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.

Abstract

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 ofcyclic 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 AMPdependent 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 AVPstimulated 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 phos-A preliminary report of this work was presented at the 17th Annual

Effects of protein kinase C activation on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule.

“…Phorbol esters such as PMA enhance PGIE2 production in MDCK cells (20) and toad bladder epithelia (7). These findings are an important consideration in our studies since Stokes and Kokko have found that exogenous PGE2 inhibits net Na absorption and VT in the rabbit CCT (21).…”

“…Schlondorff and Levine have found evidence that protein kinase C activation with phorbol esters can modulate arginine vasopressin (AVP)-stimulated water flow in toad bladder by inhibiting cyclic AMP generation (7). Yanase and Handler have recently demonstrated that activation of protein kinase C with phorbol esters inhibits apical sodium transport of A6 epithelia (8), a cell line that shares many characteristics of the mammalian distal tubule.…”

Cyclic adenosine monophosphate and diacylglycerol. Mutually inhibitory second messengers in cultured rat inner medullary collecting duct cells.

“…Activation of protein kinase C has been shown to inhibit AVP-stimulated water flow in the toad bladder (14) and in the isolated perfused rabbit cortical collecting tubule (15). Though a pre-cAMP site appears operant in the toad bladder (14), the predominant site of action of protein kinase C in both tissues is post-cAMP.…”

“…Furthermore, the other signaling limb, i.e., the effect of stimulation of PLC on cAMP generation, was not examined. Similarly, while activation of protein kinase C has been shown to inhibit AVP-stimulated water flow in the toad bladder (14) and in the rabbit cortical collecting duct (15), in neither tissue has the effect ofcAMP on hormonally stimulated PLC activity been examined. Accordingly, the present study was undertaken to examine bidirectional interactions between the AC and PLC signaling systems in a single cell type, the cultured rat inner medullary collecting tubule (RIMCT) epithelial cell.…”

Axial heterogeneity of vasopressin-receptor subtypes along the human and mouse collecting duct

“…Increased intracellular calcium and activation of PKC has been reported to inhibit AVP-induced water transport in rabbit CCD (5,50). There are several potential mechanisms by which increased intracellular calcium might inhibit AVP-stimulated water flow.…”