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PubMed1989

Long-term inhibition of angiotensin converting enzyme suppresses calcium channel agonist-induced contraction of aorta in hypertensive rats.

To elucidate functional changes in the vascular smooth muscle of spontaneously hypertensive rats (SHR) after chronic inhibition of angiotensin converting enzyme, we examined the contractile responses to different pharmacological interventions in the isolated aortas from SHR treated with a novel angiotensin converting enzyme inhibitor, CS-622 (10 mg/kg/day) for 20 weeks. In normal K+ medium, a marked contraction was elicited by increasing Ca2+ concentration from 0 to 3 mM in aortas from a control group of SHR, but not in aortas from SHR treated with CS-622. In 60 mM K+ medium, however, the sensitivity of aorta to Ca2+ was almost the same in the two groups. A calcium channel activator, CGP-28392 (10(-7) to 10(-6) M), induced a marked contraction in the aortas from control SHR, but not in the aortas from CS-622-treated SHR. When slightly depolarized in 10 or 12 mM K+ solution, the aortas from CS-622-treated SHR contracted in response to CGP-28392. The aortic sensitivity to KCl contraction was much lower in CS-622-treated SHR than in untreated SHR, whereas the sensitivity to phenylephrine contraction was little different in the two groups. These contractile profiles of aortas from CS-622-treated SHR were very similar to those from normotensive Wistar-Kyoto rats but not to those from hydralazine-treated SHR. These data suggest that contractions due to Ca2+ through voltage-dependent calcium channels are exaggerated in SHR aorta and that long-term treatment with angiotensin converting enzyme inhibitor suppresses the abnormal contractility of SHR vascular smooth muscle, probably through alterations of voltage-related functions of calcium channels.

Animals↗
PubMed1993

Altered responses to agonists after chronic in vivo atropine administration in rat parotid acini.

Salivary gland hypofunction, resulting from a variety of perturbations including prescribed medications, is associated with adverse effects on the health of the oral cavity. In the present study, we investigated the in vivo effects of chronic administration of atropine, a muscarinic antagonist, on the acute response of rat parotid acini to alpha-adrenergic and muscarinic stimulation. The regulation of intracellular pH (pHi) and cytosolic free Ca2+ ([Ca2+]i) were monitored using dual wavelength microfluorometry of the ion-sensitive fluorescent dyes, BCECF and fura-2, respectively. Chronic atropine treatment (40 mg/kg/d for 4 weeks) significantly increased the magnitude of the initial (< 30 s) agonist-induced rise in [Ca2+]i, but did not alter the sustained increase in [Ca2+]i (> 2 min). The generation of inositol trisphosphates and inositol tetrakisphosphates after 30 s of muscarinic stimulation was not significantly altered. The resting Cl- content as well as the stimulated Cl- loss, were reduced in parotid acini after chronic atropine administration. In addition, the muscarinic- and alpha-adrenergic-induced intracellular acidification was blunted, suggesting that reduced HCO3- efflux occurs in acini isolated from atropine-treated animals. Our results indicate (1) that chronic atropine treatment does not inhibit the receptor-coupled generation of inositol phosphates or the resulting rise in [Ca2+]i and (2) chronic treatment may prevent the production of saliva either by reducing the driving force for anion-dependent fluid secretion or by preventing the activation of the anion efflux pathway.

Animals↗
PubMed1998

Regulation of glucagon-like peptide-1 synthesis and secretion in the GLUTag enteroendocrine cell line.

Glucagon-like peptide-1 (GLP-1) released from the intestine is a potent stimulator of glucose-dependent insulin secretion. To elucidate the factors regulating GLP-1 secretion, we have studied the enteroendocrine GLUTag cell line. GLP-1 secretion was stimulated in a dose-dependent fashion by activation of protein kinase A or C with forskolin or phorbol 12,13-dibutyrate, respectively (by 2.3 +/- 0.5-fold at 100 microM and 4.3 +/- 0.6-fold at 0.3 microM, respectively; P < 0.01-0.001). Of the regulatory peptides tested, only glucose-dependent insulinotropic peptide stimulated the release of GLP-1 (by 2.3 +/- 0.2-fold at 0.1 microM; P < 0.001); glucagon was without effect, and paradoxically, the inhibitory neuropeptide somatostatin-14 increased secretion slightly (by 1.6 +/- 0.3-fold at 0.01 microM; P < 0.05). In tests of several neurotransmitters, only the cholinergic agonists carbachol and bethanechol stimulated peptide secretion in a dose-dependent fashion (by 2.3 +/- 0.5- and 1.7 +/- 0.3-fold at 1000 microM; P < 0.05-0.001); the beta-adrenergic agonist isoproterenol and the chloride channel inhibitor gamma-aminobutyric acid did not affect release of GLP-1. Long chain monounsaturated fatty acids (18:1), but not saturated fatty acids (16:0), also stimulated the release of GLP-1 (by 1.7 +/- 0.1-fold at 150 microM; P < 0.001). Consistent with the presence of a cAMP response element in the proglucagon gene, activation of the protein kinase A-dependent pathway with forskolin increased proglucagon messenger RNA transcript levels by 2-fold (P < 0.05); glucose-dependent insulinotropic peptide and phorbol 12,13-dibutyrate were without effect. Therefore, by comparison with results obtained using primary L cell cultures or in vivo models, GLUTag cells appear to respond appropriately to the regulatory mechanisms controlling intestinal GLP-1 secretion.

Animals↗
PubMed2003

TNF-alpha rapidly antagonizes the beta-adrenergic responses of the chloride current in guinea-pig ventricular myocytes.

The purpose of this study was to test the hypothesis that tumor necrosis factor-alpha (TNF-alpha) rapidly antagonizes the beta-adrenergic responses of the chloride current and to clarify the intracellular mechanisms responsible for the anti-adrenergic action. The whole-cell patch-clamp technique was used to monitor the anti-adrenergic effects of TNF-alpha on the cAMP-dependent chloride current (I(Cl)) recorded from isolated guinea-pig ventricular myocytes. Ramp pulses (+/-120 mV; dv/dt = +/-0.4 V/s) were applied from the holding potential of -40 mV. TNF-alpha rapidly (<15 min) inhibited the isoproterenol (Iso, 0.1 micromol/L)-induced I(Cl) in a concentration-dependent manner (30-1,000 U/ml, IC (50) = 144 U/ml, n=30). The inhibitory action of TNF-alpha was also observed when I(Cl) had been previously stimulated by 1 micromol/L forskolin (n=5). Prior exposure of myocytes to 5 microg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). However, when I(Cl) was induced by both 8-bromo-cAMP (100 micromol/L) and isobutylmethylxanthine (0.1 mmol/L), TNF-alpha (1,000 U/ml) failed to decrease I(Cl) amplitude (n=5). Prior exposure of myocytes to 5 mg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). Furthermore, despite of the presence of nitro-L-arginine methyl ester (0.1 mmol/L), a nitric oxide synthase (NOS) inhibitor, TNF-alpha reversed the Iso-induced increase in I(Cl) (n=5). These results suggest that TNF-alpha rapidly antagonizes the beta-adrenergic responses of I(Cl) by reducing cAMP concentration. This anti-adrenergic action is mediated by neither the PTX-sensitive G proteins regulatory pathway nor constitutive NOS activation.

Adrenergic beta-Agonists↗
PubMed2001

Possible mechanisms of diarrheal side effects associated with the use of a novel chemotherapeutic agent, flavopiridol.

The novel cyclin-dependent kinase inhibitor flavopiridol has recently completed Phase I trials for the treatment of refractory neoplasms. The dose-limiting toxicity observed with this agent was severe diarrhea. Because the compound otherwise showed promise, the present study sought to determine possible mechanisms underlying the diarrheal side effects. Flavopiridol was tested for its ability to modify chloride secretory responses of the human colonic epithelial cell line, T84. Studies were conducted in vitro in modified Ussing chambers. High concentrations of flavopiridol (10(-4) M), above those likely to be clinically relevant, had a direct stimulatory effect on chloride secretion, probably ascribable to an increase in cyclic AMP. Lower, clinically relevant concentrations of flavopiridol (10(-6) M) had no effect on chloride secretion by themselves but potentiated responses to the calcium-dependent secretagogue, carbachol. The drug also potentiated responses to thapsigargin and taurodeoxycholate and reversed the inhibitory effects of carbachol and epidermal growth factor on calcium-dependent chloride secretion. Pretreatment with the cyclic AMP-dependent secretagogue, forskolin, potentiated responses to flavopiridol, but not vice versa. Thus, diarrheal side effects induced by flavopiridol are likely multifactorial in origin and may involve interactions with endogenous secretagogues such as acetylcholine and bile acids. A better understanding of the diarrhea induced by flavopiridol should allow optimization of therapy with this otherwise promising drug and/or the development of related agents with improved toxicity profiles.

Antineoplastic Agents↗
PubMed2005

Benzodiazepines affect channel opening of GABA A receptors induced by either agonist binding site.

Benzodiazepines are widely used as anxiolytics, sedatives, muscle relaxants, and anticonvulsants. They allosterically modulate GABA type A (GABA(A)) receptors by increasing the apparent affinity of the agonist GABA to elicit chloride currents. Such an increase in apparent affinity of channel gating could either be caused by an increase in affinity for GABA or by a facilitation of channel opening. In the first case, conformation of the affected sites would have to be altered. In the second case, the affected sites are not necessarily altered, because diazepam could facilitate conformational changes leading to the open channel. It is controversial as to whether benzodiazepines affect only channel opening induced by the occupation of one of the two agonist binding sites or by both. We used receptors formed by concatenated subunits to selectively destroy one of the two agonist sites by point mutation. Both of the receptors harboring only one active agonist site could be stimulated by diazepam. We therefore present evidence that binding of diazepam can affect channel opening induced by either agonist binding site.

Animals↗
PubMed1999

Chloride channels and salivary gland function.

Fluid and electrolyte transport is driven by transepithelial Cl- movement. The opening of Cl- channels in the apical membrane of salivary gland acinar cells initiates the fluid secretion process, whereas the activation of Cl- channels in both the apical and the basolateral membranes of ductal cells is thought to be necessary for NaCl re-absorption. Saliva formation can be evoked by sympathetic and parasympathetic stimulation. The composition and flow rate vary greatly, depending on the type of stimulation. As many as five classes of Cl- channels with distinct gating mechanisms have been identified in salivary cells. One of these Cl- channels is activated by intracellular Ca2+, while another is gated by cAMP. An increase in the intracellular free Ca2+ concentration is the dominant mechanism triggering fluid secretion from acinar cells, while cAMP may be required for efficient NaCl re-absorption in many ductal cells. In addition to cAMP- and Ca(2+)-gated Cl- channels, agonist-induced changes in membrane potential and cell volume activate different Cl- channels that likely play a role in modulating fluid and electrolyte movement. In this review, the properties of the different types of Cl- currents expressed in salivary gland cells are described, and functions are proposed based on the unique properties of these channels.

Absorption↗
PubMed1993

Intracellular calcium ions activate a low-conductance chloride channel in smooth-muscle cells isolated from human mesenteric artery.

Calcium-activated chloride currents were studied by the patch-clamp technique in vascular smooth muscle cells (VSMC) isolated from human mesenteric arteries. Bath application of 20 mM caffeine caused the cell membrane to depolarize by a calcium-activated inward current that peaked to -654 +/- 230 pA (holding potential -50 mV). Cell-attached, at the same time inwardly directed single-channel currents were detected with an amplitude of -0.22 pA. In open-cell-attached patches channel activity was triggered by elevating [Ca2+]i to 10 microM. At -60 mV the mean amplitude of the current was -0.24 pA and the mean open time of the channels was 28 ms. Plotting the amplitude of the current versus the test potential yielded a single-channel conductance of 2.8 +/- 0.5 pS. The currents disappeared when [Cl-] was reduced from 150 mM to 5 mM at the cytosolic side of the inside-out patch at a holding potential of -60 mV (calculated reversal potential -58 mV) suggesting that the calcium-activated current was a chloride current. This suggests that, in human mesenteric VSMC, elevation of [Ca2+]i activates a low-conductance chloride channel, which may mediate the agonist-induced depolarization of the cell membrane.

Action Potentials↗
PubMed1993

Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca(2+)-mediated secretagogue-induced volume decrease in small-intestinal crypts.

The cellular volume of crypts isolated from 2- to 3-week-old mouse small intestine has been measured to assess the capacity of the epithelial cells to respond to secretagogues. Vasoactive intestinal polypeptide (VIP) or carbachol, respectively cAMP- and calcium-mediated secretagogues, produced a reduction crypt volume attributed to KCl loss through channels activated by the agonists. Consistent with the participation of separate chloride channels, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) blocked the carbachol- but not the VIP-induced volume decrease, whilst glibenclamide abolished the VIP effect without affecting the carbachol-induced volume decrease. Animals homozygous for a disrupted cftr gene, introduced by gene targeting, were also used as the source for crypt isolation. In these CFTR (-/-) crypts. VIP failed to elicit any reduction in cellular volume, while the response to carbachol was indistinguishable from that seen in crypts from age-matched control animals. These results are consistent with murine CFTR being a cAMP-activated chloride channel inhibited by glibenclamide and resistant to DIDS. A separate chloride conductance activated by calcium mobilization in small-intestinal crypts appears to be independent of CFTR.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

The electrical properties of auditory hair cells in the frog amphibian papilla.

The amphibian papilla (AP) is the principal auditory organ of the frog. Anatomical and neurophysiological evidence suggests that this hearing organ utilizes both mechanical and electrical (hair cell-based) frequency tuning mechanisms, yet relatively little is known about the electrophysiology of AP hair cells. Using the whole-cell patch-clamp technique, we have investigated the electrical properties and ionic currents of isolated hair cells along the rostrocaudal axis of the AP. Electrical resonances were observed in the voltage response of hair cells harvested from the rostral and medial, but not caudal, regions of the AP. Two ionic currents, ICa and IK(Ca), were observed in every hair cell; however, their amplitudes varied substantially along the epithelium. Only rostral hair cells exhibited an inactivating potassium current (IA), whereas an inwardly rectifying potassium current (IK1) was identified only in caudal AP hair cells. Electrically tuned hair cells exhibited resonant frequencies from 50 to 375 Hz, which correlated well with hair cell position and the tonotopic organization of the papilla. Variations in the kinetics of the outward current contribute substantially to the determination of resonant frequency. ICa and IK(Ca) amplitudes increased with resonant frequency, reducing the membrane time constant with increasing resonant frequency. We conclude that a tonotopically organized hair cell substrate exists to support electrical tuning in the rostromedial region of the frog amphibian papilla and that the cellular mechanisms for frequency determination are very similar to those reported for another electrically tuned auditory organ, the turtle basilar papilla.

4-Aminopyridine↗
PubMed1982

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary.

An in vitro perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release (IC50 = 6 X 10(-8)M and 10(-5)M respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.

Animals↗
PubMed1999

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.

Benzimidazoles↗
PubMed2001

Pharmacological treatment of the ion transport defect in cystic fibrosis.

Cystic fibrosis (CF) is a lethal monogenetic disease characterised by impaired water and ion transport over epithelia. The lung pathology is fatal and causes death in 95% of CF patients. The genetic basis of the disease is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most common mutation, DeltaF508, results in a protein that cannot properly be folded in the endoplasmic reticulum, is destroyed and hence does not reach the apical cell membrane. This paper will discuss those pharmacological approaches that are directed at correcting the defect in ion transport. At present, no clinically effective drug is available, although research has defined areas in which progress might be made. These are the following: (1) the drug 4-phenylbutyrate (4PBA) increases the expression of DeltaF508-CFTR in the cell membrane, probably by breaking the association between DeltaF508-CFTR and a chaperone; (2) a number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), are effective in activating CFTR, presumably by direct binding and also possibly by correcting the trafficking defect; (3) the isoflavone genistein can activate both wild-type and mutant CFTR, probably through direct binding to the channel; (4) purinergic agonists (ATP and UTP) can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR, but stable analogues will be required before this type of treatment has clinical significance; (5) treatment with inhaled amiloride may correct the excessive absorption of Na(+) ions and water by airway epithelial cells that appears connected to the defect in CFTR; although clinical tests have not been very successful so far, amiloride analogues with a longer half-life may give better results. The role of CFTR in bicarbonate secretion has not yet been established with certainty, but correction of the defect in bicarbonate secretion may be important in clinical treatment of the disease. Currently, major efforts are directed at developing a pharmacological treatment of the ion transport defect in CF, but much basic research remains to be done, in particular, with regard to the mechanism by which defective CFTR is removed in the endoplasmic reticulum by the ubiquitin-proteasome pathway, which is a central pathway in protein production and of significance for several other diseases apart from CF.

Amiloride↗
PubMed1989

Antagonism of ethanol effects by Ro 15-4513: an electrophysiological analysis.

Ethanol (ETH) and general anesthetics have been reported to facilitate the chloride channel opening, possibly, or at least partly, through an interaction with the GABA-benzodiazepine (BZ) receptor-gated chloride ionophore "supramolecular complex". Recently Ro 15-4513, a novel BZ ligand, has been indicated as a potent and selective antagonist of various ETH-induced behavioral and biochemical effects. However, since its precise characterization is still a matter of debate, we have tested and compared the effect of Ro 15-4513, as well as its antagonism against ETH, in two objective electrophysiological parameters, i.e., the electroencephalograph (EEG) pattern in freely moving rats and single unit activity of reticulata neurons. Ro 15-4513 produced an EEG state of alertness and antagonized the behavioral impairment and the EEG deterioration by ETH. However, while its protective action was consistent against moderate doses (2 g/kg) of ETH, it was much less evident versus higher doses (4 and 8 g/kg). On reticulata cells, Ro 15-4513 potently stimulated their spontaneous firing and reversed the depression by both ETH and Na-pentobarbital. Moreover, the beta-carboline DMCM also had similar effects. The "pure" BZ antagonist Ro 15-1788 was completely inefective against ETH, yet fully cancelled the reversing actions of Ro 15-4513 and DMCM upon ETH or Na-pentobarbital effects. It is concluded that Ro 15-4513 behaves as a BZ inverse agonist, so that its opposition to ETH and Na-pentobarbital is probably the result of its "negative" coupling with the BZ recognition site that triggers the closing of chloride channels. It suggests that BZ inverse agonists might constitute, in the near future, a new class of analeptic drugs.

Action Potentials↗
PubMed1982

A three-state model of the benzodiazepine receptor explains the interactions between the benzodiazepine antagonist Ro 15-1788, benzodiazepine tranquilizers, beta-carbolines, and phenobarbitone.

The potent benzodiazepine receptor ligands beta-carboline-3-carboxylic acid ethyl ester (beta-CCE) and the corresponding methylester (beta-CCM) administered i.v. depressed segmental dorsal root potentials in spinal cats, reversed the prolongation of dorsal root potentials by phenobarbitone, and abolished the depression of a motor performance task induced by phenobarbitone in mice; beta-CCE enhanced the low-frequency facilitation of pyramidal population spikes in the hippocampus of anaesthetized rats. These effects of beta-carbolines reflect a depression of GABAergic synaptic transmission and, thus, are diametrically opposed to the enhancing action of benzodiazepine tranquilizers. The specific benzodiazepine antagonist, Ro 15-1788, while not affecting dorsal root potentials, hippocampal population spikes or phenobarbitone-induced motor performance depression, abolished the effects of beta-CCE on the three parameters and similar effects of beta-CCM on the spinal cord and motor performance. A three-state model of the benzodiazepine receptor is proposed in which benzodiazepine tranquilizers act as agonists enhancing the function of the benzodiazepine receptor as a coupling unit between GABA receptor and chloride channel, beta-carbolines act as "inverse agonists" reducing this coupling function, and Ro 15-1788 represents a competitive antagonist blocking both the enhancing effect of agonists and the depressant effect of "inverse agonists" on GABAergic synaptic transmission.

Animals↗
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↗