Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Oct 1;287(Pt 1):31–36. doi: 10.1042/bj2870031

Stimulation of phosphatidate synthesis in endothelial cells in response to P2-receptor activation. Evidence for phospholipase C and phospholipase D involvement, phosphatidate and diacylglycerol interconversion and the role of protein kinase C.

J R Purkiss 1, M R Boarder 1
PMCID: PMC1133119  PMID: 1417783

Abstract

To investigate the stimulation of phosphatidic acid formation in bovine aortic endothelial cells by P2-purinergic agonists, we labelled AG4762 cells with [32P]P1 and stimulated in the presence of butanol. Under these conditions phospholipase D generated [32P]phosphatidylbutanol, whereas the [32P]phosphatidic acid from phospholipase C and diacylglycerol kinase was unchanged. The action of various purinergic agonists on both [32P]phosphatidic acid and [32P]phosphatidylbutanol was consistent with the presence of a P2Y receptor. The stimulation of phospholipase D was dependent on extracellular Ca2+ and was mostly transient (completed within 3 min), whereas the initial stimulation of phospholipase C was independent of extracellular Ca2+, followed by a Ca(2+)-dependent phase. The agonist stimulation of phospholipase D was dependent on protein kinase C, as judged by its sensitivity to the relatively selective protein kinase C inhibitor Ro 31-8220. These results show that purinergic-receptor-mediated stimulation of phosphatidic acid has three phases: an initial Ca(2+)-independent stimulation of phospholipase C, an early but transient Ca(2+)- and protein kinase C-dependent stimulation of phospholipase D, and a sustained Ca(2+)-dependent stimulation of phospholipase C. Using propranolol to inhibit phosphatidate phosphohydrolase, we provide evidence that phosphatidic acid derived from purinergic-receptor-mediated stimulation of the phospholipase C/diacylglycerol kinase route can itself be converted back into diacylglycerol.

Full text

PDF
31

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. Billah M. M., Anthes J. C. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J. 1990 Jul 15;269(2):281–291. doi: 10.1042/bj2690281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeynaems J. M., Pearson J. D. P2 purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms. Trends Pharmacol Sci. 1990 Jan;11(1):34–37. doi: 10.1016/0165-6147(90)90039-b. [DOI] [PubMed] [Google Scholar]
  4. Bonser R. W., Thompson N. T., Randall R. W., Garland L. G. Phospholipase D activation is functionally linked to superoxide generation in the human neutrophil. Biochem J. 1989 Dec 1;264(2):617–620. doi: 10.1042/bj2640617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burnstock G., Kennedy C. Is there a basis for distinguishing two types of P2-purinoceptor? Gen Pharmacol. 1985;16(5):433–440. doi: 10.1016/0306-3623(85)90001-1. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Cook S. J., Briscoe C. P., Wakelam M. J. The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells. Biochem J. 1991 Dec 1;280(Pt 2):431–438. doi: 10.1042/bj2800431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Eichberg J., Hauser G. Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1460–1467. doi: 10.1016/0006-291x(74)90362-3. [DOI] [PubMed] [Google Scholar]
  11. Exton J. H. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990 Jan 5;265(1):1–4. [PubMed] [Google Scholar]
  12. Hallam T. J., Pearson J. D. Exogenous ATP raises cytoplasmic free calcium in fura-2 loaded piglet aortic endothelial cells. FEBS Lett. 1986 Oct 20;207(1):95–99. doi: 10.1016/0014-5793(86)80019-9. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Houston D. A., Burnstock G., Vanhoutte P. M. Different P2-purinergic receptor subtypes of endothelium and smooth muscle in canine blood vessels. J Pharmacol Exp Ther. 1987 May;241(2):501–506. [PubMed] [Google Scholar]
  15. Huang C. F., Cabot M. C. Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway. J Biol Chem. 1990 Sep 5;265(25):14858–14863. [PubMed] [Google Scholar]
  16. Magnússon M. K., Halldórsson H., Kjeld M., Thorgeirsson G. Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4-. Biochem J. 1989 Dec 15;264(3):703–711. doi: 10.1042/bj2640703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Martin T. W. Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells. Biochim Biophys Acta. 1988 Oct 14;962(3):282–296. doi: 10.1016/0005-2760(88)90258-5. [DOI] [PubMed] [Google Scholar]
  18. Martin T. W., Michaelis K. P2-purinergic agonists stimulate phosphodiesteratic cleavage of phosphatidylcholine in endothelial cells. Evidence for activation of phospholipase D. J Biol Chem. 1989 May 25;264(15):8847–8856. [PubMed] [Google Scholar]
  19. Martin W., Cusack N. J., Carleton J. S., Gordon J. L. Specificity of P2-purinoceptor that mediates endothelium-dependent relaxation of the pig aorta. Eur J Pharmacol. 1985 Feb 5;108(3):295–299. doi: 10.1016/0014-2999(85)90452-2. [DOI] [PubMed] [Google Scholar]
  20. Martinson E. A., Trilivas I., Brown J. H. Rapid protein kinase C-dependent activation of phospholipase D leads to delayed 1,2-diglyceride accumulation. J Biol Chem. 1990 Dec 25;265(36):22282–22287. [PubMed] [Google Scholar]
  21. Mo M., Eskin S. G., Schilling W. P. Flow-induced changes in Ca2+ signaling of vascular endothelial cells: effect of shear stress and ATP. Am J Physiol. 1991 May;260(5 Pt 2):H1698–H1707. doi: 10.1152/ajpheart.1991.260.5.H1698. [DOI] [PubMed] [Google Scholar]
  22. Mullmann T. J., Siegel M. I., Egan R. W., Billah M. M. Phorbol-12-myristate-13-acetate activation of phospholipase D in human neutrophils leads to the production of phosphatides and diglycerides. Biochem Biophys Res Commun. 1990 Aug 16;170(3):1197–1202. doi: 10.1016/0006-291x(90)90520-w. [DOI] [PubMed] [Google Scholar]
  23. Murrin R. J., Boarder M. R. Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP. Mol Pharmacol. 1992 Mar;41(3):561–568. [PubMed] [Google Scholar]
  24. Owen P. J., Boarder M. R. Influence of bradykinin on diacylglycerol and phosphatidic acid accumulation in cultured bovine adrenal chromaffin cells. J Neurochem. 1991 Sep;57(3):760–768. doi: 10.1111/j.1471-4159.1991.tb08217.x. [DOI] [PubMed] [Google Scholar]
  25. Pirotton S., Raspe E., Demolle D., Erneux C., Boeynaems J. M. Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells. J Biol Chem. 1987 Dec 25;262(36):17461–17466. [PubMed] [Google Scholar]
  26. Pirotton S., Robaye B., Lagneau C., Boeynaems J. M. Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells. J Cell Physiol. 1990 Mar;142(3):449–457. doi: 10.1002/jcp.1041420303. [DOI] [PubMed] [Google Scholar]
  27. Purkiss J., Murrin R. A., Owen P. J., Boarder M. R. Lack of phospholipase D activity in chromaffin cells: bradykinin-stimulated phosphatidic acid formation involves phospholipase C in chromaffin cells but phospholipase D in PC12 cells. J Neurochem. 1991 Sep;57(3):1084–1087. doi: 10.1111/j.1471-4159.1991.tb08262.x. [DOI] [PubMed] [Google Scholar]
  28. Sandmann J., Wurtman R. J. Stimulation of phospholipase D activity in human neuroblastoma (LA-N-2) cells by activation of muscarinic acetylcholine receptors or by phorbol esters: relationship to phosphoinositide turnover. J Neurochem. 1991 Apr;56(4):1312–1319. doi: 10.1111/j.1471-4159.1991.tb11427.x. [DOI] [PubMed] [Google Scholar]
  29. Weinheimer G., Wagner B., Osswald H. Interference of phorbolesters with endothelium-dependent vascular smooth muscle relaxation. Eur J Pharmacol. 1986 Nov 4;130(3):319–322. doi: 10.1016/0014-2999(86)90285-2. [DOI] [PubMed] [Google Scholar]
  30. Yamada K., Kanaho Y., Miura K., Nozawa Y. Antigen-induced phospholipase D activation in rat mast cells is independent of protein kinase C. Biochem Biophys Res Commun. 1991 Feb 28;175(1):159–164. doi: 10.1016/s0006-291x(05)81214-8. [DOI] [PubMed] [Google Scholar]
  31. Yang S. F., Freer S., Benson A. A. Transphosphatidylation by phospholipase D. J Biol Chem. 1967 Feb 10;242(3):477–484. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES