Calcium is not involved in the cAMP-mediated stimulation of Cl? conductance in the apical membrane of Necturus gallbladder epithelium

1995 ◽  
Vol 429 (5) ◽  
pp. 647-658 ◽  
Author(s):  
G. Kottra
Keyword(s):  
Apical Membrane ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder ◽  
Cl Conductance ◽  
Stimulation Of

scholarly journals Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

1992 ◽  
Vol 99 (2) ◽  
pp. 241-262 ◽  
Author(s):  
G A Altenberg ◽  
J S Stoddard ◽  
L Reuss
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Gallbladder Epithelium ◽  
K Channels ◽  
Necturus Gallbladder ◽  
Electrophysiological Effects ◽  
Paracellular Diffusion ◽  
Cl Conductance

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.

Regulation of apical membrane ion transport in Necturus gallbladder

1992 ◽  
Vol 263 (1) ◽  
pp. C187-C193 ◽  
Author(s):  
J. L. Garvin ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder ◽  
Camp Levels ◽  
Nacl Cotransport ◽  
Cl Conductance ◽  
Membrane Potential Difference ◽  
Membrane Ion Transport

Na and Cl movement through the apical membrane of Necturus gallbladder epithelium was investigated using electrophysiological and light microscopic measurements. Changes in membrane potential difference, fractional resistance of the apical membrane, and transepithelial resistance caused by changes in apical bath Cl concentration revealed the presence of a Cl conductance in the apical membrane of control tissues that was apparently not present in the preparations studied by other investigators. This Cl conductance was blocked by bumetanide (10(-5) M) or by the inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP) action, the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 0.5 mM). Treatment of the tissues with Rp-cAMPS also eliminated bumetanide-sensitive cell swelling in the presence of ouabain and activated an amiloride-sensitive swelling, changes consistent with inhibition of NaCl cotransport and the activation of Na-H and Cl-HCO3 exchange. We conclude that the mode of NaCl entry into Necturus gallbladder epithelial cells is determined by the level of cAMP. When cAMP levels are high, entry occurs by NaCl cotransport; when cAMP levels are low, parallel exchange of Na-H and Cl-HCO3 predominates. These observations explain the previous disagreements about the mode of NaCl entry into Necturus gallbladder epithelial cells.

Furosemide blocks basolateral membrane Cl- permeability in gallbladder epithelium

1990 ◽  
Vol 258 (6) ◽  
pp. C1150-C1158 ◽  
Author(s):  
J. S. Stoddard ◽  
G. A. Altenberg ◽  
M. L. Ferguson ◽  
L. Reuss
Keyword(s):  
Anion Exchange ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Ringer Solution ◽  
Combined Effect ◽  
Gallbladder Epithelium ◽  
K Permeability ◽  
Cl Conductance ◽  
Stimulation Of ◽  
Cl Permeability

In Necturus gallbladders bathed in a NaCl Ringer solution buffered with 10 mM HCO3(-)-1% CO2, furosemide (added to the serosal solution) caused a concentration-dependent hyperpolarization of both cell membranes that was slow and reversible. At 10(-3) M furosemide, the basolateral membrane voltage (Vcs) increased significantly from -71 +/- 3 to -85 +/- 3 mV, the depolarization of Vcs elicited by a 10-fold rise in serosal [K+] increased from 34 +/- 4 to 50 +/- 1 mV, the depolarization elicited by lowering serosal [Cl-] from 98 to 8.1 mM was reduced from 15 +/- 1 to 1 +/- 1 mV, and the depolarization in response to lowering serosal [HCO3-] from 10 to 1 mM was reduced from 13 +/- 1 to 5 +/- 0.4 mV. Furosemide could in principle decrease the basolateral membrane Cl- conductance (Gcl), increase the basolateral membrane K+ conductance, or have a combined effect. To distinguish among these possibilities, we estimated the resistance of the basolateral membrane (Rb) by means of two-point intraepithelial cable analysis experiments. Furosemide increased Rb by 22%, which indicates that furosemide reduces basolateral membrane Gcl. The effect cannot be attributed to inhibition of apical membrane anion exchange by serosal addition of furosemide, because base secretion from cells to lumen is unchanged. We conclude that furosemide blocks reversibly basolateral membrane electrodiffusive Cl- permeability. A concomitant stimulation of basolateral membrane electrodiffusive K+ permeability is also possible.

Muscarinic stimulation of gallbladder epithelium. III. Antagonism of cAMP-mediated effects

1994 ◽  
Vol 267 (5) ◽  
pp. C1196-C1202 ◽  
Author(s):  
G. A. Altenberg ◽  
M. Subramanyam ◽  
L. Reuss
Keyword(s):  
Adenylate Cyclase ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Physiological Role ◽  
Electrophysiological Response ◽  
Mediated Effects ◽  
Camp Levels ◽  
Electrophysiological Effects ◽  
Cl Conductance ◽  
Stimulation Of

Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) levels in Necturus gallbladder (NGB) epithelium activates an apical membrane Cl- conductance and decreases transepithelial fluid transport (Jv). Acetylcholine (ACh), which had no effects on Jv by itself, antagonized the electrophysiological effects of forskolin (FSK) and theophylline and the decrease in Jv produced by FSK. By itself, ACh had no effects on basal cAMP levels but antagonized the increases in cAMP induced by FSK and theophylline. ACh had no effect on phosphodiesterase activity and prevented both the electrophysiological response and the elevation in cAMP by theophylline. In conclusion, the effect of ACh is mediated by inhibition of adenylate cyclase. A pertussis toxin (PTX)-sensitive G protein may mediate inhibition of adenylate cyclase because pretreatment with PTX prevented the reversal of the electrophysiological effects of FSK by ACh, and PTX catalyzed the ribosylation of cell membranes from NGB epithelium. ACh could have a physiological role in modulating the effects of secretagogues that act via elevation of cAMP levels.

Potassium-induced cell swelling in Necturus gallbladder epithelium

1988 ◽  
Vol 254 (5) ◽  
pp. C643-C650 ◽  
Author(s):  
C. W. Davis ◽  
A. L. Finn
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Basolateral Membrane ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Gallbladder Epithelium ◽  
Bathing Medium ◽  
Necturus Gallbladder ◽  
Per Se ◽  
Cl Conductance

In Necturus gallbladder epithelium, elevation of mucosal K+ to 95 mM in the presence of 10 mM Na+ resulted in cell swelling at a rate of 3.2% original volume per minute, followed by volume-regulatory shrinking. When Na+ was completely removed from or when amiloride (10(-4) M) was added to the mucosal medium, K+-induced cell swelling was abolished. In the presence of 10 mM Na+, 1 mM Ba2+ abolished and substitution of mucosal Cl- by NO-3 had no effect on K+-induced swelling. Thus solute entry following elevation of mucosal K+ is effected by separate K+ and Cl- pathways. Furthermore, substitution of 95 mM K+ for Na+ in the mucosal bathing medium leads to the development of a Cl- conductance in the basolateral membrane as long as some Na+ remains in the medium. However, cell swelling induced by mucosal dilution does not lead to the appearance of a Cl- conductance. Thus the activation of this conductance requires both swelling and membrane depolarization. These results show that 1) high mucosal K+ leads to cell swelling due to the entry of Cl- along with K+ and the Cl- can enter across either membrane, 2) the Cl- pathways require the presence of mucosal Na+, and 3) cell volume regulation is activated by an increase in volume per se, i.e., a hyposmotic exposure is not required for volume regulation to occur.

scholarly journals Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.

1990 ◽  
Vol 95 (5) ◽  
pp. 791-818 ◽  
Author(s):  
Y Segal ◽  
L Reuss
Keyword(s):  
Patch Clamp ◽  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Depolarization ◽  
Membrane Voltage ◽  
K Channel ◽  
Gallbladder Epithelium ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Necturus Gallbladder

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.

scholarly journals Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium.

1985 ◽  
Vol 85 (3) ◽  
pp. 409-429 ◽  
Author(s):  
L Reuss ◽  
K U Petersen
Keyword(s):  
Apical Membrane ◽  
Phosphodiesterase Inhibitor ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Intracellular Camp ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder ◽  
Intracellular Na Activity ◽  
Acid Extrusion ◽  
Camp Levels

The effects of elevating intracellular cAMP levels on Na+ transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 microM). During elevation of intracellular cAMP, intracellular Na+ activity (alpha Nai) and intracellular pH (pHi) decreased significantly. In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na+, was inhibited by approximately 50%. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of alpha Nai upon rapid alterations of mucosal [Na+] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was approximately 50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na+/H+ exchange. The results of measurements of pHi recovery at 10 and 100 mM mucosal [Na+] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.-U., and L. Reuss, 1983, J. Gen. Physiol., 81:705), inhibition of Na+/H+ exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na+/H+ exchange by cAMP plays a role in the regulation of pHi in other cell types.

scholarly journals Effects of changes in mucosal solution Cl- or K+ concentration on cell water volume of Necturus gallbladder epithelium.

1991 ◽  
Vol 97 (4) ◽  
pp. 667-686 ◽  
Author(s):  
C U Cotton ◽  
L Reuss
Keyword(s):  
Apical Membrane ◽  
Cell Swelling ◽  
Water Volume ◽  
Intracellular Camp ◽  
Gallbladder Epithelium ◽  
Cell Water ◽  
Cell Shrinkage ◽  
Necturus Gallbladder ◽  
Significant Change ◽  
Camp Levels

An electrophysiologic technique was used to measure changes in cell water volume in response to isosmotic luminal solution ion replacement. Intracellular Cl- activity (aCl-i) was measured and net flux determined from the changes in volume and activity. Reduction of luminal solution [Cl-] from 98 to 10 mM (Cl- replaced with cyclamate) resulted in a large fall in aCl-i with no significant change in cell water volume. Elevation of luminal solution [K+] from 2.5 to 83.5 mM (K+ replaced Na+) caused a small increase in aCl-i with no change in cell water volume. Exposure of the Necturus gallbladder epithelium to agents that increase intracellular cAMP levels (forskolin and/or theophylline) induces an apical membrane electrodiffusive Cl- permeability accompanied by a fall in aCl-i and cell shrinkage. In stimulated tissues, reduction of luminal solution [Cl-] resulted in a large fall in aCl-i and rapid cell shrinkage, whereas elevation of luminal solution [K+] caused a large, rapid cell swelling with no significant change in aCl-i. The changes in cell water volume of stimulated tissues elicited by lowering luminal solution [Cl-] or by elevating luminal solution [K+] were reduced by 60 and 70%, respectively, by addition of tetraethylammonium (TEA+) to the luminal bathing solution. From these results, we conclude that: (a) In control tissues, the fall in aCl-i upon reducing luminal solution [Cl-], without concomitant cell shrinkage, indicates that the Cl- entry mechanism is electroneutral (Cl-/HCO3-) exchange. (b) Also in control tissues, the small increase in aCl-i upon elevating luminal solution [K+] is consistent with the recent demonstration of a basolateral Cl- conductance. (c) The cell shrinkage elicited by elevation of intracellular cAMP levels results from conductive loss of Cl- (and probably K+). (d) Elevation of cAMP inhibits apical membrane Cl-/HCO-3-exchange activity by 70%. (e) The cell shrinkage in response to the reduction of mucosal solution [Cl-] in stimulated tissues results from net K+ and Cl- efflux via parallel electrodiffusive pathways. (f) A major fraction of the K+ flux is via a TEA(+)-sensitive apical membrane K+ channel.

scholarly journals cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.

1993 ◽  
Vol 102 (2) ◽  
pp. 177-199 ◽  
Author(s):  
J Copello ◽  
T A Heming ◽  
Y Segal ◽  
L Reuss
Keyword(s):  
Chloride Channels ◽  
Apical Membrane ◽  
Membrane Conductance ◽  
Intracellular Camp ◽  
Channel Blockers ◽  
Gallbladder Epithelium ◽  
Open Probability ◽  
Necturus Gallbladder ◽  
Cytosolic Side ◽  
Cl Channels

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- > SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.

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