Pierre Ripoche

Simultaneous minute by minute determination of unidirectional and net water fluxes in frog urinary bladder A reexamination of the two barriers in series hypothesis

Unidirectional and net water fluxes were simultaneously estimated in frog urinary bladder. The minute by minute tritiated water (3HOH) transepithelial flux and the net volume of fluid traversing the tissue were employed. It was observed that: (1) the time course of the increase in the 3HOH flux induced by antidiuretic hormone had a very similar pattern to that reported for the increase in the net movement. (2) Unstirred layers strongly affected the magnitude of the antidiuretic hormone-induced increase in 3HOH fluxes while the time course of the response was almost non-affected. In non-stimulated bladders 3HOH fluxes were poorly modified by medium stirring. New steady-state conditions for 3HOH fluxes were established 1 min after stirring rate modifications. (3) The simultaneously determined net water flux was not affected by a modification in the unstirred layers, indicating that the variations in the measured net water fluxes are a good estimation of the changes in the mucosal border permeability. (4) The presence of an osmotic gradient during hormonal challenge (implying net water fluxes, cell swelling and dilation of the intracellular spaces) did not modify the time course of 3HOH movements. These results suggest that the time course of the increase in water permeability is an intrinsic characteristic of the experimental system that could result from the addition of permeability units that increase in number during the development of the hormonal action.

Interactions between Na+-dependent uptake of D-glucose, phosphate and L-alanine in rat renal brush border membrane vesicles.

D-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, D-glucose and L-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na+-dependent phosphate transport was inhibited in the presence of either D-glucose (39 mM) or L-alanine (2.4 mM). In this model, with D-glucose or with L-alanine the V value of the phosphate uptake was decreased, whereas the apparent Km for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of L-glucose, D-alanine or D-glucose after phlorizin preincubation. A 30% Na+-dependent L-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. D-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, D-glucose decreased the L-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of D-glucose by L-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na+ gradient insofar as the rate of the Na+ uptake was increased with D-glucose, L-alanine or phosphate and since the absence of variations in membrane potentials did not suppress these inhibitions.

A Sodium-independent Mechanism for L-Arginine Uptake by Rat Renal Brush Border Membrane Vesicles

L-arginine accumulation in renal cortex slices or in brush border membrane vesicles differs from that of neutral amino acids and sugars, since it can occur despite a lack of sodium in the incubation medium. L-arginine uptake was measured under different conditions in order to determine the relative importance of the Na+-independent component of this transport. In contrast to D-glucose, which was accumulated into the vesicles only in the presence of a NaCl gradient, the accumulation of L-arginine was induced by NaCl, LiCl, KCl, or choline Cl gradients. The uptakes of L-arginine and D-glucose wer proportional to the membrane potential (inside less than 0), but the presence of Na+ was not required for the L-arginine uptake. No difference was observed in Kj values of the L-arginine uptake measured with low or high concentration of salt (Li+ or Na+). Jmax were also in the same range of magnitude. An influx of D-glucose or L-alanine (Na+-cotransported molecules) induced an increase of the sodium content in vesicles equilibrated with 22Na; the L-arginine uptake did not induce it. These different results suggest that the electrical potential and not the chemical gradient was the major driving force for the L-arginine uptake.

Effect of hypertonic media on the reversal of the hydrosmotic action of antidiuretic hormone in frog urinary bladder

Abstract The reversal of hydrosmotic response of isolated frog urinary bladder to oxytocin, theophylline and cyclic AMP is markedly slowed during incubation in hypertonic media. These results enforce the hypothesis that hypertonic media could exert their action by inhibiting the inactivation of some required factor more than by enhancing its activation.

Detergent extraction of membrane proteins related to the action of antidiuretic hormone.

Antidiuretic hormone (ADH) induces, in the apical plasma membrane of target cells, the insertion of intramembranous particle aggregates that probably contain water channels. A mild attack of this membrane by a polyoxyethylene nonylphenyl detergent, which reversibly depressed ADH-induced water permeability, has been found to modify aggregate structure while extracting additional proteins. This simple procedure could be a valuable approach to the problem of aggregate isolation and characterization.

Probenecid: effects on water permeability in frog urinary bladder.

Abstract The action of probenecid (probenecid is an agent known to inhibit the release of cyclic AMP by pigeon erythrocytes) on the urinary bladder of Rana esculenta has been investigated. Conspicuous modifications in the responses of the bladder to various compounds were observed; surprisingly, hydrosmotic responses to exogenous cyclic AMP and theophylline were strongly enhanced, although the response to neuropeptides was inhibited. A single primary action of probenecid on cyclic AMP transfers is suggested as a tentative explanation of these results.