Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1995 Jul;115(5):822–827. doi: 10.1111/j.1476-5381.1995.tb15006.x

Gi proteins and the response to 5-hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF.

N S Day 1, T Ge 1, J Codina 1, L Birnbaumer 1, P M Vanhoutte 1, C M Boulanger 1
PMCID: PMC1908515  PMID: 8548182

Abstract

1. The receptor-mediated release of endothelium-derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5-hydroxytryptamine (5-HT), which involves activation of pertussis toxin-sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultured endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5-HT may be associated with the loss of functional pertussis toxin-sensitive Gi proteins. 2. Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-HT with Kd of 7.2 +/- 3.5 nM and maximal binding (Bmax) of 121.4 +/- 51.3 fmol mg-1 protein. Binding of [3H]-5-HT was displaced by methiothepin (5-HT1 and 5-HT2 antagonist; Ki = 6.2 +/- 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3. Gi alpha 1 protein was expressed in cultured but not in native endothelial cells. Gi alpha 2 and Gi alpha 3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as detected by Northern and Western blot analysis. 4. In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Gi alpha 2 and the combination of Gi alpha 3-Gi alpha 1 proteins, respectively, were ADP-ribosylated by pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
822

Images in this article

825Figure 3
on p.825
825Figure 4
on p.825

Selected References

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

  1. Asano T., Semba R., Kamiya N., Ogasawara N., Kato K. Go, a GTP-binding protein: immunochemical and immunohistochemical localization in the rat. J Neurochem. 1988 Apr;50(4):1164–1169. doi: 10.1111/j.1471-4159.1988.tb10588.x. [DOI] [PubMed] [Google Scholar]
  2. Boulanger C., Hendrickson H., Lorenz R. R., Vanhoutte P. M. Release of different relaxing factors by cultured porcine endothelial cells. Circ Res. 1989 Jun;64(6):1070–1078. doi: 10.1161/01.res.64.6.1070. [DOI] [PubMed] [Google Scholar]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Codina J., Grenet D., Chang K. J., Birnbaumer L. Urea gradient/SDS-PAGE; a useful tool in the investigation of signal transducing G proteins. J Recept Res. 1991;11(1-4):587–601. doi: 10.3109/10799899109066429. [DOI] [PubMed] [Google Scholar]
  5. Cohen R. A., Shepherd J. T., Vanhoutte P. M. Inhibitory role of the endothelium in the response of isolated coronary arteries to platelets. Science. 1983 Jul 15;221(4607):273–274. doi: 10.1126/science.6574604. [DOI] [PubMed] [Google Scholar]
  6. Day N. S., Berti-Mattera L. N., Eichberg J. Muscarinic cholinergic receptor-mediated phosphoinositide metabolism in peripheral nerve. J Neurochem. 1991 Jun;56(6):1905–1913. doi: 10.1111/j.1471-4159.1991.tb03447.x. [DOI] [PubMed] [Google Scholar]
  7. Day N. S., Koutz C. A., Anderson R. E. Inositol-1,4,5-trisphosphate receptors in the vertebrate retina. Curr Eye Res. 1993 Nov;12(11):981–992. doi: 10.3109/02713689309029224. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Houston D. S., Vanhoutte P. M. Comparison of serotonergic receptor subtypes on the smooth muscle and endothelium of the canine coronary artery. J Pharmacol Exp Ther. 1988 Jan;244(1):1–10. [PubMed] [Google Scholar]
  10. Itoh H., Toyama R., Kozasa T., Tsukamoto T., Matsuoka M., Kaziro Y. Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs. J Biol Chem. 1988 May 15;263(14):6656–6664. [PubMed] [Google Scholar]
  11. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  12. Kugiyama K., Kerns S. A., Morrisett J. D., Roberts R., Henry P. D. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990 Mar 8;344(6262):160–162. doi: 10.1038/344160a0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Lochrie M. A., Simon M. I. G protein multiplicity in eukaryotic signal transduction systems. Biochemistry. 1988 Jul 12;27(14):4957–4965. doi: 10.1021/bi00414a001. [DOI] [PubMed] [Google Scholar]
  15. Moncada S., Palmer R. M., Higgs E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991 Jun;43(2):109–142. [PubMed] [Google Scholar]
  16. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  17. Ribeiro-Neto F., Mattera R., Grenet D., Sekura R. D., Birnbaumer L., Field J. B. Adenosine diphosphate ribosylation of G proteins by pertussis and cholera toxin in isolated membranes. Different requirements for and effects of guanine nucleotides and Mg2+. Mol Endocrinol. 1987 Jul;1(7):472–481. doi: 10.1210/mend-1-7-472. [DOI] [PubMed] [Google Scholar]
  18. Scherer N. M., Toro M. J., Entman M. L., Birnbaumer L. G-protein distribution in canine cardiac sarcoplasmic reticulum and sarcolemma: comparison to rabbit skeletal muscle membranes and to brain and erythrocyte G-proteins. Arch Biochem Biophys. 1987 Dec;259(2):431–440. doi: 10.1016/0003-9861(87)90509-1. [DOI] [PubMed] [Google Scholar]
  19. Schoeffter P., Hoyer D. 5-Hydroxytryptamine (5-HT)-induced endothelium-dependent relaxation of pig coronary arteries is mediated by 5-HT receptors similar to the 5-HT1D receptor subtype. J Pharmacol Exp Ther. 1990 Jan;252(1):387–395. [PubMed] [Google Scholar]
  20. Shibano T., Codina J., Birnbaumer L., Vanhoutte P. M. Guanosine 5'-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries. Biochem Biophys Res Commun. 1992 Nov 30;189(1):324–329. doi: 10.1016/0006-291x(92)91561-4. [DOI] [PubMed] [Google Scholar]
  21. Shibano T., Codina J., Birnbaumer L., Vanhoutte P. M. Pertussis toxin-sensitive G proteins in regenerated endothelial cells of porcine coronary artery. Am J Physiol. 1994 Sep;267(3 Pt 2):H979–H981. doi: 10.1152/ajpheart.1994.267.3.H979. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Ui M., Okajima F., Itoh H. ADP-ribosylation of the inhibitory guanine nucleotide regulatory protein (Ni) as a possible mechanism underlying development of beta-adrenergic responses during primary culture of rat hepatocytes. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1985;19:195–205. [PubMed] [Google Scholar]
  25. Vane J. R. The Croonian Lecture, 1993. The endothelium: maestro of the blood circulation. Philos Trans R Soc Lond B Biol Sci. 1994 Jan 29;343(1304):225–246. doi: 10.1098/rstb.1994.0023. [DOI] [PubMed] [Google Scholar]
  26. Vanhoutte P. M., Lüscher T. F., Gräser T. Endothelium-dependent contractions. Blood Vessels. 1991;28(1-3):74–83. doi: 10.1159/000158846. [DOI] [PubMed] [Google Scholar]
  27. Yokoyama M., Hirata K., Miyake R., Akita H., Ishikawa Y., Fukuzaki H. Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein. Biochem Biophys Res Commun. 1990 Apr 16;168(1):301–308. doi: 10.1016/0006-291x(90)91708-z. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES