Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C.

Many types of peptide hormone and neurotransmitter receptors mediate hydrolysis of phosphoinositides (PI) and arachidonic acid and arachidonic acid metabolite (AA) release, but the relation between these responses is not clearly defined. We have characterized bradykinin (BK)-mediated AA release and PI hydrolysis in clonal Madin-Darby canine kidney cells (MDCK-D1). Both responses occurred over a similar dose range in response to the B1 and B2 receptor agonist, BK, but not in response to the B1 receptor-selective agonist des-Arg-BK. To test whether AA release occurs via a mechanism which is sequential to and dependent upon PI hydrolysis, we used the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C. TPA treatment blocked BK-mediated PI hydrolysis in MDCK-D1 cells, while at the same time and at similar concentrations enhancing BK-mediated AA release. Thus, TPA treatment dissociated BK-mediated AA release from PI hydrolysis. In addition, treatment of MDCK-D1 cells with neomycin blocked BK-mediated hydrolysis of phosphatidylinositol bisphosphate without reducing BK-mediated AA release. BK treatment increased formation of lysophospholipids with a time course in accord with BK-mediated AA release, indicating that at least part of the BK-mediated AA release was likely derived from activation of phospholipase A2. BK-mediated lysophospholipid production was enhanced by pretreatment with TPA, suggesting that the mechanism of AA release before and after treatment with TPA was the same. BK-mediated AA release and lysophospholipid production was dependent on the presence of extracellular calcium, while the enhanced responses to BK in the presence of TPA were not dependent on the presence of extracellular calcium. TPA treatment also enhanced AA release and lysophospholipid production in response to the calcium ionophore A23187. From these data we propose that BK, acting at B2 receptors, promotes AA release in MDCK cells via a mechanism which is 1) independent of polyphosphoinositide hydrolysis by phospholipase C, 2) dependent upon influx of extracellular calcium and activation of phospholipase A2, and 3) enhanced by activation of protein kinase C.