Serosal Sodium Research Articles

The gill and homeostasis: transport under stress.

in his recent cannon lecture, Cowley ([3][1]) pointed out the parallels between Cannon's time early in the last century and our own, i.e., the challenge facing us now, as then, is that of “systems integration” and understanding how organisms actually achieve homeostasis. We have begun to

Mucosal ouabain and Na+ inhibit active Rb+(K+) absorption in normal and sodium-depleted rat distal colon

To determine the effect of mucosal sodium and mucosal ouabain on active Rb+(K+) absorption, unidirectional and net 86Rb+ fluxes were measured under voltage-clamp conditions in the distal colon of normal and sodium-depleted rats. The role of mucosal sodium (independent of serosal sodium) was evaluated in a model of Rb+(K+) absorption in which serosal ouabain markedly enhanced active Rb+(K+) absorption. In normal rats, mucosal sodium was a competitive inhibitor of Rb+(K+) absorption, and Rb+(K+) absorption consisted of a mucosal sodium-sensitive component and a mucosal sodium-insensitive component. Further, mucosal ouabain almost completely inhibited the mucosal sodium-insensitive component but did not affect the mucosal sodium-sensitive component. In sodium-depleted rats, both mucosal sodium-sensitive and mucosal sodium-insensitive fractions of Rb+(K+) absorption were also identified. Aldosterone markedly stimulated the mucosal sodium-sensitive component (1.68 ± 0.15 vs. 0.60 ± 0.10 μEq · h−1 · cm−2) but not the sodium-insensitive component (0.88 ± 0.09 vs. 0.64 ± 0.06 μEq · h−1 · cm−2) component of Rb+(K+) absorption; however, in contrast to normal animals, mucosal sodium in sodium-depleted animals was a noncompetitive inhibitor of Rb+(K+) absorption. The mucosal sodium-insensitive component of Rb+(K+) absorption in sodium-depleted animals was substantially inhibited by mucosal ouabain, but the mucosal sodium-sensitive component, unlike that in normal animals, was partially inhibited by mucosal ouabain. These studies indicate that the characteristics of the Rb+(K+) absorptive process in sodium-depleted animals differ significantly from those present in normal animals, suggesting that aldosterone induces an Rb+(K+) absorptive mechanism not present in normal animals.

Effects of cell volume changes on membrane ionic permeabilities and sodium transport in frog skin (Rana ridibunda).

1. Membrane potential and conductances and short-circuit current were continuously measured with microelectrodes and conventional electrophysiological techniques in a stripped preparation of frog skin epithelium. The effects of the removal of chloride or sodium ions and the concentration or dilution of the serosal (inner) bathing solution were studied. 2. Chloride- or sodium-free solutions produced a cell depolarization of about 30 mV in parallel with a fall in the short-circuit current. Mucosal and serosal membrane conductances both decreased and the sodium permeability of the mucosal barrier was calculated to fall to about one-half its value in standard Ringer solution. The observed decrease in the short-circuit current is probably related to the combined effect of the decrease in sodium permeability and the decrease in the driving force across the mucosal membrane. 3. The removal of chloride or sodium ions reduced the depolarization caused by serosal perfusion with high-potassium solutions (50 mM-KCl). The ratio of the change in cell membrane potential under short-circuit conditions to the change in the potassium equilibrium potential (delta Ec(s.c.)/delta EK), was 0.59 in standard Ringer solution and 0.26 and 0.24 after the removal of chloride or sodium respectively. The depolarizing effect of barium-containing solutions (2 mM-BaCl2) was also markedly reduced in chloride- or sodium-free solutions, suggesting a decrease of the potassium selectivity of the serosal membrane in these conditions. 4. Increasing the osmolality of the serosal bathing solution produced similar effects, i.e. cell depolarization, fall in the short-circuit current and membrane conductances and reduction of the depolarizing effect of high-potassium and barium solutions. On the contrary, dilution of the serosal bath produced the opposite effects, consistent with an increase in the serosal permeability to potassium. 5. The effects of chloride- or sodium-free solutions were reversed by the dilution of the serosal bath. Cells repolarized when exposed to low-osmolality solutions after being in the absence of serosal chloride or sodium. The repolarization ran in parallel with the restoration of the short-circuit current and the potassium selectivity of the serosal membrane. 6. The results show that the effects produced by the removal of sodium or chloride ions from the serosal bathing solution are most probably mediated by a reduction in cell volume. Cell volume changes would lead to changes in the serosal membrane selectivity to potassium and thus to changes in cell membrane potential and sodium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium transport in turtle bladder.

Unidirectional 45Ca fluxes were measured in the turtle bladder under open-circuit and short-circuit conditions. In the open-circuited state net calcium flux (JnetCa) was secretory (serosa to mucosa) and was 388.3 +/- 84.5 pmol.mg-1.h-1 (n = 20, P less than 0.001). Ouabain (5 X 10(-4) M) reversed JnetCa to an absorptive flux (serosal minus mucosal flux = -195.8 +/- 41.3 pmol.mg-1.h-1; n = 20, P less than 0.001). Amiloride (1 X 10(-5) M) reduced both fluxes such that JnetCa was not significantly different from zero. Removal of mucosal sodium caused net calcium absorption; removal of serosal sodium caused calcium secretion. When bladders were short circuited, JnetCa decreased to approximately one-third of control value but remained secretory (138.4 +/- 54.3 pmol.mg-1.h-1; n = 9, P less than 0.025). When ouabain was added under short-circuit conditions, JnetCa was similar in magnitude and direction to ouabain under open-circuited conditions (i.e., absorptive). Tissue 45Ca content was approximately equal to 30-fold lower when the isotope was placed in the mucosal bath, suggesting that the apical membrane is the resistance barrier to calcium transport. The results obtained in this study are best explained by postulating a Ca2+-ATPase on the serosa of the turtle bladder epithelium and a sodium-calcium antiporter on the mucosa. In this model, the energy for calcium movement would be supplied, in large part, by the Na+-K+-ATPase. By increasing cell sodium, ouabain would decrease the activity of the mucosal sodium-calcium exchanger (or reverse it), uncovering active calcium transport across the serosa.

Cytosolic calcium and the action of vasopressin in toad urinary bladder.

The effects of experimental procedures believed to increase cytosolic calcium on basal and vasopressin-stimulated osmotic water flow and transepithelial sodium transport were examined in the toad urinary bladder. Exposure of isolated toad bladders to quinidine, calcium ionophores (A23187, X537A), or low-sodium or potassium-free serosal solutions resulted in a dose-dependent decrease in the hydrosmotic response to vasopressin or exogenous adenosine 3',5'-cyclic monophosphate (cAMP). The degree of inhibition of cAMP-induced water flow induced by low-sodium or potassium-free serosal bathing media varied, and in a similar manner, with the serosal calcium concentration. The effects of quinidine sulfate (2 X 10-4 M), X537A (2 X 10(-5) M), and low serosal sodium (20 mM), but not that of A23187 (10(-5) M), were readily reversible. Exposure to quinidine (4 X 10(-4) M), A23187 (10(-5) M), X537A (5 X 10(-6) M), or low serosal sodium (2 mM) also inhibited the basal short-circuit current (SCC). Vasopressin, 4-20 mU/ml, completely overcame the inhibition of the SCC induced by quinidine, A23187, or low serosal sodium, but a submaximal dose of hormone (4 mU/ml) failed to fully reverse the inhibitory effect of X537A, 5 X 10(-6) M. These results are consistent with the view that 1) a Na-Ca exchange process operates across the basolateral surface of the granular epithelial cells of the toad urinary bladder in vivo, and 2) the level of free calcium in the granular cell cytosol plays a modulatory role in the control of apical membrane water and sodium permeability by vasopressin, and in the regulation of the basal rate of transepithelial sodium transport.

Origin of transport inhibition after omission of serosal sodium.

The omission of sodium from the serosal incubation fluid in isolated frog skins inhibits transcellular Na transport. By the use of intracellular recording with microelectrodes, it has been demonstrated that this inhibition is associated with an increase of the basolateral membrane resistance, resulting in a depolarization of the short-circuited cells. This depolarization in turn accounts for the reduction of Na entry across the apical border. The resistance changes across the outer (apical) border are small in magnitude and unrelated to the inhibition of transcellular transport. The origin of the increase in basolateral membrane resistance, presumably due to decrease of K permeability, is unclear. These data do not support the hypothesis that intracellular Ca regulates the resistance of the apical and basolateral membranes.

Regulation of the sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium: role of sodium-calcium exchange in the basolateral membrane.

Sodium movement across the luminal membrane of the toad bladder is the rate-limiting step for active transepithelial transport. Recent studies suggest that changes in intracellular sodium regulate the Na permeability of the luminal border, either directly or indirectly via increases in cell calcium induced by the high intracellular sodium. To test these proposals, we measured Na movement across the luminal membrane (th Na influx) and found that it is reduced when intracellular Na is increased by ouabain or by removal of external potassium. Removal of serosal sodium also reduced the influx, suggesting that the Na gradient across the serosal border rather than the cell Na concentration is the critical factor. Because in tissues such as muscle and nerve a steep transmembrane sodium gradient is necessary to maintain low cytosolic calcium, it is possible that a reduction in the sodium gradient in the toad bladder reduces luminal permeability by increasing the cell calcium activity. We found that the inhibition of the influx by ouabain or low serosal Na was prevented, in part, by removal of serosal calcium. To test for the existence of a sodium-calcium exchanger, we studied calcium transport in isolated basolateral membrane vesicles and found that calcium uptake was proportional to the outward directed sodium gradient. Uptake was not the result of a sodium diffusion potential. Calcium efflux from preloaded vesicles was accelerated by an inward directed sodium gradient. Preliminary kinetic analysis showed that the sodium gradient changes the Vmax but not the Km of calcium transport. These results suggest that the effect of intracellular sodium on the luminal sodium permeability is due to changes in intracellular calcium.

The effects of capsaicin on intestinal sodium and fluid transport.

Capsaicin, the pungent component of red pepper, was studied to determine its inhibitory effect on fluid and Na+ absorption using rat and hamster everted jejunal sacs. At a mucosal concentration of 140 mg% incubated for 60 min, capsaicin reduced the fluid transport into the serosal side by 14.8% in rat and 23.9% in hamster. Similarly, Na+ transport was also inhibited by 12.5% and 26.2% in rat and hamster, respectively. Such decrease in serosal sodium coincided with the increase in Na+ content of the gut wall and the intracellular Na+ concentration in the epithelial layer. It is, accordingly, concluded that capsaicin inhibits the Na+ exits through the serosal pole of the epithelium. These observations may also provide an explanation for the previously observed inhibition of glucose transport.

Solute transport process in intestinal epithelial cells.

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.

Effects of ethacrynic acid on electrolyte and fluid transport by the guinea pig gallbladder.

The effect of ethacrynic acid on fluid and electrolyte transport by the guinea pig gallbladder was investigated in vitro. 10-4M ethacrynic acid, applied to the serosal side, inhibited fluid and sodium chloride absorption. The reduction in salt absorption was accounted for by a 3 muEq/cm2h decrease in the unidirectional fluxes of Na and Cl from mucosa to serosa with no change in the fluxes from serosa to mucosa. Ethacrynic acid (10-4 M) had no effect on HCO3 - Cl exchange, PGE1-induced fluid secretion and inulin permeability. At 10-3 M, ethacrynic acid markedly increased both the serosa to mucosa fluxes of Na and Cl, and the inulin permeability. Examination by light and electron microscopy of gallbladder tissue treated with 10-3 M ethacrynic acid revealed large intracellular vacuoles and occasionally ruptured apical cell membranes. Only slight morphological changes were seen by 10-4 M ethacrynic acid with no changes in the controls and ouabain treated gallbladders. The effects of ethacrynic acid are remarkably different from those of furosemide which has been previously shown to inhibit only the HCO3 secretion leaving fluid and NaCl absorption unchanged.

Ionic and metabolic requirements for the hydroosmotic response to antidiuretic hormone in toad urinary bladder.

A study has been conducted to determine the ionic and metabolic requirements for full expression of the hydroosmotic response to antidiuretic hormone in the toad urinary bladder. By appropriate manipulation of incubation conditions it can be shown that there is a pool of serosal sodium necessary for a full hormone response. This serosal sodium pool is not related to the transepithelial sodium transport pool A full hydroosmotic response also requires serosal potassium; however, no specific anion requirement was demonstrated. Additionally, anaerobic or aerobic metabolism support a full hydroosmotic response equally well.

Interdependence of the two borders in a sodium transporting epithelium. Possible regulation by the transport pool

Specific binding of 14C-amiloride to the mucosal surface of frog skin epithelium (Rana temporaria) has been used as a measure of the number of sodium entry sites. All binding measurements were made with the mucosal surface bathed in a solution containing 1.1 mM sodium. When manipulations were used which increased the intracellular concentration of sodium the amount of amiloride bound was reduced. The manipulations included flushing the mucosal surface with solutions containing 111 mM sodium after serosal efflux was inhibited with ouabain or potassium removal. Similar results were obtained when cells were loaded with lithium. These effects on amiloride binding did not appear to depend on changes in membrane potential or upon changes in affinity of amiloride for its binding site. It appears that inhibition of serosal sodium efflux from the epithelium causes a reduction of mucosal sodium influx by making entry sites unavailable. This latter may be a result, directly or indirectly, of the sodium concentration in the sodium transport pool.

Passive sodium fluxes across toad bladder in the presence of simultaneous transepithellal gradients of concentration and potential.

Bidirectional sodium fluxes were measured across toad bladder sacs after eliminating active transport with ouabain. Transepithelial potential was clamped to 100 mV or the Nernst potential, psieq, at varying sodium concentrations, Cm, in the mucosal medium. Serosal sodium concentration, Cs, was held constant. Equations were derived for permeability, partial ionic conductance, and unidirectional fluxes as functions of Cm and Cs, based in part on the assumption that the ratio, Q, of bulk sodium permeability to tracer sodium permeability is a constant, independent of concentration and potential. The results conformed closely to these equations.

Transepithelial sodium transport and carbon dioxide production by the toad urinary bladder in the absence of serosal sodium.

1. The production of CO2 in relation to sodium transport by toad urinary bladder has been examined in the absence of sodium from the serosal medium. 2. Replacement of serosal sodium by choline increased CO2 production without stimulating short-circuit current. Replacement of serosal medium by Tris did not have this effect. 3. With serosal sodium Ringer replaced by Tris Ringer, the ratio of sodium transported to CO2 produced was not altered significantly. 4. The results therefore suggest that there is no important recycling of sodium between the serosal medium and the transporting epithelial cells.

Metabolic evidence that serosal sodium does not recycle through the active transepithelial transport pathway of toad bladder.

The possibility that sodium from the serosal bathing medium "back diffuses" into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO2 production by the tissue and enhance of stoichiometric ratio of sodium ions transported across the bladder per molecula of sodium transport dependent CO2 produced simultaneously by the bladder (JNa/JCO2). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO2 production by the bladder [(a)rate of basal, nontransport related, CO2 production (JbCO2) equals 1.54 +/- 0.52 with serosal sodium and 1.54 +/- 0.37 without serosal sodium; (b) Jb CO2 equals 2.18 +/- 0.21 with serosal sodium and 2.09 +/- 0.21 without serosal sodium; (c) 1.14 +/- 0.26 without ouabain and 1.13 +/- 0.25 with ouabain; unite of JbCO2 are nmoles mg d.w.-1 min-1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the total bladder.

Relationships between serosal medium potassium concentration and sodium transport in toad urinary bladder. II. Effects of different medium potassium concentrations on epithelial cell composition.

Epithelial cells from hemibladders incubated in potassium-free sodium Ringer's serosal medium lost potassium, both in exchange for serosal sodium and with chloride and water. Cellular sodium of mucosal origin did not change. The loss of cellular potassium, chloride and water closely followed the fall in short-circuit current (SCC). One third as much potassium, chloride and water were lost in 1 mM potassium serosal medium; SCC fell 1/3 as much. Potassium-free choline Ringer's serosal medium abolished the initial increase in SCC and reduced the fall in cellular potassiu, chloride and water and in SCC. Ouabain (10(-2)M) in potassium-free medium prevented the initial increase in SCC and the loss of cellular chloride and water. Ouabain (5 X 10(-4)M) caused loss of cellular potassium in exchange for mucosal and serosal sodium, effects different from those of absence of serosal potassium although SCC was similarly inhibited. Sodium-free mucosal medium abolished SCC and prevented the initial transient of SCC and diminished loss of cellular potassium, chloride and water on removing serosal potassium. When serosal potassium concentration was increased considerably, cells gained potassium, chloride and water, and in 116 mM potassium media, lost sodium of serosal origin. A hypothesis is advanced to explain the transients in SCC on changing serosal potassium concentration. The fall in cellular potassium, not water, probably inhibits sodium transport in media of less than 2 mM potassium.

Relationships between serosal medium potassium concentration and sodium transport in toad urinary bladder. I. Effects of different medium potassium concentrations on electrical parameters.

When serosal medium potassium was decreased from the usual concentration of 3.5 mM, the short-circuit current (SCC) of hemibladders in chambers immediately and transiently increased. The maximum SCC attained was greater the greater the decrease in serosal potassium, and was twice the initial SCC when the final serosal medium was potassium-free. The SCC then fell to its previous level for final serosal potassium concentrations greater than 2 mM and to less than its previous level for those less than 2 mM, being lowest (15% of previous levle) in potassium-free sodium Ringer's. When serosal medium potassium was increased from 3.5 mM by substituting potassium for sodium, SCC transiently decreased and then recovered to its previous level. Steady SCC was the same in serosal media of 2-116 mM potassium; conductance increased and p.d. decreased after incubation in 50-116 mM potassium serosal media. Short-circuit current and p.d. transiently increased (decreased) whenever serosal medium potassium was decreased (increased); conductance increased with any change in serosal potassium. Changing mucosal medium potassium concentration between 0 and 50 mM did not affect SCC. The initial transient increase and subsequent decrease in SCC on removing serosal potassium were partially prevented by 3.5 mM rubidium or caesium, or by 116 mM choline in the serosal medium. The transient changes in SCC were due partly to changes in transepithelial sodium transport.

Effect of transepithelial concentration gradients on the passive fluxes of sodium across toad bladder.

Bidirectional sodium fluxes across toad bladder were measured after eliminating active transport with ouabain. Mucosal sodium concentration, Cm, was progressively reduced (from 114 to 3 mM) while serosal sodium remained constant. Potential difference was maintained at zero by current passage. The ratio, Q, of the bulk permeability coefficient for sodium, P, to the tracer sodium permeability coefficient, P, was found to remain constant as Cm decreased. Equations were derived on this basis for bidirectional fluxes and for P and P as functions of Cm, which corresponded closely to the observed data. The explanation for the observed value of Q and its constancy under these conditions is uncertain.

The sodium transport pool in toad urinary bladder epithelial cells.

The sodium which equilibrates with 24-Na in epithelial cells of toad urinary bladders has been determined. With sodium Ringer's bathing both mucosal and serosal surfaces, 24-Na in the mucosal medium equilibrated with about 35 mmoles cellular sodium/kg cellular dry weight, representing about 20% of the total cellular sodium determined flame photometrically; 24-Na in the serosal medium equilibrated with 120 mmoles cellular sodium/kg cellular dry weight, about 80% of the total cellular sodium. With 24-Na in both media all cellular sodium was labeled within 30 min. In the absence of serosal sodium, total cellular sodium and that sodium which equilibrated with mucosal 24-Na in sodium Ringer's were both similar to the cellular sodium of mucosal origin which had been determined in epithelial cells exposed on both surfaces to sodium Ringer's. Sodium-free mucosal medium, and sodium Ringer's containing amiloride 10-4 or 10-3 M in the mucosal medium, both virtually completely inhibited transepithelial sodium transport. But, whereas the cellular sodium of mucosal origin fell to only 2 mmoles/kg cellular dry weight with sodium-free mucosal medium, an appreciable labeling of cellular sodium was found whether amiloride was present before, or only after, exposure of tissue to mucosal 24-Na. Rapid washing of the mucosal surface of hemibladders just before removal of epithelial cells for analysis removed most of this sodium labeled in the presence of amiloride, suggesting that the cellular sodium of mucosal origin consists of at least two fractions with only about two-thirds truly intracellular. The sodium transport pool measured directly in these experiments is appreciably smaller than any previous estimates of pool size all of which have been obtained by indirect techniques involving use of whole hemibladders rather than epithelial cells alone.

Ouabain-dependent potassium-potassium exchange in the toad bladder.

Recent results from this laboratory have indicated the existence of two potassium compartments in the isolated toad bladder. Only one of these, containing less than 10% of total intracellular potassium, appears to be related to the sodium transport system, since potassium influx at the serosal border of this compartment is coupled to the sodium efflux which occurs there. Ouabain, which specifically inhibits serosal sodium exit, has no effect on potassium fluxes and compartment sizes in bladders mounted in normal (2.5mm K) Ringer's solution. However, in the presence of this inhibitor, removal of serosal potassium results in a significant decrease in the rate coefficient for potassium efflux into the serosal medium, while an increase in serosal potassium results in a significant rise in this parameter, which appears to saturate at approximately 5mm K. This sensitivity to serosal potassium is seen neither in the absence of ouabain nor when the sodium pump is inactivated by removal of sodium from the mucosal medium. Furosemide, which also inhibits the sodium transport system, both inhibits potassium transport parameters in normal Ringer's and abolishes the potassium-sensitive potassium efflux seen in the presence of ouabain. Thus, the Na−K pump appears to operate as a K−K exchanger when the sodium system is inhibited by ouabain; this K−K exchange mechanism is inhibited by furosemide. One explanation for these results is that ouabain effects an alteration in the affinities of the transport system for sodium and potassium.