P2Y(11), a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y(2) receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+](i)) and stimulate Ca2+-activated Cl- and K+ channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl- channels. ATP, 2-methylthio-ATP, and ATP-gamma-S stimulated a 4- to 10-fold cAMP increase with EC(50) of 10-100 microM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y(2) or P1 receptors. Although UTP stimulated an (125)I(-) efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl- conductances through P2Y(2)-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+](i). Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I(sc) increase, whereas serosal ATP stimulated a large cAMP-type I(sc) response. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl- conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y(11) receptor mRNA, the only P2Y receptor acting via cAMP.     

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.     

Characterisation of three pathways for osmolyte efflux in human erythroleukemia cells

Cell volume decrease is a key step during differentiation of erythroid cells. This could arise from membrane transporter activation leading to a loss of cell osmolytes; however, the pathways involved are poorly understood. We have characterised Cl(-)-independent K(+) and (3)H-taurine efflux from the erythroleukemia cell line, K562. K(+) efflux (measured using (86)Rb(+)) from pre-loaded cells subjected to hypo-osmotic challenge demonstrated two phases, a rapid increase in K(+) efflux followed by a smaller slower increase. Swelling-activated taurine efflux only demonstrated a single phase. Both phases of K(+) efflux were significantly (P<0.05) blocked by anion channel inhibitor 5-nitro-2-(3-phenypropylamino)-benzoic acid (NPPB). However the antiestrogen, tamoxifen, only inhibited the slow late phase. The initial rapid phase had a higher IC(50) for NPPB inhibition than the slow phase, and was insensitive to protein kinases inhibitors KN-62, wortmannin and PD98059. For the slow K(+) efflux phase, the IC(50) for NPPB inhibition and the inhibition by KN-62, wortmannin, genistein or PD98059, were very similar to those measured for the hypo-osmotically-activated taurine efflux. With NPPB (100 microM) present, the slow K(+) efflux phase was further significantly decreased by the Ca(2+) chelator BAPTA-AM or by the Ca(2+)-activated K(+) channel blockers clotrimazole and charybdotoxin but not by apamin. Thus, at least 3 Cl(-)-independent pathways are involved: (a) a tamoxifen-sensitive and taurine-permeable anion channel; (b) a tamoxifen-insensitive and taurine-impermeable K(+) efflux pathway; and (c) a subtype of Ca(2+)-activated K(+) channel. Any or all of these could be involved in the cell volume decrease associated with differentiation in K562 cells.     

Low-dose PGE2 mimics the duodenal secretory response to luminal acid in mice

Luminal exposure to concentrated acid, the most accepted physiological stimulus for duodenal bicarbonate secretion (DBS), cannot be used with in vitro preparations due to potential tissue damage. We thus examined whether exposure to PGE(2), a well-characterized physiological duodenal secretagogue, could mimic the effects of acid perfusion. DBS was measured in C57/BL mice by pH-stat/back-titration and measurement of total dissolved CO(2) concentration ([CO(2)](t)). Anion transport inhibitor DIDS, anion channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), carbonic anhydrase inhibitor methazolamide, and nonselective cyclooxygenase inhibitor indomethacin were used to inhibit separate components of HCO(3)(-) secretory pathway. Baseline DBS was not altered by exposure to methazolamide (0.1 mM) but was slightly reduced by DIDS (0.5 mM). DBS and [CO(2)](t) increased after acid and PGE(2) exposure. DIDS (0.5 mM) and NPPB (0.2 mM) abolished acid-induced DBS increase. Methazolamide (0.1 mM) and DIDS inhibited acid-induced [CO(2)](t) increase. DIDS, NPPB, or methazolamide had little effect on DBS in response to high concentration PGE(2) (100 microg/ml). Low concentration PGE(2) (1 microg/ml) increased DBS that was inhibited by DIDS, NPPB, and methazolamide. Pretreatment with indomethacin (5 mg/kg) inhibited DBS induced by acid exposure but not by PGE(2). High-dose PGE(2) substantially increases DBS by a mechanism that appears to be different than secretory response to luminal acid perfusion. Secretory response to low-dose PGE(2), at least in terms of inhibitor profile, closely resembles secretion in response to perfusion of physiological acid concentrations and may be a useful stimulus for in vitro study of DBS in isolated mouse duodenum.     

Elucidation of the Mechanisms Underlying Hypo-osmotically Induced Turgor Pressure Regulation in the Marine Alga Valonia utricularis

Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 microM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 microM NPPB, regulation could only be inhibited by simultaneous external addition of 200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl(-) transport. At concentrations of about 100 microM, NPPB seems to selectively inhibit Cl(-) transporters in the tonoplast and K(+) transporters in the plasmalemma, whereas 300 microM NPPB inhibits K(+) and Cl(-) transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G(t) and G(p)) in low-Cl(-) and K(+)-free artificial seawater. Inhibition of turgor pressure regulation by 300 microM NPPB was accompanied by about 85% reduction of G(t) and G(p). Vacuolar addition of sulfate, an inhibitor of tonoplast Cl(-) transporters, together with external addition of DIDS and Ba(2+) (an inhibitor of K(+) transporters) also strongly reduced G(p) and G(t) but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.     

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.     

Osmotic swelling activates two pathways for K+ efflux in a rat hepatoma cell line.

The pathways for the efflux of K(+) from osmotically-swollen HTC rat hepatoma cells were investigated using (86)Rb(+) as a tracer for K(+). Exposure of HTC cells to a hypotonic solution (<250 mOsm kg(-1)) resulted in a transient efflux of (86)Rb(+) that reached a maximal value after approximately 1 min, and inactivated within 3 min. This initial (86)Rb(+) efflux was inhibited by charybdotoxin, clotrimazole and Ba(2+), but not by apamin or paxilline, consistent with it being via an intermediate-conductance Ca(2+)-activated K(+) channel. For cells exposed to an extracellular osmolality < 180 mOsm kg(-1) there was an additional (86)Rb(+) efflux component which was slower to activate, taking 4 - 6 min to reach a maximum, and remaining active for > 20 min. The second (86)Rb(+) efflux component was not inhibited by K(+) channel blockers but was inhibited by the anion channel blockers, tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumate. The time-courses for its activation and inactivation, as well as its dependence on the extracellular osmolality, were very similar to those observed for the hypotonically-activated efflux of the organic osmolyte, taurine. The data are consistent with the second component of (86)Rb(+) efflux and the efflux of taurine from osmotically-swollen cells occurring via a common pathway having a marked selectivity for taurine over (86)Rb(+).     

Swelling-activated Taurine and Creatine Effluxes from Rat Cortical Astrocytes are Pharmacologically Distinct

Primary cultures of rat cortical astrocytes undergo a swelling-activated loss of taurine and creatine. In this study, the pharmacological characteristics of the taurine and creatine efflux pathways were compared, and significant differences were shown to exist between the two. Both taurine and creatine effluxes were rapidly activated upon exposure of astrocytes to hypo-osmotic media, and rapidly inactivated upon their return to iso-osmotic media. The relative rates of taurine and creatine efflux depended upon the magnitude of the hypo-osmotic shock. Anion-transport inhibitors strongly inhibited taurine efflux, with the order of potency being NPPB > DIDS > niflumic acid. DIDS and NPPB had less of an inhibitory effect on creatine efflux, whereas tamoxifen and niflumic acid actually stimulated creatine efflux. These data are consistent with separate pathways for taurine and creatine loss during astrocyte swelling.     

Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa

The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.     

Effect of anion transport blockers on CFTR in the human sweat duct

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.     

Cl- channel blockers inhibit transition of quiescent (G0) fibroblasts into the cell cycle

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl- channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl- channel blocker 5-N-2-(3-phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4-aminopyridine (4-AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0-to-G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10-14 h. NPPB applied only at 0-8 or 8-14 h after serum addition inhibited entry into S phase. Cl- permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki-67-negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl- channel blockers (NPPB and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid [DIDS]) but not the K+ channel blocker (4-AP) significantly decreased the rate of reduction in number of Ki-67-negative cells. These data indicate that an increase in Cl- permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint.     

Cholinergic inhibition of electrogenic sodium absorption in the guinea pig distal colon

Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating Cl(-) secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic Na(+) absorption in distal colon. Amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive I(sc) and amiloride-sensitive (22)Na(+) absorption. The inhibitory effect of CCh was observed at approximately 0.1 microM, and maximum inhibition of approximately 70% was attained at approximately 30 microM (IC(50) = approximately 1 microM). CCh-induced inhibition of amiloride-sensitive I(sc) was almost totally abolished by 10 microM atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive I(sc), but a subsequent addition of CCh further decreased it. Also, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-Ca(2+) solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive I(sc), approximately 33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive I(sc), this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic Na(+) absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.     

Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na(+) absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I(sc) and (22)Na(+) absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I(sc) had an IC(50) value of 20-30 microM, with the maximum inhibition being approximately 50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I(sc) induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I(sc), this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na(+) absorption. This effect was probably mediated by a P2Y(2) receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.     

Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.     

Characterization of the taurine transport pathway in A6 kidney cells

We investigated the role of taurine in cell homeostasis and characterized the taurine transport pathway in cultured kidney cells (A6). The taurine concentration in A6 cells varies with the osmolarity of the culture medium, suggesting that taurine participates in cell osmolarity. Under isosmotic conditions, 14C-taurine efflux through the apical membranes (aJtaur) was 6-7 times lower than that through the basolateral membranes (bJtaur). Under hyposmotic conditions, aJtaur remained almost unchanged. On the contrary, bJtaur increased 8 times in comparison with isosmotic conditions. In hyposmotic conditions, bJtaur was inhibited by 500 microM DIDS, 50 microM NPPB, 10 microM of the two oxonol derivatives DISBAC(2)3 and WW-791, and 100 microM ketoconazole. Conversely, 100 microM 1,9-dideoxyforskolin, 10 microM tamoxifen, 100 microM niflumic acid and 50 microM verapamil had no inhibitory effects. Cell volume regulation upon hyposmotic stress was also found to be inhibited by DISBAC(2)3 (K0.5 of 5+/-1 microM) and by ketoconazole. Nystatin was used to permeabilize the apical membranes with the aim to further characterize bJtaur. 14C-taurine transepithelial fluxes in nystatin-treated cells were found to be linear over taurine concentrations ranging from 3.5 microM to 35 mM. Clamping the transepithelial voltage at positive values (serosal side) slightly stimulated the 14C-taurine transport. Similar time courses of 14C-taurine, 36Cl and 86Rb transepithelial fluxes were found under osmotic stimulation followed by DIDS inhibition in nystatin-treated cells. In whole cell patch-clamp experiments, DISBAC(2)3 application resulted in a strong and reversible decrease of the global Cl- current which was stimulated by hyposmotic stress. Our study indicates that taurine participates in the control of A6 cell osmolarity and that the transporting taurine pathway (efflux) is on the basolateral membranes. In addition to usual chloride channel blockers, oxonol was found to be a potent blocker of the taurine transport and of the swelling-activated chloride current. Using a pharmacological approach, we could not distinguish between a common or different pathway for Cl- and taurine.     

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.     

The anion transport inhibitor DIDS activates a Ba2+-sensitive K+ flux associated with hepatic exocrine secretion.

4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an anion transport inhibitor and choleretic organic anion, was used to study the relationship between putative DIDS-sensitive K channels and exocrine secretion in the isolated and bile duct cannulated perfused rat liver. Bile flow, DIDS excretion, and effluent perfusate K+ content were measured. DIDS (125 microM) caused a doubling in bile generation concomitant with its appearance in bile, confirming earlier reports. Furthermore, DIDS induced a transient increase in perfusate K+ concentration that peaked prior to the biliary parameters and, after 10 min, reversed to net uptake that fully compensated for the initial release. The K channel blocker Ba2+ (1 mM) strongly inhibited the release phase along with the accompanying choleresis and DIDS excretion. Ouabain (13.5 microM) alone was choleretic and hyperkalemic and, when applied in combination with DIDS, depressed DIDS excretion, choleresis, and DIDS-sensitive K+ uptake. To obtain further evidence for the presence of DIDS-sensitive K channels K+ flux was measured under the influence of different gradients of the cation. Perfusate K+ at 26 and 80 mM changed the DIDS-activated K+ flux from a transient outward to a sustained inward flux, and both DIDS excretion and bile flow decreased. Mean net K+ flux over 20 min DIDS perfusion changed from -1.3 +/-1.1 micromol/g with 5.9 mM K+ to -1304 +/- 55 micromol/g with 80 mM K+ in the perfusate. K+ efflux was fully and reversibly blocked by Ba2+ and influx was ouabain-insensitive, suggesting that the DIDS-activated K+ flux was channel mediated. The results show that a significant fraction of DIDS-induced bile generation is associated with K+ release that may be mediated by Ba(2+)-sensitive K channels, possibly of the inward rectifying type.     

P2Y receptor regulation of sodium transport in human mammary epithelial cells

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors. RT-PCR experiments also revealed expression of A(2b) adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (I(sc)) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na(+) channel (ENaC) alpha-, beta-, and gamma-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na(+) transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPgammaS. Basolateral stimulation with UTP produced a rapid but transient increase in I(sc) that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in I(sc) and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca(2+) mobilization evoked by P2Y receptor agonists stimulates Na(+) absorption by increasing the activity of Ca(2+)-activated K(+) channels located in the basolateral membrane.     

Effect of NYD-SP27 down-regulation on ATP-induced Ca2+-dependent pancreatic duct anion secretion in cystic fibrosis cells

Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca(2+)-dependent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I(SC)) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl(-) channel blocker, DPC or Glibenclamide (1mM), and inhibitor of Na(+)-K(+)-Cl(-) cotransporter, bumetanide (100 microM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 microM), and Ca(2+)-dependent Cl(-) channel (CaCC) blocker, DIDS (100 microM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 microM) stimulated a fast transient I(SC) increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 microM) reduced the ATP-induced I(SC) response in AT-CF cells by 77.9%. PMA (1 microM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 microM) could inhibit the ATP-induced I(SC) response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca(2+) dependent pancreatic anion secretion.