Effect of sodium transport inhibition on active phosphate transport by toad bladder

Summary The relationship between active Na transport (estimated by the short-circuit (SCC)) and active inorganic phosphate (P_i) transport was studied in the toad bladder. When SCC was inhibited by amiloride, ouabaim, or removal of K from the serosal bathing solution, active P_i transport was totally inhibited. When Na was replaced isotonically by choline in either the mucosal bathing solution or both the mucosal and serosal bathing solutions, there was no measurable SCC or active P_i transport. These experiments are compatible with the hypothesis that active P_i transport occurs only in the presence of active Na transport.     

Active sodium transport by the isolated toad bladder

Studies were made of the active ion transport by the isolated urinary bladder of the European toad, Bufo bufo, and the large American toad, Bufo marinus. The urinary bladder of the toad is a thin membrane consisting of a single layer of mucosal cells supported on a small amount of connective tissue. The bladder exhibits a characteristic transmembrane potential with the serosal surface electrically positive to the mucosal surface. Active sodium transport was demonstrated by the isolated bladder under both aerobic and anaerobic conditions. Aerobically the mean net sodium flux across the bladder wall measured with radioactive isotopes, Na²⁴ and Na²², just equalled the simultaneous short-circuit current in 42 periods each of 1 hour's duration. The electrical phenomenon exhibited by the isolated membrane was thus quantitatively accounted for solely by active transport of sodium. Anaerobically the mean net sodium flux was found to be slightly less than the short-circuit current in 21 periods of observation. The cause of this discrepancy is not known. The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions. Adrenaline failed to stimulate the short-circuit current of the toad bladder.     

Dobutamine inhibits vasopressin-mediated water transport across toad bladder epithelium

This study aimed to investigate the effect of dobutamine on water transport across toad bladder epithelium. Water flow through the membrane was measured gravimetrically in bladder sac preparations. Dobutamine had no effect on basal water transport, but partially inhibited transport stimulated by vasopressin. Similarly, dobutamine exerted no influence on the hydrosmotic response to 8-chlorophenylthio-cAMP, but interfered with the response to phosphodiesterase inhibitor 1-methyl-3-isobutyl-xanthine. These results demonstrate that this catecholamine may inhibit vasopressin-stimulated water transport at a site prior to cAMP formation. The use of propranolol was ineffective in blocking the effect of dobutamine on transport stimulated by vasopressin, indicating that beta-adrenoceptors play no role in this effect. On the other hand, phentolamine significantly reduced the effect of dobutamine, indicating the involvement of alpha-adrenoceptors in such event. Rauwolscine also inhibited the effect of dobutamine, pointing to the specific contribution of the alpha(2)-adrenoceptors to this effect. Taken together, the results of this study demonstrate that dobutamine inhibits vasopressin-stimulated water transport in toad bladders through a mechanism mediated by the stimulation of alpha(2)-adrenoceptors, thus suggesting that such a drug may exert a direct cellular effect on membrane permeability to water in transporting epithelia. The current study may provide a better understanding of the effects of dobutamine on renal function by contributing towards the elucidation of its action mechanism.     

Effect of metabolic inhibitors on vasopressin-stimulated transport systems in the toad bladder

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.     

Effects of cholinergic agents on the vasopressin-mediated water transport in the isolated toad bladder

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3′5′_cAMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3′5′ cyclic adenosine monophosphate (3′5′_cAMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M₁ and M₃, which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.     

Effect of colchicine on the osmotic water flow across the toad urinary bladder

Osmotic water movement across the toad urinary bladder in response to both vasopressin and cyclic AMP was inhibited by 10^?5 to 10^?4 M colchicine on the serosal but not on the mucosal side. This inhibitory effect was found to be time- and dose-dependent. Colchicine alone did not change basal osmotic flow and a baseline of the short-circuit current (I_sc) and also did not affect a vasopressin-induced rise of the I_sc. The inhibitory effect was not prevented by the addition of pyruvate. The osmotic water movement produced by 360 mM Urea (mucosal), 360 mM mannitol (serosal) or 2 μg/ml amphotericin B (mucosal), was not affected by 10^?4 M colchicine. These results suggest that colchicine inhibits some biological process subsequent to the formation of cyclic AMP except a directional cytoplasmic streaming process where microtubules may be involved.     

Surface hydrophobicity and water transport of the toad urinary bladder: Effects of vasopressin

Summary The present study investigated whether the hydrophobic properties (wettability) of the luminal surface of the toad urinary bladder might play a role in modulating water transport across this epithelium. In the absence of vasopressin (ADH), water transport across the tissue was low, while luminal surface hydrophobicity (water contact angle) was relatively high. Following stimulation by ADH, water transport increased and surface hydrophobicity decreased. The addition of indomethacin to inhibit ADH-induced prostaglandin synthesis did not reduce these actions of ADH. In an attempt to alter water transport in this tissue, a liposomal suspension of surface-active phospholipids was administered to the luminal surface. This addition had no detectable influence on the low basal rates of water transport, but blocked the ADH-induced stimulation of water transport. We suggest that surface-active phospholipids on the toad bladder luminal membrane may contribute to the hydrophobic characteristics of this tissue. ADH may act to decrease surface hydrophobicity, facilitating the movement of water molecules across an otherwise impermeable epithelium. This surface alteration may be associated with the appearance of water channels in the apical membrane.     

Effect of colchicine on the osmotic water flow across the toad urinary bladder.

Osmotic water movement across the toad urinary bladder in response to both vasopressin and cyclic AMP was inhibited by 10(-5) to 10(-4) M colchicine on the serosal but not on the mucosal side. This inhibitory effect was found to be time- and dose-dependent. Colchicine alone did not change basal osmotic flow and a baseline of the short-circuit current (Isc) and also did not affect a vasopressin-induced rise of the Isc. The inhibitory effect was not prevented by the addition of pyruvate. The osmotic water movement produced by 360 mM Urea (mucosal), 360 mM mannitol (serosal) or 2 mug/ml amphotericin B (mucosal), was not affected by 10(-4) M colchicine. These results suggest that colchicine inhibits some biological process subsequent to the formation of cyclic AMP except a directional cytoplasmic streaming process where microtubules may be involved.     

Effect of distension on ADH-induced osmotic water flow in toad urinary bladder

Summary We recently described a method by which the resistance to water flow of the luminal membrane of ADH-stimulated toad bladder can be quantitatively distinguished from that of barriers lying in series with it. This method requires estimates of both total bladder water permeability (assessed by transbladder osmotic water flow at constant gradient) and luminal membrane water permeability (assessed by quantitation of the frequency of ADH-induced luminal membrane particle aggregates). In the present study we examined the effect of bladder distension on transepithelial osmotic water flow before and during maximal ADH stimulation. Base-line water flow was unaffected by bladder distension, but hormonally stimulated flow increased systematically as bladders became more distended. Distension had no effect on the frequency of ADH-induced intramembranous particle aggregates. By comparing the relationships between aggregate frequency and hormonally induced water permeability in distended and undistended bladders, we found that distension appeared to enhance ADH-stimulated water flow by decreasing the resistance of the series permeability barrier while the apparent water permeability associated with each single luminal membrane aggregate was unaffected. In that bladder distension causes tissue thinning, the series resistance limiting ADH-stimulated water flow appears to be accounted for by deformable barriers within the bladder tissue itself, probably unstirred layers of water.     

Mechanism of inhibition by lithium of sodium transport in the toad bladder

Lithium transport across the urinary bladder of Bufo marinus has been studied by means of the short-circuit current technique, as well as unidirectional ion flux measurements. Exposure to lithium of the epithelial (mucosal) surface of this preparation led to a slow, progressive decrease of ion transport, with increasing discrepancy between short-circuit current and lithium influx; in fact there was still an appreciable lithium influx across bladder exposed to amiloride even though short-circuit current was suppressed. Ohmic conductance and sodium efflux barely increased under these circumstances. Upon replacement of lithium by sodium on the epithelial side, the preparations recovered slowly indeed, and residual lithium could be detected in bladder tissue for more than 2 hr while the rate of sodium extrusion at the basal-lateral cell border was slowed down. Recovery from exposure to lithium was accelerated by vasopressin and amphotericin, both of which facilitate sodium entry at the apical border of the epithelium. Thus the lasting deleterious influence of lithium on sodium transport might result from the fact that this ion, once trapped in the cytoplasm, closes the sodium channels.     

Effect of carbenoxolone sodium on steroid-induced sodium transport in the toad bladder: Further studies

The licorice derivative, carbenoxolone sodium, is a potent inhibitor of the enzyme 11β-hydroxysteroid dehydrogenase. When this enzyme is suppressed or is absent, endogenous glucocorticoids induce mineralocorticoid-like sodium retention by the kidney. Carbenoxolone sodium administered in vivo to an adrenalectomized rat has also recently been shown to enhance the mineralocorticoid response to submaximal concentrations of aldosterone, deoxycorticosterone (DOC) and 11-dehydrocorticosterone (compound A). In the present studies conducted on the urinary bladder isolated from the Dominican toad, Bufo marinus, a concentration of carbenoxolone sodium shown previously to increase glucocorticoid-induced sodium transport (2.5 × 10⁻⁵ M) did not appear to alter the response to submaximal concentrations of aldosterone 10⁻⁸ M, DOC 10⁻⁷ M, or compound A 10⁻⁵ M. These findings are consistent with the view that in the whole animal carbenoxolone sodium may modify additional steroid metabolic pathways and/or physiological processes in several organs to produce the enhanced renal response to mineralocorticoids and compound A.     

Insulin-stimulated sodium transport in toad urinary bladder

Mammalian and teleost insulins increase active sodium transport by the toad urinary bladder at subnanomolar concentrations. This stimulation is evident within 15 min and persists for hours. Porcine proinsulin and a cross-linked derivative of bovine insulin are less effective than porcine insulin in stimulating the short-circuit current (SCC), indicating the specificity appropriate for activation of sodium transport through an insulin receptor. The initial stimulation by insulin of the SCC is not blocked by pretreatment with actinomycin D, puromycin, cycloheximide, or tunicamycin. However, in the presence of any one of these inhibitors the sustained increase in SCC is blocked and the rise is short-lived, lasting only 45 to 90 min. In amphotericin-treated bladders, the addition of insulin did not further stimulate SCC.     

Amide transport channels across toad urinary bladder

Summary Urea and other small amides cross the toad urinary bladder by a vasopressinsensitive pathway which is independent of somotic water flow. Amide transport has characteristics of facilitated transport: saturation, mutual inhibition between amides, and selective depression by agents such as phloretin. The present studies were designed to distinguish among several types of transport including (1) movement thought a fixed selective membrane channel and (2) movement via a mobile carrier. The former wold be characterized by co-transport (acceleration of labele amide flow in the direction of net flow in the opposite direction). Mucosal to serosal (MS) and serosal to mucosal (SM) permeabilities of labeled amides were determined in paired bladers. Unlabeled methylurea, a particularly potent inhibitor of amide movement, was added to either the M or S bath, while osmotic water flow was eliminated by addition of ethylene glycol to the opposite bat. Co-transport of labeled methylurea and, to a lesser degree, acetamide and urea with unlabeled methylurea was observed. Co-transport of the nonamides ethylene glycol and ethanol could not be demonstrated. Methylurea did not alter water permeability or transmembrane electrical resistance. The demonstration of co-transport is consistent with the presence of ADH-sensitive amide-selective channcels rather than a mobile carrier.