R. C. de Sousa

Particle aggregates in plasma and intracellular membranes of toad bladder (granular cell).

Freeze-fracture of granular cells of toad urinary bladder (Bufo marinus) reveals the presence, hitherto undescribed, of intramembranous particle aggregates in intracytoplasmic structures (tubules, vacuoles and vesicles) both in resting and vasopressin-stimulated epithelia.

Osmotic water flow across the abdominal skin of the toad Bufo marinus: effect of vasopressin and isoprenaline

1. Net water flow Jw, was measured across the abdominal skin of the toad Bufo marinus with a volumetric, automatic technique that allows for averaging Jw over time intervals as short as 1 sec.

Patterns of membrane organization in toad bladder epithelium: A freeze-fracture study

Two cell-types of toad bladder epithelium show uncommon plasma membrane organization in freeze-fractured specimens. One type, the granular cell, contains a plasma membrane in which the A-face is poorly particulate luminally while the B-face discloses multiple large particles at this site. In contrast, the lateral and basal portions of the granular-cell membrane are typical in that more particles occupy the A-face than the B-face. In the other cell-type, which is mitochondriarich, the plasma membrane, luminally, laterally, and basally, contains rod-shaped and a few glubular particles in the A-face. We suggest that these two peculiar membrane organizations by considered in the localization of both vasopressin and aldosterone action in toad bladder.

Inhibitory and stimulatory effects of amiloride analogues on sodium transport in frog skin

SummaryEffects of amiloride analogues on Na transport were studied in isolated skins of the frogRana ridibunda. The pattern of structure-activity relationship of these compounds showed that both the −NH2 group at position 5 and Cl at position 6 of the pyrazine ring of the amiloride molecule were important for their biological activity. The paramount role of the groups at position 5 was further demonstrated by the striking properties of an analogue resulting from dimethylation of that −NH2 group. A stimulation of Na transport, opposite to the effect of amiloride itself, was observed in this instance. The increase in Na transport could already be seen at 10−6m and was equivalent to the measured increase in Na influx, reversible, dose-dependent, and additive to the natriferic action of oxytocin. Such characteristics resemble those reported with “external” agents like propranolol and La3+. Furthermore, mutual inhibition was observed between the stimulatory effects of this analogue and those of propranolol or La3+. These results suggest that the analogue may be considered as another “external” agent acting at sites of the external membrane distinct from those activated by cAMP but similar to the Ca sites described by Herrera and Curran (Herrera, F.C., Curran, P.F. 1963.J. Gen. Physiol.46:999).

Cyclic AMP levels in isolated frog skin epithelium: Effects of phosphodiesterase inhibitors, oxytocin and catecholamines

Direct measurements of cyclic AMP were performed in the isolated epithelium of frog skin. Phosphodiesterase inhibitors (methylxanthines, papaverine) and activators of adenylyl cyclase (oxytocin, catecholamines) significantly increased the cyclic AMP content. Propranolol completely blocked the generation of cAMP induced by beta-adrenergic agonists but had little or no effect on that induced by oxytocin. Phentolamine enhanced the cAMP production by adrenalin and noradrenalin. At supramaximal concentrations, oxytocin and isoproterenol produced similar increments in cAMP, while exposure to both agents roughly doubled the increase in cAMP. The results suggest the presence of independent receptors for oxytocin and catecholamines in frog skin, with additive effects on cAMP generation.

Vasopressin-like effects of a hallucinogenic drug-harmaline-on sodium and water transport

SummaryTo determine if harmala alkaloids affect transport systems other than (Na +K)-ATPase, effects of harmaline on Na and water fluxes were studied in amphibian skins. Net Na flux was evaluated from short-circuit current, and water flux monitored with automatic, volumetric methods. At 2 to 5mm, harmaline consistently inhibited SCC and prevented the natriferic effects of oxytocin and norepinephrine. However, at 0.1 to 0.5mm, harmaline produced an increase in SCC inhibitable with amiloride. The stimulatory effects of harmaline and oxytocin were either nonadditive or additive depending on whether the hallucinogen was present in the inner solution or in the outer solution bathing the skin, respectively. Water flow was not modified by harmaline on the outer medium. In contrast, addition of the drug to the inner medium elicited a conspicuous, sustained, vasopressin-like, hydrosmotic effect, comparable to and competitive with those of vasopressin and norepinephrine. The ensemble of these results suggests that harmaline may affect three distinct transport systems: (i) the Na pump; (ii) the cyclic nucleotide system; (iii) the Na entry pathway at the outer membrane of the skin that is also activated by agents such as diphenylhydantoin, lanthanides and propranolol.

Cytochalasin B and water transport

SummaryA morpho-functional study of the effects of cytochalasin B (CB) on Na and water transport was made in amphibian epithelia. The functional studies confirmed the dissociation of the natriferic and hydrosmotic effects of vasopressin in toad urinary bladders exposed to CB and showed in addition that the block of the hydrosmotic effect was reversible and could still be induced in epithelia maximally stimulated with the hormone. Scanning electron microscopy revealed that CB, per se, did not alter the apical surface of the bladders. An almost total loss of microvilli of granular cells was seen, however, if CB was associated with vasopressin and an osmotic gradient. The results suggest two points: a) the block of the hydrosmotic flow induced by CB is due to factors beyond the apical membrane; b) microfilaments may be important mechanochemical transducers in the chain of events leading to the hydrosmotic effect of vasopressin.

Vasopressin-like effects of psychotropic drugs in amphibian epithelia

SummaryAmphibian epithelia have been used as models for studying the effects of psychotropic drugs on membrane transport. Several of these agents added to the internal or to the external media, at concentrations greater than 10−3m, had inhibitory, “ouabainlike” effects on Na transport. In contrast, stimulatory, “vasopressin-like” effects were seen at lower concentrations. The stimulation was additive to that of oxytocin if the drug was present in the external solution but nonadditive if in the internal solution. On water transport, harmala alkaloids had a vasopressinomimetic action in toad skin, while inhibition was seen with Li and amitriptyline. To account for these multiple effects, it is hypothesized that psychotropic drugs act on the following cell targets: the Na pump, the cyclic nucleotide system, microtubules, and membrane calcium sites at the outer barrier of the epithelium. Direct, biochemical evidence is needed to substantiate this hypothesis.

Forskolin mimics the hydrosmotic action of vasopressin in the urinary bladder of toads Bufo marinus.

Net water flow JW was measured across the urinary bladder of toads Bufo marinus and averaged over periods of 1 min by means of a volumetric, automatic technique. The diterpene forskolin, an activator of adenylate cyclase bypassing the hormonal receptor subunit, induced a rapid, reversible, dose‐dependent increase in osmotic water permeability, Pf, very similar to that induced by vasopressin. At 1.1 microM, forskolin induced a half‐maximal response. At 5 microM forskolin caused a near maximal response and Pf increased from 1.66 +/‐ 0.15 to 66.6 +/‐ 2.99 microns s‐1. In bladders pre‐exposed to 5 microM‐forskolin, further significant increases in Pf were obtained by their subsequent exposure to vasopressin, cyclic AMP, theophylline or serosal hypertonicity. The similarity of the forskolin and vasopressin actions was further demonstrated by the finding that substances causing enhancement (quercetin) or inhibition (trifluoperazine, vanadate, silver, cobalt, manganese and Ca2+‐free Ringer solution) of the vasopressin response, induced parallel changes in the forskolin response. Three agents, however, induced dissimilar effects on vasopressin and forskolin: high K+ potentiated vasopressin but inhibited forskolin; methohexital and diamide inhibited vasopressin but had no effect on forskolin. The forskolin‐induced hydrosmotic response can be viewed as a new criterion for ascertaining the messenger role of cycle AMP in the the hydrosmotic effect of vasopressin.

Effects of Ag+ on frog skin: Interactions with oxytocin, amiloride and ouabain

Different biological effects of Ag+ (10−4 M) were found depending on its presence in the outer or the inner solution bathing the frog skin. A marked increase in the electrical conductance and an interference with the action of oxytocin and amiloride were found only when Ag+ was added to the outer solution. Results suggest that Ag+ affects several transport processes, in particular the permeability of the Na entry pathways.