Guinea Pig Gallbladder Epithelium Research Articles

An Anion Channel in Guinea Pig Gallbladder Epithelial Cells Is Highly Permeable to HCO−3

In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl− and HCO−3, is elicited in the presence of a high intracellular concentration of adenosine 3′-5′-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pdcm). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine–Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl− channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10−5 M), but not by other anionic inhibitors. Measurements of the pdcm value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl− and HCO−3. The selectivity of the channel was gluconate < SO2−4 < Cl− < Br− < I− < HCO−3 < SCN− and the PHCO3/PCl was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO−3 conductance activated by cAMP.

Large-conductance K + channels and their regulation in epithelial cells of guinea pig gall-bladder

Ion secretion in guinea pig gall-bladder epithelium can be electrogenic. Therefore ion channels were studied utilizing the patch-clamp technique. Using the cell-attached configuration on dissociated cells, K + channels with a single-channel conductance of about 160 pS, for negative voltage values, were identified. An inward rectification was usually observed. The channel open probabilities at physiological voltage values were low (less than 0.1) and increased 3- to 4-fold with depolarization of 20–30 mV. Using inside-out configuration and symmetrical 145 mM KCI, the solI V curve was linear and was not modified by the presence of Mg 2+ and ATP on the cytoplasmic side. The channels were selective for K + over Na + ( P Na + / P K + = 0.05). They were activated by increased levels of [Ca 2+] or pH on the cytoplasmic side. Data suggest that the activity of these channels is not only influenced by voltage, [Ca 2+], pH but also by unidentified cytoplasmic factors responsible for the inward rectification.

Localization of large-conductance K + channels of guinea pig gall-bladder epithelium studied using isolated cells or primary cell cultures

Localization of large-conductance K + channels of guinea pig gall-bladder epithelium studied using isolated cells or primary cell cultures

Electrogenic bicarbonate secretion in gallbladder: induction by barium via neuronal, possibly VIP-ergic pathways.

In guinea-pig gallbladder epithelium, cAMP converts electroneutral HCO3- secretion into an electrogenic process. The effects of blood side Ba2+ (5 mmol/l) on HCO3- transport were investigated in vitro, using pH-stat and voltage clamp techniques to determine unidirectional fluxes of HCO3- and transepithelial electrical characteristics. Serosal, not mucosal addition of Ba2+ elevated short-circuit current (Isc), transepithelial potential difference, and tissue conductance; it inhibited the absorptive HCO3- flux while leaving the secretory flux unchanged. The Isc effect of Ba2+ was inhibited or prevented by tetrodotoxin; D- and L-propranolol; the Cl- channel blocker 4-N-methyl-N-phenylaminothiophene-3-carboxylic acid; the intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)ocytl ester; noradrenaline, by a yohimbine-sensitive action; somatostatin; HCO3(-)-free solutions. Thus Ba2+ appeared to release a neurotransmitter that gives rise to cAMP synthesis sufficient to turn part of electroneutral HCO3- secretion electrogenic. In a search for the involved signalling pathways, the H1-receptor antagonist, cetirizine, largely and hexamethonium, atropine, atenolol, indomethacin, and trifluoperazine entirely failed to antagonize the Isc effect of Ba2+. Similarly, carbachol, dobutamine, salbutamol, and serotonin were unable to mimic the action of Ba2+ and Isc effects of histamine were small and short-lived. By contrast, vasoactive intestinal peptide (VIP; 3 x 10(-7) mol/l) completely transformed HCO3- secretion into an electrogenic process. The VIP receptor antagonist (4Cl-DPhe6, Leu17) VIP, delayed and reduced the Isc responses to Ba2+ and VIP. As guinea-pig gallbladder epithelial cells possess cAMP-coupled VIP receptors close to VIPergic neurons, Ba2+ is likely to act by releasing VIP from neural terminals.

Convective movement of Ca2+ across guinea pig gallbladder epithelium.

Recently, much interest has developed in biliary calcium because of its importance in the pathogenesis and composition of gallstones. While much progress has been made in understanding the thermodynamic factors that control biliary calcium concentrations, little is known about the kinetic factors that control the movement of calcium across the gallbladder epithelium. These studies measure guinea pig gallbladder epithelial permeability to Ca2+ during in vivo convective water movement across the membrane. Water movement, ranging from -15.2 (absorption) to 6.3 microliters.min-1.cm-2 (water entry), was induced by placing hypotonic, isotonic, and hypertonic solutions into the gallbladder lumen. Calcium movement was found to be directly and linearly related to water flow, indicating that Ca2+ moved with the convective water flow, presumably across paracellular channels. The slope of this relationship (0.602), representing the concentration of calcium in the fluid translocated across the gallbladder epithelium, was only about half that of plasma or luminal contents, indicating that calcium movement across the membrane was restricted. The mean sieving coefficient (1 - r) of guinea pig gallbladder, calculated from this slope, was approximately 0.5, indicating that the epithelium is only moderately permeable to Ca2+. The results suggest that intraluminal chelation of Ca2+ for the possible prevention and/or treatment of calcium-containing gallstones is a potentially feasible therapeutic modality.

Naloxone-insensitive transport effects of loperamide in guinea-pig gallbladder epithelium

The effects of the antidiarrheal drug, loperamide, on HCO3 and Na transport across guinea-pig gallbladder epithelium were investigated using Ussing-chamber methods. Under basal conditions, mucosal loperamide (10(-4) mol/l) moderately lowered both the absorptive (JHCO3ms) and the secretory HCO3 flux (JHCO3sm) (pH-stat method), most likely by changing paracellular HCO3 flow. Exposure to serosal prostaglandin E1 (10(-6) mol/l) abolished Na absorption and turned HCO3 secretion electrogenic. The associated short-circuit current (Isc) was inhibited by loperamide in a concentration-dependent manner; mucosal addition (threshold at 3 x 10(-6) mol/l) of the drug was more effective. Inhibition of Isc was related to a decrease in JHCO3sm, but exceeded the drop in JHCO3net. The effects on JHCO3sm and Isc were mimicked by [Met5]enkephalin. Naloxone (10(-6) mol/l) was unable to influence the effects of loperamide and [Met5]enkephalin on Isc. There were no pro-absorptive effects of loperamide on unidirectional Na fluxes. We conclude that antisecretory properties of loperamide are solely due to inhibition of electrogenic HCO3 secretion, an effect unrelated to opiate receptor binding.

Selective blockage of cell membrane K conductance by an antisecretory agent in guinea-pig gallbladder epithelium.

Loperamide inhibits PGE1-induced electrogenic HCO3 secretion in guinea-pig gallbladder. Underlying changes in epithelial cell membrane properties were investigated using intracellular microelectrode techniques in vitro. In the absence of PGE1, mucosal loperamide (10(-4) mol/l) reversibly depolarized both cell membranes by approximately 6 mV. The apparent ratio of membrane resistances (Ra/Rb) remained unchanged and so did voltage responses to luminal Cl removal and Na reduction. The depolarizing response to elevation of luminal K concentration from 5 to 76 mmol/l was decreased from 13 to 8 mV. In the presence of 1 PGE1, the apical membrane is mainly permeable to Cl and HCO3. Under these conditions, loperamide reduced membrane potentials by approximately 10 mV, Ra/Rb remaining constant at approximately 0.4. Effects on voltage responses to changes in luminal Na or K concentration were unchanged. Responses to luminal Cl removal (transient depolarization) were greatly enhanced (from 22 to 42 mV) as predictable from the fall in K permeability that hinders Cl efflux from cell into lumen. Less marked but significant effects were obtained with 10(-5) mol/l (mucosal side) and serosal loperamide (10(-4) mol/l). We suggest that loperamide inhibits electrogenic HCO3 secretion by reducing apical membrane K permeability. The resulting depolarization diminishes the driving force for conductive anion efflux from cell into lumen. This conclusion is supported by the ability of luminal K elevation to mimick loperamide inhibition of the secretory flux of HCO3 (pH-stat experiments).

Electrogenic bicarbonate secretion by guinea pig gallbladder epithelium: apical membrane exit

Guinea pig gallbladder epithelium secretes HCO3- by electroneutral mechanisms, resulting in transepithelial Cl- -HCO3- exchange. Adenosine 3',5'-cyclic monophosphate (cAMP) converts HCO3- secretion into an electrogenic process. This transformation was examined using voltage-clamp, pH-stat, and microelectrode techniques. Prostaglandin E1 (PGE1; 10(-6) M) was used to raise intracellular cAMP levels. It increased short-circuit current (Isc) by approximately 1.8 mumol.cm-2.h-1, an effect dependent on serosal HCO3- and, partly, on mucosal Cl-. Mucosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10(-3) M) halved Isc, but only in Cl- containing solutions. PGE1 increased the secretory HCO3- flux from approximately 2.0 to approximately 2.7 mumol.cm-2.h-1 and reduced the absorptive HCO3- flux from approximately 1.1 to approximately 0.5 mumol.cm-2.h-1, with net HCO3- secretion accounting for the increase in Isc. During single-cell impalements, PGE1 depolarized the apical membrane by greater than 10 mV (transiently in the absence of HCO3-) and decreased the apparent ratio of membrane resistances (Ra/Rb) from 5-8 to a value close to zero. These effects were largely reduced in magnitude and rapidity by removing Cl- and HCO3- from both sides of the epithelium. Ion substitutions in the luminal perfusate revealed substantial Cl- and HCO3- permeabilities at the apical membrane under PGE1 conditions. Our results indicate that, in the presence of PGE1 (cAMP), HCO3- crosses the apical membrane by two different routes. A SITS-sensitive fraction leaves the cell in exchange for luminal Cl-, which, in turn, recycles into the lumen by electrodiffusion. The remaining HCO3- exits through a HCO3- conductive pathway.

Electroneutral secretion of bicarbonate by guinea pig gallbladder epithelium.

Transepithelial HCO3- movement in guinea pig gallbladder was investigated in vitro. Absorptive (JHCO3ms) and secretory (JHCO3sm) HCO3- fluxes, determined by use of the pH-stat method were approximately 1.0 and 2.1 mumol X cm-2 X h-1, respectively. The resultant net secretion equaled in magnitude, and balanced electrically, the excess in net absorption of Cl- over that of Na+ X JHCO3sm was dependent on luminal Cl- and serosal Na+; it was inhibited by mucosal 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10(-3) M) and serosal ouabain (3 X 10(-5) M) but not by serosal amiloride (10(-3) M) and scarcely by bilateral methazolamide (10(-4) M). JHCO3ms was reduced by mucosal Cl- but enhanced by serosal Cl-; it was dependent on mucosal Na+ and inhibited by mucosal amiloride and bilateral methazolamide. Our findings are consistent with a model in which 1) serosal HCO3- enters the cell in cotransport with Na+ and is then extruded into the lumen by Cl-(-)HCO3- exchange at the apical membrane; 2) mucosal HCO3- enters the cell secondary to apical membrane Na+-H+ exchange and recycles into the lumen via Cl-(-)HCO3- exchange or is, to a lesser extent, absorbed across the basolateral membrane.

Na/H exchange at the apical membrane of guinea-pig gallbladder epithelium: properties and inhibition by cyclic AMP.

The properties and, in particular, the cAMP-sensitivity of apical Na/H exchange in guinea-pig gallbladder epithelium were investigated using gravimetric, pH-stat, and microelectrode techniques. Proton secretion was Na-dependent, inhibited by ouabain and amiloride, and insensitive to changes in apical membrane potential. It was markedly reduced by 8-Br-cAMP and PGE1. PGE1 also attenuated the changes in intracellular Na activity produced by luminal Na removal and restoration. Our results suggest inhibition of Na/H exchange by cAMP. In conjunction with the cAMP-induced rise in apical membrane Cl permeability shown previously, this effect can account for inhibition of NaCl absorption by cAMP.

Prevalence of vasoactive intestinal peptide (VIP) in the regulation of cyclic AMP production in digestive epithelia: a specific feature of human

We have previously shown that VIP, a potent stimulator of water and electrolytes secretion in human intestine, acts through its binding to specific receptors and the stimulation of cyclic AMP production in epithelial cells. We report here the occurence in epithelial cells of human stomach and gallbladder of receptors for VIP that are similar for the followings: 1) two classes of binding sites, a small number with high affinity (Kd = 1.3 × 10-9M) and a large number with low affinity (Kd = 1.6 × 10-8M); 2) positive non linear coupling with a cyclic AMP producing system; 3) effectiveness of VIP at low physiological doses (3 × 10-12M - 10-8M). Secretin does not exhibit any specific receptor in these epithelia and stimulates cyclic AMP production only at suprapharmacological doses (10-7-10-5M) through its binding to the VIP receptor. The presence of a VIP receptor together with the absence of a specific secretin receptor in stomach and gallbladder epithelia is a character specific of human. Indeed, we show that rat gastric epithelium exhibits a cyclic AMP system sensitive specifically to secretin and not to VIP and that guinea pig gallbladder epithelium exhibits both a VIP- and a secretin-sensitive cyclic AMP system. Conclusion. Peptide receptors have been characterized in epithelial cells isolated from digestive organs. Their species specificity emphasizes the importance of experiments carried out in human.

Presence of cholesterol ester synthetase activity in guinea pig gallbladder epithelium

This study is the first to demonstrate the presence of cholesteryl ester synthetase activity in the gallbladder epithelium. Using epithelium of the guinea pig gallbladder, the study demonstrated that the enzyme was localized mainly in the particulate fraction. The enzyme required CoA and ATP for activity and displayed a pH optimum of 7.0. The uptake of biliary cholesterol by gallbladder (shown by other investigators) and the presence of cholesteryl ester synthetase activity (demonstrated in this study) suggest that the gallbladder epithelium has an active role which might be important in conditions of cholesterol supersaturation in bile.