Effects of mucosal sodium removal on cell volume in Necturus gallbladder epithelium

1985 ◽  
Vol 249 (3) ◽  
pp. C304-C312 ◽  
Author(s):  
C. W. Davis ◽  
A. L. Finn
Keyword(s):  
Cell Volume ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Complete Removal ◽  
Sodium Concentration ◽  
Inhibitory Effect ◽  
Cell Swelling ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder ◽  
Sodium Removal

Necturus gallbladder epithelium transports sodium and chloride by a process that first involves the cellular entry of each ion across the apical membrane in an electrically silent process. In this paper we present results from cell volume and fluid flux measurements in the presence of different inhibitors and at normal and reduced sodium concentrations, which bear on the process by which ionic entry is effected. We find that reduction of mucosal sodium to a concentration of 10 mM has no effect on either cell volume or on the rate of transepithelial fluid transport, whereas the complete removal of sodium causes a significant decrease in cell volume in addition to its known inhibitory effect on fluid transport. Amiloride had no effect on cell volume at normal sodium concentrations but markedly reduced it when the sodium concentration was reduced to 10 mM. Amiloride, bumetanide, and dipyridamole markedly and reversibly inhibited fluid transport. Finally, the addition of ouabain to the serosal medium induced cell swelling, which was prevented by the removal of potassium from the mucosal medium. These results indicate that the process of sodium entry at the apical membrane is complicated and likely includes both cotransport (NaCl or Na-K-2Cl) and parallel exchange (Na-H and Cl-HCO3) transport mechanisms, and that the proportion of NaCl transported by the different mechanisms varies with the conditions.

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 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.

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.

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 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.

Coupled NaCl entry into Necturus gallbladder epithelial cells

1982 ◽  
Vol 243 (3) ◽  
pp. C140-C145 ◽  
Author(s):  
A. C. Ericson ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Cell Volume ◽  
Apical Membrane ◽  
Ionic Composition ◽  
Basolateral Membrane ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Bathing Solution ◽  
Parallel Operation ◽  
Solute Content

NaCl entry into Necturus maculosus gallbladder epithelial cells was studied by determination of the rate of fluid movement into the cell when the Na+-K+-ATPase was inhibited by 10(-4) M ouabain in the serosal bathing solution. The cell swelling was due to continuing entrance of NaCl into the cell across the apical membrane, which increased the solute content of the cell; the resultant rise in cell osmolality induced water flow and cell swelling. The rate of swelling was 4.3% of the cell volume per minute, equivalent to a volume flow across the apical membrane of 1.44 x 10(-6) cm/s, similar in magnitude to the normal rate of fluid absorption by the gallbladder. We determined the mechanism of NaCl entry by varying the ionic composition of the mucosal bath; when most of the mucosal Na+ or Cl- was replaced, cell volume did not increase during pump inhibition. The rate of NaCl entry was a saturable function of Na+ or Cl- in the mucosal bathing solution with K1/2 values of 26.6 mM for Na+ and 19.5 mM for Cl-. The mode of NaCl entry was probably not the parallel operation of Na+-H+ and Cl(-)-HCO-3 exchangers because of the lack of effect of bicarbonate removal or of the inhibitors amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. NaCl entry was reversibly inhibited by bumetanide in the mucosal bathing solution. Transepithelial NaCl and water absorption is the result of the coupled, carrier-mediated movement of NaCl into the cell across the apical membrane and the active extrusion of Na+ by the Na+-K+-ATPase in the basolateral membrane.

scholarly journals Interactions of sodium transport, cell volume, and calcium in frog urinary bladder.

1987 ◽  
Vol 89 (5) ◽  
pp. 687-702 ◽  
Author(s):  
C W Davis ◽  
A L Finn
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cell Volume Regulation ◽  
Positive Control ◽  
Negative Control ◽  
Cell Swelling ◽  
Ionophore A23187 ◽  
Intracellular Ca

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.

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.

Regulation of NaCl entry into Necturus gallbladder epithelium by protein kinase C

1994 ◽  
Vol 266 (2) ◽  
pp. C531-C535 ◽  
Author(s):  
R. Dausch ◽  
K. R. Spring
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Nacl Transport ◽  
Control Value ◽  
Necturus Gallbladder ◽  
Kinase C

The role of protein kinase C in the regulation of the mode of NaCl entry into Necturus gallbladder epithelial cells was determined from the rate and magnitude of ouabain-induced cell swelling in the presence of inhibitors. Stimulation of protein kinase C by phorbol ester increased the rate of cell swelling from the control value of 2.9% to 4.7%/min and caused the predominant apical membrane transport mechanism for NaCl to switch from bumetanide-sensitive Na-Cl cotransport to amiloride-sensitive parallel exchange. Na-Cl cotransport could be restored as the predominant mode of NaCl entry by treatment of stimulated tissues with the kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and calphostin C. Therefore the mechanism of NaCl transport across the apical membrane can be controlled by the activity of protein kinase C.

Effects of apical membrane Cl(-)-formate exchange on cell volume in rabbit proximal tubule

1990 ◽  
Vol 258 (3) ◽  
pp. F530-F536 ◽  
Author(s):  
L. Schild ◽  
P. S. Aronson ◽  
G. Giebisch
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Volume Changes ◽  
Proximal Tubule Cells ◽  
Volume Increase ◽  
Tubule Lumen ◽  
Stimulation Of

We used real-time recordings of cell volume changes to test for the role of the Cl(-)-formate exchanger in mediating NaCl entry across the apical membrane of rabbit proximal tubule cells. In the absence of extracellular Cl-, 0.5 and 5 mM formate in the tubule lumen induced an increase in cell volume of 1 and 9%, respectively. Formate-induced cell swelling was reduced by alkalinizing the tubule lumen or by addition of luminal amiloride (2 mM), indicating that the increase in cell volume results from the intracellular accumulation of Na-formate via nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. The cell volume increase induced by 0.5 mM formate was potentiated (from 1 to 4%) by Cl-, as expected for a formate-mediated stimulation of NaCl uptake via parallel Cl(-)-formate exchange and Na(+)-H+ exchange across the apical membrane. By contrast, the cell volume increase induced by 5 mM formate was attenuated (from 9 to 4%) by Cl-. The attenuating effect of Cl- on formate-induced cell swelling required the operation of the apical membrane Cl(-)-formate exchanger. The effect of 1:1 Cl(-)-formate exchange to attenuate formate-induced cell swelling can be explained if the cell possesses a volume-activated anion exit pathway, most likely at the basolateral cell membrane, that is capable of mediating the efflux of Cl- but not formate from the cell.

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