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76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Purification and characterization.

Exocytotic processes play a major role in the hormonal control of water permeability in the amphibian urinary bladder. Different treatments such as antidiuretic hormone (ADH) stimulation, incubation with phorbol ester or mild detergent and mechanical stretch of the bladder, consistently induce a liberation of two major polypeptides of 76 and 14 kDa molecular mass into the luminal medium. Each of these polypeptides represents 3 to 5% of the total protein of epithelial cell homogenates and 20 to 50% of the released material. Proportions of released 76 kDa polypeptide in urinary bladders of toads (Bufo marinus) and frogs (Rana esculenta) were similar but, in the frog extracts, two bands ("doublet") were resolved at the level of 76 kDa. In high performance liquid chromatography (HPLC), using gel filtration and ion exchange chromatography, the frog 76 kDa protein was resolved into two polypeptides of 80,000 to 100,000 and 60,000 to 80,000 daltons while the 14 kDa protein included two polypeptides, each with a molecular mass of approximately 14,000 daltons. Isoelectric focusing of the material released during a mechanical stretch of the tissue ("stretch extract") or of isolated purified proteins from the frog urinary bladder showed that the 14 kDa polypeptides were resolved in two major groups of polypeptides, one in the range of pH 7.4 to 7.8, the other at pH 5.6. The lower band of the 76 kDa doublet also comprised some diffuse bands (5.0 less than pI less than 5.2) while the other polypeptide of the doublet presented a sharp band at pH 6.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Stimulation of sodium transport by cobalt ions in the skin of amphibia against a background of aldosterone action].

Sodium transport across toad Bufo marinus and frog Rana temporaria skin was enhanced by aldosterone added to Ringer solution at the basal plasma membrane. Cobalt ions applied to the outer surface of amphibian skin have increased sodium transport stimulated by aldosterone. The regulatory sites of the sodium channel macromolecule seem to be located on both surfaces of the channel apical membrane.

Aldosterone↗

Stereoselective blockade of amphibian epithelial sodium channels by amiloride analogs.

The blockade of amphibian epithelial sodium channels by amiloride has been shown to not be a simple diffusion-limited encounter but rather a selective binding process with the rate of approach of the molecule for the channel site determined by the side chain at position 2 of the pyrazine ring and the stability of the blocking complex provided largely by the bond of the ligand at position 6. The presence of such a putative channel-associated receptor suggests that the sodium channel might have chiral recognition for its blockers. Using the short-circuit technique to measure the amiloride-sensitive sodium current through the apical membrane of the toad urinary bladder and frog skins, we evaluated the sodium channel-blocking potency of three enantiomeric pairs of amiloride analogs with chiral centers on the side chain. We demonstrated the stereoselectivity in sodium channel blockade of those analogs and confirmed the existence of a second attachment site on the channel for interacting with the lipophilic ligand on the side chain of the analog molecule to enhance its blocking activity. Depending on the separation between the chiral carbon and the pyrazine ring and the chemical groups on the chiral carbon, the eudismic ratio for the 50% inhibition constant of the enantiomers, i.e., the ratio of the sodium channel-blocking potency of the more active enantiomer to that of the less active enantiomer, was found to range from slightly more than 1 to more than 100.

Amiloride↗

NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis.

The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mOsM) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed.

Adaptation, Physiological↗