Novel effects of minocycline on Ca(2+)-dependent Cl(-) secretion in human airway epithelial Calu-3 cells

The present study concerns previously unreported effects of the antibiotic minocycline on the transepithelial Cl(-) transport in Calu-3 cells, which display electrophysiological properties consistent with human airway serous cells. Basolateral 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 200 microM) augmented Cl(-) secretion, which was detected as a 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 microM, a Cl(-) channel blocker)-sensitive short-circuit current (I(sc)). The DIDS-induced I(sc) was composed of Ca(2+)-activated K(+) (K(Ca)) channel-dependent and -independent components. The former was selectively inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM, 10 microM), charybdotoxin (ChTx, 100 nM), clotrimazole (10 microM), basolateral Ca(2+) removal, and basolateral minocycline (IC(50) = 20 microM). The latter was attenuated by basolateral BaCl (5 mM). In contrast, forskolin (10 microM)-induced I(sc), which is insensitive to BAPTA/AM and ChTx, was unaffected by minocycline (100 microM). ATP-induced I(sc) was partially inhibited by basolateral but not by apical minocycline. I(sc) due to basolateral application of ionomycin (1 microM) was markedly suppressed by NPPB and basolateral Ca(2+) removal. These inhibitory effects were mimicked by minocycline applied only from the basolateral side of the monolayer. In the basolateral absence of Ca(2+), 1-ethyl-2-benzimdazolinone (500 microM), a K(Ca) channel opener, generated a sustained I(sc) sensitive to ChTx. Minocycline had no significant effect on the ChTx-sensitive component of the I(sc). It is concluded that minocycline inhibits K(Ca) channel-dependent Cl(-) secretion via a blockade of Ca(2+) influx across the basolateral membrane from the extracellular side.     

NPPB block of the intermediate-conductance Ca2+-activated K+ channel

We have shown that the Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) also blocks the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) current in human leukemic HL-60 and glioblastoma GL-15 cell lines. The macroscopic IK(Ca) current was activated by ionomycin plus 1-EBIO, and identified as intermediate conductance by being fully blocked by charybdotoxin, clotrimazole, nitrendipine (L-type Ca(2+) channel blocker), and NS1619 (BK(Ca) channel opener), but not by D-tubocurarine or TEA. The IK(Ca) current was blocked by NPPB in a reversible dose-dependent manner, with an IC(50) of 39 microM in HL-60 and 125 microM in GL-15 cells. The block of the IK(Ca) current was also recorded at the single channel level in excised inside-out patches. As expected, NPPB also blocked the volume-activated Cl(-) current expressed by GL-15 cells, with an IC(50) of 44 microM. The functional implications of IK(Ca) current block by NPPB are discussed.     

Bioelectric toxicity caused by chlorpromazine in human lung epithelial cells

Endogeneous and exogeneous amine-containing substances possess pneumophilic properties. Among them, tricyclic amphiphilic amine drugs like neuroleptics intensively accumulate in the lung cell membrane and occasionally cause severe respiratory disorders. In the present study, we examined the bioelectric toxicity of chlorpromazine (CPZ), a commonly used neuroleptic, in human lung epithelial cells. CPZ concentration-dependently inhibited the isoproterenol (ISO)-generated short-circuit current (I(sc)) sensitive to a nonselective K(+) channel blocker, clotrimazole (30 microM), but insensitive to a selective Ca(2+)-activated K(+) (K(Ca)) channel blocker, charybdotoxin (ChTx, 100 nM). The effects of apical CPZ on the ISO-induced responses were greater than those of basolateral CPZ. Forskolin- and 8-bromo-cyclic AMP-induced I(sc) were partially prevented by CPZ. Nystatin permeabilization of the monolayers revealed that CPZ attenuated the basolateral K(+) current elicited by ISO more than that elicited by forskolin and that the apical Cl(-) current elicited by forskolin was instead potentiated by CPZ, although it inhibited the ISO-induced Cl(-) current. 1-Ethyl-2-benzimdazolinone (1-EBIO, a K(Ca) channel opener, 500 microM)- and ionomycin (Ca(2+) ionophore, 1 microM)-evoked Cl(-) secretions were also sensitive to CPZ. These results indicate that CPZ inhibits transepithelial Cl(-) transport, affecting at least two different targets: the beta-adrenergic receptor and the basolateral K(+) channels (especially the K(Ca) channel). Electrostatic interactions at the inner surface of the membrane between the protonated amines of CPZ and negatively charged portions of the plasma membrane may be involved in the mechanisms.     

Palmatine, a protoberberine alkaloid, inhibits both Ca(2+)- and cAMP-activated Cl(-) secretion in isolated rat distal colon

BACKGROUND AND PURPOSE: The protoberberine alkaloid berberine has been reported to inhibit colonic Cl(-) secretion. However, it is not known if other protoberberine alkaloids share these effects. We have therefore selected another protoberberine alkaloid, palmatine, to assess its effects on active ion transport across rat colonic epithelium. EXPERIMENTAL APPROACH: Rat colonic mucosa was mounted in Ussing chambers and short circuit current (I (SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cAMP content was determined by an enzyme immunoassay. Intracellular Ca(2+) concentration was measured with Fura-2 AM. KEY RESULTS: Palmatine inhibited carbachol-induced Ca(2+)-activated Cl(-) secretion and the carbachol-induced increase of intracellular Ca(2+) concentration. Palmatine also inhibited cAMP-activated Cl(-) secretion induced by prostaglandin E(2) (PGE(2)) or forskolin. Palmatine prevented the elevation of intracellular cAMP by forskolin. Determination of apical Cl(-) currents showed that palmatine suppressed the forskolin-stimulated, apical cAMP-activated Cl(-) current but not the carbachol-stimulated apical Ca(2+)-activated Cl(-) current. Following permeabilization of apical membranes with nystatin, we found that palmatine inhibited a carbachol-stimulated basolateral K(+) current that was sensitive to charybdotoxin and resistant to chromanol 293B. However, the forskolin-stimulated basolateral K(+) current inhibited by palmatine was specifically blocked by chromanol 293B and not by charybdotoxin. CONCLUSIONS AND IMPLICATIONS: Palmatine attenuated Ca(2+)-activated Cl(-) secretion through inhibiting basolateral charybdotoxin-sensitive, SK4 K(+) channels, whereas it inhibited cAMP-activated Cl(-) secretion by inhibiting apical CFTR Cl(-) channels and basolateral chromanol 293B-sensitive, KvLQT1 K(+) channels.     

Phenanthrolines--a new class of CFTR chloride channel openers

1. A number of phenanthrolines and benzoquinolines were examined for their ability to activate epithelial chloride secretion by measuring short circuit current (SCC) using the mouse colon epithelium. 1,10 phenanthroline stimulated electrogenic chloride secretion with an EC(50) of 612+/-10 microM and a Hill slope of 4.9+/-0.3. A similar pharmacology was demonstrated by both 1,7 and 4,7 phenanthrolines, 7,8 benzoquinoline and phenanthridine. 2. Evidence that the increase in SCC caused by 1,10 phenanthroline was due to chloride secretion is based upon (a) inhibition of the current by furosemide, (b) failure of cystic fibrosis (CF) colons to respond and (c) an associated net flux of (36)Cl(-). 3. 1,10 Phenanthroline affected neither the generation of cyclic AMP or the concentration of intracellular Ca(2+) in colonic epithelial cells. 4. 1,10 phenanthroline affected the chloride conductance of the apical membrane, as shown by an increase in chloride current in 'apical membrane only' preparations in the presence of an apical to basolateral chloride gradient. The increase in chloride current was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and was not present in CF colons. 5. Additionally, 1,10 phenanthroline activated basolateral K(+) channels, both Ca(2+)- and cyclic AMP-sensitive channels, as shown by inhibitor studies with charybdotoxin (ChTX) and XE991, and after the apical membrane was permeabilized with nystatin. 6. The phenanthrolines and benzoquinolines described here, with dual actions affecting CFTR and basolateral K(+) channels, may constitute useful lead compounds for adjunct therapy in CF.     

Contribution of K+ channels to relaxation induced by 17beta-estradiol but not by progesterone in isolated rat mesenteric artery rings

17beta-Estradiol and progesterone were found to relax various vascular beds through multiple mechanisms. However, the exact ionic mechanisms underlying the acute relaxant responses to both hormones are incompletely understood. This study was aimed to examine the possible role of K channel activation in the relaxation induced by both hormones in isolated rat mesenteric artery rings. Isometric tension of each ring was measured with Grass force displacement transducers. In rat endothelium-denuded rings preconstricted by 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F (U46619), the relaxation induced by 17beta-estradiol was partially inhibited by tetrapentylammonium, 4-aminopyridine, iberiotoxin, BaCl, and tertiapin-Q but not by tetraethylammonium, charybdotoxin, apamin, or glibenclamide. In contrast, these putative K channel blockers, except for glibenclamide, did not affect the relaxant response to progesterone. In 4 x 10(-2) K -preconstricted rings, the K channel blockers lost their inhibitory effects on 17beta-estradiol-induced relaxation. Endothelium did not seem to be involved in the effects of K channel blockers on 17beta-estradiol-mediated relaxation. Nifedipine-induced relaxation was not inhibited but was instead enhanced by tetrapentylammonium, iberiotoxin, 4-aminopyridine, and BaCl2. The above results indicate that in rat mesenteric artery rings, nonselective activation of K channels contributes partially to the relaxation induced by 17beta-estradiol. These K channels involved in the estrogen response appeared to be sensitive to inhibition by K(Ca), K, and K(IR) channel blockers. Lack of effect of K channel blockers on progesterone-induced relaxation suggests that these K channels play little or no role. The present findings provide pharmacological evidence for an additional mechanism contributing to acute vasorelaxation induced by 17beta-estradiol.     

Sodium reabsorption in thick ascending limb of Henle's loop: effect of potassium channel blockade in vivo

1. Based on previous in vitro studies, inhibition of K(+) recycling in thick ascending limb (TAL) is expected to lower Na(+) reabsorption through (i) reducing the luminal availability of K(+) to reload the Na(+)-2Cl(-)-K(+) cotransporter and (ii) diminishing the lumen positive transepithelial potential difference which drives paracellular cation transport. 2. This issue was investigated in anaesthetized rats employing microperfusion of Henle's loop downstream from late proximal tubular site with K(+)-free artificial tubular fluid in nephrons with superficial glomeruli. 3. The unselective K(+) channel blocker Cs(+) (5 - 40 mM) dose-dependently increased early distal tubular delivery of fluid and Na(+) with a maximum increase of approximately 20 and 185%, respectively, indicating predominant effects on water-impermeable TAL. 4. The modest inhibition of Na(+) reabsorption in response to the 15 mM of Cs(+) but not the enhanced inhibition by 20 mM Cs(+) was prevented by luminal K(+) supplementation. Furthermore, pretreatment with 20 mM Cs(+) did not attenuate the inhibitory effect of furosemide (100 microM) on Na(+)-2Cl(-)-K(+) cotransport. 5. Neither inhibitors of large (charybdotoxin 1 microM) nor low (glibenclamide 250 microM; U37883A 100 microM) conductance K(+) channels altered loop of Henle fluid or Na(+) reabsorption. 6. The intermediate conductance K(+) channel blockers verapamil and quinine (100 microM) modestly increased early distal tubular Na(+) but not fluid delivery, indicating a role for this K(+) channel in Na(+) reabsorption in TAL. As observed for equieffective concentrations of Cs(+) (15 mM), Na(+) reabsorption was preserved by K(+) supplementation. 7. The results indicate that modest inhibition of K(+) channels lowers the luminal availability of K(+) and thus transcellular Na(+) reabsorption in TAL. More complete inhibition lowers paracellular Na(+) transport probably by reducing or even abolishing the lumen positive transepithelial potential difference. Under the latter conditions, transcellular Na(+) transport may be restored by paracellular K(+) backleak.     

Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation

The effects of 1-ethyl-2-benzimidazolinone (1-EBIO) and riluzole on human prostate cancer cells, LNCaP and PC-3, were evaluated using rubidium (86Rb(+)) efflux and proliferation assays. 1-EBIO and riluzole evoked concentration-dependent increases in 86Rb(+) efflux from LNCaP and PC-3 cells that were sensitive to inhibition by intermediate-conductance Ca(2+)-activated K(+) channel (IK(Ca)) blockers clotrimazole and charybdotoxin. Blockers of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, iberiotoxin, or small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel, apamin or scyllatoxin, had no effect. Concurrently, both 1-EBIO and riluzole evoked concentration-dependent increases in proliferation from human prostate cancer cell lines (LNCaP and PC-3 cells). Clotrimazole and charybdotoxin, but not iberiotoxin, apamin or scyllatoxin, inhibited 1-EBIO- and riluzole-evoked increases in proliferation from LNCaP and PC-3 cells. N-(3-(trifluoromethyl)phenyl)-N'-(2-hydroxy-5-chlorophenyl)urea (NS-1608) and 2-amino-5-(2-fluorophenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BK(Ca) channel openers had no effect on LNCaP and PC-3 proliferation. These results demonstrate that IK(Ca) channels play an important role in the regulation of human prostate cancer cell proliferation.     

Effects of Ca(2+) channel blockers on amiloride-sensitive Na(+) permeable channels and Na(+) transport in fetal rat alveolar type II epithelium

A beta-adrenergic agonist (beta-agonist), terbutaline, stimulated amiloride-sensitive Na(+) absorption in fetal rat alveolar type II epithelium, contributing to the clearance of lung fluid. Cytosolic Ca(2+) plays an important role in terbutaline-stimulated Na(+) absorption, since Ca(2+)-activated, amiloride-sensitive Na(+)-permeable channels are involved in transcellular Na(+) absorption and terbutaline stably elevates the cytosolic Ca(2+) concentration by stimulating Ca(2+) influx. Therefore, we studied whether Ca(2+) channel blockers (Ni(2+), verapamil, and nifedipine) affect terbutaline-stimulated transcellular Na(+) absorption. Ni(2+) partially blocked the channel responsible for the terbutaline-stimulated Na(+) absorption at the Na(+) entry pathway across the apical membrane of the epithelium, but did not diminish the terbutaline-stimulated transcellular Na(+) absorption. By measuring the capacity of the Na(+),K(+)-pump activity, we determined that the rate-limiting step of the terbutaline-stimulated transcellular Na(+) absorption was the extrusion step across the basolateral membrane by the Na(+),K(+)-pump. The other Ca(2+) channel blockers, verapamil and nifedipine, had effects identical to those of Ni(2+). Based upon these observations, we conclude that, in the beta-agonist-stimulated fetal rat alveolar type II epithelium, Ca(2+) channel blockers diminish amiloride-sensitive channels, but do not affect transcellular Na(+) absorption, since under the beta-agonist-stimulated condition the Na(+),K(+)-pump is the rate-limiting step in Na(+) transport.     

Regulation of mastoparan-induced increase of paracellular permeability in T84 cells by RhoA and basolateral potassium channels

Mastoparan, a polypeptide known to activate heterotrimeric GTP-binding proteins, enhances the transport of Ca2+ and K+ across membranes. In the present study we investigated the influence of mastoparan on transepithelial resistance (TER) and on short circuit current (SCC) of the intestinal cell line T84. Mastoparan decreased the TER by 80% of baseline and induced a SCC of 8.34+/-1.38 microAcm(-2). The changes in paracellular conductance were estimated using the nystatin technique and showed that mastoparan increased the paracellular conductance 4-fold. Basolateral Cl(-)-free medium, or blockade of the basolateral Cl(-) uptake via the Na+/K+/2Cl(-) co-transporter with bumetanide, reduced SCC of T84 cells, but did not abolish the effect of mastoparan on the TER. Luminal addition of the Cl(-)-channel blocker DIDS or NPPB had no effect on the increase in SCC. In contrast, blocking the basolateral K(+)-channels by 2mM Ba2+ inhibited both the resistance decrease and elevation of the SCC, and further inhibited the mastoparan-induced increase in intracellular free Ca2. This indicates that mastoparan acts primarily via activating K+ channels with a secondary Cl(-) secretion and Ca2+ influx. Reduction of intracellular free Ca2+ did not alter the effect of mastoparan on TER. Stimulation with mastoparan led to a biphasic rearrangement of actin filaments and increased globular actin content in T84 cells. Depolymerization of actin filaments also correlated with inactivation of Rho-proteins, which are known regulators of the cytoskeleton. Mastoparan induced a 2-fold increase in GDI-complexed Rho.We conclude that mastoparan-induced changes in paracellular permeability are mediated via enhanced basolateral K+ conductance and Rho-protein inactivation. A secondary increase in intracellular Ca2+ or direct interaction of small GTPases with the cytoskeleton are likely mediators of the remodeling of the cytoskeleton with subsequent changes in paracellular permeability.     

Chloride channel blockers decrease intracellular pH in cultured renal epithelial LLC-PK1 cells.

The effects of chloride channel blockers upon intracellular pH (pHi) were examined in renal epithelial monolayers of LLC-PK1 cells. A significant intracellular acidification was found with addition of 100 microM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), niflumic acid, flufenamate and diphenylamine-2-carboxylate (DPC) but not with 4,4'-diisothiocyanatostilbene-2-2'disulphonic acid (DIDS). The effects of these agents upon pHi was dose-dependent with apparent K0.5 values of: 16.7 +/- 0.3 microM, 34.2 +/- 0.9 microM and 740 +/- 13 microM for niflumic acid, flufenamate and DPC respectively. The results indicate that at concentrations commonly used to block channel activity these chloride channel blockers have profound effects upon pHi.     

Involvement of endogenous prostaglandin in emodin-evoked rat colonic anion secretion

It has been reported that emodin is able to promote gastrointestinal motility and stimulate large intestinal water secretion; however, the mechanism is still not clear. The aim of the present study is to examine the effects of emodin on the rat colonic transepithelial ion transport and the underlying mechanism. The study was carried out by means of the short circuit current (I(SC)) recording. Basolateral application of emodin induced a concentration-dependent I(SC) increase, and the EC(50) was 76.0 micromol/l. Pretreatment with epithelial Na(+) channel blocker, amiloride (10 micromol/l), did not affect the I(SC) responses elicited by emodin, but removal of extracellular Cl(-) or apical pretreatment with Cl(-) channel blocker, glibenclamide (1 mmol/l) inhibited emodin-elicited I(SC) responses by 76.3% and 83.8% respectively. Inhibiting basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC) with bumetanide (100 micromol/l) decreased emodin-induced I(SC) from 118.1+/-6.7 microA/cm(2) to 16.7+/-2.0 microA/cm(2), which was reduced by 85.9%. Basolateral pretreatment with neuronal Na(+) channel blocker tetrodotoxin (TTX) (1 micromol/l) did not affect emodin-induced I(SC) increase, but pretreatment with indomethacin (10 micromol/l) alone or with both TTX and indomethacin significantly decreased emodin-induced I(SC) increase by 88.4 and 81.2%, respectively. The present study demonstrated that emodin was able to stimulate rat colonic epithelial Cl(-) secretion, which was predominantly mediated by endogenous prostaglandin release.     

Xe991 reveals differences in K(+) channels regulating chloride secretion in murine airway and colonic epithelium

The cognitive enhancer XE991 interacts with K(+) channels consisting of KCNQ2 and KCNQ3 heteromultimers to block the M-current. XE991 can also block KCNQ1 K(+) channels expressed in oocytes, but sensitivity is reduced when the channels are coexpressed with minK (KCNE1). The purpose of the study was to examine the interaction of XE991 with other types of K(+) channel, especially those in the basolateral membranes of murine epithelia. K(+) channel blockade was measured by the inhibition of chloride secretion resulting from depolarization. XE991 inhibited the chloride secretory current in colonic epithelia by an interaction with basolateral K(+) channels when forskolin was used as the stimulus. However, when 1-ethyl-2-benzimidazolinone (EBIO) was used to stimulate chloride secretion, XE991 was ineffective unless charybdotoxin was also present. Because EBIO also activates Ca(2+)-sensitive K(+) channels, whereas forskolin activates only cAMP-sensitive K(+) channels, it is concluded that the latter are the targets for XE991. XE991 had effects similar to those of 293B on epithelial chloride transport, for which the target is known to be KCNQ1/KCNE3 multimers. mRNA for both these components of the cAMP-sensitive K(+) channels were found in high abundance in the colon, whereas KCNE1 was barely detectable. Furthermore, both XE991 and 293B were active in colonic epithelia from KCNE1 knockout mice. By contrast, in nasal epithelium, the forskolin sensitive chloride secretory current was barely sensitive to XE991 but was sensitive to clofilium. Xenopus laevis oocytes in which both KCNQ1 and KCNE3 had been expressed were significantly more sensitive to XE991 than oocytes expressing only KCNQ1.     

Inhibition by chloride channel blockers of anion secretion in cultured epididymal epithelium and intact epididymis of rats.

1. Anion secretion by primary monolayer cultures of rat epididymal cells was studied by the short circuit current technique. 2. Monolayers had a transepithelial potential difference of 1.34 mV, apical side negative and a short circuit current of 2.45 microA cm-2. The transepithelial resistance was 504 omega cm2. 3. Addition of anthracene-9-carboxylate (9-AC) to the apical side caused a biphasic response, a decrease followed by an increase in the short circuit current (SCC) which then returned to the basal level. Addition of 9-AC to the basolateral side also caused a biphasic response but the increase in current was sustained. 4. Addition of diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused an inhibition of the SCC when added to the apical or basolateral side. 5. When the epithelium was stimulated with adrenaline (0.23 microM, basolaterally), the SCC rose to a peak value of 10.36 microA cm-2 and then stabilized at 3.82 microA cm-2 after 15 min. Addition of 9-AC to the apical side caused a triphasic response: a decrease, reversal to the original level followed by a slow inhibition which was sustained. The inhibition achieved at the steady state was concentration-dependent with an apparent IC50 value of 2.51 mM. Addition of 9-AC to the basolateral side produced a similar response but a time lag of 20 s was observed. 6. DPC and NPPB also caused the SCC to decrease when added to the apical side of the monolayers stimulated with adrenaline. The IC50 values 0.148 mM and 0.049 mM for DPC and NPPB, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological evidence for the activation of K(+) channels by diclofenac

The involvement of K(+) channels in the antinociceptive action of diclofenac was assessed in the formalin test. Local administration of diclofenac produced a dose-dependent antinociceptive effect due to a local action because drug administration in the contralateral paw was ineffective. Pretreatment of the injured paw with glibenclamide and tolbutamide (ATP-sensitive K(+) channel inhibitors), charybdotoxin and apamin (large- and small-conductance Ca(2+)-activated K(+) channel blockers, respectively), 4-aminopyridine or tetraethylammonium (voltage-dependent K(+) channel inhibitors) prevented diclofenac-induced antinociception. Given alone, K(+) channel inhibitors did not modify formalin-induced nociceptive behavior. Pinacidil (an ATP-sensitive K(+) channel opener) also produced antinociception which was blocked by glibenclamide. The peripheral antinociceptive effect of morphine (positive control) was blocked by glibenclamide and 4-aminopyridine but not by charybdotoxin or apamin. The results suggest that the peripheral antinociceptive effect of diclofenac may result from the activation of several types of K(+) channels, which may cause hyperpolarization of peripheral terminals of primary afferents.     

Anion secretion evoked by Pasteurella multocida toxin across rat colon

Stimulation of muscarinic receptors is known to have a biphasic effect on colonic Cl(-) secretion: a short-lasting activation, which is followed by a long-lasting inhibition. In order to find out, which role Gq proteins play in both processes, Pasteurella multocida toxin was used, a known activator of G alpha q. This toxin (1.5 microg/ml) had a dual action on short-circuit current (Isc) across rat distal colon: it stimulated transiently Isc and subsequently down-regulated the Isc evoked by Ca2+-dependent secretagogues such as acetylcholine or ATP. The inactive mutant (P. multocida toxin C1165S), which does not stimulate G alpha q), was ineffective. Cl(-) dependence and sensitivity against bumetanide, a blocker of the Na+-K+-2Cl(-) cotransporter, confirmed that the increase in Isc evoked by the toxin represented Cl(-) secretion. The effect of P. multocida toxin was suppressed by YM-254890 (10(-7) M), a blocker of G alpha q. Experiments with apically permeabilized tissues revealed that the secretory response to P. multocida toxin was concomitant with an increase in basolateral K+ conductance as it is observed for other agonists inducing Ca2+-dependent anion secretion. Consequently, these results suggest that Gq proteins are not only involved in the activation of secretion, e.g. after stimulation of muscarinic or purinergic receptors, but also play a central role in the long-term down-regulation of intestinal secretion after activation of these types of receptors.     

Involvement of inwardly rectifying K+ channels in secretory responses of human ileal mucosa

In acute secretory diarrhoea the primary event driving fluid secretion is a transcellular, electrogenic, serosal to mucosal transport of chloride ions. Such transport requires the maintenance of an electrically negative cell membrane voltage, which is achieved through a basolateral outward leakage of potassium ions. The aim of this study was to investigate the nature of K(+) channel involvement in facilitating secretory processes in the human ileum. Muscle-stripped mucosal preparations of human ileal mucosa were set up in Ussing chambers for recording short-circuit current and transmucosal conductance. Escherichia coli heat-stable toxin and vasoactive intestinal peptide (VIP) produced concentration-dependent increases in short-circuit current. Responses to the heat-stable toxin were unaffected by basolateral application of 4-aminopyridine (5 mM), glibenclamide (10 microM) or a combination of charybdotoxin (0.3 microM) plus apamin (0.3 microM). However, basolateral barium (0.2-5 mM) caused a concentration-dependent inhibition. Responses to VIP were similarly affected by barium (0.05-1 mM). These results suggested that electrogenic chloride transport by human ileal mucosa required the presence of basolateral K(+) channels. The use of selective K(+)-channel inhibitors and low concentrations of barium suggested that the channels involved might be of the inwardly rectifying type.     

Differences in the actions of some blockers of the calcium-activated potassium permeability in mammalian red cells

1. The actions of some inhibitors of the Ca2+-activated K+ permeability in mammalian red cells have been compared. 2. Block of the permeability was assessed from the reduction in the net loss of K+ that followed the application of the Ca2+ ionophore A23187 (2 microM) to rabbit red cells suspended at a haematocrit of 1% in a low potassium solution ([K]0 0.12-0.17 mM) at 37 degrees C. Net movement of K+ was measured using a K+-sensitive electrode placed in the suspension. 3. The concentrations (microM +/- s.d.) of the compounds tested causing 50% inhibition of K+ loss were: quinine, 37 +/- 3; cetiedil, 26 +/- 1; the cetiedil congeners UCL 1269, UCL 1274 and UCL 1495, approximately 150, 8.2 +/- 0.1, 0.92 +/- 0.03 respectively; clotrimazole, 1.2 +/- 0.1; nitrendipine, 3.6 +/- 0.5 and charybdotoxin, 0.015 +/- 0.002. 4. The characteristics of the block suggested that compounds could be placed in two groups. For one set (quinine, cetiedil, and the UCL congeners), the concentration-inhibition curves were steeper (Hill coefficient, nH, > or = 2.7) than for the other (clotrimazole, nitrendipine, charybdotoxin) for which nH approximately 1. 5. Compounds in the first set alone became less active on raising the concentration of K+ in the external solution to 5.4 mM. 6. The rate of K+ loss induced by A23187 slowed in the presence of high concentrations of cetiedil and its analogues, suggesting a use-dependent component to the inhibitory action. This was not seen with clotrimazole. 7. The blocking action of the cetiedil analogue UCL 1274 could not be overcome by an increase in external Ca2+ and its potency was unaltered when K+ loss was induced by the application of Pb2+ (10 microM) rather than by A23187. 8. These results, taken with the findings of others, suggest that agents that block the red cell Ca2+-activated K+ permeability can be placed in two groups with different mechanisms of action. The differences can be explained by supposing that clotrimazole and charybdotoxin act at the outer face of the channel whereas cetiedil and its congeners may block within it, either at or near the K+ binding site that determines the flow of K+.     

4-Chloro-benzo[F]isoquinoline (CBIQ) activates CFTR chloride channels and KCNN4 potassium channels in Calu-3 human airway epithelial cells

1 Calu-3 cells have been used to investigate the actions of 4-chloro-benzo[F]isoquinoline (CBIQ) on short-circuit current (SCC) in monolayers, whole-cell recording from single cells and by patch clamping. 2 CBIQ caused a sustained, reversible and repeatable increase in SCC in Calu-3 monolayers with an EC50 of 4.0 microm. Simultaneous measurements of SCC and isotopic fluxes of 36Cl- showed that CBIQ caused electrogenic chloride secretion. 3 Apical membrane permeabilisation to allow recording of basolateral membrane conductance in the presence of a K+ gradient suggested that CBIQ activated the intermediate-conductance calcium-sensitive K(+)-channel (KCNN4). Permeabilisation of the basolateral membranes of epithelial monolayers in the presence of a Cl- gradient suggested that CBIQ activated the Cl(-)-channel CFTR in the apical membrane. 4 Whole-cell recording in the absence of ATP/GTP of Calu-3 cells showed that CBIQ generated an inwardly rectifying current sensitive to clotrimazole. In the presence of the nucleotides, a more complex I/V relation was found that was partially sensitive to glibenclamide. The data are consistent with the presence of both KCNN4 and CFTR in Calu-3. 5 Isolated inside-out patches from Calu-3 cells revealed clotrimazole-sensitive channels with a conductance of 12 pS at positive potentials after activation with CBIQ and demonstrating inwardly rectifying properties, consistent with the known properties of KCNN4. Cell-attached patches showed single channel events with a conductance of 7 pS and a linear I/V relation that were further activated by CBIQ by an increase in open state probability, consistent with known properties of CFTR. It is concluded that CBIQ activates CFTR and KCNN4 ion channels in Calu-3 cells.     

Study of the involvement of K+ channels in the peripheral antinociception of the kappa-opioid receptor agonist bremazocine

The involvement of the nitric oxide (NO)/cyclic GMP pathway in the molecular mechanisms of antinociceptive drugs like morphine has been previously shown by our group. Additionally, it is known that the desensitisation of nociceptors by K(+) channel opening should be the final target for several analgesic drugs including nitric oxide donors and exogenous micro-opioid receptor agonists. In our previous study, we demonstrated that bremazocine, a kappa-opioid receptor agonist, induces peripheral antinociception by activating nitric oxide/cyclic GMP pathway. In the current study, we assessed whether bremazocine is capable to activate K(+) channels eliciting antinociception. Bremazocine (20, 40 and 50 microg) dose-dependently reversed the hyperalgesia induced in the rat paw by local injection of carrageenan (250 microg) or prostaglandin E(2) (2 microg), measured by the paw pressure test. Using the selective kappa-opioid receptor antagonist nor-binaltorphimine (Nor-BNI, 200 microg/paw), it was confirmed that bremazocine (50 microg/paw) acts specifically on the kappa-opioid receptors present at peripheral sites. Prior treatment with the ATP-sensitive K(+) channel blockers glibenclamide (40, 80 and 160 microg) and tolbutamide (40, 80 and 160 microg) did not antagonise the antinociceptive effect of bremazocine (50 microg). The same results were obtained when we used prostaglandin E(2) (2 microg) as the hyperalgesic stimulus. The supposed participation of other types of K(+) channels was tested using the Ca(2+)-activated K(+) channel blockers dequalinium (12.5, 25 and 50 microg) and charybdotoxin (0.5, 1 and 2 microg) and different types of the non-selective K(+) channel blockers tetraethylammonium (25, 50 and 100 microg) and 4-aminopyridine (10, 25 and 50 microg). None of the K(+) channel blockers reversed the antinociceptive effect of bremazocine. On the basis of these results, we suggest that K(+) channels are not involved in the peripheral antinociceptive effect of bremazocine, although this opioid receptor agonist induces nitric oxide/cGMP pathway activation.