In the rat pheochromocytoma cell line PC-12, bradykinin (BK) stimulated phosphatidylinositol hydrolysis by 4–5-fold and, additionally, intracellular cAMP accumulation by approx. 1.6-fold. EC50 values for BK were 3 nM and 2 nM respectively. The BK-induced increase in cAMP accumulation was paralleled by a 1.6-fold increase in protein kinase A (PKA) activity. The time course of BK-stimulated inositol phosphate formation was rapid (t1/2 < 1 min), whereas the BK-induced cAMP accumulation was lagging (t1/2 approx. 6 min). The effect of BK on the cAMP pathway was independent of pertussis toxin, excluding an indirect stimulation of adenylate cyclase via βγ-complexes from Gi or Go proteins. Two different protein kinase C (PKC) inhibitors, bisindolylmaleimide and Ro 31-820, failed to prevent BK-induced cAMP accumulation, and exclude PKC as mediator of BK action on adenylate cyclase. In contrast, the stimulatory effect of BK on cAMP accumulation was completely abolished by two calmodulin antagonists, chlorpromazine and ophiobolin, suggesting an indirect, Ca2+/calmodulin-mediated effect of BK on the cAMP pathway. In addition, exposure of PC-12 cells to BK resulted in a translocation of the PKC isoforms α, δ, ϵ and ζ displaying different kinetics. The BK-induced translocations of the PKCs α and δ were rapid and biphasic, whereas the PKCs ϵ and ζ revealed a slower and slightly transient translocation in response to BK. The BK-elicited translocation of PKCϵ, but not that of the PKCs α, δ and ζ, was prevented by two different inhibitors of adenylate cyclase, 2′,5′-dideoxyadenosine and MDL-12,330A, as well as the PKA inhibitor adenosine 3′:5′-monophosphothioate. These findings suggest that the BK-induced translocation of novel (n)PKCϵ is mediated via the cAMP pathway. Since nPKCϵ appears to regulate neurite outgrowth in PC-12 cells [Hundke, McMahon, Dadgar and Messing (1995) J. Biol. Chem. 270, 30134–30140] our results provide evidence for a novel signalling mechanism that might be involved in BK-induced neuronal differentiation of PC-12 cells.

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