuan-lin; Liu, Wei-dong; Zhao, Lei; Si, Jun-qiang

    2014-08-01

    Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. We investigated the cellular mechanisms underlying vasomotion in the cerebral basilar artery (BA) of Wistar rats. Pressure myograph video microscopy was used to study the changes in cerebral artery vessel diameter. The main results of this study were as follows: (1) The diameters of BA and middle cerebral artery (MCA) were 314.5±15.7 μm (n=15) and 233.3±10.1 μm (n=12) at 10 mmHg working pressure (P<0.05), respectively. Pressure-induced vasomotion occurred in BA (22/28, 78.6%), but not in MCA (4/31, 12.9%) from 0 to 70 mmHg working pressure. As is typical for vasomotion, the contractile phase of the response was more rapid than the relaxation phase; (2) The frequency of vasomotion response and the diameter were gradually increased in BA from 0 to 70 mmHg working pressure. The amplitude of the rhythmic contractions was relatively constant once stable conditions were achieved. The frequency of contractions was variable and the highest value was 16.7±4.7 (n=13) per 10 min at 60 mmHg working pressure; (3) The pressure-induced vasomotion of the isolated BA was attenuated by nifedipine, NFA, 18β-GA, TEA or in Ca(2+)-free medium. Nifedipine, NFA, 18β-GA or Ca(2+)-free medium not only dampened vasomotion, but also kept BA in relaxation state. In contrasts, TEA kept BA in contraction state. These results suggest that the pressure-induced vasomotion of the isolated BA results from an interaction between Ca(2+)-activated Cl(-) channels (CaCCs) currents and K(Ca) currents. We hypothesize that vasomotion of BA depends on the depolarizing of the vascular smooth muscle cells (VSMCs) to activate CaCCs. Depolarization in turn activates voltage-dependent Ca(2+) channels, synchronizing contractions of adjacent cells through influx of extracellular calcium and the flow of calcium through gap junctions. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates K(Ca) channels and hyperpolarizes VSMCs, which provides a negative feedback loop for regenerating the contractile cycle. PMID:25135715

  14. Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

    PubMed

    Hussy, N

    1992-12-01

    1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1283407

  15. The secret life of CFTR as a calcium-activated chloride channel.

    PubMed

    Billet, Arnaud; Hanrahan, John W

    2013-11-01

    cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca(2+)-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca(2+)-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations. PMID:23959675

  16. Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

    PubMed Central

    Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

    2015-01-01

    All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

  17. Both barium and calcium activate neuronal potassium currents.

    PubMed Central

    Ribera, A B; Spitzer, N C

    1987-01-01

    Amphibian spinal neurons in culture possess both rapidly inactivating and sustained calcium-dependent potassium current components, similar to those described for other cells. Divalent cation-dependent whole-cell outward currents were isolated by subtracting the voltage-dependent potassium currents recorded from Xenopus laevis neurons in the presence of impermeant cadmium (100-500 microM) from the currents produced without cadmium but in the presence of permeant divalent cations (50-100 microM). These concentrations of permeant ions were low enough to avoid contamination by macroscopic inward currents through calcium channels. Calcium-dependent potassium currents were reduced by 1 microM tetraethylammonium. These currents can also be activated by barium or strontium. Barium as well as calcium activated outward currents in young neurons (6-8 hr) and in relatively mature neurons (19-26 hr in vitro). However, barium influx appeared to suppress the sustained voltage-dependent potassium current in most cells. Barium also activated at least one class of potassium channels observed in excised membrane patches, while blocking others. The blocking action may have masked and hindered detection of the stimulatory action of barium in other systems. PMID:2442762

  18. The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster

    PubMed Central

    Schleede, Justin; Blair, Seth S.

    2015-01-01

    The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues. PMID:26440503

  19. Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

    PubMed Central

    Félétou, Michel

    2009-01-01

    The three subtypes of calcium-activated potassium channels (KCa) of large, intermediate and small conductance (BKCa, IKCa and SKCa) are present in the vascular wall. In healthy arteries, BKCa channels are preferentially expressed in vascular smooth muscle cells, while IKCa and SKCa are preferentially located in endothelial cells. The activation of endothelial IKCa and SKCa contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na+/K+-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H2O2) hyperpolarize and relax the underlying smooth muscle cells by activating BKCa. In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BKCa. Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle KCa could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IKCa may prevent restenosis and that of BKCa channels sepsis-dependent hypotension. PMID:19187341

  20. Calcium Activities During Different Ion Exchange Separation Procedures

    NASA Astrophysics Data System (ADS)

    Zhang, Z.; Zhu, H.; Liu, Y.; Liu, F.; Zhang, C.; Sun, W.

    2014-12-01

    Calcium is a major element and participates in many geological processes. Investigations on stable calcium isotopic compositions of natural geological samples provide a great powerful tool to understand all kinds of those geological processes from a view of the field of isotope geochemistry. With the development of modern instruments and chemical separation techniques, calcium isotopic compositions could be determined even more precisely if the column chemistry brings no deviation. Usually, Calcium is separated from matrix elements using cation resin columns and the related chemical separation techniques seem to be robust. However, more detailed work still need to be done on matrix effects and calcium isotopic fractionations on column chemistry or during elution processes. If calcium is run on TIMS instruments, the interference effect could be lower and easier controlled, thus, the requirement to the chemistry is relatively not critic, but calcium fractionation on filaments could be much difficult to monitor. If calcium is run on MC-ICP-MS instruments, the interference effect could be huge and is really difficult to be recognized and subtracted, the requirement to the chemistry is much more critical in order to get a real result of the sample, but the instrument fractionation could be easier to monitor. Here we investigate calcium activities on several kinds of cation resins under different column/acid conditions. We seek to find a good balance between recovery and interference effect on column chemistry and are intend to set up a better chemical separation procedure to satisfy the instrument requirements for calcium. In addition, Calcium isotopic fractionation on column will also be discussed further here based on our previous and ongoing results.

  1. Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II.

    PubMed

    Marathe, Nisha; Rangaswami, Hema; Zhuang, Shunhui; Boss, Gerry R; Pilz, Renate B

    2012-01-01

    Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD. PMID:22117068

  2. Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development.

    PubMed

    Green, Judith L; Moon, Robert W; Whalley, David; Bowyer, Paul W; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K; Howell, Steven A; Grainger, Munira; Jones, Hayley M; Ansell, Keith H; Chapman, Timothy M; Taylor, Debra L; Osborne, Simon A; Baker, David A; Tatu, Utpal; Holder, Anthony A

    2016-03-01

    Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771

  3. Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development

    PubMed Central

    Moon, Robert W.; Whalley, David; Bowyer, Paul W.; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K.; Howell, Steven A.; Grainger, Munira; Jones, Hayley M.; Ansell, Keith H.; Chapman, Timothy M.; Taylor, Debra L.; Osborne, Simon A.; Baker, David A.; Tatu, Utpal

    2015-01-01

    Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771

  4. Chloride Test

    MedlinePlus

    ... Addison disease, or increased salt intake. If both chloride and sodium levels are high in a person on a ... anything else I should know? Drugs that affect sodium blood levels will also cause changes in chloride. In addition, swallowing large amounts of baking soda ...

  5. Novel Roles for Chloride Channels, Exchangers, and Regulators in Chronic Inflammatory Airway Diseases

    PubMed Central

    Sala-Rabanal, Monica; Yurtsever, Zeynep; Berry, Kayla N.; Brett, Tom J.

    2015-01-01

    Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases. PMID:26612971

  6. Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons.

    PubMed

    Bock, Tobias; Stuart, Greg J

    2016-03-01

    Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels onN-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive. PMID:26936985

  7. The Anti-fibrotic Actions of Relaxin Are Mediated Through a NO-sGC-cGMP-Dependent Pathway in Renal Myofibroblasts In Vitro and Enhanced by the NO Donor, Diethylamine NONOate

    PubMed Central

    Wang, Chao; Kemp-Harper, Barbara K.; Kocan, Martina; Ang, Sheng Yu; Hewitson, Tim D.; Samuel, Chrishan S.

    2016-01-01

    GMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p < 0.01 vs. vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p < 0.05 vs. the respective treatments alone). Conclusion: These findings confirmed that RLX mediates its TGF-β1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO. PMID:27065874

  8. Calcium-calmodulin does not alter the anion permeability of the mouse TMEM16A calcium-activated chloride channel

    PubMed Central

    Yu, Yawei; Kuan, Ai-Seon

    2014-01-01

    The transmembrane protein TMEM16A forms a Ca2+-activated Cl− channel that is permeable to many anions, including SCN−, I−, Br−, Cl−, and HCO3−, and has been implicated in various physiological functions. Indeed, controlling anion permeation through the TMEM16A channel pore may be critical in regulating the pH of exocrine fluids such as the pancreatic juice. The anion permeability of the TMEM16A channel pore has recently been reported to be modulated by Ca2+-calmodulin (CaCaM), such that the pore of the CaCaM-bound channel shows a reduced ability to discriminate between anions as measured by a shift of the reversal potential under bi-ionic conditions. Here, using a mouse TMEM16A clone that contains the two previously identified putative CaM-binding motifs, we were unable to demonstrate such CaCaM-dependent changes in the bi-ionic potential. We confirmed the activity of CaCaM used in our study by showing CaCaM modulation of the olfactory cyclic nucleotide–gated channel. We suspect that the different bi-ionic potentials that were obtained previously from whole-cell recordings in low and high intracellular [Ca2+] may result from different degrees of bi-ionic potential shift secondary to a series resistance problem, an ion accumulation effect, or both. PMID:24981232

  9. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

    PubMed

    Forrest, Abigail S; Joyce, Talia C; Huebner, Marissa L; Ayon, Ramon J; Wiwchar, Michael; Joyce, John; Freitas, Natalie; Davis, Alison J; Ye, Linda; Duan, Dayue D; Singer, Cherie A; Valencik, Maria L; Greenwood, Iain A; Leblanc, Normand

    2012-12-15

    Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 μM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. PMID:23034390

  10. Calcium-activated chloride channels do not contribute to the odorant transduction current in the marine teleost Isacia conceptionis.

    PubMed

    Osorio, R; Schmachtenberg, O

    2013-11-01

    This study compared the contribution of the Ca²⁺-activated Cl⁻ conductance to the electroolfactogram (EOG) evoked by different odorant classes between the marine Cabinza grunt Isacia conceptionis and rainbow trout Oncorhynchus mykiss. The Ca²⁺-activated Cl⁻ channel blocker niflumic acid significantly diminished odorant responses in O. mykiss, but had no effect on the EOG in I. conceptionis, supporting the notion that Ca²⁺-activated Cl⁻ channels may not operate as odorant transduction current amplifiers in this marine teleost. PMID:24580677

  11. Chloride channels in stroke

    PubMed Central

    Zhang, Ya-ping; Zhang, Hao; Duan, Dayue Darrel

    2013-01-01

    Vascular remodeling of cerebral arterioles, including proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs), is the major cause of changes in the cross-sectional area and diameter of the arteries and sudden interruption of blood flow or hemorrhage in the brain, ie, stroke. Accumulating evidence strongly supports an important role for chloride (Cl−) channels in vascular remodeling and stroke. At least three Cl− channel genes are expressed in VSMCs: 1) the TMEM16A (or Ano1), which may encode the calcium-activated Cl− channels (CACCs); 2) the CLC-3 Cl− channel and Cl−/H+ antiporter, which is closely related to the volume-regulated Cl− channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR), which encodes the PKA- and PKC-activated Cl− channels. Activation of the CACCs by agonist-induced increase in intracellular Ca2+ causes membrane depolarization, vasoconstriction, and inhibition of VSMC proliferation. Activation of VRCCs by cell volume increase or membrane stretch promotes the production of reactive oxygen species, induces proliferation and inhibits apoptosis of VSMCs. Activation of CFTR inhibits oxidative stress and may prevent the development of hypertension. In addition, Cl− current mediated by gamma-aminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death. This review focuses on the functional roles of Cl− channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Cl− channels as new targets for the prevention and treatment of stroke. PMID:23103617

  12. Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways.

    PubMed

    Gianotti, Ambra; Ferrera, Loretta; Philp, Amber R; Caci, Emanuela; Zegarra-Moran, Olga; Galietta, Luis J V; Flores, Carlos A

    2016-06-15

    Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice. PMID:27063443

  13. Vinyl chloride

    Integrated Risk Information System (IRIS)

    Vinyl chloride ; CASRN 75 - 01 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  14. Methyl chloride

    Integrated Risk Information System (IRIS)

    Methyl chloride ; CASRN 74 - 87 - 3 ( 07 / 17 / 2001 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for

  15. Ethyl chloride

    Integrated Risk Information System (IRIS)

    Ethyl chloride ; CASRN 75 - 00 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  16. Benzyl chloride

    Integrated Risk Information System (IRIS)

    Benzyl chloride ; CASRN 100 - 44 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  17. Hydrogen chloride

    Integrated Risk Information System (IRIS)

    Hydrogen chloride ; CASRN 7647 - 01 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogeni

  18. Mepiquat chloride

    Integrated Risk Information System (IRIS)

    Mepiquat chloride ; CASRN 24307 - 26 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogen

  19. Allyl chloride

    Integrated Risk Information System (IRIS)

    Allyl chloride ; CASRN 107 - 05 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  20. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  1. Trypsin-Sensitive, Rapid Inactivation of a Calcium-Activated Potassium Channel

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

    Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

  2. Calcium-activated potassium channels in insect pacemaker neurons as unexpected target site for the novel fumigant dimethyl disulfide.

    PubMed

    Gautier, Hélène; Auger, Jacques; Legros, Christian; Lapied, Bruno

    2008-01-01

    Dimethyl disulfide (DMDS), a plant-derived insecticide, is a promising fumigant as a substitute for methyl bromide. To further understand the mode of action of DMDS, we examined its effect on cockroach octopaminergic neurosecretory cells, called dorsal unpaired median (DUM) neurons, using whole-cell patch-clamp technique, calcium imaging and antisense oligonucleotide strategy. At low concentration (1 microM), DMDS modified spontaneous regular spike discharge into clear bursting activity associated with a decrease of the amplitude of the afterhyperpolarization. This effect led us to suspect alterations of calcium-activated potassium currents (IKCa) and [Ca(2+)](i) changes. We showed that DMDS reduced amplitudes of both peak transient and sustained components of the total potassium current. IKCa was confirmed as a target of DMDS by using iberiotoxin, cadmium chloride, and pSlo antisense oligonucleotide. In addition, we showed that DMDS induced [Ca(2+)](i) rise in Fura-2-loaded DUM neurons. Using calcium-free solution, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxy-phenyl)ethyl]-acetamide (LOE 908) [an inhibitor of transient receptor potential (TRP)gamma], we demonstrated that TRPgamma initiated calcium influx. By contrast, omega-conotoxin GVIA (an inhibitor of N-type high-voltage-activated calcium channels), did not affect the DMDS-induced [Ca(2+)](i) rise. Finally, the participation of the calcium-induced calcium release mechanism was investigated using thapsigargin, caffeine, and ryanodine. Our study revealed that DMDS-induced elevation in [Ca(2+)](i) modulated IKCa in an unexpected bell-shaped manner via intracellular calcium. In conclusion, DMDS affects multiple targets, which could be an effective way to improve pest control efficacy of fumigation. PMID:17942746

  3. Long-term correlation in single calcium-activated potassium channel kinetics

    NASA Astrophysics Data System (ADS)

    Campos de Oliveira, R. A.; Barbosa, C. T. F.; Consoni, L. H. A.; Rodrigues, A. R. A.; Varanda, W. A.; Nogueira, R. A.

    2006-05-01

    Ion channels are protein molecules found in biological membranes, which can assume distinct open and closed conformational states, a phenomenon called ion channel kinetics. The transitions from one state to another are dependent on the potential energy barrier that separates them and can be controlled by the electrical field, ions and/or drugs. Both Markovian and fractal models have been used for modeling the ion channel kinetics. Ion single channel records are characterized by successive openings and closings, which are correlated in time. Here the rescaled range analysis ( R/S Hurst analysis) is used to test for the occurrence of long-term correlation in the kinetics of a calcium-activated potassium channel of Leydig cells. A Hurst coefficient H=0.640±0.064 ( n=5) was found for the single calcium-activated potassium channel clamped at -80 mV and exposed to a free Ca 2+ concentration equal to 10 nM. This numerical value indicates the presence of long-term correlation (memory) in this kinetic process. However, when the R/ S analysis was applied to ion channel data simulated using Markovian and fractal models, it could not account for the long-term correlation previously found in the experimental data. In summary, in this work we show that: (i) opening and closing dwell times for the single calcium-activated potassium channel of Leydig cells present long-term correlation and (ii) Markovian and fractal models, which describe well the dwell time distributions, are not adequate to describe the memory found in the kinetics of this channel.

  4. Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease

    PubMed Central

    Tao, Jie; Lan, Zhen; Wang, Yunman; Hei, Hongya; Tian, Lulu; Pan, Wanma; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca2+, possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus.

  5. Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease.

    PubMed

    Tao, Jie; Lan, Zhen; Wang, Yunman; Hei, Hongya; Tian, Lulu; Pan, Wanma; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca(2+), possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus. PMID:27445840

  6. Calcium-activated potassium channels mask vascular dysfunction associated with oxidized LDL exposure in rabbit aorta.

    PubMed

    Bocker, J M; Miller, F J; Oltman, C L; Chappell, D A; Gutterman, D D

    2001-05-01

    Endothelium-dependent vasodilation is impaired in atherosclerosis. Oxidized low density lipoprotein (ox-LDL) plays an important role, possibly through alterations in G-protein activation. We examined the effect of acute exposure to ox-LDL on the dilator responses of isolated rabbit aorta segments. We sought also to evaluate the specificity of this dysfunction for dilator stimuli that traditionally operate through a Gi-protein mechanism. Aortic segments were prepared for measurement of isometric tension. After contraction with prostaglandin F2alpha, relaxation to thrombin, adenosine diphosphate (ADP), or the endothelium-independent agonists, sodium nitroprusside (SNP) or papaverine was examined. Maximal relaxation to thrombin was impaired in the presence of ox-LDL (17.7+/-3.7% p<0.05) compared to control (no LDL) (52.6+/-4.0%). Ox-LDL did not affect maximal relaxation to ADP or SNP. However, in the presence of charybdotoxin (CHTX: calcium-activated potassium channel inhibitor) ox-LDL impaired relaxation to ADP (17.4+/-3.2%). CHTX did not affect control (no LDL) responses to ADP (69.6+/-5.0%) or relaxation to thrombin or papaverine. In conclusion, ox-LDL impairs relaxation to thrombin, but in the case of ADP, calcium-activated potassium channels compensate to maintain this relaxation. PMID:11605770

  7. Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates

    SciTech Connect

    Schallreuter, Karin U.; University of Bradford ). E-mail: K.Schallreuter@bradford.ac.uk; Gibbons, Nicholas C.J.; Elwary, Souna M.; Parkin, Susan M.; Wood, John M.

    2007-04-20

    The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5 x 10{sup -3}M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. {sup 45}Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H{sub 2}O{sub 2}-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8 m{sup 2} surface area with its calcium gradient in the 10{sup -3}M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.

  8. Blockade of swelling-induced chloride channels by phenol derivatives.

    PubMed Central

    Gschwentner, M.; Jungwirth, A.; Hofer, S.; Wöll, E.; Ritter, M.; Susanna, A.; Schmarda, A.; Reibnegger, G.; Pinggera, G. M.; Leitinger, M.; Frick, J.; Deetjen, P.; Paulmichl, M.

    1996-01-01

    1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein. PMID:8733574

  9. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

    PubMed Central

    Gründemann, Jan; Clark, Beverley A.

    2015-01-01

    Summary Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  10. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation.

    PubMed

    Gründemann, Jan; Clark, Beverley A

    2015-09-22

    Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  11. Properties of a calcium-activated K(+) current on interneurons in the developing rat hippocampus.

    PubMed

    Aoki, T; Baraban, S C

    2000-06-01

    Calcium-activated potassium currents have an essential role in regulating excitability in a variety of neurons. Although it is well established that mature CA1 pyramidal neurons possess a Ca(2+)-activated K(+) conductance (I(K(Ca))) with early and late components, modulation by various endogenous neurotransmitters, and sensitivity to K(+) channel toxins, the properties of I(K(Ca)) on hippocampal interneurons (or immature CA1 pyramidal neurons) are relatively unknown. To address this problem, whole-cell voltage-clamp recordings were made from visually identified interneurons in stratum lacunosum-moleculare (L-M) and CA1 pyramidal cells in hippocampal slices from immature rats (P3-P25). A biphasic calcium-activated K(+) tail current was elicited following a brief depolarization from the holding potential (-50 mV). Analysis of the kinetic properties of I(K(Ca)) suggests that an early current component differs between these two cell types. An early I(K(Ca)) with a large peak current amplitude (200.8 +/- 13.2 pA, mean +/- SE), slow time constant of decay (70.9 +/- 3.3 ms), and relatively rapid time to peak (within 15 ms) was observed on L-M interneurons (n = 88), whereas an early I(K(Ca)) with a small peak current amplitude (112.5 +/- 7.3 pA), a fast time constant of decay (39.4 +/- 1.6 ms), and a slower time-to-peak (within 26 ms) was observed on CA1 pyramidal neurons (n = 85). Removal of extracellular calcium or addition of inorganic Ca(2+) channel blockers (cadmium, nickel, or cobalt) was used to demonstrate the calcium dependence of these currents. Addition of norepinephrine, carbachol, and a variety of channel toxins (apamin, iberiotoxin, verruculogen, paxilline, penitrem A, and charybdotoxin) were used to further distinguish between I(K(Ca)) on these two hippocampal cell types. Verruculogen (100 nM), carbachol (100 microM), apamin (100 nM), TEA (1 mM), and iberiotoxin (50 nM) significantly reduced early I(K(Ca)) on CA1 pyramidal neurons; early I(K(Ca)) on L

  12. Diabetic state-induced activation of calcium-activated neutral proteinase in mouse skeletal muscle.

    PubMed

    Kobayashi, S; Fujihara, M; Hoshino, N; Kimura, I; Kimura, M

    1989-12-01

    The effect of a diabetic state in the diabetic KK-CAy mouse on calcium activated neutral proteinase (CANP) of hind-limb skeletal muscles was investigated. In the diabetic state, there was an increased sensitivity to activation of CANP by calcium (Ca). In addition, there was an enhancement of maximal activity of the enzyme. The effect was induced by secondary modification of the diabetic state, but not genetical factors. Several lines of evidence suggest that the CANP is responsible for 92 K dalton protein in diabetic skeletal muscles. Among the evidence are the following: a) The 92 K band in the diabetic muscles was lower than in the prediabetic mouse and restored by the addition of 2 mM EDTA and 2 mM EGTA. b) The band was reduced by increasing the Ca content and neutral pH in the non-diabetic normal muscles. c) E-64-C, a CANP inhibitor, restored the 92 K component reduced by the diabetic state. Since the band in denervated muscles was not changed by the Ca chelating agents, the reduction of the band in the diabetic muscles is related with musculotrophic factors, not diabetic neuropathy. These results suggest that diabetic amyotrophy may be regarded as a phenomenon linked to an increase in intracellular Ca ions and an increase in CANP activity. PMID:2561275

  13. A coarse-grained model to study calcium activation of the cardiac thin filament

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Schwartz, Steven

    2015-03-01

    Familial hypertrophic cardiomyopathy (FHC) is one of the most common heart disease caused by genetic mutations. Cardiac muscle contraction and relaxation involve regulation of crossbridge binding to the cardiac thin filament, which regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin (cTn) and tropomyosin (Tm). An atomistic model of cTn complex interacting with Tm has been studied by our group. A more realistic model requires the inclusion of the dynamics of actin filament, which is almost 6 times larger than cTn and Tm in terms of atom numbers, and extensive sampling of the model becomes very resource-demanding. By using physics-based protein united-residue force field, we introduce a coarse-grained model to study the calcium activation of the thin filament resulting from cTn's allosteric regulation of Tm dynamics on actin. The time scale is much longer than that of all-atom molecular dynamics simulation because of the reduction of the degrees of freedom. The coarse-grained model is a good template for studying cardiac thin filament mutations that cause FHC, and reduces the cost of computational resources.

  14. Chaotic model and memory in single calcium-activated potassium channel kinetics

    NASA Astrophysics Data System (ADS)

    Bandeira, Heliovânio T.; Barbosa, Catão T. F.; Campos De Oliveira, Regina A.; Aguiar, José F.; Nogueira, Romildo A.

    2008-09-01

    Ion channels are pores formed by proteins and responsible for carrying ion fluxes through cellular membranes. The ion channels can assume conformational states thereby controlling ion flow. Physically, the conformational transitions from one state to another are associated with energy barriers between them and are dependent on stimulus, such as, electrical field, ligands, second messengers, etc. Several models have been proposed to describe the kinetics of ion channels. The classical Markovian model assumes that a future transition is independent of the time that the ion channel stayed in a previous state. Others models as the fractal and the chaotic assume that the rate of transitions between the states depend on the time that the ionic channel stayed in a previous state. For the calcium activated potassium channels of Leydig cells the R/S Hurst analysis has indicated that the channels are long-term correlated with a Hurst coefficient H around 0.7, showing a persistent memory in this kinetic. Here, we applied the R /S analysis to the opening and closing dwell time series obtained from simulated data from a chaotic model proposed by L. Liebovitch and T. Tóth [J. Theor. Biol. 148, 243 (1991)] and we show that this chaotic model or any model that treats the set of channel openings and closings as independent events is inadequate to describe the long-term correlation (memory) already described for the experimental data.

  15. Correction of Chloride Transport and Mislocalization of CFTR Protein by Vardenafil in the Gastrointestinal Tract of Cystic Fibrosis Mice

    PubMed Central

    Dhooghe, Barbara; Noël, Sabrina; Bouzin, Caroline; Behets-Wydemans, Gaëtane; Leal, Teresinha

    2013-01-01

    Although lung disease is the major cause of mortality in cystic fibrosis (CF), gastrointestinal (GI) manifestations are the first hallmarks in 15–20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5) inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg) used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF

  16. Chloride in diet

    MedlinePlus

    ... found in table salt or sea salt as sodium chloride. It is also found in many vegetables. Foods ... Nutrition Board. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate. National Academy Press, Washington, DC: 2005. ...

  17. Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase: Characterization of the Dimer Interface

    SciTech Connect

    Yang,M.; Horii, K.; Herr, A.; Kirley, T.

    2006-01-01

    Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile{sup 170}, Ser{sup 172}, and Ser{sup 226} with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys{sup 30}, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

  18. Distribution of High-Conductance Calcium-Activated Potassium Channels in Rat Vestibular Epithelia

    PubMed Central

    Schweizer, Felix E.; Savin, David; Luu, Cindy; Sultemeier, David R.; Hoffman, Larry F.

    2011-01-01

    Voltage- and calcium-activated potassium channels (BK) are important regulators of neuronal excitability. BK channels seem to be crucial for frequency tuning in nonmammalian vestibular and auditory hair cells. However, there are a paucity of data concerning BK expression in mammalian vestibular hair cells. We therefore investigated the localization of BK channels in mammalian vestibular hair cells, specifically in rat vestibular neuroepithelia. We find that only a subset of hair cells in the utricle and the crista ampullaris express BK channels. BK-positive hair cells are located mainly in the medial striolar region of the utricle, where they constitute at most 12% of hair cells, and in the central zone of the horizontal crista. A majority of BK-positive hair cells are encapsulated by a calretinin-positive calyx defining them as type I cells. The remainder are either type I cells encapsulated by a calretinin-negative calyx or type II hair cells. Surprisingly, the number of BK-positive hair cells in the utricle peaks in juvenile rats and declines in early adulthood. BK channels were not found in vestibular afferent dendrites or somata. Our data indicate that BK channel expression in the mammalian vestibular system differs from the expression pattern in the mammalian auditory and the nonmammalian vestibular system. The molecular diversity of vestibular hair cells indicates a functional diversity that has not yet been fully characterized. The predominance of BK-positive hair cells within the medial striola of juvenile animals suggests that they contribute to a scheme of highly lateralized coding of linear head movements during late development. PMID:19731297

  19. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

    PubMed Central

    Castillo, Karen; Contreras, Gustavo F.; Pupo, Amaury; Torres, Yolima P.; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-01-01

    Being activated by depolarizing voltages and increases in cytoplasmic Ca2+, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

  20. Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP).

    PubMed

    Inomata, M; Hayashi, M; Nakamura, M; Imahori, K; Kawashima, S

    1985-08-01

    Some endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (muCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for muCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between muCANP and mCANP. In all cases, muCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs. The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the autodigestion. The large subunit of muCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested muCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact muCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of muCANP. No such transition was observed for mCANP. PMID:2999095

  1. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

  2. 17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder

    PubMed Central

    2016-01-01

    Purpose: To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively. Methods: Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 μg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements. Results: Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment. Conclusions: These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression. PMID:27032553

  3. Melaminium chloride hemihydrate.

    PubMed

    Janczak, J; Perpétuo, G J

    2001-09-01

    The crystals of a new melaminium salt, 2,4,6-triamino-1,3,5-triazin-1-ium chloride hemihydrate, C(3)H(7)N(6)(+).Cl(-).0.5H(2)O, are built up from single-protonated melaminium residues, chloride anions and water molecules. The protonated melaminium cations lie on a twofold axis, while the chloride anions and water molecule lie on the m plane. The melaminium residues are interconnected by N-H...N hydrogen bonds, forming chains parallel to the (001) plane. The chains of melaminium residues form a three-dimensional network through hydrogen-bond interactions with chloride anions and water molecules. PMID:11588391

  4. Calcium-activated inward spike after-currents in bursting neurone R15 of Aplysia.

    PubMed Central

    Lewis, D V

    1988-01-01

    after-currents seemed dependent upon calcium influx, they were not suppressed by depolarizing commands whose voltage exceeded the calcium equilibrium potential at the soma as indicated by suppression of the calcium-activated potassium current, or IK(Ca), observed during the depolarization. However, if the extracellular calcium was lowered to 1 mM, large depolarizations did suppress the DAC. 7. Dopamine blocked the after-currents when applied to the axo-dendritic area but not when applied to the soma. Similarly, synaptic inhibition of long duration blocked the after-currents.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2457678

  5. Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

    PubMed

    Amjad, Asma; Hernandez-Clavijo, Andres; Pifferi, Simone; Maurya, Devendra Kumar; Boccaccio, Anna; Franzot, Jessica; Rock, Jason; Menini, Anna

    2015-04-01

    Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca(2+) concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca(2+) in isolated mouse vomeronasal sensory neurons in the absence of intracellular K(+). In whole-cell recordings, the average current in 1.5 µM Ca(2+) and symmetrical Cl(-) was -382 pA at -100 mV. Ion substitution experiments and partial blockade by commonly used Cl(-) channel blockers indicated that Ca(2+) activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca(2+)-activated Cl(-) channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca(2+)-activated Cl(-) channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca(2+)-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca(2+)-activated Cl(-) currents in mouse vomeronasal sensory neurons. PMID:25779870

  6. Phosphonium chloride for thermal storage

    NASA Technical Reports Server (NTRS)

    Sutton, J. G.; Heimlich, P. F.; Tepper, E. H.

    1972-01-01

    Development of systems for storage of thermal energy is discussed. Application of phosphonium chloride for heat storage through reversible dissociation is described. Chemical, physical, and thermodynamic properties of phosphonium chloride are analyzed and dangers in using phosphonium chloride are explained.

  7. Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

    PubMed

    Yuhas, W A; Fuchs, P A

    1999-11-01

    Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell. PMID:10573868

  8. Chloride flux in phagocytes.

    PubMed

    Wang, Guoshun

    2016-09-01

    Phagocytes, such as neutrophils and macrophages, engulf microbes into phagosomes and launch chemical attacks to kill and degrade them. Such a critical innate immune function necessitates ion participation. Chloride, the most abundant anion in the human body, is an indispensable constituent of the myeloperoxidase (MPO)-H2 O2 -halide system that produces the potent microbicide hypochlorous acid (HOCl). It also serves as a balancing ion to set membrane potentials, optimize cytosolic and phagosomal pH, and regulate phagosomal enzymatic activities. Deficient supply of this anion to or defective attainment of this anion by phagocytes is linked to innate immune defects. However, how phagocytes acquire chloride from their residing environment especially when they are deployed to epithelium-lined lumens, and how chloride is intracellularly transported to phagosomes remain largely unknown. This review article will provide an overview of chloride protein carriers, potential mechanisms for phagocytic chloride preservation and acquisition, intracellular chloride supply to phagosomes for oxidant production, and methods to measure chloride levels in phagocytes and their phagosomes. PMID:27558337

  9. Mercuric chloride poisoning

    MedlinePlus

    ... Mercuric chloride is a very poisonous form of mercury. It is a type of mercury salt. There are different types of mercury poisonings . This article discusses poisoning from swallowing mercuric ...

  10. Hydrogen chloride test set

    NASA Technical Reports Server (NTRS)

    Workman, G. L.

    1976-01-01

    Detector uses tertiary amine, which makes reaction fairly specific for relatively small highly polarized hydrogen chloride molecule. Reaction is monitored by any microbalance capable of measuring extremely small mass differences in real time.

  11. Strontium-89 Chloride

    MedlinePlus

    ... ask your doctor or pharmacist for more information.Strontium-89 chloride is in a class of drugs known as radioisotopes. It delivers radiation to cancer sites and ultimately decreases bone pain. The length of treatment depends on the ...

  12. Increasing O-GlcNAcylation Level on Organ Culture of Soleus Modulates the Calcium Activation Parameters of Muscle Fibers

    PubMed Central

    Cieniewski-Bernard, Caroline; Montel, Valerie; Berthoin, Serge; Bastide, Bruno

    2012-01-01

    O-N-acetylglucosaminylation is a reversible post-translational modification which presents a dynamic and highly regulated interplay with phosphorylation. New insights suggest that O-GlcNAcylation might be involved in striated muscle physiology, in particular in contractile properties such as the calcium activation parameters. By the inhibition of O-GlcNAcase, we investigated the effect of the increase of soleus O-GlcNAcylation level on the contractile properties by establishing T/pCa relationships. We increased the O-GlcNAcylation level on soleus biopsies performing an organ culture of soleus treated or not with PUGNAc or Thiamet-G, two O-GlcNAcase inhibitors. The enhancement of O-GlcNAcylation pattern was associated with an increase of calcium affinity on slow soleus skinned fibers. Analysis of the glycoproteins pattern showed that this effect is solely due to O-GlcNAcylation of proteins extracted from skinned biopsies. We also characterized the O-GlcNAcylated contractile proteins using a proteomic approach, and identified among others troponin T and I as being O-GlcNAc modified. We quantified the variation of O-GlcNAc level on all these identified proteins, and showed that several regulatory contractile proteins, predominantly fast isoforms, presented a drastic increase in their O-GlcNAc level. Since the only slow isoform of contractile protein presenting an increase of O-GlcNAc level was MLC2, the effect of enhanced O-GlcNAcylation pattern on calcium activation parameters could involve the O-GlcNAcylation of sMLC2, without excluding that an unidentified O-GlcNAc proteins, such as TnC, could be potentially involved in this mechanism. All these data strongly linked O-GlcNAcylation to the modulation of contractile activity of skeletal muscle. PMID:23110217

  13. Large-conductance calcium-activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains.

    PubMed

    Kaufmann, Walter A; Ferraguti, Francesco; Fukazawa, Yugo; Kasugai, Yu; Shigemoto, Ryuichi; Laake, Petter; Sexton, Joseph A; Ruth, Peter; Wietzorrek, Georg; Knaus, Hans-Günther; Storm, Johan F; Ottersen, Ole Petter

    2009-07-10

    Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells. PMID:19412945

  14. Chloride removal from vitrification offgas

    SciTech Connect

    Slaathaug, E.J.

    1995-06-01

    This study identified and investigated techniques of selectively purging chlorides from the low-level waste (LLW) vitrification process with the purge stream acceptable for burial on the Hanford Site. Chlorides will be present in high concentration in several individual feeds to the LLW Vitrification Plant. The chlorides are highly volatile in combustion type melters and are readily absorbed by wet scrubbing of the melter offgas. The Tank Waste Remediation System (TWRS) process flow sheets show that the resulting chloride rich scrub solution is recycled back to the melter. The chlorides must be purged from the recycle loop to prevent the buildup of excessively high chloride concentrations.

  15. Strontium-89 Chloride

    MedlinePlus

    ... doctor if you have or have ever had bone marrow disease, blood disorders, or kidney disease.you should know that strontium-89 chloride may interfere with the normal menstrual cycle (period) in women and may stop sperm production in men. However, ...

  16. Lithium thionyl chloride battery

    SciTech Connect

    Saathoff, D.J.; Venkatasetty, H.V.

    1982-10-19

    The discharge rate and internal conductivity of electrochemical cell including a lithium anode, and a cathode and an electrolyte including LiAlCl4 and SOC2 is improved by the addition of an amount of a mixture containing AlCl3 and butyl pyridinium chloride.

  17. PHOTOOXIDATION OF ALLYL CHLORIDE

    EPA Science Inventory

    The photooxidation of allyl chloride was studied by irradiation either in 100-L Teflon bags or in a 22.7-cu m Teflon smog chamber in the presence of added NOx. In the absence of added hydrocarbons, the reaction involves a Cl atom chain, which leads to a highly reactive system. A ...

  18. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  19. Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture.

    PubMed

    Enomoto, K; Furuya, K; Maeno, T; Edwards, C; Oka, T

    1991-01-01

    Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2(+)-gated K+ current. The characteristics of the Ca2(+)-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 microM of the intracellular Ca2+, but it was independent of the membrane potential. Charybdotoxin reduced the activity of the Ca2(+)-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2(+)-activated K+ channels. PMID:1710671

  20. Calcium activated K⁺ channels in the electroreceptor of the skate confirmed by cloning. Details of subunits and splicing.

    PubMed

    King, Benjamin L; Shi, Ling Fang; Kao, Peter; Clusin, William T

    2016-03-01

    Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K(+) channels, first described in 1974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel α isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intra-cellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1α expressed in the skate. One of the β subunits (β4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues. PMID:26687710

  1. The calcium-activated potassium channel KCa3.1 is an important modulator of hepatic injury.

    PubMed

    Sevelsted Møller, Linda; Fialla, Annette Dam; Schierwagen, Robert; Biagini, Matteo; Liedtke, Christian; Laleman, Wim; Klein, Sabine; Reul, Winfried; Koch Hansen, Lars; Rabjerg, Maj; Singh, Vikrant; Surra, Joaquin; Osada, Jesus; Reinehr, Roland; de Muckadell, Ove B Schaffalitzky; Köhler, Ralf; Trebicka, Jonel

    2016-01-01

    The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury. PMID:27354175

  2. The calcium-activated potassium channel KCa3.1 is an important modulator of hepatic injury

    PubMed Central

    Sevelsted Møller, Linda; Fialla, Annette Dam; Schierwagen, Robert; Biagini, Matteo; Liedtke, Christian; Laleman, Wim; Klein, Sabine; Reul, Winfried; Koch Hansen, Lars; Rabjerg, Maj; Singh, Vikrant; Surra, Joaquin; Osada, Jesus; Reinehr, Roland; de Muckadell, Ove B. Schaffalitzky; Köhler, Ralf; Trebicka, Jonel

    2016-01-01

    The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury. PMID:27354175

  3. [Degradation of succinylcholine chloride].

    PubMed

    Németh, G; Török, I; Paál, T

    1993-05-01

    Quantitative thin-layer chormatographic method has been developed for the investigation of the degradation of injection formulations containing succinylcholinium chloride. The method is based on the denistometric determination of the main degradation product, choline at 430 nm after visualization with iodine vapour. The stability of the injection was investigated under various storage conditions and it has been stated that considerable decomposition takes place during as short a period as one week. PMID:8362654

  4. Chloride channels as drug targets

    PubMed Central

    Verkman, Alan S.; Galietta, Luis J. V.

    2013-01-01

    Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABAA (γ-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery. PMID:19153558

  5. Modulation of Chloride Channel Functions by the Plant Lignan Compounds Kobusin and Eudesmin

    PubMed Central

    Jiang, Yu; Yu, Bo; Fang, Fang; Cao, Huanhuan; Ma, Tonghui; Yang, Hong

    2015-01-01

    Plant lignans are diphenolic compounds widely present in vegetables, fruits, and grains. These compounds have been demonstrated to have protective effect against cancer, hypertension and diabetes. In the present study, we showed that two lignan compounds, kobusin and eudesmin, isolated from Magnoliae Flos, could modulate intestinal chloride transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs). The compounds activated CFTR channel function in both FRT cells and in HT-29 cells. The modulating effects of kobusin and eudesmin on the activity of CaCCgie (CaCC expressed in gastrointestinal epithelial cells) were also investigated, and the result showed that both compounds could stimulate CaCCgie-mediated short-circuit currents and the stimulation was synergistic with ATP. In ex vivo studies, both compounds activated CFTR and CaCCgie chloride channel activities in mouse colonic epithelia. Remarkably, the compounds showed inhibitory effects toward ANO1/CaCC-mediated short-circuit currents in ANO1/CaCC-expressing FRT cells, with IC50 values of 100 μM for kobusin and 200 μM for eudesmin. In charcoal transit study, both compounds mildly reduced gastrointestinal motility in mice. Taken together, these results revealed a new kind of activity displayed by the lignan compounds, one that is concerned with the modulation of chloride channel function. PMID:26635857

  6. External action of di- and polyamines on maxi calcium-activated potassium channels: an electrophysiological and molecular modeling study.

    PubMed Central

    Weiger, T M; Langer, T; Hermann, A

    1998-01-01

    In this study we compared polyamines to various diamines, and we modeled flexibility as well as hydrophobicity properties of these molecules to examine possible structural differences that could explain their external effects on the channels. The natural polyamines (putrescine, cadaverine, spermidine, spermine) and diamines increasing in CH2 chain length from C2 to C12 were used to probe maxi calcium-activated potassium (BK) channels in GH3 pituitary tumor cells when applied extracellularly. In single-channel recordings we found polyamines as well as diamines up to 1,10-diaminodecane to be ineffective in altering channel current amplitudes or kinetics. In contrast, 1,12-diamino dodecane (1,12-DD) was found to be a reversible blocker, with a blocking site at an electrical distance (z delta) of 0.72 within the channel. It reduced single-channel current amplitude, mean channel open time, and channel open probability. In computer simulations structural data, such as flexibility, hydration, and log D values, were calculated. 1,12-DD showed the largest flexibility of all diamines (minimum N-N distance 9.9 A) combined with a marked hydrophobicity due to a 4-5 A hydrophobic intersegment between hydrophilic ends in the molecule, as confirmed by GRID water probe maps and a log D value of -1.82 at pH 7.2. We propose that the amount of hydration of the molecule, more than its flexibility, constitutes an essential parameter for its ability to act as a channel blocker. PMID:9533685

  7. Calcium-activated afterhyperpolarizations regulate synchronization and timing of epileptiform bursts in hippocampal CA3 pyramidal neurons.

    PubMed

    Fernández de Sevilla, David; Garduño, Julieta; Galván, Emilio; Buño, Washington

    2006-12-01

    Calcium-activated potassium conductances regulate neuronal excitability, but their role in epileptogenesis remains elusive. We investigated in rat CA3 pyramidal neurons the contribution of the Ca(2+)-activated K(+)-mediated afterhyperpolarizations (AHPs) in the genesis and regulation of epileptiform activity induced in vitro by 4-aminopyridine (4-AP) in Mg(2+)-free Ringer. Recurring spike bursts terminated by prolonged AHPs were generated. Burst synchronization between CA3 pyramidal neurons in paired recordings typified this interictal-like activity. A downregulation of the medium afterhyperpolarization (mAHP) paralleled the emergence of the interictal-like activity. When the mAHP was reduced or enhanced by apamin and EBIO bursts induced by 4-AP were increased or blocked, respectively. Inhibition of the slow afterhyperpolarization (sAHP) with carbachol, t-ACPD, or isoproterenol increased bursting frequency and disrupted burst regularity and synchronization between pyramidal neuron pairs. In contrast, enhancing the sAHP by intracellular dialysis with KMeSO(4) reduced burst frequency. Block of GABA(A-B) inhibitions did not modify the abnormal activity. We describe novel cellular mechanisms where 1) the inhibition of the mAHP plays an essential role in the genesis and regulation of the bursting activity by reducing negative feedback, 2) the sAHP sets the interburst interval by decreasing excitability, and 3) bursting was synchronized by excitatory synaptic interactions that increased in advance and during bursts and decreased throughout the subsequent sAHP. These cellular mechanisms are active in the CA3 region, where epileptiform activity is initiated, and cooperatively regulate the timing of the synchronized rhythmic interictal-like network activity. PMID:16971683

  8. The large-conductance calcium-activated potassium channel holds the key to the conundrum of familial hypokalemic periodic paralysis

    PubMed Central

    Kim, Sung-Jo; Kang, Sun-Yang; Yi, Jin Woong; Kim, Seung-Min

    2014-01-01

    Purpose Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant channelopathy characterized by episodic attacks of muscle weakness and hypokalemia. Mutations in the calcium channel gene, CACNA1S, or the sodium channel gene, SCN4A, have been found to be responsible for HOKPP; however, the mechanism that causes hypokalemia remains to be determined. The aim of this study was to improve the understanding of this mechanism by investigating the expression of calcium-activated potassium (KCa) channel genes in HOKPP patients. Methods We measured the intracellular calcium concentration with fura-2-acetoxymethyl ester in skeletal muscle cells of HOKPP patients and healthy individuals. We examined the mRNA and protein expression of KCa channel genes (KCNMA1, KCNN1, KCNN2, KCNN3, and KCNN4) in both cell types. Results Patient cells exhibited higher cytosolic calcium levels than normal cells. Quantitative reverse transcription polymerase chain reaction analysis showed that the mRNA levels of the KCa channel genes did not significantly differ between patient and normal cells. However, western blot analysis showed that protein levels of the KCNMA1 gene, which encodes KCa1.1 channels (also called big potassium channels), were significantly lower in the membrane fraction and higher in the cytosolic fraction of patient cells than normal cells. When patient cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the altered subcellular distribution of BK channels remained unchanged. Conclusion These findings suggest a novel mechanism for the development of hypokalemia and paralysis in HOKPP and demonstrate a connection between disease-associated mutations in calcium/sodium channels and pathogenic changes in nonmutant potassium channels. PMID:25379045

  9. CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

    PubMed

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

    2016-08-01

    Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons. PMID:27097551

  10. Oxomemazine hydro-chloride.

    PubMed

    Siddegowda, M S; Butcher, Ray J; Akkurt, Mehmet; Yathirajan, H S; Ramesh, A R

    2011-08-01

    IN THE TITLE COMPOUND [SYSTEMATIC NAME: 3-(5,5-dioxo-phen-othia-zin-10-yl)-N,N,2-trimethyl-propanaminium chloride], C(18)H(23)N(2)O(2)S(+)·Cl(-), the dihedral angle between the two outer aromatic rings of the phenothia-zine unit is 30.5 (2)°. In the crystal, the components are linked by N-H⋯Cl and C-H⋯Cl hydrogen bonds and C-H⋯π inter-actions. PMID:22090928

  11. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ferric chloride. 184.1297 Section 184.1297 Food... GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III) chloride, FeC13, CAS Reg. No. 7705-08-0) may be prepared from iron and chlorine or from ferric oxide and hydrogen chloride. The...

  12. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ferric chloride. 184.1297 Section 184.1297 Food... Specific Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III) chloride... hydrogen chloride. The pure material occurs as hydroscopic, hexagonal, dark crystals. Ferric...

  13. 21 CFR 184.1297 - Ferric chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ferric chloride. 184.1297 Section 184.1297 Food... Specific Substances Affirmed as GRAS § 184.1297 Ferric chloride. (a) Ferric chloride (iron (III) chloride... hydrogen chloride. The pure material occurs as hydroscopic, hexagonal, dark crystals. Ferric...

  14. Benzalkonium Chloride and Glaucoma

    PubMed Central

    Kaufman, Paul L.; Kiland, Julie A.

    2014-01-01

    Abstract Glaucoma patients routinely take multiple medications, with multiple daily doses, for years or even decades. Benzalkonium chloride (BAK) is the most common preservative in glaucoma medications. BAK has been detected in the trabecular meshwork (TM), corneal endothelium, lens, and retina after topical drop installation and may accumulate in those tissues. There is evidence that BAK causes corneal and conjunctival toxicity, including cell loss, disruption of tight junctions, apoptosis and preapoptosis, cytoskeleton changes, and immunoinflammatory reactions. These same effects have been reported in cultured human TM cells exposed to concentrations of BAK found in common glaucoma drugs and in the TM of primary open-angle glaucoma donor eyes. It is possible that a relationship exists between chronic exposure to BAK and glaucoma. The hypothesis that BAK causes/worsens glaucoma is being tested experimentally in an animal model that closely reflects human physiology. PMID:24205938

  15. A Synthetic Chloride Channel Relaxes Airway Smooth Muscle of the Rat

    PubMed Central

    Yau, Kwok-hei; Mak, Judith Choi-wo; Leung, Susan Wai-sum; Yang, Dan; Vanhoutte, Paul M.

    2012-01-01

    Synthetic ion channels may have potential therapeutic applications, provided they possess appropriate biological activities. The present study was designed to examine the ability of small molecule-based synthetic Cl– channels to modulate airway smooth muscle responsiveness. Changes in isometric tension were measured in rat tracheal rings. Relaxations to the synthetic chloride channel SCC-1 were obtained during sustained contractions to KCl. The anion dependency of the effect of SCC-1 was evaluated by ion substitution experiments. The sensitivity to conventional Cl– transport inhibitors was also tested. SCC-1 caused concentration-dependent relaxations during sustained contractions to potassium chloride. This relaxing effect was dependent on the presence of extracellular Cl– and HCO3−. It was insensitive to conventional Cl– channels/transport inhibitors that blocked the cystic fibrosis transmembrane conductance regulator and calcium-activated Cl– channels. SCC-1 did not inhibit contractions induced by carbachol, endothelin-1, 5-hydroxytryptamine or the calcium ionophore A23187. SCC-1 relaxes airway smooth muscle during contractions evoked by depolarizing solutions. The Cl– conductance conferred by this synthetic compound is distinct from the endogenous transport systems for chloride anions. PMID:23049786

  16. Reactor-chromatographic determination of vinyl chloride in polyvinyl chloride

    SciTech Connect

    Berezkin, V.G.

    1986-08-01

    The authors carry out a chromatographic study of the volatile products that evolve when various grades of domestic polyvinyl chloride are heated, to determine the concentration of residual monomer. To find vinyl chloride in complex mixtures of air pollutants the authors used sorptive reaction concentration of impurities. This new combination of methods is based on preliminary separation at the sampling stage of impurities that interfere in the analysis, followed by concentration of the desired components in a trap with an adsorbent, and chromatographic determination of the concentrated trace materials. The method obtains low vinyl chloride concentrations (down to 10/sup -4/-10/sup -5/ wt. %) with +/-5 relative error.

  17. Role of calcium-activated potassium channels in the regulation of basal and agonist-elevated tones in isolated conduit arteries. Short communication.

    PubMed

    Pataricza, J; Márton, Z; Hegedus, Z; Krassói, Irén; Kun, A; Varró, A; Papp, J Gy

    2004-01-01

    Functional role of calcium-activated potassium (KCa) channels on the basal and agonist-elevated arterial tones was investigated in isolated rabbit aorta, porcine and canine coronary arteries as well as in human internal mammary artery. The vascular tones enhanced by contractile agents were increased further by preincubation of these conduit blood vessels with selective (charybdotoxin or iberiotoxin) or nonselective (tetraethylammonium) inhibitors of KCa channels. The basal tone (without an agonist) was increased only in the canine coronary artery. The results indicate a feed-back regulatory role of KCa channels counteracting the vasospasm of conduit arteries. PMID:16438119

  18. Studies Update Vinyl Chloride Hazards.

    ERIC Educational Resources Information Center

    Rawls, Rebecca

    1980-01-01

    Extensive study affirms that vinyl chloride is a potent animal carcinogen. Epidemiological studies show elevated rates of human cancers in association with extended contact with the compound. (Author/RE)

  19. An XAFS study of nickel chloride in the ionic liquid 1-ethyl-3-methyl imidazolium chloride/ aluminum chloride

    SciTech Connect

    D Roeper; G Cheek; K Pandya; W OGrady

    2011-12-31

    Nickel chloride was studied with cyclic voltammetry and X-ray absorption spectroscopy in acidic and basic aluminum chloride/1-ethyl-3-methyl imidazolium chloride (EMIC) ionic liquids. Acidic melts display metal stripping peaks which are not observed in the basic melt. EXAFS analysis shows that the nickel is tetrahedrally coordinated with chloride ions in the basic solution. In the acidic solution the nickel is coordinated by six chloride ions that are also associated with aluminum ions.

  20. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... GRAS § 184.1138 Ammonium chloride. (a) Ammonium chloride (NH4Cl, CAS Reg. No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The less soluble sodium salt... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Ammonium chloride. 184.1138 Section 184.1138...

  1. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  2. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  3. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  4. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg. No. 7447-40-7) is a white... manufacturing practice. Potassium chloride may be used in infant formula in accordance with section 412(g)...

  5. 21 CFR 184.1622 - Potassium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium chloride. 184.1622 Section 184.1622 Food... Specific Substances Affirmed as GRAS § 184.1622 Potassium chloride. (a) Potassium chloride (KCl, CAS Reg... levels not to exceed current good manufacturing practice. Potassium chloride may be used in...

  6. Regeneration of zinc chloride hydrocracking catalyst

    DOEpatents

    Zielke, Clyde W.

    1979-01-01

    Improved rate of recovery of zinc values from the solids which are carried over by the effluent vapors from the oxidative vapor phase regeneration of spent zinc chloride catalyst is achieved by treatment of the solids with both hydrogen chloride and calcium chloride to selectively and rapidly recover the zinc values as zinc chloride.

  7. CHLORIDE WASHER PERFORMACE TESTING

    SciTech Connect

    Coughlin, J; David Best, D; Robert Pierce, R

    2007-11-30

    Testing was performed to determine the chloride (Cl-) removal capabilities of the Savannah River National Laboratory (SRNL) designed and built Cl- washing equipment intended for HB-Line installation. The equipment to be deployed was tested using a cerium oxide (CeO2) based simulant in place of the 3013 plutonium oxide (PuO2) material. Two different simulant mixtures were included in this testing -- one having higher Cl- content than the other. The higher Cl- simulant was based on K-Area Interim Surveillance Inspection Program (KIS) material with Cl- content approximately equal to 70,000 ppm. The lower Cl- level simulant was comparable to KIS material containing approximately 8,000-ppm Cl- content. The performance testing results indicate that the washer is capable of reducing the Cl- content of both surrogates to below 200 ppm with three 1/2-liter washes of 0.1M sodium hydroxide (NaOH) solution. Larger wash volumes were used with similar results - all of the prescribed test parameters consistently reduced the Cl- content of the surrogate to a value below 200 ppm Cl- in the final washed surrogate material. The washer uses a 20-micron filter to retain the surrogate solids. Tests showed that 0.16-0.41% of the insoluble fraction of the starting mass passed through the 20-micron filter. The solids retention performance indicates that the fissile masses passing through the 20-micron filter should not exceed the waste acceptance criteria for discard in grout to TRU waste. It is recommended that additional testing be pursued for further verification and optimization purposes. It is likely that wash volumes smaller than those tested could still reduce the Cl- values to acceptable levels. Along with reduced wash volumes, reuse of the third wash volume (in the next run processed) should be tested as a wash solution minimization plan. A 67% reduction in the number of grouted paint pails could be realized if wash solution minimization testing returned acceptable results.

  8. Chloride in vesicular trafficking and function.

    PubMed

    Stauber, Tobias; Jentsch, Thomas J

    2013-01-01

    Luminal acidification is of pivotal importance for the physiology of the secretory and endocytic pathways and its diverse trafficking events. Acidification by the proton-pumping V-ATPase requires charge compensation by counterion currents that are commonly attributed to chloride. The molecular identification of intracellular chloride transporters and the improvement of methodologies for measuring intraorganellar pH and chloride have facilitated the investigation of the physiology of vesicular chloride transport. New data question the requirement of chloride for pH regulation of various organelles and furthermore ascribe functions to chloride that are beyond merely electrically shunting the proton pump. This review surveys the currently established and proposed intracellular chloride transporters and gives an overview of membrane-trafficking steps that are affected by the perturbation of chloride transport. Finally, potential mechanisms of membrane-trafficking modulation by chloride are discussed and put into the context of organellar ion homeostasis in general. PMID:23092411

  9. Stability of succinylcholine chloride injection.

    PubMed

    Schmutz, C W; Mühlebach, S F

    1991-03-01

    The stability of succinylcholine chloride injection prepared by a hospital pharmacy was studied under a wide variety of conditions. Batches of succinylcholine chloride injection 10 mg/mL containing sodium chloride, methyl-4-hydroxybenzoate, hydrochloric acid, and water were prepared. Samples were tested for the effect of initial pH (3.0 and 4.2) and sterilization (steam treatment at 100 degrees C for 30 minutes and 121 degrees C for 20 minutes) on stability after three weeks; long-term stability under refrigeration (12, 17, and 23 months of storage at 4 degrees C); and the effect of storage temperature (4-6 degrees C, 20-26 degrees C, 35 degrees C, and 70 degrees C) and light exposure at various intervals up to 12 months. Samples were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Unlike heating at 121 degrees C, heating at 100 degrees C produced no significant loss of succinylcholine chloride, independent of the initial pH. Succinylcholine chloride was hydrolyzed only minimally over 23 months if the solution was stored at 4-6 degrees C. A 10% loss of drug content occurred if solutions were kept at 20-26 degrees C for five months, at 35 degrees C for one month, or at 70 degrees C for one day. Initial degradation was slowed if the solution was protected from light. The assessments by TLC proved to be more sensitive than the HPLC measurements. Succinylcholine chloride injection sterilized at 100 degrees C for 30 minutes can be stored for up to five months at room temperature if protected from light. The preparation is stable for at least two years under refrigeration. PMID:2028996

  10. Calcium-activated non-selective cation currents are involved in generation of tonic and bursting activity in dopamine neurons of the substantia nigra pars compacta.

    PubMed

    Mrejeru, Ana; Wei, Aguan; Ramirez, Jan Marino

    2011-05-15

    Nigral dopamine neurons are transiently activated by high frequency glutamatergic inputs relaying reward-predicting sensory information. The tonic firing pattern of dopamine cells responds to these inputs with a transient burst of spikes that requires NMDA receptors. Here, we show that NMDA receptor activation further excites the cell by recruiting a calcium-activated non-selective cation current (ICAN) capable of generating a plateau potential. Burst firing in vitro is eliminated after blockade of ICAN with flufenamic acid, 9-phenanthrol, or intracellular BAPTA. ICAN is likely to be mediated by a transient receptor potential (TRP) channel, and RT-PCR was used to confirm expression of TRPM2 and TRPM4mRNA in substantia nigra pars compacta.We propose that ICAN is selectively activated during burst firing to boost NMDA currents and allow plateau potentials. This boost mechanism may render DA cells vulnerable to excitotoxicity. PMID:21486760

  11. Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle.

    PubMed

    Pringle, T D; Harrelson, J M; West, R L; Williams, S E; Johnson, D D

    1999-12-01

    Steers of known percentage Brahman (B) and Angus (A) breeding (100% A, n = 6; F1 B x A, n = 6; and 100% B, n = 6) were used to determine the effect of calcium chloride injection on the calpain proteinase system and meat tenderness. The steers were slaughtered in six replications (at either 9 or 14 mm of backfat, determined ultrasonically), with each breed type represented. Calpains and calpastatin activities were measured on fresh, prerigor longissimus muscle samples. Carcass data were collected after a 24-h chill, and the short loin (IMPS #180), top sirloin (IMPS #184), and top round (IMPS #168) were removed from both sides of each carcass. The cuts from the right side were then injected at 5% (wt/wt) with CaCl2 solution (2.2%). Longissimus muscle calpain and calpastatin activities were also measured at 48 h postmortem from the injected and control sides of each carcass. Warner-Bratzler shear force was measured on steaks from the three subprimals aged 1, 2, 5, 15, or 31 d. Marbling scores and USDA quality grades were higher (P<.05) in A than in F1 B x A and B carcasses. Calpastatin activity was higher (P<.05) in muscle from B than in muscle from A and F1 B x A steers, and postmortem storage (O vs 48 h) and CaCl2 injection reduced (P<.05) the activity of the calpains and calpastatin. Strip loin and top sirloin steaks from A and F1 B x A steers were more tender (P<.05) than steaks from B steers; however, top round steak tenderness did not differ (P>.05) across breed type. Calcium injection improved strip loin and top sirloin steak tenderness, but it did not affect top round steak tenderness. Collectively, these data show that CaC12 injection can be used to improve meat tenderness, with similar responses shown in cattle containing 0, 50, and 100% B inheritance. However, even with CaCl2 injection, B steaks are less tender than their A and F1 B x A counterparts. PMID:10641869

  12. Disruption of vascular Ca2+-activated chloride currents lowers blood pressure

    PubMed Central

    Heinze, Christoph; Seniuk, Anika; Sokolov, Maxim V.; Huebner, Antje K.; Klementowicz, Agnieszka E.; Szijártó, István A.; Schleifenbaum, Johanna; Vitzthum, Helga; Gollasch, Maik; Ehmke, Heimo; Schroeder, Björn C.; Hübner, Christian A.

    2014-01-01

    High blood pressure is the leading risk factor for death worldwide. One of the hallmarks is a rise of peripheral vascular resistance, which largely depends on arteriole tone. Ca2+-activated chloride currents (CaCCs) in vascular smooth muscle cells (VSMCs) are candidates for increasing vascular contractility. We analyzed the vascular tree and identified substantial CaCCs in VSMCs of the aorta and carotid arteries. CaCCs were small or absent in VSMCs of medium-sized vessels such as mesenteric arteries and larger retinal arterioles. In small vessels of the retina, brain, and skeletal muscle, where contractile intermediate cells or pericytes gradually replace VSMCs, CaCCs were particularly large. Targeted disruption of the calcium-activated chloride channel TMEM16A, also known as ANO1, in VSMCs, intermediate cells, and pericytes eliminated CaCCs in all vessels studied. Mice lacking vascular TMEM16A had lower systemic blood pressure and a decreased hypertensive response following vasoconstrictor treatment. There was no difference in contractility of medium-sized mesenteric arteries; however, responsiveness of the aorta and small retinal arterioles to the vasoconstriction-inducing drug U46619 was reduced. TMEM16A also was required for peripheral blood vessel contractility, as the response to U46619 was attenuated in isolated perfused hind limbs from mutant mice. Out data suggest that TMEM16A plays a general role in arteriolar and capillary blood flow and is a promising target for the treatment of hypertension. PMID:24401273

  13. Laser using lead chloride vapor

    NASA Technical Reports Server (NTRS)

    Chen, C. J.

    1975-01-01

    By applying electric discharge, lead chloride vapor in tube is dissociated into lead and chlorine atoms. Population inversion of lead atoms is attained subsequently by second discharge, before chemical recombination of lead and chlorine has occurred. Optimum time interval between two discharges is required for maximum laser output.

  14. CHEMILUMINESCENT MONITOR FOR VINYL CHLORIDE

    EPA Science Inventory

    A monitor for vinyl chloride monomer (VCM) in ambient air was constructed using commercially available components of a gas chromatograph (GC) coupled with a chemiluminescence ozone analyzer slightly modified to make it suitable for use as a GC detector. The specificity for VCM is...

  15. 21 CFR 582.5446 - Manganese chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5446 Manganese chloride. (a) Product. Manganese chloride. (b) Conditions of use....

  16. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Specifications for Dairy Plants Approved for USDA Inspection and Grading Service 1 Quality Specifications for Raw Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  17. Method for the abatement of hydrogen chloride

    DOEpatents

    Winston, S.J.; Thomas, T.R.

    1975-11-14

    A method is described for reducing the amount of hydrogen chloride contained in a gas stream by reacting the hydrogen chloride with ammonia in the gas phase so as to produce ammonium chloride. The combined gas stream is passed into a condensation and collection vessel, and a cyclonic flow is created in the combined gas stream as it passes through the vessel. The temperature of the gas stream is reduced in the vessel to below the condensation temperature of ammonium chloride in order to crystallize the ammonium chloride on the walls of the vessel. The cyclonic flow creates a turbulence which breaks off the larger particles of ammonium chloride which are, in turn, driven to the bottom of the vessel where the solid ammonium chloride can be removed from the vessel. The gas stream exiting from the condensation and collection vessel is further cleaned and additional ammonium chloride is removed by passing through additional filters.

  18. Method for the abatement of hydrogen chloride

    DOEpatents

    Winston, Steven J.; Thomas, Thomas R.

    1977-01-01

    The present invention provides a method for reducing the amount of hydrogen chloride contained in a gas stream by reacting the hydrogen chloride with ammonia in the gas phase so as to produce ammonium chloride. The combined gas stream is passed into a condensation and collection vessel and a cyclonic flow is created in the combined gas stream as it passes through the vessel. The temperature of the gas stream is reduced in the vessel to below the condensation temperature of ammonium chloride in order to crystallize the ammonium chloride on the walls of the vessel. The cyclonic flow creates a turbulence which breaks off the larger particles of ammonium chloride which are, in turn, driven to the bottom of the vessel where the solid ammonium chloride can be removed from the vessel. The gas stream exiting from the condensation and collection vessel is further cleaned and additional ammonium chloride is removed by passing through additional filters.

  19. Microbial reductive dehalogenation of vinyl chloride

    DOEpatents

    Spormann, Alfred M [Stanford, CA; Muller, Jochen A [Baltimore, MD; Rosner, Bettina M [Berlin, DE; Von Abendroth, Gregory [Mannheim, DE; Meshulam-Simon, Galit [Los Angeles, CA; McCarty, Perry L [Stanford, CA

    2014-02-11

    Compositions and methods are provided that relate to the bioremediation of chlorinated ethenes, particularly the bioremediation of vinyl chloride by Dehalococcoides-like organisms. An isolated strain of bacteria, Dehalococcoides sp. strain VS, that metabolizes vinyl chloride is provided; the genetic sequence of the enzyme responsible for vinyl chloride dehalogenation; methods of assessing the capability of endogenous organisms at an environmental site to metabolize vinyl chloride; and a method of using the strains of the invention for bioremediation.

  20. Microbial reductive dehalogenation of vinyl chloride

    DOEpatents

    Spormann, Alfred M.; Muller, Jochen A.; Rosner, Bettina M.; Von Abendroth, Gregory; Meshulam-Simon, Galit; McCarty, Perry L

    2011-11-22

    Compositions and methods are provided that relate to the bioremediation of chlorinated ethenes, particularly the bioremediation of vinyl chloride by Dehalococcoides-like organisms. An isolated strain of bacteria, Dehalococcoides sp. strain VS, that metabolizes vinyl chloride is provided; the genetic sequence of the enzyme responsible for vinyl chloride dehalogenation; methods of assessing the capability of endogenous organisms at an environmental site to metabolize vinyl chloride; and a method of using the strains of the invention for bioremediation.

  1. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The less soluble sodium salt separates out at elevated temperatures, and ammonium chloride is recovered... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Ammonium chloride. 184.1138 Section 184.1138...

  2. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The less soluble sodium salt separates out at elevated temperatures, and ammonium chloride is recovered... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Ammonium chloride. 184.1138 Section 184.1138...

  3. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The less soluble sodium salt separates out at elevated temperatures, and ammonium chloride is recovered... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Ammonium chloride. 184.1138 Section 184.1138...

  4. 21 CFR 184.1138 - Ammonium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... No. 12125-02-9) is produced by the reaction of sodium chloride and an ammonium salt in solution. The less soluble sodium salt separates out at elevated temperatures, and ammonium chloride is recovered... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Ammonium chloride. 184.1138 Section 184.1138...

  5. 21 CFR 182.8252 - Choline chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Choline chloride. 182.8252 Section 182.8252 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8252 Choline chloride. (a) Product. Choline chloride....

  6. 21 CFR 582.1193 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium chloride. 582.1193 Section 582.1193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1193 Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This...

  7. 21 CFR 582.6193 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium chloride. 582.6193 Section 582.6193 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium chloride. (a) Product. Calcium chloride. (b) Conditions of use. This substance is...

  8. 7 CFR 58.434 - Calcium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Calcium chloride. 58.434 Section 58.434 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards... Material § 58.434 Calcium chloride. Calcium chloride, when used, shall meet the requirements of the...

  9. 21 CFR 582.5622 - Potassium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Potassium chloride. 582.5622 Section 582.5622 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5622 Potassium chloride. (a) Product. Potassium chloride. (b) Conditions of use....

  10. 21 CFR 582.5622 - Potassium chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Potassium chloride. 582.5622 Section 582.5622 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5622 Potassium chloride. (a) Product. Potassium chloride. (b) Conditions of use....

  11. 21 CFR 582.5622 - Potassium chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Potassium chloride. 582.5622 Section 582.5622 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5622 Potassium chloride. (a) Product. Potassium chloride. (b) Conditions of use....

  12. 21 CFR 582.5622 - Potassium chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Potassium chloride. 582.5622 Section 582.5622 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5622 Potassium chloride. (a) Product. Potassium chloride. (b) Conditions of use....

  13. 21 CFR 582.5622 - Potassium chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Potassium chloride. 582.5622 Section 582.5622 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5622 Potassium chloride. (a) Product. Potassium chloride. (b) Conditions of use....

  14. 21 CFR 184.1426 - Magnesium chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Magnesium chloride. 184.1426 Section 184.1426 Food... Specific Substances Affirmed as GRAS § 184.1426 Magnesium chloride. (a) Magnesium chloride (MgC12·6H2O, CAS... mineral bischofite. It is prepared by dissolving magnesium oxide, hydroxide, or carbonate in...

  15. 21 CFR 182.8985 - Zinc chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Zinc chloride. 182.8985 Section 182.8985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8985 Zinc chloride. (a) Product. Zinc chloride. (b)...

  16. 21 CFR 582.5985 - Zinc chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Zinc chloride. 582.5985 Section 582.5985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance...

  17. 21 CFR 182.8985 - Zinc chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Zinc chloride. 182.8985 Section 182.8985 Food and... CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance is generally recognized as safe when used...

  18. 21 CFR 582.5985 - Zinc chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Zinc chloride. 582.5985 Section 582.5985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance...

  19. 21 CFR 582.5985 - Zinc chloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Zinc chloride. 582.5985 Section 582.5985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance...

  20. 21 CFR 182.8985 - Zinc chloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Zinc chloride. 182.8985 Section 182.8985 Food and... CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance is generally recognized as safe when used...

  1. 21 CFR 582.5985 - Zinc chloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Zinc chloride. 582.5985 Section 582.5985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance...

  2. 21 CFR 582.5985 - Zinc chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Zinc chloride. 582.5985 Section 582.5985 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance...

  3. 21 CFR 182.8985 - Zinc chloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Zinc chloride. 182.8985 Section 182.8985 Food and... CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance is generally recognized as safe when used...

  4. 21 CFR 182.8985 - Zinc chloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Zinc chloride. 182.8985 Section 182.8985 Food and... CONSUMPTION (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients § 182.8985 Zinc chloride. (a) Product. Zinc chloride. (b) Conditions of use. This substance is generally recognized as safe when used...

  5. REMOVAL OF CHLORIDE FROM AQUEOUS SOLUTIONS

    DOEpatents

    Schulz, W.W.

    1959-08-01

    The removal of chlorides from aqueons solutions is described. The process involves contacting the aqueous chloride containing solution with a benzene solution about 0.005 M in phenyl mercuric acetate whereby the chloride anions are taken up by the organic phase and separating the organic phase from the aqueous solutions.

  6. 46 CFR 151.50-34 - Vinyl chloride (vinyl chloride monomer).

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Vinyl chloride (vinyl chloride monomer). 151.50-34 Section 151.50-34 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES BARGES CARRYING BULK LIQUID HAZARDOUS MATERIAL CARGOES Special Requirements § 151.50-34 Vinyl chloride (vinyl chloride monomer). (a)...

  7. An XAFS Study of Tantalum Chloride in the Ionic Liquid 1-ethyl-3-methyl Imidazolium Chloride/ aluminum Chloride

    SciTech Connect

    D Roeper; K Pandya; G Cheek; W OGrady

    2011-12-31

    Tantalum chloride was studied with extended X-ray absorption fine structure spectroscopy (XAFS) in acidic and basic aluminum chloride/1-ethyl-3-methyl imidazolium chloride ionic liquids (ILs). Anhydrous Ta2Cl10 is more soluble in the basic solution than in the acidic solution and the X-ray absorption data shows that the coordination shell of chlorides around the tantalum is larger in the basic solution. In the acidic solution, tantalum has five chlorides in its coordination shell while in the basic solution; the tantalum is coordinated by seven chlorides. This indicates that the Lewis acidity of the tantalum chloride causes the Ta to coordinate differently in the acidic and the basic solutions.

  8. Shock compression of polyvinyl chloride

    NASA Astrophysics Data System (ADS)

    Neogi, Anupam; Mitra, Nilanjan

    2016-04-01

    This study presents shock compression simulation of atactic polyvinyl chloride (PVC) using ab-initio and classical molecular dynamics. The manuscript also identifies the limits of applicability of classical molecular dynamics based shock compression simulation for PVC. The mechanism of bond dissociation under shock loading and its progression is demonstrated in this manuscript using the density functional theory based molecular dynamics simulations. The rate of dissociation of different bonds at different shock velocities is also presented in this manuscript.

  9. Chloride flux out of Yellowstone National Park

    USGS Publications Warehouse

    Norton, D.R.; Friedman, I.

    1985-01-01

    Monitoring of the chloride concentration, electrical conductivity, and discharge was carried out for the four major rivers of Yellowstone National Park from September 1982 to January 1984. Chloride flux out of the Park was determined from the measured values of chloride concentration and discharge. The annual chloride flux from the Park was 5.86 ?? 1010 g. Of this amount 45% was from the Madison River drainage basin, 32% from the Yellowstone River basin, 12% from the Snake River basin, and 11% from the Falls River basin. Of the annual chloride flux from the Yellowstone River drainage basin 36% was attributed to the Yellowstone Lake drainage basin. The geothermal contribution to the chloride flux was determined by subtracting the chloride contribution from rock weathering and atmospheric precipitation and is 94% of the total chloride flux. Calculations of the geothermal chloride flux for each river are given and the implications of an additional chloride flux out of the western Park boundary discussed. An anomalous increase in chloride flux out of the Park was observed for several weeks prior to the Mt. Borah earthquake in Central Idaho on October 28, 1983, reaching a peak value shortly thereafter. It is suggested that the rise in flux was a precursor of the earthquake. The information in this paper provides baseline data against which future changes in the hydrothermal systems can be measured. It also provides measurements related to the thermal contributions from the different drainage basins of the Park. ?? 1985.

  10. Chloride flux out of Yellowstone National Park

    NASA Astrophysics Data System (ADS)

    Norton, Daniel R.; Friedman, Irving

    1985-12-01

    Monitoring of the chloride concentration, electrical conductivity, and discharge was carried out for the four major rivers of Yellowstone National Park from September 1982 to January 1984. Chloride flux out of the Park was determined from the measured values of chloride concentration and discharge. The annual chloride flux from the Park was 5.86 × 10 10 g. Of this amount 45% was from the Madison River drainage basin, 32% from the Yellowstone River basin, 12% from the Snake River basin, and 11% from the Falls River basin. Of the annual chloride flux from the Yellowstone River drainage basin 36% was attributed to the Yellowstone Lake drainage basin. The geothermal contribution to the chloride flux was determined by subtracting the chloride contribution from rock weathering and atmospheric precipitation and is 94% of the total chloride flux. Calculations of the geothermal chloride flux for each river are given and the implications of an additional chloride flux out of the western Park boundary discussed. An anomalous increase in chloride flux out of the Park was observed for several weeks prior to the Mt. Borah earthquake in Central Idaho on October 28, 1983, reaching a peak value shortly thereafter. It is suggested that the rise in flux was a precursor of the earthquake. The information in this paper provides baseline data against which future changes in the hydrothermal systems can be measured. It also provides measurements related to the thermal contributions from the different drainage basins of the Park.

  11. Salt, chloride, bleach, and innate host defense.

    PubMed

    Wang, Guoshun; Nauseef, William M

    2015-08-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

  12. Salt, chloride, bleach, and innate host defense

    PubMed Central

    Wang, Guoshun; Nauseef, William M.

    2015-01-01

    Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

  13. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    PubMed Central

    Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

    2014-01-01

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress. PMID:25387672

  14. Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα.

    PubMed

    Moon, Thomas M; Tykocki, Nathan R; Sheehe, Jessica L; Osborne, Brent W; Tegge, Werner; Brayden, Joseph E; Dostmann, Wolfgang R

    2015-12-17

    PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease. PMID:26687482

  15. Tetrahydrobiopterin regulates cyclic GMP-dependent electrogenic Cl- secretion in mouse ileum in vitro.

    PubMed Central

    Rolfe, V E; Brand, M P; Heales, S J; Lindley, K J; Milla, P J

    1997-01-01

    1. Basal electrogenic Cl- secretion, measured as the short-circuit current (Isc), was variable in ileum removed from tetrahydrobiopterin (BH4)-deficient hph-1 mice and wild-type controls in vitro, although values were not significantly different. 2. The basal nitrite release and mucosal cyclic guanosine 3',5'-monophosphate (cyclic GMP) production were similar in control and BH4-deficient ileum. 3. Mucosally added Escherichia coli heat-stable toxin (STa, 55 ng ml-1) increased the nitrite release, cyclic GMP levels and the Isc in control ileum, but its secretory actions were reduced in BH4-deficient ileum. 4. L-Arginine (1 mM) increased the nitrite release, cyclic GMP production and the Isc in control ileum, but the actions were reduced in BH4-deficient ileum. 5. Serosal carbachol (1 mM) stimulated maximum short-circuit currents of similar magnitude in both control and BH4-deficient ileum, whilst nitrite release and cyclic GMP production were minimal. 6. E. coli STa and L-arginine increased electrogenic Cl- secretion across intact mouse ileum in vitro by releasing nitric oxide and elevating mucosal cyclic GMP. The inhibition of these processes in the hph-1 mouse ileum suggests that BH4 may be a target for the modulation of electrogenic transport, and highlight the complexity of the interactions between nitric oxide and cyclic GMP in the gut. PMID:9306277

  16. A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii

    PubMed Central

    Avelar, Gabriela Mól; Glaser, Talita; Leonard, Guy; Richards, Thomas A.; Ulrich, Henning

    2015-01-01

    Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K+ channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K+ selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K+ channel and the rhodopsin–guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K+ channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus. PMID:26150416

  17. A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

    PubMed

    Avelar, Gabriela Mól; Glaser, Talita; Leonard, Guy; Richards, Thomas A; Ulrich, Henning; Gomes, Suely L

    2015-09-01

    Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus. PMID:26150416

  18. Detection of calcium activity in human monocytes by the fura-2 fluorescence method: in vitro differentiation sensitizes cells to dihydropyridine calcium channel modulators

    NASA Astrophysics Data System (ADS)

    Oraevsky, Alexander A.; Cabello, Olga A.; Shan, Qin; Tittel, Frank K.; Henry, Philip D.

    1994-07-01

    Dihydropyridine (DHP) calcium channel blockers have been shown to suppress atherogenesis in various species and controlled angiographic trials suggest that these drugs may retard the progression of occlusive coronary disease in humans. Because mononuclear leukocytes play a key role in the formation of early and advanced atheromatous lesions, we determined effects of DHP calcium channel modulators on calcium uptake by cells of the monocytic lineage. Human peripheral blood monocytes were evaluated before and after undergoing in vitro differentiation induced by two days of culture with fetal calf serum and FMLP. Changes in intracellular calcium activity were estimated with fura-2, a fluorescent calcium indicator. Freshly isolated (unactivated) monocytes were insensitive to DHP drugs both in the presence and absence of high potassium membrane depolarization. In contrast, nisoldipine, a DHP calcium channel blocker, and BAY K 8644, a DHP calcium channel activator, decreased and increased calcium uptake by KC1-depolarized differentiated monocytes. Results suggest that differentiation of monocytes to macrophages may involve a change in the expression and/or regulation of DHP- sensitive calcium channels.

  19. Improvement of spatial learning by facilitating large-conductance calcium-activated potassium channel with transcranial magnetic stimulation in Alzheimer's disease model mice.

    PubMed

    Wang, Furong; Zhang, Yu; Wang, Li; Sun, Peng; Luo, Xianwen; Ishigaki, Yasuhito; Sugai, Tokio; Yamamoto, Ryo; Kato, Nobuo

    2015-10-01

    Transcranial magnetic stimulation (TMS) is fragmentarily reported to be beneficial to Alzheimer's patients. Its underlying mechanism was investigated. TMS was applied at 1, 10 or 15 Hz daily for 4 weeks to young Alzheimer's disease model mice (3xTg), in which intracellular soluble amyloid-β is notably accumulated. Hippocampal long-term potentiation (LTP) was tested after behavior. TMS ameliorated spatial learning deficits and enhanced LTP in the same frequency-dependent manner. Activity of the large conductance calcium-activated potassium (Big-K; BK) channels was suppressed in 3xTg mice and recovered by TMS frequency-dependently. These suppression and recovery were accompanied by increase and decrease in cortical excitability, respectively. TMS frequency-dependently enhanced the expression of the activity-dependently expressed scaffold protein Homer1a, which turned out to enhance BK channel activity. Isopimaric acid, an activator of the BK channel, magnified LTP. Amyloid-β lowering was detected after TMS in 3xTg mice. In 3xTg mice with Homer1a knocked out, amyloid-β lowering was not detected, though the TMS effects on BK channel and LTP remained. We concluded that TMS facilitates BK channels both Homer1a-dependently and -independently, thereby enhancing hippocampal LTP and decreasing cortical excitability. Reduced excitability contributed to amyloid-β lowering. A cascade of these correlated processes, triggered by TMS, was likely to improve learning in 3xTg mice. PMID:26051398

  20. Model Vestibular Nuclei Neurons Can Exhibit a Boosting Nonlinearity Due to an Adaptation Current Regulated by Spike-Triggered Calcium and Calcium-Activated Potassium Channels

    PubMed Central

    Schneider, Adam D.

    2016-01-01

    In vitro studies have previously found a class of vestibular nuclei neurons to exhibit a bidirectional afterhyperpolarization (AHP) in their membrane potential, due to calcium and calcium-activated potassium conductances. More recently in vivo studies of such vestibular neurons were found to exhibit a boosting nonlinearity in their input-output tuning curves. In this paper, a Hodgkin-Huxley (HH) type neuron model, originally developed to reproduce the in vitro AHP, is shown to produce a boosting nonlinearity similar to that seen in vivo for increased the calcium conductance. Indicative of a bifurcation, the HH model is reduced to a generalized integrate-and-fire (IF) model that preserves the bifurcation structure and boosting nonliearity. By then projecting the neuron model’s phase space trajectories into 2D, the underlying geometric mechanism relating the AHP and boosting nonlinearity is revealed. Further simplifications and approximations are made to derive analytic expressions for the steady steady state firing rate as a function of bias current, μ, as well as the gain (i.e. its slope) and the position of its peak at μ = μ*. Finally, although the boosting nonlinearity has not yet been experimentally observed in vitro, testable predictions indicate how it might be found. PMID:27427914

  1. Chloride Transporting CLC Proteins1

    NASA Astrophysics Data System (ADS)

    Pusch, Michael

    In the early 1980s, Chris Miller and colleagues described a curious "double-barreled" chloride channel from the electric organ of Torpedo fish reconstituted in planar lipid bilayers (Miller and White, 1980). Single-channel openings occurred in "bursts" separated by long closures. A single burst was characterized by the presence of two open conductance levels of equal size and the gating (i.e., openings and closings) during a burst could be almost perfectly described as a superposition of two identical and independent conductances that switched between open and closed states with voltage-dependent rates α and β (Hanke and Miller, 1983) (Fig. 8.1).

  2. Oxomemazine hydro­chloride

    PubMed Central

    Siddegowda, M. S.; Butcher, Ray J.; Akkurt, Mehmet; Yathirajan, H. S.; Ramesh, A. R.

    2011-01-01

    In the title compound [systematic name: 3-(5,5-dioxo­phen­othia­zin-10-yl)-N,N,2-trimethyl­propanaminium chloride], C18H23N2O2S+·Cl−, the dihedral angle between the two outer aromatic rings of the phenothia­zine unit is 30.5 (2)°. In the crystal, the components are linked by N—H⋯Cl and C—H⋯Cl hydrogen bonds and C—H⋯π inter­actions. PMID:22090928

  3. An XAFS Study of Niobium chloride in the ionic liquid 1-ethyl-3-methyl imidazolium chloride/ aluminum chloride

    SciTech Connect

    D Roeper; K Pandya; G Cheek; W OGrady

    2011-12-31

    Niobium chloride was studied with extended X-ray absorption fine structure spectroscopy (EXAFS) in acidic and basic aluminum chloride/1-ethyl-3-methyl imidazolium chloride (EMIC) ionic liquids. Although anhydrous Nb2Cl10 is more soluble in the basic melt than in the acidic melt, the EXAFS data shows that the coordination shell around the niobium does not change in the different ionic liquids. Both the acidic and basic melts show a coordination of five chlorides in the first shell. This indicates that in this series of ionic liquids, the Nb2Cl10 breaks up into two NbCl5 entities in both the acidic and the basic melts.

  4. Production of anhydrous aluminum chloride composition

    DOEpatents

    Vandergrift, G.F. III; Krumpelt, M.; Horwitz, E.P.

    1981-10-08

    A process is described for producing an anhydrous aluminum chloride composition from a water-based aluminous material such as a slurry of aluminum hydroxide in a multistage extraction process in which the aluminum ion is first extracted into an organic liquid containing an acidic extractant and then extracted from the organic phase into an alkali metal chloride or chlorides to form a melt containing a mixture of chlorides of alkali metal and aluminum. In the process, the organic liquid may be recycled. In addition, the process advantageously includes an electrolysis cell for producing metallic aluminum and the alkali metal chloride or chlorides may be recycled for extraction of the aluminum from the organic phase.

  5. Chloride transport in human red cells.

    PubMed Central

    Dalmark, M

    1975-01-01

    1. The chloride equilibrium flux (chloride self-exchange) was determined by measuring the rate of 36Cl efflux from radioactively labelled human red cells. The cellular chloride concentration was varied between 5 and 700 mM by the nystatin technique (Cass & Dalmark, 1973). The chloride transport capacity was not affected by the nystatin technique. 2. The chloride equilibrium flux showed saturation kinetics in the pH range between 6-2 and 9-2 (0 degrees C). The chloride transport decreased at chloride concentrations higher than those which gave the maximum transport. 3. The apparent half-saturation constant, (K1/2), depended on the pH and whether the chloride transport was perceived as a function of the chloride concentration in the medium or in the cell water. The (K1/2)m increased and the (K1/2)c decreased with increasing pH. The dependence of the chloride transport on the chloride concentration was described by Michaelis-Menten kinetics at pH 7-2, but at values of pH outside pH 7-8 S-shaped or steeper graphs were observed. 4. The chloride equilibrium flux varied with the pH at constant chloride concentration in the medium (pH 5-7-9-5). The transport had a bell-shaped pH dependence at chloride concentrations below 200 mM. At chloride concentrations between 300 and 600 mM the chloride transport increased with increasing pH to reach a plateau around pH 8. The position of the acidic branches of the pH graphs was independent of the chloride concentration (25-600 mM), but the position of the alkaline branches moved towards higher values of pH with increasing chloride concentration (5-150 mM). Thus, the position of the pH optimum increased with increasing chloride concentration. The chloride transport at low pH values was a function of the inverse second power of the hydrogen ion concentration. The pK of the groups which caused the inhibition was approximately 6 and independent of the temperature (0-18 degrees C). 5. The chloride equilibrium flux as a function of

  6. [Sodium chloride 0.9%: nephrotoxic crystalloid?].

    PubMed

    Dombre, Vincent; De Seigneux, Sophie; Schiffer, Eduardo

    2016-02-01

    Sodium chloride 0.9%, often incorrectly called physiological saline, contains higher concentration of chloride compared to plasma. It is known that the administration of sodium chloride 0.9% can cause hyperchloremic metabolic acidosis in a reproducible manner. The elevated chloride concentration in 0.9% NaCl solution can also adversely affect renal perfusion. This effect is thought to be induced by hyperchloremia that causes renal artery vasoconstriction. For these reasons, the use of 0.9% NaCl solution is raising attention and some would advocate the use of a more "physiological" solution, such as balanced solutions that contain a level of chloride closer to that of plasma. Few prospective, randomized, controlled trials are available today and most were done in a perioperative setting. Some studies suggest that the chloride excess in 0.9% NaCl solution could have clinical consequences; however, this remains to be established by quality randomized controlled trials. PMID:26999998

  7. Indium-111 chloride imaging with ununited fractures

    SciTech Connect

    Sayle, B.A.; Fawcett, H.D.; Yudt, W.M.; Wang, S.C.; Mader, J.T.; Cierny, G. 3d.

    1987-03-01

    Twenty patients with ununited fractures and a suspicion of infection had In-111 chloride imaging. Surgically obtained cultures were positive for infection in 12 and negative in eight patients. In-111 chloride images were positive in all 12 patients with infection but also were positive in six of the patients with negative cultures. It is not possible to differentiate infected from noninfected ununited fractures by In-111 chloride imaging.

  8. Metal chloride cathode for a battery

    NASA Technical Reports Server (NTRS)

    Bugga, Ratnakumar V. (Inventor); Distefano, Salvador (Inventor); Bankston, C. Perry (Inventor)

    1991-01-01

    A method of fabricating a rechargeable battery is disclosed which includes a positive electrode which contains a chloride of a selected metal when the electrode is in its active state. The improvement comprises fabricating the positive electrode by: providing a porous matrix composed of a metal; providing a solution of the chloride of the selected metal; and impregnating the matrix with the chloride from the solution.

  9. Arsenic removal by ferric chloride

    SciTech Connect

    Hering, J.G.; Chen, P.Y.; Wilkie, J.A.; Elimelech, M.; Liang, S.

    1996-04-01

    Bench-scale studies were conducted in model freshwater systems to investigate how various parameters affected arsenic removal during coagulation with ferric chloride and arsenic adsorption onto preformed hydrous ferric oxide. Parameters included arsenic oxidation state and initial concentration, coagulant dosage or adsorbent concentration, pH, and the presence of co-occurring inorganic solutes. Comparison of coagulation and adsorption experiments and of experimental results with predictions based on surface complexation modeling demonstrated that adsorption is an important (though not the sole) mechanism governing arsenic removal during coagulation. Under comparable conditions, better removal was observed with arsenic(V) [As(V)] than with arsenic(III) [As(III)] in both coagulation and adsorption experiments. Below neutral pH values, As(III) removal-adsorption was significantly decreased in the presence of sulfate, whereas only a slight decrease in As(V) removal-adsorption was observed. At high pH, removal-adsorption of As(V) was increased in the presence of calcium. Removal of As(V) during coagulation with ferric chloride is both more efficient and less sensitive than that of As(III) to variations in source water composition.

  10. Enrofloxacin hydro-chloride dihydrate.

    PubMed

    Miranda-Calderón, Jorge E; Gutiérrez, Lilia; Flores-Alamo, Marcos; García-Gutiérrez, Ponciano; Sumano, Héctor

    2014-04-01

    The asymmetric unit of the title compound, C19H23FN3O3 (+)·Cl(-)·2H2O [systematic name: 4-(3-carb-oxy-1-cyclo-propyl-6-fluoro-4-oxo-1,4-di-hydro-quin-o-lin-7-yl)-1-ethyl-piperazin-1-ium chloride dihydrate], consists of two independent monocations of the protonated enrofloxacin, two chloride anions and four water mol-ecules. In the cations, the piperazinium rings adopt chair conformations and the dihedral angles between the cyclo-propyl ring and the 10-membered quinoline ring system are 56.55 (2) and 51.11 (2)°. An intra-molecular O-H⋯O hydrogen bond is observed in each cation. In the crystal, the components are connected via O-H⋯Cl, N-H⋯Cl and O-H⋯O hydrogen bonds, and a π-π inter-action between the benzene rings [centroid-centroid distance = 3.6726 (13) Å], resulting in a three-dimensional array. PMID:24826167

  11. Irreversible gettering of thionyl chloride

    SciTech Connect

    LeRoy Whinnery; Steve Goods; George Buffleben; Tim Sheppodd

    1999-11-01

    The authors have successfully demonstrated the irreversible gettering of SOCl{sub 2} by ZnO/ASZMTEDA carbon over a modest temperature range. While thionyl chloride decomposition was slow below {minus}20 C, lower temperatures are expected to be less of a problem than at higher temperatures. The approximately 30 cc of thionyl chloride in a typical D-cell would require 50 g of ZnO and 107 g of ASZMTEDA carbon. Fortunately, since it is unlikely to happen at all, it is common practice to assume only one cell will fail (leak) in a given battery pack. So, one charge of getter can protect the whole battery pack. In summary, ZnO/ASZMTEDA carbon fulfills all of the requirements of an ideal getter including: irreversible binding or reaction with SOCl{sub 2}, high volumetric uptake capacity, high efficiency, non-volatile, air stable, insensitive to poisoning, non-toxic, cheap, non-corrosive, and the gettering product is not a liquid or oil that could block further flow or accessibility. Future work in this area includes incorporation of the ZnO and carbon into a structural open-celled porous monolith, as well as, gettering for other types of batteries (e.g., Li/MnO{sub 2}).

  12. Chloride ingress in cement paste and mortar

    SciTech Connect

    Jensen, O.M.; Hansen, P.F.; Coats, A.M.; Glasser, F.P.

    1999-09-01

    In this paper chloride ingress in cement paste and mortar is followed by electron probe microanalysis. The influence of several paste and exposure parameters on chloride ingress are examined (e.g., water-cement ratio, silica fume addition, exposure time, and temperature). The measurements are modelled on Fick's law modified by a term for chloride binding. Inclusion of chloride binding significantly improves the profile shape of the modelled ingress profiles. The presence of fine aggregate and formation of interfacial transition zones at paste-aggregate boundaries does not significantly affect diffusion rates.

  13. Production of chlorine from chloride salts

    DOEpatents

    Rohrmann, Charles A.

    1981-01-01

    A process for converting chloride salts and sulfuric acid to sulfate salts and elemental chlorine is disclosed. A chloride salt and sulfuric acid are combined in a furnace where they react to produce a sulfate salt and hydrogen chloride. Hydrogen chloride from the furnace contacts a molten salt mixture containing an oxygen compound of vanadium, an alkali metal sulfate and an alkali metal pyrosulfate to recover elemental chlorine. In the absence of an oxygen-bearing gas during the contacting, the vanadium is reduced, but is regenerated to its active higher valence state by separately contacting the molten salt mixture with an oxygen-bearing gas.

  14. Fabrication Of Metal Chloride Cathodes By Sintering

    NASA Technical Reports Server (NTRS)

    Bugga, Ratnakumar V.; Di Stefano, Salvador; Bankston, C. Perry

    1992-01-01

    Transition-metal chloride cathodes for use in high-temperature rechargeable sodium batteries prepared by sintering transition-metal powders mixed with sodium chloride. Need for difficult and dangerous chlorination process eliminated. Proportions of transition metal and sodium chloride in mixture adjusted to suit specific requirements. Cathodes integral to sodium/metal-chloride batteries, which have advantages over sodium/sulfur batteries including energy densities, increased safety, reduced material and thermal-management problems, and ease of operation and assembly. Being evaluated for supplying electrical power during peak demand and electric vehicles.

  15. The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling*

    PubMed Central

    Bi, Dan; Toyama, Kazuyoshi; Lemaître, Vincent; Takai, Jun; Fan, Fan; Jenkins, David P.; Wulff, Heike; Gutterman, David D.; Park, Frank; Miura, Hiroto

    2013-01-01

    The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca2+ concentration ([Ca2+]i) and was enhanced by elevating [Ca2+]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca2+]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca2+]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca2+]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca2+-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis. PMID:23609438

  16. Inactivation of Endothelial Small/Intermediate Conductance of Calcium-Activated Potassium Channels Contributes to Coronary Arteriolar Dysfunction in Diabetic Patients

    PubMed Central

    Liu, Yuhong; Xie, An; Singh, Arun K; Ehsan, Afshin; Choudhary, Gaurav; Dudley, Samuel; Sellke, Frank W; Feng, Jun

    2015-01-01

    Background Diabetes is associated with coronary arteriolar endothelial dysfunction. We investigated the role of the small/intermediate (SKCa/IKCa) conductance of calcium-activated potassium channels in diabetes-related endothelial dysfunction. Methods and Results Coronary arterioles (80 to 150 μm in diameter) were dissected from discarded right atrial tissues of diabetic (glycosylated hemoglobin = 9.6±0.25) and nondiabetic patients (glycosylated hemoglobin 5.4±0.12) during coronary artery bypass graft surgery (n=8/group). In-vitro relaxation response of precontracted arterioles was examined in the presence of the selective SKCa/IKCa activator NS309 and other vasodilatory agents. The channel density and membrane potential of diabetic and nondiabetic endothelial cells was measured by using the whole cell patch-clamp technique. The protein expression and distribution of the SKCa/IKCa in the human myocardium and coronary arterioles was examined by Western blotting and immunohistochemistry. Our results indicate that diabetes significantly reduced the coronary arteriolar response to the SKCa/IKCa activator NS309 compared to the respective responses of nondiabetic vessels (P<0.05 versus nondiabetes). The relaxation response of diabetic arterioles to NS309 was prevented by denudation of endothelium (P=0.001 versus endothelium-intact). Diabetes significantly decreased endothelial SKCa/IKCa currents and hyperpolarization induced by the SKCa/IKCa activator NS309 as compared with that of nondiabetics. There were no significant differences in the expression and distribution of SKCa/IKCa proteins in the coronary microvessels. Conclusions Diabetes is associated with inactivation of endothelial SKCa/IKCa channels, which may contribute to endothelial dysfunction in diabetic patients. PMID:26304940

  17. Calcium-Activated SK Channels Influence Voltage-Gated Ion Channels to Determine the Precision of Firing in Globus Pallidus Neurons

    PubMed Central

    Deister, Christopher A.; Chan, C. Savio; Surmeier, D. James; Wilson, Charles J.

    2012-01-01

    Globus pallidus (GP) neurons fire rhythmically in the absence of synaptic input, suggesting that they may encode their inputs as changes in the phase of their rhythmic firing. Action potential afterhyperpolarization (AHP) enhances precision of firing by ensuring that the ion channels recover from inactivation by the same amount on each cycle. Voltage-clamp experiments in slices showed that the longest component of the GP neuron’s AHP is blocked by apamin, a selective antagonist of calcium-activated SK channels. Application of 100 nm apamin also disrupted the precision of firing in perforated-patch and cell-attached recordings. SK channel blockade caused a small depolarization in spike threshold and made it more variable, but there was no reduction in the maximal rate of rise during an action potential. Thus, the firing irregularity was not caused solely by a reduction in voltage-gated Na+ channel availability. Subthreshold voltage ramps triggered a large outward current that was sensitive to the initial holding potential and had properties similar to the A-type K+ current in GP neurons. In numerical simulations, the availability of both Na+ and A-type K+ channels during autonomous firing were reduced when SK channels were removed, and a nearly equal reduction in Na+ and K+ subthreshold-activated ion channel availability produced a large decrease in the neuron’s slope conductance near threshold. This change made the neuron more sensitive to intrinsically generated noise. In vivo, this change would also enhance the sensitivity of GP neurons to small synaptic inputs. PMID:19571136

  18. The small conductance calcium-activated potassium channel 3 (SK3) is a molecular target for Edelfosine to reduce the invasive potential of urothelial carcinoma cells.

    PubMed

    Steinestel, Konrad; Eder, Stefan; Ehinger, Konstantin; Schneider, Juliane; Genze, Felicitas; Winkler, Eva; Wardelmann, Eva; Schrader, Andres J; Steinestel, Julie

    2016-05-01

    Metastasis is the survival-determining factor in urothelial carcinoma (UC) of the urinary bladder. The small conductance calcium-activated potassium channel 3 (SK3) enhances tumor cell invasion in breast cancer and malignant melanoma. Since Edelfosine, a glycerophospholipid with antitumoral properties, effectively inhibits SK3 channel activity, our goal was to evaluate SK3 as a potential molecular target to inhibit the gain of an invasive phenotype in UC. SK3 protein expression was analyzed in 208 tissue samples and UC cell lines. Effects of Edelfosine on SK3 expression and intracellular calcium levels as well as on cell morphology, cell survival and proliferation were assessed using immunoblotting, potentiometric fluorescence microscopy, and clonogenic/cell survival assay; furthermore, we analyzed the effect of Edelfosine and SK3 RNAi knockdown on tumor cell migration and invasion in vitro and in vivo. We found that SK3 is strongly expressed in muscle-invasive UC and in the RT112 cellular tumor model. Higher concentrations of Edelfosine have a strong antitumoral effect on UC cells, while 1 μM effectively inhibits migration/invasion of UC cells in vitro and in vivo comparable to the SK3 knockdown phenotype. Taken together, our results show strong expression of SK3 in muscle-invasive UC, consistent with the postulated role of the protein in tumor cell invasion. Edelfosine is able to effectively inhibit migration and invasion of UC cells in vitro and in vivo in an SK3-dependent way, pointing towards a possible role for Edelfosine as an antiinvasive drug to effectively inhibit UC cell invasion and metastasis. PMID:26619845

  19. Activation of endothelial and epithelial KCa2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

    PubMed Central

    Kroigaard, Christel; Dalsgaard, Thomas; Nielsen, Gorm; Laursen, Britt E; Pilegaard, Hans; Köhler, Ralf; Simonsen, Ulf

    2012-01-01

    BACKGROUND AND PURPOSE Small (KCa2) and intermediate (KCa3.1) conductance calcium-activated potassium channels (KCa) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of KCa2.3 and KCa3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries. EXPERIMENTAL APPROACH Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. KCa2 and KCa3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques. RESULTS While KCa2.3 expression was similar, KCa3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive KCa2.3 and KCa3.1 proteins were found in both endothelium and epithelium. KCa currents were present in HSAEpi cells and sensitive to the KCa2.3 blocker UCL1684 and the KCa3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the KCa2.3/ KCa3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the KCa2 channel blocker apamin, while the KCa3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01–1 µM). CONCLUSIONS AND IMPLICATIONS KCa2.3 and KCa3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. KCa2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease. PMID:22506557

  20. The small-conductance calcium-activated potassium channel is a key modulator of firing and long-term depression in the dorsal striatum

    PubMed Central

    Hopf, F. Woodward; Seif, Taban; Mohamedi, Maysha L.; Chen, Billy T.; Bonci, Antonello

    2011-01-01

    The striatum is considered to be critical for the control of goal-directed action, with the lateral dorsal striatum (latDS) being implicated in modulation of habits and the nucleus accumbens thought to represent a limbic–motor interface. Although medium spiny neurons from different striatal subregions exhibit many similar properties, differential firing and synaptic plasticity could contribute to the varied behavioral roles across subregions. Here, we examined the contribution of small-conductance calcium-activated potassium channels (SKs) to action potential generation and synaptic plasticity in adult rat latDS and nucleus accumbens shell (NAS) projection neurons in vitro. The SK-selective antagonist apamin exerted a prominent effect on latDS firing, significantly decreasing the interspike interval. Furthermore, prolonged latDS depolarization increased the interspike interval and reduced firing, and this enhancement was reversed by apamin. In contrast, NAS neurons exhibited greater basal firing rates and less regulation of firing by SK inhibition and prolonged depolarization. LatDS neurons also had greater SK currents than NAS neurons under voltage-clamp. Importantly, SK inhibition with apamin facilitated long-term depression (LTD) induction in the latDS but not the NAS, without alterations in glutamate release. In addition, SK activation in the latDS prevented LTD induction. Greater SK function in the latDS than in the NAS was not secondary to differences in sodium or inwardly rectifying potassium channel function, and apamin enhancement of firing did not reflect indirect action through cholinergic interneurons. Thus, these data demonstrate that SKs are potent modulators of action potential generation and LTD in the dorsal striatum, and could represent a fundamental cellular mechanism through which habits are regulated. PMID:20497469

  1. Modulation by purines of calcium-activated non-selective cation channels in the outer hair cells of the guinea-pig cochlea.

    PubMed Central

    Van den Abbeele, T; Tran Ba Huy, P; Teulon, J

    1996-01-01

    1. The cell-attached and cell-free configurations of the patch-clamp technique were used to investigate whether external ATP and its derivatives modulate channel activity in outer hair cells freshly isolated from the guinea-pig cochlea. 2. Submicromolar concentrations of ATP stimulated a non-selective cation channel with a conductance of about 25 pS. The ATP-elicited stimulation was partly blocked by the membrane-permeant blocker 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), and mimicked by the calcium ionophore, ionomycin, suggesting that the channel activated by ATP is identical to a previously reported calcium-activated non-selective (CAN) cation channel. 3. The P2x agonist beta, gamma-methylene-ATP (beta, gamma-MeATP, 10 microM) and the P2Y agonist 2-methyl-thio-ATP (2-MeSATP, 1 microM) both activated CAN channels. The effect of ATP was inhibited by the P2 antagonist suramin but not by the P2Y antagonist Reactive Blue 2. These results suggest that both purinergic receptors are involved in the ATP-evoked response and that internal calcium acts as a second messenger for opening CAN channels. 4. In contrast, adenosine inhibited CAN channels. This effect was reproduced by the A2 agonist 5'-N-ethylcarboxyamidoadenosine (NECA) and the permeant cAMP analogue 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), but not by the A1 agonist N6-cyclo-hexyladenosine (CHA). CAN channels were also inhibited when the catalytic subunit of protein kinase A was applied internally on inside-out patches, suggesting that adenosine A2 receptor downregulates CAN channels via a cAMP-dependent phosphorylation. Images Figure 10 PMID:8814608

  2. Cutaneous blood flow during intradermal NO administration in young and older adults: roles for calcium-activated potassium channels and cyclooxygenase?

    PubMed

    Fujii, Naoto; Meade, Robert D; Minson, Christopher T; Brunt, Vienna E; Boulay, Pierre; Sigal, Ronald J; Kenny, Glen P

    2016-06-01

    Nitric oxide (NO) increases cutaneous blood flow; however, the underpinning mechanism(s) remains to be elucidated. We hypothesized that the cutaneous blood flow response during intradermal administration of sodium nitroprusside (SNP, a NO donor) is regulated by calcium-activated potassium (KCa) channels and cyclooxygenase (COX) in young adults. We also hypothesized that these contributions are diminished in older adults given that aging can downregulate KCa channels and reduce COX-derived vasodilator prostanoids. In 10 young (23 ± 5 yr) and 10 older (54 ± 4 yr) adults, cutaneous vascular conductance (CVC) was measured at four forearm skin sites infused with 1) Ringer (Control), 2) 50 mM tetraethylammonium (TEA), a nonspecific KCa channel blocker, 3) 10 mM ketorolac, a nonspecific COX inhibitor, or 4) 50 mM TEA + 10 mM ketorolac via intradermal microdialysis. All skin sites were coinfused with incremental doses of SNP (0.005, 0.05, 0.5, 5, and 50 mM each for 25 min). During SNP administration, CVC was similar at the ketorolac site (0.005-50 mM, all P > 0.05) relative to Control, but lower at the TEA and TEA + ketorolac sites (0.005-0.05 mM, all P < 0.05) in young adults. In older adults, ketorolac increased CVC relative to Control during 0.005-0.05 mM SNP administration (all P < 0.05), but this increase was not observed when TEA was coadministered (all P > 0.05). Furthermore, TEA alone did not modulate CVC during any concentration of SNP administration in older adults (all P > 0.05). We show that during low-dose NO administration (e.g., 0.005-0.05 mM), KCa channels contribute to cutaneous blood flow regulation in young adults; however, in older adults, COX inhibition increases cutaneous blood flow through a KCa channel-dependent mechanism. PMID:27053645

  3. Cognitive recovery by chronic activation of the large-conductance calcium-activated potassium channel in a mouse model of Alzheimer's disease.

    PubMed

    Wang, Li; Kang, Huicong; Li, Yongzhi; Shui, Yuan; Yamamoto, Ryo; Sugai, Tokio; Kato, Nobuo

    2015-05-01

    We previously showed that activity of the large conductance calcium-activated potassium (Big-K; BK) channels is suppressed in 3xTg Alzheimer disease (AD) model mice. However, its behavioral significance is not known. In the present report, ventricular injection of the BK channel activator isopimaric acid (ISO) was conducted to examine whether BK channel activation ameliorates cognition in 3xTg mice. The novel object recognition (NOR) test revealed that chronic injection of ISO improved non-spatial memory in 3xTg mice. In the Morris water maze, the probe test demonstrated an improved spatial memory after ISO injection. Electrophysiological underpinnings of the ISO effect were then examined in slices obtained from the mice after behavior. At hippocampal CA1 synapses, the basic synaptic transmission was abnormally elevated and long-term potentiation (LTP) was partially suppressed in 3xTg mice. These were both recovered by ISO treatment. We then confirmed suppressed BK channel activity in 3xTg mice by measuring the half-width of evoked action potentials. This was also recovered by ISO treatment. We previously showed that the recovery of BK channel activity accompanies reduction of neuronal excitability in pyramidal cells. Here again, pyramidal cell excitability, as assessed by calculating the frequency of evoked spikes, was elevated in the 3xTg mouse and was normalized by ISO. ELISA experiments demonstrated an ISO-induced reduction of Aβ1-42 content in hippocampal tissue in 3xTg mice. The present study thus suggests a potential therapeutic utility of BK channel activators for AD. PMID:25577958

  4. A small conductance calcium-activated K+ channel in C. elegans, KCNL-2, plays a role in the regulation of the rate of egg-laying.

    PubMed

    Chotoo, Cavita K; Silverman, Gary A; Devor, Daniel C; Luke, Cliff J

    2013-01-01

    In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. PMID:24040423

  5. A Small Conductance Calcium-Activated K+ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

    PubMed Central

    Chotoo, Cavita K.; Silverman, Gary A.; Devor, Daniel C.; Luke, Cliff J.

    2013-01-01

    In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson’s disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. PMID:24040423

  6. XAFS Studies of Ni Ta and Nb Chlorides in the Ionic Liquid 1-Ethyl-3-Methyl Imidazolium Chloride / Aluminum Chloride

    SciTech Connect

    W OGrady; D Roeper; K Pandya; G Cheek

    2011-12-31

    The structures of anhydrous nickel, niobium, and tantalum chlorides have been investigated in situ in acidic and basic ionic liquids (ILs) of 1-methyl-3-ethylimidazolium chloride (EMIC)/AlCl{sub 3} with X-ray absorption spectroscopy (XAS). The coordination of NiCl{sub 2} changes from tetrahedral in basic solution to octahedral in acidic solution. The NiCl{sub 2} is a strong Lewis acid in that it can induce the AlCl{sub 3} to share its chlorides in the highly acidic IL, forming a structure with six near Cl{sup -} ions and eight further distant Al ions which share the chloride ions surrounding the Ni{sup 2+}. When Nb{sub 2}Cl{sub 10}, a dimer, is added to the acidic or basic solution, the dimer breaks apart and forms two species. In the acid solution, two trigonal bipyramids are formed with five equal chloride distances, while in the basic solution, a square pyramid with four chlorides forming a square base and one shorter axial chloride bond. Ta{sub 2}Cl{sub 10} is also a dimer and divides into half in the acidic solution and forms two trigonal bipyramids. In the basic solution, the dimer breaks apart but the species formed is sufficiently acidic that it attracts two additional chloride ions and forms a seven coordinated tantalum species.

  7. Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines

    PubMed Central

    Kim, Minseo; Lee, Ho K.; Kim, Jin-Hee; Jeong, Jin-Hyun; Namkung, Wan

    2015-01-01

    The expression levels of anoctamin 1 (ANO1, TMEM16A), a calcium-activated chloride channel (CaCC), are significantly increased in several tumors, and inhibition of ANO1 is known to reduce cell proliferation and migration. Here, we performed cell-based screening of a collection of natural products and drug-like compounds to identify inhibitors of ANO1. As a result of the screening, idebenone, miconazole and plumbagin were identified as novel ANO1 inhibitors. Electrophysiological studies showed that idebenone, a synthetic analog of coenzyme Q10, completely blocked ANO1 activity in FRT cells expressing ANO1 without any effect on intracellular calcium signaling and CFTR, a cAMP-regulated chloride channel. The CaCC activities in PC-3 and CFPAC-1 cells expressing abundant endogenous ANO1 were strongly blocked by idebenone. Idebenone inhibited cell proliferation and induced apoptosis in PC-3 and CFPAC-1 cells, but not in A549 cells, which do not express ANO1. These data suggest that idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy. PMID:26196390

  8. Chloride substitution in sodium borohydride

    SciTech Connect

    Ravnsbaek, Dorthe B.; Rude, Line H.; Jensen, Torben R.

    2011-07-15

    The dissolution of sodium chloride and sodium borohydride into each other resulting in formation of solid solutions of composition Na(BH{sub 4}){sub 1-x}Cl{sub x} is studied. The dissolution reaction is facilitated by two methods: ball milling or combination of ball milling and annealing at 300 deg. C for three days of NaBH{sub 4}-NaCl samples in molar ratios of 0.5:0.5 and 0.75:0.25. The degree of dissolution is studied by Rietveld refinement of synchrotron radiation powder X-ray diffraction (SR-PXD) data. The results show that dissolution of 10 mol% NaCl into NaBH{sub 4}, forming Na(BH{sub 4}){sub 0.9}Cl{sub 0.1}, takes place during ball milling. A higher degree of dissolution of NaCl in NaBH{sub 4} is obtained by annealing resulting in solid solutions containing up to 57 mol% NaCl, i.e. Na(BH{sub 4}){sub 0.43}Cl{sub 0.57}. In addition, annealing results in dissolution of 10-20 mol% NaBH{sub 4} into NaCl. The mechanism of the dissolution during annealing and the decomposition pathway of the solid solutions are studied by in situ SR-PXD. Furthermore, the stability upon hydrogen release and uptake were studied by Sieverts measurements. - Graphical Abstract: Dissolution of sodium chloride and sodium borohydride into each other resulting in formation of solid solutions of composition Na(BH{sub 4}){sub 1-x}Cl{sub x} is studied. Dissolution is facilitated by two methods: ball milling or annealing at 300 deg. C for three days of NaBH{sub 4}-NaCl samples. Sample compositions and dissolution mechanism are studied by Rietveld refinement of synchrotron radiation powder X-ray diffraction data. Highlights: > Studies of dissolution of sodium chloride and sodium borohydride into each other. > Solid state diffusion facilitated by mechanical and thermal treatments. > Dissolution is more efficiently induced by heating than by mechanical treatment. > Mechanism for dissolution studied by Rietveld refinement of in situ SR-PXD data.

  9. Glutamate-gated Chloride Channels*

    PubMed Central

    Wolstenholme, Adrian J.

    2012-01-01

    Glutamate-gated chloride channels (GluCls) are found only in protostome invertebrate phyla but are closely related to mammalian glycine receptors. They have a number of roles in these animals, controlling locomotion and feeding and mediating sensory inputs into behavior. In nematodes and arthropods, they are targeted by the macrocyclic lactone family of anthelmintics and pesticides, making the GluCls of considerable medical and economic importance. Recently, the three-dimensional structure of a GluCl was solved, the first for any eukaryotic ligand-gated anion channel, revealing a macrocyclic lactone-binding site between the channel domains of adjacent subunits. This minireview will highlight some unique features of the GluCls and illustrate their contribution to our knowledge of the entire Cys loop ligand-gated ion channel superfamily. PMID:23038250

  10. Sodium-metal chloride batteries

    NASA Technical Reports Server (NTRS)

    Ratnakumar, B. V.; Attia, A. I.; Halpert, G.

    1992-01-01

    It was concluded that rapid development in the technology of sodium metal chloride batteries has been achieved in the last decade mainly due to the: expertise available with sodium sulfur system; safety; and flexibility in design and fabrication. Long cycle lives of over 1000 and high energy densities of approx. 100 Wh/kg have been demonstrated in both Na/FeCl2 and Na/NiCl2 cells. Optimization of porous cathode and solid electrolyte geometries are essential for further enhancing the battery performance. Fundamental studies confirm the capabilities of these systems. Nickel dichloride emerges as the candidate cathode material for high power density applications such as electric vehicle and space.