Stimulation of cyclic GMP production in cultured endothelial cells of the pig by bradykinin, adenosine diphosphate, calcium ionophore A23187 and nitric oxide

Abstract

1. The effects of bradykinin, adenosine diphosphate, calcium ionophore A23187 and nitric oxide on the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) were investigated in cultured aortic endothelial cells of the pig. 2. Bradykinin (10-7 M), adenosine diphosphate (3 x 10-5 M), nitric oxide (2 x 10-6 M) and A23187 (10-6 M) stimulated the production of cyclic GMP. This stimulation reached a maximum within 1 min and declined rapidly with the first three agonists whereas that induced by A23187 was long-lasting. 3. These concentrations of bradykinin, A23187 and nitric oxide had no effect on cyclic AMP production. However, adenosine diphosphate (3 x 10-5 M) slightly but significantly enhanced its production by about 1.7 fold. 4. The basal content of cyclic GMP in endothelial cells was significantly reduced by haemoglobin (10-5 M, a scavenger of endothelium-derived relaxing factor(s)) and methylene blue (10-5 M, and inhibitor of the activation of soluble guanylate cyclase) and was significantly enhanced by superoxide dismutase (500 u ml-1, a scavenger of superoxide anions). The basal content of cyclic GMP was not affected by N(G)-monomethyl-L-arginine (10-5 M, a specific inhibitor of the formation of nitric oxide from L-arginine) and was slightly but significantly increased by its D-enantiomer, N(G)-monomethyl-D-arginine. 5. The production of cyclic GMP stimulated by bradykinin, adenosine diphosphate, A23187 and nitric oxide was inhibited by haemoglobin (10-5 M) and methylene blue (10-5 M) but was unaffected by superoxide dismutase (500 u ml-1). 6. The production of cyclic GMP stimulated by bradykinin, adenosine diphosphate or A23187, but not that stimulated by nitric oxide, was significantly reduced by N(G)-monomethyl-L-arginine (10-5 M). The production of cyclic GMP evoked by nitric oxide, but not that induced by the other three agents, was enhanced significantly by N(G)-monomethyl-D-arginine by about 1.5 fold. 7. These data indicate that the endothelium-dependent vasodilators bradykinin, adenosine diphosphate and A23187 activate the production of cyclic GMP in endothelial cells via the synthesis of nitric oxide, which in turn stimulates the soluble guanylate cyclase.