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Results for “Chloride Channel Agonists”

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PubMed2005

Differential ion current activation by human 5-HT(1A) receptors in Xenopus oocytes: evidence for agonist-directed trafficking of receptor signalling.

The subject of the present study was the functional and pharmacological characterization of human 5-HT(1A) receptor regulation of ion channels in Xenopus oocytes. Activation of the heterologously expressed human 5-HT(1A) receptor induced two distinct currents in Xenopus oocytes, consisting of a smooth inward current (I(smooth)) and an oscillatory calcium-activated chloride current, I(Cl(Ca)). 5-HT(1A) receptor coupling to both ionic responses as well as to co-expressed inward rectifier potassium (GIRK) channels was pharmacologically characterized using 5-HT(1A) receptor agonists. The relative order of efficacy for activation of GIRK current was 5-HT approximately F 13714 approximately L 694,247 approximately LY 228,729>flesinoxan approximately (+/-)8-OH-DPAT. In contrast, flesinoxan and (+/-)8-OH-DPAT typically failed to activate I(Cl(Ca)). The other ligands behaved as full or partial agonists, exhibiting an efficacy rank order of 5-HT approximately L 694,247>F 13714 approximately LY 228,729. The pharmacological profile of I(smooth) activation was completely distinct: flesinoxan and F 13714 were inactive and rather exhibited an inhibition of this current. I(smooth) was activated by the other agonists with an efficacy order of L 694,247>5-HT approximately LY 228,729>(+/-)8-OH-DPAT. Moreover, activation of I(smooth) was not affected by application of pertussis toxin or the non-hydrolyzable GDP-analogue, guanosine-5'-O-(2-thio)-diphosphate (GDP betaS), suggesting a GTP binding protein-independent pathway. Together, these results suggest the existence of distinct and agonist-specific signalling states of this receptor.

Animals↗

PubMed1997

Inhibition of intestinal Cl- secretion by clotrimazole: direct effect on basolateral membrane K+ channels.

We evaluated the effects of clotrimazole and clofibrate on Ca(2+)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO) to activate the Ca(2+)-dependent K+ channel (KCa) in these cells to induce a sustained Cl- secretory current (Isc). Clotrimazole potently inhibited the KCa-dependent Isc, with an inhibition constant (Ki) of 0.27 +/- 0.02 microM. Clofibrate also inhibited the 1-EBIO-induced Isc albeit with lower affinity (Ki = 6.5 +/- 1.2 microM). Clotrimazole (10 microM) inhibited the Isc response to the Ca(2+)-mediated agonist, carbachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with Ki values of 5.2 +/- 1.0 and 6.7 +/- 1.1 microM, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and clofibrate on the basolateral K+ (IK) and apical Cl- (ICl) currents following stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced IK without affecting ICl. We determined the effects of clotrimazole and clofibrate on KCa using 86Rb+ uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced 86Rb+ uptake, with Ki values of 0.31 +/- 0.08 and 10.8 +/- 5.5 microM, respectively. Similarly, clotrimazole inhibited the Ca(2+)-induced 86Rb+ uptake with a Ki of 0.51 +/- 0.15 microM. Charybdotoxin inhibited both the 1-EBIO- and Ca(2+)-induced 86Rb+ uptakes with similar affinities (Ki values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggesting 1-EBIO and Ca2+ activate the same channel (KCa) in this assay. In excised, single-channel recordings both clotrimazole and clofibrate inhibited KCa, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal KCa, thereby inhibiting Cl- secretion. These results suggest that clotrimazole may be useful as an antidiarrheal.

Benzimidazoles↗

PubMed1989

Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor.

The inhibitory postsynaptic glycine receptor (GlyR) of rat spinal cord is an oligomeric transmembrane protein which forms an agonist-gated anion channel. Expression in Xenopus oocytes of its mol. wt 48,000 subunit generated glycine-gated chloride channels which were analysed by voltage clamp. The agonist and antagonist response properties as well as the desensitization characteristics of these 48 kd subunit receptors resembled GlyRs expressed from spinal cord poly(A)+ RNA. These data indicate that the 48 kd subunit is capable of assembling into a functional receptor homo-oligomer which displays the pharmacology characteristic of the spinal cord GlyR.

Animals↗

PubMed1988

Altered gamma-aminobutyric acid/benzodiazepine interaction after chronic diazepam exposure.

Binding to components of the GABA/benzodiazepine receptor complex was examined in cortical membranes from rats treated for 3 weeks with continuously releasing diazepam pellet implants. Chronic diazepam treatment resulted in a decrease in the ability of GABA to inhibit benzodiazepine inverse agonist binding. The amount of binding of benzodiazepine agonist, antagonist, and inverse agonist to benzodiazepine recognition sites was unaltered by the chronic treatment, as were the potencies of these benzodiazepine ligands in inhibiting agonist (3H-flunitrazepam) binding. Chloride channel binding (35S-TBPS) was also unchanged by chronic diazepam treatment. A change in GABA/benzodiazepine coupling and/or a decreased effectiveness of GABA may be responsible for the desensitization of GABA/benzodiazepine interaction.

Animals↗

PubMed2001

Expression and biological significance of Ca2+-activated ion channels in human keratinocytes.

In whole-cell recordings from HaCaT keratinocytes, ATP, bradykinin, and histamine caused a biphasic change of the membrane potential consisting of an initial transient depolarization, followed by a pronounced and long-lasting hyperpolarization. Flash photolysis of caged IP3 mimicked the agonist-induced voltage response, suggesting that intracellular Ca2+ release and subsequent opening of Ca2+-activated ion channels serve as the common transduction mechanism. In contrast, cAMP- and PKC-dependent pathways were not involved in the electrophysiological effects of the extracellular signaling molecules. The depolarization was predominantly mediated by a DIDS- and niflumic acid-sensitive Cl- current, whereas a charybdotoxin- and clotrimazole-sensitive K+ current underlay the prominent hyperpolarization. Consistent with the electrophysiological data, RT-PCR showed that HaCaT keratinocytes express two types of Ca2+-activated Cl- channels, CaCC2 and CaCC3 (CLCA2), as well as the Ca2+-activated K+ channel hSK4. That the pronounced hSK4-mediated hyperpolarization bears significance on the growth and differentiation properties of keratinocytes is suggested by RNase protection assays showing that hSK4 mRNA expression is strongly down-regulated under conditions that allow keratinocyte differentiation. hSK4 might thus play a role in linking changes in membrane potential to the biological fate of keratinocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

Effect of intracerebroventricular picrotoxin and muscimol on intravenous bupivacaine toxicity. Evidence supporting central nervous system involvement in bupivacaine cardiovascular toxicity.

BACKGROUND: The authors have previously shown that administration of bupivacaine into the cerebral ventricles of rabbits results in CNS-mediated cardiac dysrhythmias and that these CNS-mediated dysrhythmias could be terminated by CNS administration of a drug (midazolam) that enhances GABAergic activity. The goal of the current investigation was to determine whether the CNS-mediated cardiotoxicity that occurs after direct CNS administration of bupivacaine contributes to bupivacaine's cardiovascular toxicity following intravenous administration. METHODS: Three groups of rabbits were pretreated by intracerebroventricular administration of mock CSF (control), muscimol (a GABA agonist), or picrotoxin (a GABA chloride channel blocker). A fourth group received intravenous hexamethonium as a pretreatment. After pretreatment, all groups received intravenous bupivacaine at 1.5 mg.kg-1.min-1 and the bupivacaine dose and plasma concentration at the onset of dysrhythmias and cardiovascular collapse were determined. It was hypothesized that pretreatment with muscimol and hexamethonium would increase the threshold for cardiac dysrhythmias, while pretreatment with picrotoxin would decrease the threshold for dysrhythmias. RESULTS: The bupivacaine plasma concentration required to produce cardiac dysrhythmias was significantly greater in the muscimol and hexamethonium groups compared to control. The bupivacaine plasma concentration at the onset of dysrhythmias was not different from control in the picrotoxin group. The bupivacaine plasma concentration that produced cardiovascular collapse did not differ from control in any of the groups. CONCLUSIONS: It was concluded that the data generally support the hypothesis that bupivacaine-mediated cardiac dysrhythmias are, at least in part, mediated by CNS actions of the drug.

Animals↗

PubMed1982

Effect of barbiturates on the GABA receptor of cat primary afferent neurones.

1. The effects of the barbiturate anaesthetics, pentobarbitone and thiopentone, on the membrane properties and the gamma-aminobutyric acid (GABA)-induced responses of cat primary afferent neurones were studied with intracellular recording and voltageclamp techniques.2. At low concentrations (10(-7)-10(-5) M) both barbiturates slightly enhanced and prolonged GABA-induced depolarizations or currents without affecting the membrane properties. At these concentrations, barbiturates have no effect on the apparent dissociation constant of the GABA-GABA receptor interaction or the reversal potential for GABA-induced depolarizations or currents.3. At high concentrations (10(-4)-10(-3) M) barbiturates produced a few millivolts reduction in the resting membrane potential. Voltage-clamp analysis revealed that the depolarization was associated with one of the three types of conductance change, i.e., an initial increase followed by a decrease (40% of neurones examined), only an increase (40%) and only a decrease (20%).4. Analysis in different ionic media indicated that the depolarization with a reduced membrane resistance is associated with an increased chloride conductance and that the one with an increased membrane resistance is accompanied by a reduction in potassium conductance. Bath-application of GABA (10(-3) M) or picrotoxin (10(-5) M) inhibited the increase in chloride conductance but not the reduction in potassium conductance.5. Barbiturates at these high concentrations initially caused a marked augmentation and prolongation of GABA responses; this was followed by a depression. The depressant action did not appear to be voltage-dependent. These actions of barbiturates were not accompanied by changes in the apparent dissociation constant of the GABA-current dose-response curve or the reversal potential for GABA currents. In addition, the single exponential decay of GABA current was not changed despite a marked prolongation of its decay time.6. Picrotoxin (10(-5) M) antagonized the depressant effect of barbiturates at high concentrations on GABA currents, and barbiturates (5 x 10(-6) M) reduced the inhibitory action of picrotoxin (5 x 10(-6) M) on the GABA-currents.7. From all these results, it is suggested that the site of barbiturate actions on GABA-responses is mainly the allosteric site (the ionic conductance regulatory subunit) but not the agonist recognition site or the chloride channels linked with GABA receptors.

Action Potentials↗

PubMed2006

Treating chronic constipation : How should we interpret the recommendations?

Chronic constipation is a multisymptom gastrointestinal motility disorder that negatively impacts the lives of those affected. New recommendations on diagnosing and treating this condition have recently been published. This review aims to assist clinicians in applying these recommendations in clinical practice. Although lifestyle interventions and bulking agents help some patients with constipation, data are lacking to support their efficacy in those with chronic constipation. If empirical treatment of patients with chronic constipation fails, osmotic laxatives (e.g. lactulose and polyethylene glycol), a serotonin (5-HT4) receptor agonist (e.g. tegaserod) or a chloride channel activator (e.g. lubiprostone) can be considered. Osmotic laxatives, though effective at increasing stool frequency, are often associated with adverse effects such as bloating and diarrhoea and typically do not effectively relieve the multiple symptoms of chronic constipation. Use of tegaserod and the recently approved lubiprostone is supported by high-quality evidence, and these agents have acceptable safety profiles in patients with chronic constipation. Recent advances in the definition of chronic constipation and in its pharmacological management have led to refinements in identifying the appropriate treatment needs of patients with this condition. It is hoped that these advancements will help clinicians select effective medical regimens for patients with chronic constipation.

Chronic Disease↗

PubMed1999

Characterization of chloride currents and their noradrenergic modulation in rat taste receptor cells.

Taste receptor cells contain a heterogeneous array of voltage-dependent ion conductances that are essential components for the transduction of gustatory stimuli. Although mechanistic roles have been proposed for several cationic conductances, the understanding of anionic currents is rudimentary. This study characterizes biophysical and pharmacological properties of chloride currents in rat posterior taste cells using whole cell patch-clamp recording technique. Taste cells express a heterogeneous array of chloride currents that displayed strong outward rectification, contained both calcium-dependent and calcium-independent components, and achieved a maximal conductance of almost 1 nS. Reversal potentials altered predictably with changes in chloride concentration. Currents were sensitive to inhibition by the chloride channel pharmacological agents DIDS, SITS, and niflumic acid but were insensitive to 9-AC. Adrenergic enhancement of chloride currents, present in other cell types, was tested on taste cells with the beta-adrenergic agonist isoproterenol (ISP). ISP enhanced the outwardly rectifying portion of the chloride current. This enhancement was calcium dependent and was blocked by the beta-adrenergic antagonist propranolol. Collectively these observations suggest that chloride currents may participate not only in usually ascribed functions such as stabilization of the membrane potential and volume regulation but additionally play active modulatory roles in the transduction of gustatory stimuli.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Dual action of isoflurane on the gamma-aminobutyric acid (GABA)-mediated currents through recombinant alpha(1)beta(2)gamma(2L)-GABA(A)-receptor channels.

Isoflurane (ISO) increased the agonist-induced chloride flux through the gamma-aminobutyric acid A receptor (GABA(A)R). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABA(A)R. We tested the hypothesis that, in addition to a blocking effect, ISO increases gamma-aminobutyric acid (GABA)-gated currents through recombinant GABA(A)R channels. HEK293 cells were transfected with rat cDNA for alpha(1),beta(2),gamma(2L) subunits. Currents elicited by 1 mM or 0. 01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABA(A)R. The balance between potentiation and block depends on the concentrations of both ISO and GABA.

Anesthetics, Inhalation↗

PubMed1993

Enhancement of gamma-aminobutyric acidA receptor function and binding by the volatile anesthetic halothane.

The volatile general anesthetics halothane and enflurane increased muscimol-stimulated 36Cl- efflux via gamma-aminobutyric acid (GABA)A receptors in rat brain cortical slices and also increased basal 36Cl- efflux in the absence of GABA agonist. The effects occurred in the clinical range of anesthetic concentrations (0.56-1.7 mM halothane and 0.46-1.4 mM enflurane). Both anesthetics induced a slow onset increase in basal 36Cl- efflux rate when added alone with no exogenous GABA agonist. This direct effect of halothane had a biphasic dependence on anesthetic concentrations, with a maximal effect in the range 1.1 to 1.7 mM. Replacing extracellular calcium with magnesium or blocking voltage-gated calcium entry with cobalt (200 microM) altered the direct halothane effect, shifting the concentration-dependence curve to the right. Halothane direct potentiation of chloride flux in the absence of GABA agonist was blocked by the GABAA chloride channel antagonist picrotoxin but not by the GABAA receptor antagonist bicuculline. The halothane potentiation of the muscimol response was detectable at concentrations of 0.56 mM halothane in the assay buffer, and was linear with concentration up to 2.8 mM. The effect was more pronounced at low GABA agonist concentrations, apparently due to an increase in GABA affinity. Lowering the extracellular calcium concentration to micromolar levels did not affect halothane potentiation of muscimol responses. Halothane at similar concentrations increased the high-affinity binding of [3H]muscimol to GABAA receptor sites in rat brain cortical membranes in a calcium-independent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

GABAB receptor-mediated inhibition of spontaneous action potential discharge in rat supraoptic neurons in vitro.

To elucidate the role of GABAB receptors in the regulation of the electrical activity of magnocellular neurons of the supraoptic nucleus (SON), the effects of GABAB agonist and antagonist on the firing rate of spontaneous action potentials were studied in SON slice preparations of rats by extracellular recordings. In the presence of the gamma-amino butyric acid (GABA)-gated chloride channel blocker, picrotoxin, the selective GABAB agonist, baclofen, reduced the firing rate of action potentials in both phasic and non-phasic neurons in a dose-dependent manner. The reduction in the firing rate induced by baclofen was reversed by the selective GABAB antagonist, 2-hydroxy saclofen (2OH-saclofen), also in a dose-dependent manner. In non-phasic neurons, 2OH-saclofen significantly increased the firing rate and the effect was additive to the effect of picrotoxin. In phasic neurons, 2OH-saclofen alone did not increase the firing rate, but it reversed suppression of the firing induced by increasing extracellular Ca2+ concentration to 2.1 mM. Baclofen also reduced the firing rate of non-phasic neurons of virgin and lactating female rats, indicating that the GABAB receptor-mediated inhibition is not confined to SON neurons of male rats. The evidence indicates that activation of GABAB receptors inhibits electrical activity of SON neurons of both male and female rats and that GABAB receptors may play an important role in the inhibitory regulation of the electrical activity of SON neurons by GABA.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1990

Stimulation of luteinizing hormone release by gamma-aminobutyric acid (GABA) agonists: mediation by GABAA-type receptors and activation of chloride and voltage-sensitive calcium channels.

The mechanism by which gamma-aminobutyric acid (GABA) stimulates the release of LH was analyzed in cultured female rat pituitary cells. In 3-h incubations, GABA (1-100 microM) caused a dose-dependent increase in LH release, with the maximal response about 16% of that evoked by 10 nM GnRH. GABA action was independent of the GnRH receptor, since 1 microM GnRH antagonist [( N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10] GnRH), which completely inhibits GnRH action, did not affect the response to GABA. In studies on the effects of GABA receptor agonists and antagonists, 4,5,6,7-tetrahydoisoxazolo-[5,4-c]pyridin-3(2H)-one (THIP) and muscimol (GABAA agonists) gave similar response patterns, with the same maximal stimulation as GABA but much higher potencies. In contrast, the GABAB receptor agonist baclofen did not stimulate LH release. The GABAA receptor antagonist SR95531 caused dose-dependent inhibition of the LH-releasing effects of GABA and muscimol (10 microM), with complete blockade at 10 microM SR95531. T-Butylbicyclophosphorothionate, an inhibitor of the GABAA receptor-associated chloride channel, also dose-dependently reduced the releasing effect of 100 microM GABA. These results indicate that GABA action is mediated by the chloride channel-associated GABAA receptor. However, the other GABAA receptor antagonists, including bicuculline, picrotoxin, and strychnine, did not attenuate the LH-releasing effect of 100 microM GABA in concentrations up to 100 microM, suggesting that GABA action is mediated by nonclassical GABAA receptors. Incubation in the presence of nifedipine (1 microM) or in calcium-free medium inhibited the LH-releasing action of GABA, indicating that calcium influx through voltage-sensitive calcium channels (VSCC) is required for GABA-induced LH release. Such entry of Ca2+ would result from activation of VSCC by depolarization due to the increased Cl- conductance caused by GABAA receptor activation. In cell perfusion studies, the actions of GABA and muscimol were attenuated or abolished after repetitive stimulation, consistent with desensitization of the GABA receptors. These findings have demonstrated that the stimulation of LH release by GABA is independent of GnRH action, occurs via binding to nonclassical GABAA receptors, which rapidly desensitize, and is mediated by the activation of VSCC.

Amino Acid Sequence↗

PubMed1999

1-EBIO stimulates Cl- secretion by activating a basolateral K+ channel in the mouse jejunum.

We investigated the effects of 1-ethyl-2-benzimidazolinone (1-EBIO) on ion transport in the mouse jejunum through the use of the short-circuit (Isc) current technique and the application of the patch-clamp technique to isolated jejunal crypts. In HCO3- Ringer's, 1-EBIO stimulated a dose-dependent (EC50 964 micromol/l), bumetanide-sensitive increase in Isc consistent with stimulation of Cl- secretion. In contrast, in Cl(-)-free HCO3-Ringer's containing glucose, 1-EBIO (500 micromol/l) did not increase the phloridzin (100 micromol/l) sensitive Isc, suggesting that electrogenic Na+ absorption was unaltered. Measurement of the membrane potential (Vm) with the perforated-patch technique indicated that in isolated crypts, 1-EBIO caused a reversible hyperpolarization of Vm and an increase in the change in Vm associated with step changes in bath K+, consistent with an increase in K+ conductance. In on-cell patch experiments with KCI Ringer's in the patch pipette and crypts bathed with NaCl Ringer's, 1-EBIO (500 micromol/l) increased the open probability (NPo; 0.01+/-0.01 to 0.45+/-0.11, n=7) of an inwardly rectified intermediate conductance (g) channel. In inside-out patches with KCl Ringer's in the patch pipette and KCI Ringer's containing 100 nmol/l Ca2+ in the bath, the current-voltage relationship of the channel was inwardly rectified (g of 10 and 52 pS at -Vp of 100 and -100 mV, respectively) and reversed at 0 mV (n=5). Replacement of bath K+ with Na+ shifted the reversal potential toward the equilibrium potential for K+. In the presence of 1-EBIO, reducing the bath Ca2+ from 200 nmol/l to nominally Ca(2+)-free conditions decreased NPo from 0.90+/-0.27 to 0.07+/-0.03 (n=3). We conclude that in the mouse jejunum, I-EBIO does not stimulate electrogenic Na+ absorption. It does, however, stimulate secretion primarily through the activation of a basolateral, intermediate conductance Ca(2+)-sensitive K+ channel.

Animals↗

PubMed2000

Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of DeltaF508-CFTR is as important as the trafficking defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis.

3T3 Cells↗

PubMed2002

Ca2+-activated Cl- channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection.

Little is known of the excitatory mechanisms that contribute to the tonic contraction of the corpus cavernosum smooth muscle in the flaccid state. We used patch-clamp electrophysiology to investigate a previously unidentified inward current in freshly isolated rat and human corporal myocytes. Phenylephrine (PE) contracted cells and activated whole cell currents. Outward current was identified as large-conductance Ca(2+)-activated K(+) current. The inward current elicited by PE was dependent on the Cl(-) gradient and was inhibited by niflumic acid, indicative of a Ca(2+)-activated Cl(-) (Cl(Ca)) current. Furthermore, spontaneous transient outward and inward currents (STOCs and STICs, respectively) were identified in both rat and human corporal myocytes and derived from large-conductance Ca(2+)-activated K(+) and Cl(Ca) channel activity. STICs and STOCs were inhibited by PE and A-23187, and combined 8-bromoadenosine cAMP and 8-bromoadenosine cGMP decreased their frequency. When studied in vivo, chloride channel blockers transiently increased intracavernosal pressure and prolonged nerve-evoked erections. This report reveals for the first time Cl(Ca) current in rat and human corpus cavernosum smooth muscle cells and demonstrates its key functional role in the regulation of penile erection.

Adrenergic alpha-Agonists↗

PubMed1997

ATP-induced Cl- secretion with suppressed Na+ absorption in rabbit tracheal epithelium.

The effect of extracellular ATP on ion transport of rabbit tracheal epithelium was examined using an Ussing chamber. Isoproterenol (10(-8)-10(-5) M) did not alter the electrophysiological properties across the tracheal epithelium. Apically applied ATP induced an initial transient increase in short circuit current (SCC) followed by a decline to below the prior baseline. The initial increase by ATP (10(-4) M) was significantly inhibited by a Cl(-) -channel inhibitor diphenylamine-2-carboxylate (DPC, 5 x 10(-4) M) and Cl(-) -substitution with gluconate in the bath solution, while a cystic fibrosis transmembrane regulator (CFTR) Cl(-) -channel inhibitor glibenclamide (10(-4) M), a Na(+)-channel inhibitor amiloride (10(-4) M) and a K(+) -channel inhibitor quinidine (10(-4) M) all failed to alter it. The decline in SCC by ATP was abolished by amiloride, while DPC or Cl-substitution with gluconate in the bath solution did not alter it. Ca(2+)-removal from the bath solutions did not significantly alter the initial increase nor the decline by ATP. Ionomycin (10(-5) M) induced an initial transient increase in SCC, to a degree similar to that by ATP alone. A calmodulin antagonist W-7 reduced the SCC baseline and abolished SCC increase by ATP. These findings indicate that ATP activates Ca(2+)-dependent Cl(-) -channels with an inhibition of Na -channel activity or absorption in rabbit tracheal epithelium.

Adenosine Triphosphate↗

PubMed2006

Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium channels in human airway epithelial cells.

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The most common mutation, DeltaF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of DeltaF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca(2+)-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca(2+)-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca(2+)-activated K(+) channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K(+) channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca(2+)-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca(2+)-activated secretion.

Benzimidazoles↗