The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.
The factors responsible for the cell membrane hyperpolarization elicited in Necturus gallbladder epithelium on Cl- removal from the mucosal bathing solution were evaluated with conventional and ion-sensitive microelectrode techniques. Cl- removal causes reversal of apical Cl- -HCO3- exchange, resulting in a fall in intracellular Cl- activity (aiCl) and an increase in intracellular pH (pHi). Concomitantly, the cell membranes hyperpolarize to values close to the K+ equilibrium potential (EK), aiNa falls, and aiK rises. The observed changes in membrane voltage are not attributable to a pHi-dependent increase in cell membrane K+ permeability (PK), because 1) the cell membrane resistances increased and 2) elevating solution partial pressure of CO2 (PCO2) to counterbalance the cellular alkalinization on mucosal Cl- removal caused a further hyperpolarization of the cell membranes to values greater than EK. This additional hyperpolarization was related to the activity of the Na+ pump, inasmuch as it was accompanied by an increase in aiNa and was ouabain sensitive. These results are consistent with, but do not prove, pump electrogenicity. During the period of Cl- removal from the mucosal bathing solution, the cell membrane depolarization caused by raising serosal K+ concentration was increased, whereas the depolarization caused by lowering serosal Cl- concentration was decreased, compared with substitutions under control conditions. These results indicate that mucosal Cl- removal causes a decrease in basolateral PCl, which we speculate could be due to a decrease in cell volume. We conclude that the hyperpolarization of the cell membranes on mucosal Cl- removal is primarily due to the combined effects of the fall in basolateral PCl and the increase in basolateral ECl.
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.
The transmural electric PD of bladders bathed by Na2SO4 Ringer was not affected by amphotericin (5 x 10(-6) M, mucosal) but the PD followed the direction for K+ diffusion in the presence of a transmural K+ gradient. Increases in bathing solution K+ increased conductance. Ouabain pretreatment did not affect drug-induced changes in PD or conductance. Unidirectional fluxes of radiolabeled Na+ and K+ but not SO42- across the short-circuited bladder were increased by amphotericin. Ninety percent of the rise in the serosal-to-mucosal flow of Na+ disappeared when mucosal Na+ was replaced by choline. Amphotericin induced a 20-fold increase in mucosal-to-serosal K+ flux but K+ serosal-to-mucosal flow increased 200-fold. This flux asymmetry persisted for 110 min, was abolished by pre- or posttreatment with ouabain, and was immeasurable when bathing solution K+ was increased from 2.4 to 59 meq/liter. With 2.4 meq K+/liter the ratio of active Na+ reabsorption to K+ secretion was 8 to 1, but K+ secretion was not closely linked to Na+ transport. The results suggest that amphotericin induces a paracellular K+-selective path, Na+ isotope exchange, and K+ secretion.
In all animal cells, the presence of nondiffusible anions in the intracellular compartment creates a Gibbs-Donnan equilibrium which tends to produce colloid-osmotic swelling. Such a swelling is prevented by so-called mechanisms of cell volume maintenance. In addition, epithelial cells face unique and potentially severe challenges for cell volume regulation during changes in transport rate. "Homocellular" regulatory mechanisms are intrinsic mechanisms set to maintain cell volume and composition in the face of changes in transepithelial transport rate. Amiloride addition to the solution bathing the apical surface of toad or frog urinary bladder epithelium induces a rapid fall in basolateral membrane conductance with no change in cell volume. In conclusion, the results discussed in this section provide convincing demonstration of several instances of cross-talk in tight epithelia. In a later section we will discuss the sensing and transduction systems involved in these responses.
The effective thickness of the unstirred fluid layer (USL) adjacent to an epithelial barrier can be estimated from the time course for the accumulation or depletion of a solute at the membrane surface. In 1985 we reported an unstirred layer thickness of approximately 70 microns for Necturus gallbladder epithelium. In our earlier studies the delay caused by noninstantaneous bulk solution mixing was not taken into account and thus the USL thickness was systematically overestimated. In the present studies we describe an analysis of the time course of solute arrival at the membrane surface that takes into account noninstantaneous bulk solution mixing. We also describe a simple technique to monitor the accumulation or depletion of a solute at the membrane surface. The time course for the change in the concentration of either tetramethylammonium (TMA+) or tetrabutylammonium (TBA+) upon elevation of bulk solution concentration is sensed at the membrane surface with an ion-sensitive microelectrode. Because of the high selectivity of the ion-sensitive resin for TMA+ or TBA+ over other monovalent cations in the solution (Na+ and K+), a low concentration (1-2 mM) of the probe can be used. By measuring the time course of the arrival of first one probe and then the other, under identical superfusion conditions, sufficient information is obtained to eliminate multiple fits to the data, obtained when only one probe is used. Neglecting bulk solution mixing caused an error greater than 50% in estimated apparent USL thickness. The effective thickness of the USL depends critically upon chamber geometry, flow rate, and the position of superfusion and suction pipettes. Under our experimental conditions the effective USL at the mucosal surface of Necturus gallbladder epithelium was approximately 40 microns.
Cell volume regulation occurs in both tight, Na+-transporting epithelia (e.g., frog skin) and in leaky. NaCl-transporting epithelia (e.g. amphibian gallbladder). In tight epithelia volume regulation occurs only in response to cell swelling, i.e. only regulatory volume decrease (RVD) is observed, whereas in leaky epithelia cell volume regulation has been observed in response to osmotic challenges that either swell or shrink the cells. In other words, both RVD and regulatory volume increase (RVI) are present. Both volume regulatory responses involve stimulation of ion transport in a polarized fashion: in RVD the response is basolateral KCl efflux, whereas in RVI it is apical membrane NaCl uptake. The loss of KCl during RVD appears to result in most instances from increases in basolateral electrodiffusive K+ and Cl-permeabilities. In gallbladder, concomitant activation of coupled KCl efflux may also occur. The RVI response includes activation of apical membrane cation (Na+/H+) and anion (Cl-/HCO-3) exchangers. It is presently unclear whether the net ion fluxes resulting from activation of these transporters, during either RVD or RVI, account for the measured rates of restoration of cell volume. In gallbladder epithelium, RVD is inhibited by agents which disrupt microfilaments or interfere with the Ca2+-calmodulin system. These pharmacologic effects are absent in RVI. Some steps in the chain of events resulting in either RVI or RVD have been established, but the signals involved remain largely unknown. There is reason to suspect a role of intracellular pH in the case of RVI and of membrane insertion of transporters in the case of RVD, possibly with causal roles of both intracellular Ca2+ and the cytoskeleton in the latter.
'/%3e%3cuse xlink:href='%23a' transform='scale(-1 1)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(72)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(-72)'/%3e%3cuse xlink:href='%23c' transform='rotate(144)'/%3e%3c/svg%3e)