GPROTEIN KINASE-C MODULATES RECEPTOR-INDEPENDENT ACTIVATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE

The intracellular regulation of nitric oxide synthase has been the foc us of intense investigation. Bioassay studies using vascular rings hav e suggested that protein kinase C inhibits endothelium-dependent vascu lar relaxation. However, information regarding the effects of protein kinase C on the synthesis of nitric oxide in endothelial cells is not available. Therefore, we investigated the effects of protein kinase C to regulate receptor-independent activation of nitric oxide synthase a ctivity in cultured bovine pulmonary artery endothelial cells. Activat ion of protein kinase C by phorbol 12-myristate 13-acetate or 1,2-dioc tanoyl-sn-glycerol inhibited receptor-dependent and receptor-independe nt nitric oxide synthase activity. The inhibition of nitric oxide synt hase by protein kinase C was concentration dependent and markedly blun ted by staurosporine. The inhibition of protein kinase C by staurospor ine alone enhanced basal nitric oxide synthase activity. Furthermore, depletion of protein kinase C enhanced both basal and agonist-stimulat ed nitric oxide synthase activity. These studies indicate that protein kinase C modulates the activity of the constitutive Ca2+/calmodulin-d ependent endothelial nitric oxide synthase in the basal state and foll owing agonist stimulation through direct inhibition of the enzyme as w ell as receptor desensitization. These direct regulatory effects of pr otein kinase C on endothelial nitric oxide synthase activity may have important implications in the physiologic regulation of vascular tone.