1 Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2y-and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2 Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of a, £ and 4, while no immunoreactivity was found for f, y, (, 1 and 0 isoforms. PKC-a was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a .1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-s was always in a Triton X-100 insoluble membrane fraction, while PKC-C was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3 Treatment with PMA for 6 h led to a 90% downregulation of PKC-a, while the immunoreactivity to the e and 4 isoforms remained largely unchanged. 4 After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2u-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-a was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with the phospholipase C response.
5Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U-and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-a) and not Ca2+ insensitive isoforms (such as PKC-e), had no effect on the PGI2 response. 6 The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y-and P2u-purinoceptors. Both downregulation and inhibition studies show that PKC-a is not responsible for the regulation of the response to P2-purinergic stimulation, and imply that the response is mediated by PKC-e (PKC-C is unresponsive to PMA), or an as yet uncharacterized PKC isoform.