Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.
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 alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha 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-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta 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-alpha, while the immunoreactivity to the epsilon and zeta 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-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition 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-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), 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-alpha is not responsible for the regulation of the response to P2-purinergic stimulation, and imply that the response is mediated by PKC-epsilon (PKC-zeta is unresponsive to PMA), or an as yet uncharacterized PKC isoform.
Patel
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Department of Cell Physiology and Pharmacology, University of Leicester.
Brown
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C
Boarder
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MR
D015121:6-Ketoprostaglandin F1 alpha / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D015536:Down-Regulation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000201:enzymology*; D011464:Epoprostenol / Q000096:biosynthesis / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D007527:Isoenzymes; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D015121:6-Ketoprostaglandin F1 alpha; D015544:Inositol 1,4,5-Trisphosphate; D011464:Epoprostenol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate