Water Flow In Toad Bladder Research Articles

Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells.

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.

Inhibition of vasopressin-stimulated water flow in toad bladder by phorbol myristate acetate, dioctanoylglycerol, and RHC-80267. Evidence for modulation of action of vasopressin by protein kinase C.

The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium-phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C.

1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin: a new photoaffinity label for hydroosmotic hormone receptors. Characterization of the ligand and irreversible stimulation of hydroosmotic water flow in toad bladder by photoaffinity labeling.

The photoreactive analog of vasopressin [1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin [( Asu1,6, Phe (p-N3)3]AVP) has been synthesized. This analog retains a high binding affinity for the vasopressin receptor in plasma membranes from bovine kidney inner medulla (apparent dissociation constant, KD = 8.5 X 10(-9) M). [Asu1,6, Phe (p-N3)3] AVP was found to be biologically active in triggering the characteristic increase in toad bladder permeability to water. Photolysis of the analog in the presence of the toad bladder results in a hydroosmotic response which persists, in spite of repeated washings, for more than 18 h. The irreversible stimulation of the bladder is inhibited when photolysis is carried out in the presence of vasopressin. Our findings indicate that with photoactivation [Asu1,6, Phe(p-N3)3]AVP binds covalently to hormonal receptors and forms an active hormone-receptor complex. This analog, therefore, is a suitable tool for studies of hydroosmotic receptor function and for receptor isolation.

Pressure effects on the ADH-induced initiation of water flow in toad bladder.

Earlier studies employed colchicine to demonstrate the need for microtubules in the ADH-induced initiation of increased water permeability in toad bladder. We have used colchicine and hydrostatic pressure together to determine whether formed or growing microtubules are required for initiation of the ADH response in Bufo marinus. When ADH and 8,000 psi were administered simultaneously, the ADH-induced increase in water flow was inhibited while under pressure by 107 +/- 7% (n = 6). Application of 8,000 psi for 10 min before ADH administration resulted in an increased initiation of the ADH osmotic response over the non-pressure-treated control (average acceleration, rate of water flow increase during first 3 min after ADH stimulation, 1.25 +/- 0.27 vs. 0.43 +/- 0.12 mg X cm-2 X min-2, n = 6). In addition, the inhibition of the ADH response brought about by colchicine incubation was overcome with pretreatment of the colchicine-incubated bladders with 8,000 psi for 10 min (average 3-min acceleration, 0.18 +/- 0.04 vs. 1.13 +/- 0.06 mg X cm-2 X min-2, respectively). Repeating the experiments with dibutyryl cAMP gave similar results. We interpret these data as suggesting that growing microtubules are required for initiation. The proposed model is as follows. Pressure removes colchicine inhibition by introducing, through disassembly of formed microtubules, more colchicine-free tubulin subunits. These subunits are then available following decompression to reassemble when the tissue is challenged with hormone.

Calcium flow-independent actions of calcium channel blockers in toad urinary bladder.

A role for transmembrane calcium movement in vasopressin stimulation of its target cell has been postulated based on studies with calcium entry blockers such as verapamil. We examined the effect of three sets of structurally different calcium blockers--D600 (an analogue of verapamil), diltiazem, and nifedipine--on water flow in toad bladder. D600 (200 microM), diltiazem (200 microM), and nifedipine (60 microM) inhibited vasopressin-induced water flow but enhanced adenosine 3',5'-cyclic monophosphate (cAMP)-induced water flow, suggesting that the drugs inhibit cAMP generation in response to vasopressin but enhance the response to exogenous cAMP by inhibiting phosphodiesterase activity. In the case of vasopressin stimulation, inhibition of cAMP generation appears to be the overriding effect. This was confirmed by measurements of cAMP content and the protein kinase ratio (-cAMP/+cAMP), which were significantly lower in bladders receiving both D600 and vasopressin than in those receiving vasopressin alone. Furthermore the drugs inhibited activation of adenylate cyclase by vasopressin in cell homogenates and inhibited phosphodiesterase in both homogenates and membrane-free supernatants. Thus these "calcium channel blockers" can directly alter cAMP metabolism in settings where movement of calcium should be irrelevant. The close correlation between the biochemical and transport effects of these agents suggests that their effect on water flow may occur by a direct effect on cellular enzymes or the membranes in which they reside and not by altering local calcium concentrations.

Inhibitory effects of 1-iodo-3-aminomethyl-5,6,7,8-tetrahydro-2-naphthol (ONO-3122) and prostaglandin H2 on vasopressin-induced osmotic water flow in toad bladder.

Both 1-iodo-3-aminomethyl-5,6,7,8-tetrahydro-2-naphthol (ONO-3122), which increases endogenous PGH2, and PGH2 itself, significantly depressed vasopressin-induced osmotic water flow in the toad bladder. These results suggest that ONO-3122 increases endogenous PGH2 synthesis, and that PGH2 and/or its metabolites inhibit vasopressin-induced water flow.

Effect of hydrostatic pressure on ADH induced osmotic water flow in toad bladder.

The effect of hydrostatic pressure (HP) on antidiuretic hormone (ADH) stimulated osmotic water flow (Jv) across the toad urinary bladder was evaluated. Jv for ADH-stimulated bladders was significantly reduced by an elevation of the serosal HP gradient to 1 cm H2O. Subsequent elimination of the HP gradient resulted in a recovery of Jv. Serosal HP also caused a reversible increase in sucrose permeability (P sucrose). For ADH-treated bladders fixed with glutaraldehyde during serosal HP exposure, subsequent exposure to a mucosal or serosal HP gradient caused acceleration or inhibition of Jv, respectively. The reduction in ADH-associated Jv with serosal HP was apparently caused by a back-flux of water through a paracellular pathway. Jv and P sucrose were not affected by mucosal HP during ADH stimulation. The results suggest a specific sensitivity of a paracellular pathway to a small serosal HP gradient in bladders with ADH-stimulated water flow. The reversibility of this effect on P sucrose suggests that the elements comprising the apical junctions are dynamic structures capable of recovering at least some of their permeability properties.

Stimulation of osmotic water flow in toad bladder by prostaglandin E1. Evidence for different compartments of cyclic AMP.

The effect of prostaglandin E1 (PGE1) on osmotic water flow across toad bladder and cyclic AMP content of the mucosal epithelial cells has been determined under basal conditions and in the presence of either theophylline or antidiuretic hormone (ADH); Under basal conditions and with PGE1 concentrations from 10(-8) to 10(-5) M no evidence of stimulation of water flow was observed, and with 10(-7) M PGE1 a significant inhibition was foundmcyclic AMP content under control conditions was 8 pmol/mg protein. It was 9 at 10(-8) M PGE1, 13 at 10(-7) M, 16 at 10(-6) M, and 23 at 10(-5) M. In the presence of theophylline, 10(-8) and 10(-7) M PGE1 inhibited the theophylline-induced water flow as expected. In contrast, 10(-6) and 10(-5) M PGE1 enhanced the rate of water flow. Theophylline increased cyclic AMP content from 8 to 18 pmol/mg protein. PGE1 in the presence of theophylline caused marked increases in cyclic AMP content; The content was 23 at 10(-7) M, 41 at 10(-6) M, and 130 at 10(-5) M; Thus PGE1 stimulates theophylline-induced water flow at cyclic AMP concentrations somewhere between 23 and 41 pmol/mg. Further evidence along these lines was obtained from experiments in which the effects of PGE1 on ADH-induced water flow were studied. Inhibitory effects of PGE1 were not observed at concentrations of PGE1 which raised the level of intracellular cyclic AMP to 30 pmol/mg protein or higher. These results were obtained despite the fact that all four concentrations of PGE1 tested were found capable of inhibiting ADH-induced water flow under appropriate conditions or, in other words, were inhibiting the adenylate cyclase controlling water flow, Thus the increase in cyclic AMP content in response to PGE1 is not derived from this enzyme. Thus the stimulation of water flow by PGE1 in the presence of theophylline is thought to be caused by cyclic AMP spilling over from one compartment to the water flow compartment. No evidence was obtained to directly suggest spillover into the sodium transport compartment. Furthermore evidence is discussed to suggest that most of the cyclic AMP generated in the tissue does not originate from the enzyme controlling sodium transport. As cyclic AMP-stimulated water flow and sodium transport are thought to occur in one cell type, the granular cells, distinct pools of cyclic AMP are thought to be present in one and the same cell type. Thus one pool controls water flow and one controls sodium transport. With high concentrations of PGE1 in the presence of theophylline or high concentrations of ADH, the adenylate cyclase responsible for water flow is inhibited; However, PGE1 can stimulate a tissue adenylate cyclase to sufficiently high levels that cyclic AMP spills over into the "water flow compartment" and thus stimulates water flow.