Mucosal Sodium Concentration Research Articles

Time course studies on phosphate transfer in frog urinary bladder

Unidirectional 32P-phosphate and 3H-mannitol fluxes were simultaneously measured, at two minutes intervals, in frog urinary bladders. The spontaneous or externally imposed transepithelial potential (PD) and short circuit current (SCC) were also recorded in most experiments. It was observed that: (1) Phosphate transfer was rapidly and reversibly modified by changes in mucosal sodium concentration in open circuit conditions. (2) Between four and six minutes after changing mucosal NaCl concentration, phosphate fluxes reached a new steady-state value. (3) The observed correlation between the Na-dependent phosphate flux and the Na-dependent transmembrane potential was high (r = 0.99, N = 12). (4) In open circuit conditions, the mucosa-to-serosa unidirectional phosphate fluxes were inhibited by 10(-5) M amiloride, while the serosa-to-mucosa movements were increased. (5) On the contrary, no effects of mucosal NaCl concentration or amiloride on the mucosa-to-serosa phosphate fluxes were detected in short circuit conditions. (6) The transepithelial phosphate transfer was linearly related to phosphate concentration and insensitive to arsenate (10(-3) M) action. (7) An externally imposed PD was less effective for driving a phosphate movement than the one depending on Na, suggesting some type of coupling between Na+ and phosphate transports. (8) The mucosa-to-serosa phosphate fluxes were reduced by parathyroid hormone and oxytocin. Maximum inhibition was observed four minutes after the hormonal action. It is concluded that the transepithelial PD plays a major role in phosphate handling in frog urinary bladder.

Exploration of apical sodium transport mechanisms in an epithelial model by network thermodynamic simulation of the effect of mucosal sodium depletion: II. An apical sodium channel and amiloride blocking

This paper is the second part of a modeling study on apical sodium transport mechanisms in tight epithelia. In the first part (this issue) we explored three expressions for the apical membrane sodium permeability (PapNa) and showed that only a PapNa which varies as a function of sodium concentration allows simulation of the well known saturation of the short-circuit current with increasing mucosal sodium concentration. However, the ad hoc expressions used have no mechanistic interpretation. We show here that if, instead of an ad hoc expression, one includes a one-site, two-barrier sodium channel in the apical membrane, the model also simulates this saturation. In addition, the equivalent apical sodium permeability computed from the simulations appears to be very similar to the phenomenological equation used by Fuchs et al. (1977) to fit the decrease of the apical sodium permeability with increasing mucosal sodium. The apical sodium channel simulated here is thus a possible mechanism for the feedback effect of the mucosal and intracellular sodium concentrations on the apical sodium permeability. This channel also allows the simulation of the competitive inhibition of the sodium current by amiloride, and the concomitant inhibition of the apical sodium permeability.

Exploration of apical sodium transport mechanisms in an epithelial model by network thermodynamic simulation of the effect of mucosal sodium depletion: I. Comparison of three different apical sodium permeability expressions

A model based on that of Koefoed-Johnsen & Ussing (1958) and elaborated by Hviid Larsen (1978) and Lew et al. (1979), is designed using network thermodynamic theory and used to simulate experiments performed on epithelia. Three different expressions for the apical sodium permeability are tested for their ability to reproduce the saturation of the short-circuit current with increasing mucosal sodium concentration. Using the parameters from the previous models, the sodium entry step is shown to be the rate limiting step. If the apical sodium permeability is constant, there is no saturation of the short-circuit current with increasing mucosal sodium. The saturation of the short-circuit current is simulated with versions of the model which include a variable apical sodium permeability. The phenomenological expressions used for the variable permeabilities are those proposed by Fuchs et al. (1977) and Civan & Bookman (1982). They describe the so-called feedback effect of the mucosal and intracellular sodium concentrations.

Phenamil: An irreversible inhibitor of sodium channels in the toad urinary bladder

Several new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current, Isc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 microM after irradiation with ultraviolet light greater than or equal to 320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) amino-methylene] pyrazinecarboxamide, phenamil, irreversibly inhibited Isc at concentrations of 0.05 to 5 microM when added to the mucosal solution. Irreversible inhibition of Isc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH; 3) mucosal sodium concentration. For example, 5 microM phenamil irreversibly inhibited Isc by 38% in 103 mM Na at pH 8.6 and nearly 75% in 30 mM Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of less than or equal to 10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density.

Passive cation permeability of turtle colon: Evidence for a negative interaction between intracellular sodium and apical sodium permeability

The role of intracellular sodium in the regulation of apical sodium permeability was investigated in an electrically "tight" epithelium, the turtle colon. In the presence of low mucosal sodium (3 mM) and serosal ouabain, an inhibitor of the basolateral sodium pump, the apical membrane retained a substantial amiloride-sensitive, sodium conductance and the basolateral membrane exhibited a barium-sensitive potassium conductance in parallel with a significant sodium (and lithium) conductance. In the presence of a high mucosal sodium concentration (114 mM), however, inhibition of active sodium absorption by ouabain led to a disappearance of the amiloride-sensitive, transepithelial conductance that was due, at least in part, to a virtual abolition of the apical sodium permeability. Two lines of evidence indicate that this permeability decrease was dependent upon an increase in intracellular sodium content. First, raising the mucosal sodium concentration from 3-114 mM in the presence of ouabain reversibly inhibited the amiloride-sensitive conductance. The time course of the decline in conductance paralleled the apparent intracellular accumulation of sodium in exchange for potassium, which was monitored as a transient deflection in the amiloride-sensitive, short-circuit current. Second, the inhibitory effect of mucosal sodium-addition was markedly attenuated by serosal barium, which prevented the accumulation of sodium by blocking the electrically coupled, basolateral potassium exit. These results support the notion of a "negative feedback" effect of intracellular sodium on the apical sodium permeability.

Effects of changes in serosal chloride on electrical properties of toad urinary bladder.

Conventional microelectrode and tracer flux techniques were used to study the effects of reduction in serosal chloride concentration ([Cl]s) on the electrical properties of toad urinary bladder epithelium. Reduction in [Cl]s resulted in a transient change in transepithelial potential (Vms) (and of apical and basolateral membrane potentials) that was inversely dependent on the base-line values of those potentials. In all cases, however, there was a decrease in transepithelial resistance (Rt) that was explained by an increase in the sodium conductance of the apical membrane. In tissues in which the transepithelial potential increased, there was a rise in the active mucosal-to-serosal sodium flux. The increase in conductance was directly related to the increase in short-circuit current. The changes in Vms and Rt brought about by reduction in [Cl]s were prevented by agents known to modify sodium transport, including low mucosal sodium concentration, addition of amiloride or amphotericin B to the mucosal solution, or of ouabain to the serosal solution. The results are best explained by a primary effect of chloride reduction on sodium extrusion across the basolateral membrane, with a secondary increase in apical sodium conductance. In addition, the data provide new evidence for the existence of a basolateral chloride conductance pathway.

Influence of mucosal sodium concentration on electrical resistances and potentials of toad bladder.

This study was undertaken to delineate the upper limits of paracellular resistance in toad bladder, and the effect of mucosal [Na+] <10 mM on cellular resistance and epithelial, apical, and basallateral potentials. These parameters were determined at mucosal Na+ of 100, 20, 10, 7, 3, and 1 mM. There was an enormous increase in paracellular or junctional resistance which, at 1 mM Na+, was over fourfold that determined at 100 mM. There was no effect on cellular resistance. A primary effect of low mucosal Na+ on apical potential independent of effect on basal-lateral potential was not demonstrable. Although apical membrane potential responds to mucosal [Na+], the response is less than that seen with pure Na+ sensitive electrode. The response of basal-lateral potential to mucosal [Na+] suggest that, to some considerable extent, ψαb may be the result of Na+ pump activity. However, basal-lateral K+ diffusion potentials may contribute to ψαb and both membrane potentials may be influenced by shunt current flow. The data also support the concept that Na+ penetration of apical and basal-lateral membranes is unidirectional, from mucosal solution to cell to serosal solution. Shunt resistance varies inversely with epithelial potential and mucosal Na+. This is probably required to maintain large transepithelial concentration gradients.

Microelectrode studies in toad urinary bladder epithelium. effects of Na concentration changes in the mucosal solution on equivalent electromotive forces.

Microelectrode techniques were employed to measure membrane potentials, the electrical resistance of the cell membranes, and the shunt pathway, and to compute the equivalent electromotive forces (EMF) at both cell borders in toad urinary bladder epithelium before and after reductions in mucosal sodium concentration. Basal electrical parameters were not significantly different from those obtained with impalements from the serosal side, indicating that mucosal impalements do not produce significant leaks in the apical membrane. A decrease in mucosal Na concentration caused the cellular resistance to increase and both apical and basolateral EMF to depolarize. When Na was reduced from 112 to 2.4 mM in bladders with spontaneously different baseline values of transepithelial potential difference (Vms), a direct relationship was found between the change in Vms brought about by the Na reduction and the base-line Vms before the change. A direct relationship was also found by plotting the change in EMF at the apical or basolateral border caused by a mucosal Na reduction with the corresponding base-line EMF before the change. These results indicate that resting apical membrane EMF (and, therefore, resting apical membrane potential) is determined by the Na selectivity of the apical membrane, whereas basolateral EMF is at least in part the result of rheogenic Na transport. These results are consistent with data of others that suggested a link between the activity of the basolateral Na pump and apical Na conductance.

Interrelationships of sodium transport and carbon dioxide production by the toad bladder: Response to changes in mucosal sodium concentration, to vasopressin and to availability of metabolic substrate

Active sodium transport and CO2 production were measured simultaneously in toad bladders mounted in membrane chambers. The rate of sodium transport was varied by changing the concentration of sodium in the mucosal bath (substitution with choline), by adding vasopressin, by adding metabolic substrates and by adding malonate, and the ratio of the change of sodium transport and CO2 production was determined Mean values for deltaNa/deltaCO2 (equiv/mole) were: Na in equilibrium choline 18.3 +/- 1.1; vasopressin 15.5 +/- 2.8; and pyruvate (corrected for the increment in "nontransport" CO2) 15.4 +/- 3.5. Based on previously determined values for the respiratory quotient (R.Q.), calculated mean values for deltaNa/deltaO2 ranged between 15.5 and 18.5 equiv/mole. It appears that basal metabolism does not contribute to metabolism supporting sodium transport when the rate of sodium transport is varied. "Transport" metabolism appears much more responsive to changes in the availability of endogenous and exogenous substrates than does "nontransport" metabolism. We conclude that "transport" and "nontransport" metabolism are functionally separated in the toad bladder.

The electrical properties and active ion transport across the urinary bladder of the urodele, Amphiuma means.

1. The electrical properties and the active transport processes of the isolated urinary bladder of the urodele, Amphiuma means, were studied by mounting this tissue as a flat sheet between two halves of a lucite chamber. The mean transepithelial potential difference was 70-2 +/- 2-3 mV (serosa positive), the mean short-circuit current was 10-9 +/- 0-5 micrionA/mg of dry weight and the mean transepithelial d.c. resistance was 6540 +/- 374 omega mg of dry weight. 2. The short-circuit current (Isc) accounted for 92% of the net 22Na+ flux from the mucosa to the serosa. The difference resulted from a transport of 36Cl- in the same direction as sodium. 3. The active sodium transport exhibited typical saturation kinetics, having a Km of 15-4 m-equiv/l. and approaching zero order at 60-70 m-equiv/l. The transepithelial potential difference increased linearly with the log of the mucosal sodium concentration at a rate of 50-3 mV per tenfold concentration change. 4. In the absence of the major anions (HCO3- and Cl-) from the bathing solutions, the electrical properties and the sodium influx decreased to less than 40% of their control values. The presence of only one of these two anions in the serosal bathing solution was sufficient to maintain these parameters. 5. Amiloride (10(-5)M) and ouabain (10(-6)M) inhibited the sodium transport 97% and 85% respectively. Amphotericin B (10(-6)M) stimulated the sodium transport 47%. Furosemide (10(-3)M) inhibited the chloride transport 43%. The sodium transport was insensitive to the action of two enurohypophyseal peptides tested, lysine-vasotocin and pitocin.

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.

Acidification of urine by the isolated urinary bladder of the toad.

Acidification of urine by the isolated urinary bladder of the toad.