Electrogenic bicarbonate secretion by prairie dog gallbladder

2007 ◽  
Vol 292 (6) ◽  
pp. G1683-G1694 ◽  
Author(s):  
A. James Moser ◽  
A. Gangopadhyay ◽  
N. A. Bradbury ◽  
K. W. Peters ◽  
R. A. Frizzell ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Impedance Analysis ◽  
Membrane Resistance ◽  
Disulfonic Acid ◽  
Prairie Dog ◽  
Anion Secretion ◽  
Ion Substitution

Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current ( Isc; 171 ± 21 μA/cm2) and lumen-negative potential difference (−10.1 ± 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to Isc. In response to 2 μM forskolin, Isc exceeded 270 μA/cm2, and impedance estimates of the apical membrane resistance decreased from 200 Ω·cm2 to 13 Ω·cm2. The forskolin-induced Isc was dependent on extracellular HCO3− and was blocked by serosal 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl− ion substitution had little effect. Serosal trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba2+ reduced Isc, consistent with the inhibition of cAMP-dependent K+ channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO3− entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K+ channel activation.

scholarly journals Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels

2019 ◽  
Author(s):  
Yoshio Takei ◽  
Marty K.S. Wong ◽  
Masaaki Ando
Keyword(s):  
Hormonal Regulation ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Intestinal Epithelia ◽  
Mucosal Side

AbstractThe intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, reports on studies on the hormonal regulation of HCO3- secretion are just emerging. Here, we showed that guanylin (GN) applied to the mucosal side of intestinal epithelia increased HCO3- secretion in seawater-acclimated eels. The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time-course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4’-dinitrostilbene-2,2’-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. However, mucosal DNDS and diphenylamine-2-carboxylic acid (DPC) failed to inhibit the GN effect, showing that apical AE and Cl- channel are not involved. To identify molecular species of possible transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, those of Slc26a3a, b (DRAa, b) and Slc26a6a, c (Pat-1a, c) on the apical membrane of the intestinal epithelial cells, and those of Sls4a4a (NBCe1a), Slc4a7 (NBCn1), Slc4a10a (NBCn2a) and Slc26a1 (Sat-1) on the basolateral membrane were candidate transporters involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAa, b and basolateral DNDS-sensitive NBCela, n1, n2a to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

Apical adenosine activates an amiloride-sensitive conductance in human intestinal cell line HT29cl.19A

1997 ◽  
Vol 272 (3) ◽  
pp. C931-C936 ◽  
Author(s):  
H. Bouritius ◽  
J. A. Groot
Keyword(s):  
Adenosine Receptor ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Intestinal Cell ◽  
Cation Conductance ◽  
Intestinal Cell Line ◽  
Stimulation Of

We studied the effects of stimulation of the apical adenosine receptor on ion transport by HT29cl.19A cells with the conventional microelectrode technique. Adenosine (100 microM) caused an increase in the transepithelial potential (3.6 +/- 0.4 mV) and equivalent short-circuit current (I(sc), 21 +/- 3 microA/cm2), a transient depolarization of the apical membrane potential (14 +/- 2 mV), and a decrease in the apical membrane resistance. The increase in I(sc) was additive to the effect of forskolin or basolateral addition of a maximal concentration of adenosine. Bumetanide, applied after adenosine, caused a further depolarization (7 +/- 2 mV) concomitant with a decrease in I(sc) (-13 +/- 2 microA/cm2) and an increase in the basolateral membrane resistance. Substitution of Cl- with gluconate or Na+ with N-methylglucamine reduced the response to adenosine by >60%. The response was also reduced by a low concentration of amiloride. We conclude that stimulation of the apical adenosine receptor activated a cation conductance in the apical membrane.

Effect of cAMP agonists on cell pH and anion transport by cultured rat inner medullary collecting duct cells

1996 ◽  
Vol 270 (1) ◽  
pp. F131-F140 ◽  
Author(s):  
C. Zhang ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Anion Transport ◽  
Disulfonic Acid ◽  
Anion Secretion ◽  
Inner Medullary Collecting Duct ◽  
Cell Ph ◽  
Medullary Collecting Duct

The rat inner medullary collecting duct is capable of secreting anions. We previously showed that adenosine 3',5'-cyclic monophosphate (cAMP) stimulates anion secretion; the apical membrane anion exit pathway activated by cAMP appears to be the cystic fibrosis transmembrane conductance regulator Cl- channel. The present experiments were designed to test the hypothesis that the entry pathway across the basolateral membrane is a Cl-/HCO3- exchanger operating in parallel with an Na+/H+ exchanger. We investigated the mechanism by measuring cell Cl-, cell pH, and short-circuit current under a variety of conditions designed to uncover these pathways. cAMP agonists caused little change in cell Cl-, but they produced a consistent intracellular acidification. This acidification was dependent on HCO3-, but not on Cl-, and was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The presence of the basolateral Cl-/HCO3- exchanger was demonstrated by several maneuvers, and its activity was inhibited by DIDS. Applied to the basolateral solution, DIDS did not inhibit the cAMP-dependent anion current but actually stimulated it. We conclude that cAMP-stimulated anion secretion does not require activation of the basolateral Cl-/HCO3- exchanger. The transporter responsible for Cl- entry across the basolateral membrane remains unknown and is not inhibited by a variety of anion transport inhibitors, including DIDS, bumetanide, and hydrochlorothiazide. The cell acidification induced by cAMP appears to be independent of acid secretion and is the result of activation of one or more HCO3- exit pathways that are resistant to DIDS but are inhibited by a nonspecific anion transport inhibitor, 5-nitro-2-(3-phenylpro-pylamino) benzoic acid. We present a revised model for anion transport by the rat inner medullary collecting duct.

Electrophysiology and noise analysis of K+-depolarized epithelia of frog skin

1985 ◽  
Vol 249 (5) ◽  
pp. C421-C429 ◽  
Author(s):  
J. Tang ◽  
F. J. Abramcheck ◽  
W. Van Driessche ◽  
S. I. Helman
Keyword(s):  
Frog Skin ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Na Channel ◽  
Channel Density

Epithelia of frog skin bathed either symmetrically with a sulfate-Ringer solution or bathed asymmetrically and depolarized with a 112 mM K+ basolateral solution (Kb+) were studied with intracellular microelectrode techniques. Kb+ depolarization caused an initial decrease of the short-circuit current (Isc) with a subsequent return of the Isc toward control values in 60-90 min. Whereas basolateral membrane resistance (Rb) and voltage were decreased markedly by high [Kb+], apical membrane electrical resistance (Ra) was decreased also. After 60 min, intracellular voltage averaged -27.3 mV, transcellular fractional resistance (fRa) was 86.8%, and Ra and Rb were decreased to 36.1 and 13.0%, of their control values, respectively. Amiloride-induced noise analysis of the apical membrane Na+ channels revealed that Na+ channel density was increased approximately 72% while single-channel Na+ current was decreased to 39.9% of control, roughly proportional to the decrease of apical membrane voltage (34.0% of control). In control and Kb+-depolarized epithelia, the Na+ channel density exhibited a phenomenon of autoregulation. Inhibition of Na+ entry (by amiloride) caused large increases of Na+ channel density toward saturating values of approximately 520 X 10(6) channels/cm2 in Kb+-depolarized tissues.

Sodium and chloride transport across the isolated porcine gallbladder

1989 ◽  
Vol 257 (1) ◽  
pp. C45-C51 ◽  
Author(s):  
S. M. O'Grady ◽  
P. J. Wolters
Keyword(s):  
Apical Membrane ◽  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Disulfonic Acid ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Conductive Pathway

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.

Diluting segment in kidney of dogfish shark. II. Electrophysiology of apical membranes and cellular resistances

1990 ◽  
Vol 258 (2) ◽  
pp. R409-R417 ◽  
Author(s):  
S. C. Hebert ◽  
P. A. Friedman
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Primary Resistance ◽  
Diluting Segment ◽  
Dogfish Shark

Diluting segments from the bundle zone of the dogfish shark kidney were perfused in vitro and the electrophysiological characteristics of this segment investigated using conventional microelectrodes and cable analysis. In 21 tubules perfused with symmetrical Ringer solutions the average transepithelial voltage (Vte), transepithelial conductance (Gte), and equivalent short circuit current (Isc) were 8.7 +/- 0.6 mV, 91.3 +/- 10.2 mS/cm2, and 641 +/- 48 microA/cm2, respectively. Microelectrode impalements in 52 cells yielded values for the basolateral membrane voltage (Vb) and an estimated apical membrane fractional resistance (fRa) of -57.5 +/- 1.3 mV and 0.896 +/- 0.008, respectively. All of these parameters were distributed in a Gaussian manner. Liminal furosemide (10(-4) M) abolished Isc, hyperpolarized apical membrane voltage (Va) and Vb, increased Gte, and reduced fRa. The apical membrane was predominantly conductive to K+: increasing luminal K+ from 5 to 49.7 mM resulted in an apical depolarization of 41.2 mV and a fall in fRa and luminal Ba2+ (1 mM) depolarized Va by 14.3 mV and increased fRa. The apical transference number for K+ was 0.74 +/- 0.07. The cellular and paracellular resistances were estimated from the effects of luminal Ba2+ on fRa and Gte. The cell conductance represented approximately 45% of Gte, with the primary resistance barrier located at the apical membrane: apical membrane resistance was 59.7 +/- 16.0 and basolateral membrane resistance was 5.9 +/- 2.3 omega.cm2. From these resistance values together with the passive permeability (PNa/PCl) of 2.5 determined previously, the ratio of net Cl- absorption to net transcellular Na+ absorption was determined to be 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium transport and intracellular sodium activity in cultured human nasal epithelium

1991 ◽  
Vol 261 (2) ◽  
pp. C319-C331 ◽  
Author(s):  
N. J. Willumsen ◽  
R. C. Boucher
Keyword(s):  
Apical Membrane ◽  
Driving Forces ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Nasal Epithelium ◽  
Airway Epithelia ◽  
Human Nasal Epithelium ◽  
Intracellular Na Activity

Human airway epithelia are predominantly Na(+)-absorbing epithelia. To investigate the mechanisms for Na+ absorption across airway epithelia, the driving forces and paths for Na+ translocation across each membrane were examined with double-barreled Na(+)-selective microelectrodes in cultured human nasal epithelium (HNE). Under control conditions, intracellular Na+ activity (acNa) was 23 +/- 1 mM (n = 44 preparations, 393 impalements). Amiloride (10(-4) M) hyperpolarized the apical membrane and increased the fractional apical membrane resistance but did not affect acNa. Exposure to Na(+)-free luminal solution induced bioelectric responses similar to amiloride but also reduced acNa to 8 +/- 1 mM. Reduction of luminal Na+ concentration ([Na+]) in the presence of amiloride also reduced acNa without further changes in bioelectric parameters. Reduction of serosal [Na+] decreased aNac, a response blocked by bumetanide (10(-4) M). Ouabain (10(-4) M, serosal) led to a reduction in equivalent short-circuit current (Ieq) and increase in acNa. We conclude that 1) acNa is higher in HNE than in most mammalian epithelial cells, 2) the apical membrane expresses a conductive Na+ path, and 3) the basolateral membrane transports Na+ via the Na(+)-K(+)-adenosinetriphosphatase and a Na(+)-K(+)-2Cl- cotransport system.

scholarly journals Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

2005 ◽  
Vol 288 (5) ◽  
pp. G956-G963 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Isolated Cells ◽  
Rat Ileum ◽  
Anion Secretion ◽  
Cl Secretion ◽  
Ion Absorption ◽  
Isotonic Buffer

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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