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. 1993 Nov;92(5):2168–2172. doi: 10.1172/JCI116818

The G proteins of the G alpha i and G alpha q family couple the bradykinin receptor to the release of endothelium-derived relaxing factor.

J K Liao 1, C J Homcy 1
PMCID: PMC288395  PMID: 8227332

Abstract

Bradykinin stimulates diverse functions in endothelial cells including the release of endothelium-derived relaxing factor (EDRF). Little is known, however, regarding the identity of the G protein(s) involved. Here we demonstrate that G proteins of the G alpha i and G alpha q family are coupled to the bradykinin receptor (BKR) in bovine aortic endothelial cells by using specific antisera directed against the COOH-terminal region of G alpha i2 (P4), G alpha i3 (EC), and G alpha q (QL). These antisera are specific since their effects are blocked by the decapeptides from which they were derived. The degree of receptor-G protein coupling was assessed by the formation of high affinity agonist binding sites (HABS) and GTP hydrolysis. In a concentration-dependent manner, the QL antisera reduced HABS and GTPase activity by 65 and 60%, respectively, and effectively abolished them in membranes from pertussis toxin-treated cells. The combination of P4 and EC antisera produced a loss of HABS (41%) and GTPase activity (40%) comparable to the effects of pertussis toxin. These findings indicate that G alpha i and G alpha q proteins mediate the cellular responses to bradykinin in bovine aortic endothelial cells and support the observation that bradykinin-stimulated EDRF release is relatively insensitive to pertussis toxin.

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Selected References

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

  1. Amatruda T. T., 3rd, Steele D. A., Slepak V. Z., Simon M. I. G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5587–5591. doi: 10.1073/pnas.88.13.5587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aragay A. M., Katz A., Simon M. I. The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells. J Biol Chem. 1992 Dec 15;267(35):24983–24988. [PubMed] [Google Scholar]
  3. Camps M., Carozzi A., Schnabel P., Scheer A., Parker P. J., Gierschik P. Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature. 1992 Dec 17;360(6405):684–686. doi: 10.1038/360684a0. [DOI] [PubMed] [Google Scholar]
  4. Carlson K. E., Brass L. F., Manning D. R. Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. J Biol Chem. 1989 Aug 5;264(22):13298–13305. [PubMed] [Google Scholar]
  5. Cerione R. A., Staniszewski C., Benovic J. L., Lefkowitz R. J., Caron M. G., Gierschik P., Somers R., Spiegel A. M., Codina J., Birnbaumer L. Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J Biol Chem. 1985 Feb 10;260(3):1493–1500. [PubMed] [Google Scholar]
  6. Flavahan N. A. Atherosclerosis or lipoprotein-induced endothelial dysfunction. Potential mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation. 1992 May;85(5):1927–1938. doi: 10.1161/01.cir.85.5.1927. [DOI] [PubMed] [Google Scholar]
  7. Flavahan N. A., Shimokawa H., Vanhoutte P. M. Pertussis toxin inhibits endothelium-dependent relaxations to certain agonists in porcine coronary arteries. J Physiol. 1989 Jan;408:549–560. doi: 10.1113/jphysiol.1989.sp017475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldstein R. H., Wall M. Activation of protein formation and cell division by bradykinin and des-Arg9-bradykinin. J Biol Chem. 1984 Jul 25;259(14):9263–9268. [PubMed] [Google Scholar]
  9. Gutowski S., Smrcka A., Nowak L., Wu D. G., Simon M., Sternweis P. C. Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones. J Biol Chem. 1991 Oct 25;266(30):20519–20524. [PubMed] [Google Scholar]
  10. Huff R. M., Neer E. J. Subunit interactions of native and ADP-ribosylated alpha 39 and alpha 41, two guanine nucleotide-binding proteins from bovine cerebral cortex. J Biol Chem. 1986 Jan 25;261(3):1105–1110. [PubMed] [Google Scholar]
  11. Ignarro L. J., Byrns R. E., Buga G. M., Wood K. S. Mechanisms of endothelium-dependent vascular smooth muscle relaxation elicited by bradykinin and VIP. Am J Physiol. 1987 Nov;253(5 Pt 2):H1074–H1082. doi: 10.1152/ajpheart.1987.253.5.H1074. [DOI] [PubMed] [Google Scholar]
  12. Im M. J., Gray C., Rim A. J. Characterization of a phospholipase C activity regulated by the purified Gh in reconstitution systems. J Biol Chem. 1992 May 5;267(13):8887–8894. [PubMed] [Google Scholar]
  13. Jacobs M., Plane F., Bruckdorfer K. R. Native and oxidized low-density lipoproteins have different inhibitory effects on endothelium-derived relaxing factor in the rabbit aorta. Br J Pharmacol. 1990 May;100(1):21–26. doi: 10.1111/j.1476-5381.1990.tb12045.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kalan J. M., Roberts W. C. Significance of cardiac weight in patients having coronary artery bypass grafting for angina pectoris. Am J Cardiol. 1988 Jul 1;62(1):36–40. doi: 10.1016/0002-9149(88)91361-6. [DOI] [PubMed] [Google Scholar]
  15. Kanaho Y., Takahashi K., Tomita U., Iiri T., Katada T., Ui M., Nozawa Y. A protein kinase C inhibitor, staurosporine, activates phospholipase D via a pertussis toxin-sensitive GTP-binding protein in rabbit peritoneal neutrophils. J Biol Chem. 1992 Nov 25;267(33):23554–23559. [PubMed] [Google Scholar]
  16. Katada T., Oinuma M., Ui M. Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities. J Biol Chem. 1986 Jun 25;261(18):8182–8191. [PubMed] [Google Scholar]
  17. Katz A., Wu D., Simon M. I. Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. Nature. 1992 Dec 17;360(6405):686–689. doi: 10.1038/360686a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Liao J. K., Homcy C. J. Specific receptor-guanine nucleotide binding protein interaction mediates the release of endothelium-derived relaxing factor. Circ Res. 1992 May;70(5):1018–1026. doi: 10.1161/01.res.70.5.1018. [DOI] [PubMed] [Google Scholar]
  20. Liao J. K., Homey C. J. The release of endothelium-derived relaxing factor via alpha 2-adrenergic receptor activation is specifically mediated by Gi alpha 2. J Biol Chem. 1993 Sep 15;268(26):19528–19533. [PubMed] [Google Scholar]
  21. Logothetis D. E., Kurachi Y., Galper J., Neer E. J., Clapham D. E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 1987 Jan 22;325(6102):321–326. doi: 10.1038/325321a0. [DOI] [PubMed] [Google Scholar]
  22. McKenzie F. R., Kelly E. C., Unson C. G., Spiegel A. M., Milligan G. Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. Biochem J. 1988 Feb 1;249(3):653–659. doi: 10.1042/bj2490653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McKenzie F. R., Milligan G. Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2. Biochem J. 1990 Apr 15;267(2):391–398. doi: 10.1042/bj2670391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murray M. A., Heistad D. D., Mayhan W. G. Role of protein kinase C in bradykinin-induced increases in microvascular permeability. Circ Res. 1991 May;68(5):1340–1348. doi: 10.1161/01.res.68.5.1340. [DOI] [PubMed] [Google Scholar]
  25. Pang I. H., Sternweis P. C. Purification of unique alpha subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized beta gamma subunits. J Biol Chem. 1990 Oct 25;265(30):18707–18712. [PubMed] [Google Scholar]
  26. Shenker A., Goldsmith P., Unson C. G., Spiegel A. M. The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem. 1991 May 15;266(14):9309–9313. [PubMed] [Google Scholar]
  27. Shimokawa H., Flavahan N. A., Vanhoutte P. M. Loss of endothelial pertussis toxin-sensitive G protein function in atherosclerotic porcine coronary arteries. Circulation. 1991 Feb;83(2):652–660. doi: 10.1161/01.cir.83.2.652. [DOI] [PubMed] [Google Scholar]
  28. Shimokawa H., Flavahan N. A., Vanhoutte P. M. Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries. Possible dysfunction of a pertussis toxin-sensitive G protein. Circ Res. 1989 Sep;65(3):740–753. doi: 10.1161/01.res.65.3.740. [DOI] [PubMed] [Google Scholar]
  29. Simon B. C., Cunningham L. D., Cohen R. A. Oxidized low density lipoproteins cause contraction and inhibit endothelium-dependent relaxation in the pig coronary artery. J Clin Invest. 1990 Jul;86(1):75–79. doi: 10.1172/JCI114718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
  32. Tanner F. C., Noll G., Boulanger C. M., Lüscher T. F. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries. Role of scavenger receptor and endothelium-derived nitric oxide. Circulation. 1991 Jun;83(6):2012–2020. doi: 10.1161/01.cir.83.6.2012. [DOI] [PubMed] [Google Scholar]
  33. Taylor S. J., Chae H. Z., Rhee S. G., Exton J. H. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature. 1991 Apr 11;350(6318):516–518. doi: 10.1038/350516a0. [DOI] [PubMed] [Google Scholar]
  34. Tkachuk V. A., Voyno-Yasenetskaya T. A. Two types of G proteins involved in regulation of phosphoinositide turnover in pulmonary endothelial cells. Am J Physiol. 1991 Oct;261(4 Suppl):118–122. doi: 10.1152/ajpheart.1991.261.4.118. [DOI] [PubMed] [Google Scholar]
  35. Tropea M. M., Gummelt D., Herzig M. S., Leeb-Lundberg L. M. B1 and B2 kinin receptors on cultured rabbit superior mesenteric artery smooth muscle cells: receptor-specific stimulation of inositol phosphate formation and arachidonic acid release by des-Arg9-bradykinin and bradykinin. J Pharmacol Exp Ther. 1993 Feb;264(2):930–937. [PubMed] [Google Scholar]
  36. Voyno-Yasenetskaya T. A., Tkachuk V. A., Cheknyova E. G., Panchenko M. P., Grigorian G. Y., Vavrek R. J., Stewart J. M., Ryan U. S. Guanine nucleotide-dependent, pertussis toxin-insensitive regulation of phosphoinositide turnover by bradykinin in bovine pulmonary artery endothelial cells. FASEB J. 1989 Jan;3(1):44–51. doi: 10.1096/fasebj.3.1.2535990. [DOI] [PubMed] [Google Scholar]
  37. Wange R. L., Smrcka A. V., Sternweis P. C., Exton J. H. Photoaffinity labeling of two rat liver plasma membrane proteins with [32P]gamma-azidoanilido GTP in response to vasopressin. Immunologic identification as alpha subunits of the Gq class of G proteins. J Biol Chem. 1991 Jun 25;266(18):11409–11412. [PubMed] [Google Scholar]
  38. Wilkie T. M., Scherle P. A., Strathmann M. P., Slepak V. Z., Simon M. I. Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10049–10053. doi: 10.1073/pnas.88.22.10049. [DOI] [PMC free article] [PubMed] [Google Scholar]

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