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. 1996 May;118(1):123–130. doi: 10.1111/j.1476-5381.1996.tb15374.x

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

V Patel 1, C Brown 1, M R Boarder 1
PMCID: PMC1909475  PMID: 8733584

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allsup D. J., Boarder M. R. Comparison of P2 purinergic receptors of aortic endothelial cells with those of adrenal medulla: evidence for heterogeneity of receptor subtype and of inositol phosphate response. Mol Pharmacol. 1990 Jul;38(1):84–91. [PubMed] [Google Scholar]
  2. Booyse F. M., Sedlak B. J., Rafelson M. E., Jr Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb Diath Haemorrh. 1975 Dec 15;34(3):825–839. [PubMed] [Google Scholar]
  3. Bowden A., Patel V., Brown C., Boarder M. R. Evidence for requirement of tyrosine phosphorylation in endothelial P2Y- and P2U- purinoceptor stimulation of prostacyclin release. Br J Pharmacol. 1995 Nov;116(6):2563–2568. doi: 10.1111/j.1476-5381.1995.tb17208.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burch R. M., Axelrod J. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6374–6378. doi: 10.1073/pnas.84.18.6374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carter T. D., Hallam T. J., Cusack N. J., Pearson J. D. Regulation of P2y-purinoceptor-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br J Pharmacol. 1988 Dec;95(4):1181–1190. doi: 10.1111/j.1476-5381.1988.tb11754.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter T. D., Hallam T. J., Pearson J. D. Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+. Biochem J. 1989 Sep 1;262(2):431–437. doi: 10.1042/bj2620431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis P. D., Hill C. H., Keech E., Lawton G., Nixon J. S., Sedgwick A. D., Wadsworth J., Westmacott D., Wilkinson S. E. Potent selective inhibitors of protein kinase C. FEBS Lett. 1989 Dec 18;259(1):61–63. doi: 10.1016/0014-5793(89)81494-2. [DOI] [PubMed] [Google Scholar]
  8. Dekker L. V., Parker P. J., McIntyre P. Biochemical properties of rat protein kinase C-eta expressed in COS cells. FEBS Lett. 1992 Nov 9;312(2-3):195–199. doi: 10.1016/0014-5793(92)80934-9. [DOI] [PubMed] [Google Scholar]
  9. Felder C. C., Dieter P., Kinsella J., Tamura K., Kanterman R. Y., Axelrod J. A transfected m5 muscarinic acetylcholine receptor stimulates phospholipase A2 by inducing both calcium influx and activation of protein kinase C. J Pharmacol Exp Ther. 1990 Dec;255(3):1140–1147. [PubMed] [Google Scholar]
  10. Hallam T. J., Pearson J. D., Needham L. A. Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production. Biochem J. 1988 Apr 1;251(1):243–249. doi: 10.1042/bj2510243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hecker M., Lückhoff A., Busse R. Modulation of endothelial autacoid release by protein kinase C: feedback inhibition or non-specific attenuation of receptor-dependent cell activation? J Cell Physiol. 1993 Sep;156(3):571–578. doi: 10.1002/jcp.1041560317. [DOI] [PubMed] [Google Scholar]
  12. Huwiler A., Fabbro D., Pfeilschifter J. Possible regulatory functions of protein kinase C-alpha and -epsilon isoenzymes in rat renal mesangial cells. Stimulation of prostaglandin synthesis and feedback inhibition of angiotensin II-stimulated phosphoinositide hydrolysis. Biochem J. 1991 Oct 15;279(Pt 2):441–445. doi: 10.1042/bj2790441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jelsema C. L., Axelrod J. Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3623–3627. doi: 10.1073/pnas.84.11.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kast R., Fürstenberger G., Marks F. Activation of cytosolic phospholipase A2 by transforming growth factor-alpha in HEL-30 keratinocytes. J Biol Chem. 1993 Aug 5;268(22):16795–16802. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  17. Lustig K. D., Sportiello M. G., Erb L., Weisman G. A. A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP. Biochem J. 1992 Jun 15;284(Pt 3):733–739. doi: 10.1042/bj2840733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marais R. M., Parker P. J. Purification and characterisation of bovine brain protein kinase C isotypes alpha, beta and gamma. Eur J Biochem. 1989 Jun 1;182(1):129–137. doi: 10.1111/j.1432-1033.1989.tb14809.x. [DOI] [PubMed] [Google Scholar]
  19. Martin T. W., Wysolmerski R. B. Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells. J Biol Chem. 1987 Sep 25;262(27):13086–13092. [PubMed] [Google Scholar]
  20. Martiny-Baron G., Kazanietz M. G., Mischak H., Blumberg P. M., Kochs G., Hug H., Marmé D., Schächtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976. J Biol Chem. 1993 May 5;268(13):9194–9197. [PubMed] [Google Scholar]
  21. Motte S., Pirotton S., Boeynaems J. M. Heterogeneity of ATP receptors in aortic endothelial cells. Involvement of P2y and P2u receptors in inositol phosphate response. Circ Res. 1993 Mar;72(3):504–510. doi: 10.1161/01.res.72.3.504. [DOI] [PubMed] [Google Scholar]
  22. Needham L., Cusack N. J., Pearson J. D., Gordon J. L. Characteristics of the P2 purinoceptor that mediates prostacyclin production by pig aortic endothelial cells. Eur J Pharmacol. 1987 Feb 10;134(2):199–209. doi: 10.1016/0014-2999(87)90166-x. [DOI] [PubMed] [Google Scholar]
  23. Needleman P., Minkes M. S., Douglas J. R., Jr Stimulation of prostaglandin biosynthesis by adenine nucleotides. Profile of prostaglandin release by perfused organs. Circ Res. 1974 Apr;34(4):455–460. doi: 10.1161/01.res.34.4.455. [DOI] [PubMed] [Google Scholar]
  24. Olivier A. R., Parker P. J. Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester. J Cell Physiol. 1992 Aug;152(2):240–244. doi: 10.1002/jcp.1041520204. [DOI] [PubMed] [Google Scholar]
  25. Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties. Proc Natl Acad Sci U S A. 1989 May;86(9):3099–3103. doi: 10.1073/pnas.86.9.3099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parker P. J., Marais R. M. Purification of protein kinase C isotypes from bovine brain. Methods Enzymol. 1991;200:234–241. doi: 10.1016/0076-6879(91)00143-k. [DOI] [PubMed] [Google Scholar]
  27. Pearson J. D., Slakey L. L., Gordon J. L. Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells. Biochem J. 1983 Jul 15;214(1):273–276. doi: 10.1042/bj2140273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pucéat M., Hilal-Dandan R., Strulovici B., Brunton L. L., Brown J. H. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. J Biol Chem. 1994 Jun 17;269(24):16938–16944. [PubMed] [Google Scholar]
  29. Purkiss J. R., Wilkinson G. F., Boarder M. R. Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells. Br J Pharmacol. 1994 Mar;111(3):723–728. doi: 10.1111/j.1476-5381.1994.tb14797.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sa G., Murugesan G., Jaye M., Ivashchenko Y., Fox P. L. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J Biol Chem. 1995 Feb 3;270(5):2360–2366. doi: 10.1074/jbc.270.5.2360. [DOI] [PubMed] [Google Scholar]
  31. Schaap D., Parker P. J. Expression, purification, and characterization of protein kinase C-epsilon. J Biol Chem. 1990 May 5;265(13):7301–7307. [PubMed] [Google Scholar]
  32. Silk S. T., Clejan S., Witkom K. Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes. J Biol Chem. 1989 Dec 25;264(36):21466–21469. [PubMed] [Google Scholar]
  33. Ways D. K., Cook P. P., Webster C., Parker P. J. Effect of phorbol esters on protein kinase C-zeta. J Biol Chem. 1992 Mar 5;267(7):4799–4805. [PubMed] [Google Scholar]
  34. Wijkander J., Sundler R. Regulation of arachidonate-mobilizing phospholipase A2 by phosphorylation via protein kinase C in macrophages. FEBS Lett. 1992 Oct 26;311(3):299–301. doi: 10.1016/0014-5793(92)81124-5. [DOI] [PubMed] [Google Scholar]
  35. Wilkinson G. F., McKechnie K., Dainty I. A., Boarder M. R. P2Y purinoceptor and nucleotide receptor-induced relaxation of precontracted bovine aortic collateral artery rings: differential sensitivity to suramin and indomethacin. J Pharmacol Exp Ther. 1994 Feb;268(2):881–887. [PubMed] [Google Scholar]
  36. Wilkinson G. F., Purkiss J. R., Boarder M. R. Differential heterologous and homologous desensitization of two receptors for ATP (P2y purinoceptors and nucleotide receptors) coexisting on endothelial cells. Mol Pharmacol. 1994 Apr;45(4):731–736. [PubMed] [Google Scholar]
  37. Wilkinson G. F., Purkiss J. R., Boarder M. R. The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C. Br J Pharmacol. 1993 Mar;108(3):689–693. doi: 10.1111/j.1476-5381.1993.tb12862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zavoico G. B., Hrbolich J. K., Gimbrone M. A., Jr, Schafer A. I. Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester. J Cell Physiol. 1990 Jun;143(3):596–605. doi: 10.1002/jcp.1041430326. [DOI] [PubMed] [Google Scholar]

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