Dopaminergic inhibition of vasopressin-stimulated water flow in the toad bladder: Evidence for local formation of dopamine

Summary Dopamine administration increases renal excretion of water and Na. It remains uncertain whether these effects of dopamine are the result of a hemodynamic effect or the consequence of a direct cellular action. We investigated the effect of dopamine on water transport by the isolated toad bladderin vitro. Dopamine failed to alter baseline water flow but caused a significant inhibition of arginine vasopressin (AVP) or cyclic adenosine monophosphate (AMP) stimulated water flow. The effect of dopamine on stimulated water flow was not due to activation of adrenergic, adrenergic, or cholinergic receptors. The selective antagonists of dopamine, metoclopramide and apomorphine, prevented the effect of dopamine on AVP-stimulated water flow. These observations suggest the existence of a dopaminergic receptor in the toad bladder.l-Dopa also inhibited AVP-stimulated water flow. The effect ofl-Dopa could be prevented by metoclopramide, thus suggesting thatl-Dopa is converted to dopamine by an aromatic amino acid decarboxylase present in the toad bladder. To investigate this possibility we measured the effect of the decarboxylase inhibitor, carbidopa, on the¹⁴CO₂ production generated by decarboxylation of¹⁴Cl-Dopa in isolated toad bladder epithelial cells. Isolated toad bladder epithelial cells generated significant amounts of¹⁴CO₂ from¹⁴Cl-Dopa. This effect could be blocked by carbidopa, thus suggesting the existence of an aromatic amino acid decarboxylase system in the toad bladder. Carbidopa also prevented the inhibitory effect ofl-Dopa on AVP-stimulated water flow, suggesting thatl-Dopa needs to be converted to dopamine to inhibit water flow. These data suggest the existence of a dopaminergic receptor in the toad bladder. These data also suggest that dopamine can be formed locally in the toad bladder and can thus serve as a local modulator of water transport.     

The effect of acid-base changes on vasopressin-stimulated water flow in toad urinary bladder

Water flow was measured gravimetrically in the presence and absence of vasopressin across the toad urinary bladder. Four groups of toads in different states of acid-base balance were used; a normal group, a group in NH₄Cl induced metabolic acidosis, respiratory acidosis, and a group in NaHCO₃ induced metabolic alkalosis. Vasopressin induced water flow was significantly reduced during metabolic acidosis and respiratory acidosis. Metabolic alkalosis had no effect on the hydro-osmotic response to vasopressin. Dibutyryl cyclic-AMP-stimulated water flow on the other hand was not affected by either a metabolic or respiratory acidosis. Treatment with indomethacin was able to reverse the observed reduction in the vasopressin-stimulated water flow response in the toad bladder during metabolic and respiratory acidosis. We conclude that the vasopressin stimulated water flow is altered during acidosis and evidence suggests that prostaglandins may be involved in the observed reduction in vasopressin-stimulated water flow.     

Enhancing effects of angiotensin I on the vasopressin-stimulated water flow of toad bladder through increased cyclic AMP in mucosal cells

The effects of angiotensins I and II on 10 mU/ml vasopressin-stimulated water flow across toad bladder were examined. Angiotensin I at concentrations of 10(-6) and 10(-7) M enhanced the water flow, but angiotensin II failed to do so at these concentrations. Angiotensin I had no effect on 5 mM cyclic AMP-stimulated water flow. After being preincubated for 30 min with angiotensin II, angiotensin I failed to have any stimulatory effect on vasopressin-stimulated water flow. At 10(-6) M angiotensin I significantly enhanced vasopressin-stimulated cyclic AMP content in bladder mucosal cells. These results indicate that angiotensin I enhances vasopressin-stimulated water flow by increasing cyclic AMP production in bladder cells and that angiotensin II may possibly interfere with angiotensin I in a competitive manner.     

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.     

Barriers to water flow in vasopressin-treated toad urinary bladder

Summary Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.     

Cell determinants of vasopressin-stimulated water flow

I present a technique that permits evaluation of the permeability to water of the luminal membrane of the toad urinary bladder, independently of constraints to water flow imposed by the remainder of the tissue. This technique essentially depends on fixation of the luminal membrane with 1% glutaraldehyde for 5 min, and subsequent elimination of cytosolic constraints by decreasing the tonicity of the serosal bath to 1/2 normal strength. The increased hydraulic conductivity found with serosal hypotonicity is readily reversible, as the bladder returns to an isotonic serosal bath. By evaluating water flow in luminally fixed bladders during bathing in normal and hypotonic bath, one may identify the relative contribution of the luminal membrane and the "cytosol" on water flow. Using this technique, I found that the effect of the prostaglandin inhibitor Naproxen to increase vasopressin-stimulated water flow is due to increased luminal membrane permeability. The effect of histidine to increase vasopressin-stimulated water flow, however, depends on increased permeability of both the luminal membrane as well as the underlying structures. The action of serosal hypertonicity to induce water flow is due to an increased luminal permeability. However, serosal hypertonicity decreases "cytosolic" permeability, so that its overall function is a composite effect of its action at the luminal membrane and the "cytosolic" level.     

Nocodazole inhibition of the vasopressin-induced water permeability increase in toad urinary bladder

Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 μ/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an inhibition of 50% seen with a 3-h exposure of the tissue to colchicine (0.1 mM) prior to stimulation with vasopressin. Application of nocodazole (1 μ/ml) 3 min after hormonal stimulation shows no inhibition of the response at one-half hour past stimulation. These data support the view that microtubules are involved in the vasopressin-induced increase in water permeability in toad bladder and also indicate that this involvement is limited to the period prior to or directly after stimulation.     

Localization of barriers to water flow in toad urinary bladder

Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional "postluminal" site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze-fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane. The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a "postluminal" barrier that is functionally in series with the luminal membrane. We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin-stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water-permeant channels in the luminal membrane. Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2 and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B-elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of luminal membrane water permeability by water flow in toad urinary bladder

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed "aggrephores") which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder

Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.     

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.     

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.     

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.     

Regulation of the formation and water permeability of endosomes from toad bladder granular cells

Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers.     

Barium inhibition of sodium ion transport in toad bladder

The effect of Ba²⁺ on Na⁺ transport and electrical characteristics of toad bladder was determined from change produced in short circuit current (I_sc, epithelial, apical and basal-lateral potentials (ψ_t, ψ_a, ψ_b), epithelial and membrane resistances (R_t, R_a, R_b) and shunt resistance (R_s). Mucosal Ba²⁺ had no effect. Serosal Ba²⁺ reduced I_sc, ψ_t, ψ_a, and ψ_b, but had no effect on R_t, R_a, R_b and R_s. Minimal effective Ba²⁺ concentration was 5 · 10^?5 M. The phenomenon was reversed by Ba²⁺ removal, but not by 86 mM serosal K⁺. Ba²⁺ inhibition of I_sc did not impair the response to vasopressin which was quantitatively the same as controls. ψ_a with Ba²⁺ equalled ψ_b. After Ba²⁺ inhibition, ouabain produced no further decrease in ψ_t and I_sc. Ba²⁺ exposure after ouabain did not decrease ψ_t and I_sc. The results suggest that Ba²⁺ inhibits the basal-lateral electrogenic Na⁺ pump.     

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.