Muscarinic stimulation of gallbladder epithelium. II. Fluid transport, cell volume, and ion permeabilities

1993 ◽  
Vol 265 (6) ◽  
pp. C1613-C1619 ◽  
Author(s):  
G. A. Altenberg ◽  
M. Subramanyam ◽  
L. Reuss
Keyword(s):  
Cell Volume ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Time Dependent ◽  
Water Volume ◽  
K Channels ◽  
Membrane Conductances ◽  
Muscarinic Stimulation ◽  
Stimulation Of

Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.

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.

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−].

Time-dependent apical membrane K+ and Na+ selectivity in cultured kidney cells

1987 ◽  
Vol 253 (1) ◽  
pp. C1-C6 ◽  
Author(s):  
S. R. Thomas ◽  
E. Mintz
Keyword(s):  
Apical Membrane ◽  
Time Dependent ◽  
Kidney Cells ◽  
K Channels ◽  
Membrane Selectivity ◽  
Conductive Pathway ◽  
Group I ◽  
A6 Epithelia ◽  
Group Ii ◽  
Apical Membranes

Intracellular microelectrodes were used to study apical membrane selectivity to Na+ and K+ of cultured toad kidney cells (A6) grown on permeable supports. Membrane selectivity was tested by responses of apical membrane potential to replacement of Na+ by K+ or tetraethylammonium and by addition of amiloride to perfusion solutions. The A6 epithelia fell into two groups: those with K+-selective apical membranes, lack of amiloride sensitivity, and near-zero transepithelial potential (group I); and those with Na+-selective apical membranes and a serosa-positive, amiloride-sensitive transepithelial potential (Vm----s; group II). The transition from group I to group II behavior appeared definitive and time dependent, occurring approximately 10 days after plating onto filters. Transepithelial measurements under sterile conditions showed that overnight incubation with aldosterone (10(-7) M), after development of tight junctions (transepithelial resistance elevated) but before development of significant Vm----s, induced the switch from group I to group II behavior. Apical addition of Ba2+, a known blocker of K+ channels, unexpectedly reduced transepithelial resistance (Rm----s) in group I and group II A6, suggesting that it not only blocked K+ channels (when they are present) but may also open a parallel conductive pathway. In summary, after approximately 10 days in culture, apical membranes of A6 epithelia undergo a switch from K+ to Na+ selectivity, overnight incubation with aldosterone can trigger this change, and finally, Ba2+ may open a paracellular conductive pathway.

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)

Increased fluid absorption and cell volume in isolated rabbit proximal straight tubules after in vivo DOCA administration

1981 ◽  
Vol 241 (5) ◽  
pp. F502-F508 ◽  
Author(s):  
M. A. Knepper ◽  
M. B. Burg
Keyword(s):  
Proximal Tubule ◽  
Cell Volume ◽  
Fluid Transport ◽  
Sodium Intake ◽  
Plasma Concentrations ◽  
Dietary Sodium ◽  
Fluid Absorption ◽  
Direct Stimulation ◽  
Stimulation Of

To investigate whether mineralocorticoids affect the intrinsic capacity of the proximal tubule to absorb sodium and fluid, rabbits were chronically treated a number of ways to systematically vary plasma concentrations of mineralocorticoid hormones. The rate of fluid absorption and tubule dimensions were measured in superficial S2 segments from these rabbits. Chronic administration of deoxycorticosterone acetate (DOCA) was associated with a 67% increase in fluid absorption and a 29% increase in cell volume per unit tubule length. However, neither adrenalectomy nor low sodium diet significantly affected either fluid absorption or cell volume. Furthermore, marked dietary sodium restriction prevented the response to DOCA. We conclude that the DOCA-induced increases in fluid absorption and cell volume do not result from a direct stimulation of the proximal tubular cells by the steroid but more likely are responses to systemic effects of DOCA administration that are dependent on the level of sodium intake. Thus, we find no evidence for a direct mineralocorticoid stimulation of sodium and fluid transport by the S2 portion of the proximal tubule.

Voltage dependence and barium sensitivity of colonic K secretion in renal failure

1989 ◽  
Vol 256 (3) ◽  
pp. F490-F496 ◽  
Author(s):  
E. L. Siga ◽  
R. S. Martin ◽  
C. Ibarra ◽  
D. Veron ◽  
F. Ibarra ◽  
...  
Keyword(s):  
Renal Failure ◽  
Active Transport ◽  
Voltage Dependence ◽  
Apical Membrane ◽  
Distal Colon ◽  
K Channels ◽  
K Secretion ◽  
Stimulation Of

Net colonic K secretion (JKnet) is increased in rats and humans with chronic renal failure (CRF). To study whether transepithelial potential difference (PD), active transport forces and/or luminal K conductance play a role in this adaptation, experiments were performed in the colon of control, K-adapted, and CRF rats. Under basal conditions the PD in vivo in CRF was greater than in controls and not different from K-adapted rats. JKnet was comparable in vivo in CRF and K-adapted rats and was greater than in controls. Amiloride (10 microM) reduced PD and JKnet in K-adapted and CRF rats to levels comparable to controls. Under in vitro short-circuited conditions serosal-to-mucosal K flux (JKs----m) in distal colon was significantly increased in K-adapted and CRF animals compared with control, whereas barium caused a significant reduction in JKs----m in all groups of animals. The barium-sensitive component of K secretion was greater, however, in the two experimental groups (-0.2 +/- 0.02 and -0.2 +/- 0.07 in K-adapted and CRF animals, respectively, vs. -0.08 +/- 0.02 microeq.h-1.cm-2 in controls, P less than 0.05). However, luminal barium failed to completely inhibit the increase in K secretion observed in the experimental groups. These data suggest that an increase in PD that results in a rise in luminal negativity, stimulation of active transport, and an increase in barium-sensitive K channels and barium-insensitive pathways in apical membrane of distal colon participate in the mechanism by which net K secretion is increased in the large intestine of subjects with CRF.

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.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

2003 ◽  
Vol 284 (4) ◽  
pp. C897-C909 ◽  
Author(s):  
Sasha Blaug ◽  
Jodi Rymer ◽  
Stephen Jalickee ◽  
Sheldon S. Miller
Keyword(s):  
Membrane Potential ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Extracellular Nucleotides ◽  
Basolateral Membranes ◽  
Apical Side ◽  
Mammary Epithelia

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were −4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4 Ω · cm2, respectively. The apical membrane potential was −43.9 ± 1.7 mV, and the ratio of apical to basolateral membrane resistance ( R A/ R B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC50 of ∼24 μM (apical) and ∼30 μM (basal). Apical ATP/UTP (100 μM) depolarized apical membrane potential by 17.6 ± 0.8 mV ( n = 7) and decreased R A/ R B by a factor of ≈3. The addition of adenosine to either side (100 μM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca2+ concentration (427 ± 206 nM; 15–25 μM ATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 ± 3 μl · cm−2 · h−1( n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y2 purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca2+-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

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.

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