A role for heterotrimeric GTP-binding proteins and ERK1/2 in insulin-mediated, nitric-oxide-dependent, cyclic GMP production in human umbilical vein endothelial cells.
OBJECTIVE
Insulin is known to stimulate endothelial nitric oxide synthesis, although much remains unknown about the intracellular mechanisms involved. This study aims to examine, in human endothelial cells, the specific contribution of heterotrimeric Gi proteins and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in insulin signalling upstream of nitric-oxide-dependent cyclic GMP production.


METHODS
Human umbilical vein endothelial cells were treated with 1 nmol/l insulin in the presence or absence of inhibitors of tyrosine kinases (erbstatin), Gi proteins (pertussis toxin) or ERK1/2 (PD098059 or U0126), and nitric oxide production was examined by quantification of intracellular cyclic GMP. Activation/phosphorylation of ERK1/2 by insulin was examined by immunoblotting with specific antibodies, and direct association of the insulin receptor with Gi proteins was examined by immunoprecipitation.


RESULTS
Treatment of cells with a physiological concentration of insulin (1 nmol/l) for 5 min increased nitric-oxide-dependent cyclic GMP accumulation by 3.3-fold, and this was significantly inhibited by erbstatin. Insulin-stimulated cyclic GMP production was significantly reduced by pertussis toxin and by the inhibitors of ERK1/2, PD098059 and U0126. Immunoblotting indicated that insulin stimulated the phosphorylation of ERK1/2 after 5 min and 1 h, and that this was completely abolished by pertussis toxin, but insensitive to the nitric oxide synthase inhibitor L-NAME. No direct interaction of the insulin receptor beta with Gialpha2 could be demonstrated by immunoprecipitation.


CONCLUSIONS
This study demonstrates, for the first time, that nitric oxide production induced by physiologically relevant concentrations of insulin, is mediated by the post-receptor activation of a pertussis-sensitive GTP-binding protein and subsequent downstream activation of the ERK1/2 cascade.
Konopatskaya
O
O
Institute for Biomedical and Clinical Science, Peninsula Medical School, Exeter, EX1 2LU, UK. jackie.whatmore@pms.ac.uk
Shore
A C
AC
Tooke
J E
JE
Whatmore
J L
JL
D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006131:Growth Inhibitors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D006133:Growth Substances; D006873:Hydroquinones; D007328:Insulin; D009569:Nitric Oxide; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020962:Heterotrimeric GTP-Binding Proteins; D006152:Cyclic GMP; C048212:erbstatin
Circ Res. 2003 Dec 12;93(12 ):1233-40
14615286
J Cell Physiol. 1990 Sep;144(3):538-45
2118147
Nature. 1970 Aug 15;227(5259):680-5
5432063
J Biol Chem. 1990 Oct 5;265(28):16873-9
1698770
J Biol Chem. 2000 Mar 10;275(10):7167-75
10702285
FEBS Lett. 1989 Dec 18;259(1):67-70
2557246
J Clin Endocrinol Metab. 1978 Sep;47(3):699-702
400730
Diabetes. 1990 Sep;39(9):1009-16
2166696
Biochim Biophys Acta. 1993 Nov 7;1179(2):213-23
8218364
FASEB J. 2001 Nov;15(13):2508-14
11689477
Exp Clin Endocrinol Diabetes. 1999;107(2):126-32
10320053
J Biol Chem. 2003 Dec 12;278(50):49929-35
14523027
Cell Signal. 2001 Nov;13(11):777-85
11583913
J Clin Invest. 1973 Nov;52(11):2745-56
4355998
Eur J Biochem. 1995 Feb 1;227(3):597-611
7867619
J Clin Invest. 1994 Sep;94(3):1172-9
8083357
Biochem Med Metab Biol. 1990 Dec;44(3):292-3
2126944
J Clin Invest. 1986 Jul;78(1):1-5
2873150
J Clin Invest. 1994 Dec;94(6):2511-5
7989610
EMBO J. 1991 Apr;10(4):885-92
1849075
J Biol Chem. 1998 Feb 13;273(7):4220-6
9461619
Diabetes. 2000 May;49(5):684-7
10905473
Biochem J. 1998 Aug 1;333 ( Pt 3):471-90
9677303
Hypertension. 1997 Nov;30(5):1128-34
9369266
Nature. 1996 Feb 29;379(6568):840-4
8587610
J Immunol. 1998 Nov 1;161(9):4852-8
9794418
Circulation. 2000 Apr 4;101(13):1539-45
10747347
Biochem J. 2000 Oct 1;351(Pt 1):95-105
10998351
Diabetes. 1992 Sep;41(9):1076-83
1499861
Biochem Biophys Res Commun. 1992 Jul 31;186(2):1151-7
1497649
Nature. 1987 May 21-27;327(6119):229-32
3106832
Am J Physiol Endocrinol Metab. 2004 Jun;286(6):E896-901
14722023
Curr Top Microbiol Immunol. 1992;175:69-96
1628499
J Clin Invest. 1996 Aug 15;98(4):894-8
8770859
Biochem J. 1986 Apr 1;235(1):145-9
3017298
Cell. 1995 Jan 27;80(2):249-57
7834744
J Physiol. 1996 Feb 1;490 ( Pt 3):775-81
8683475
J Immunol Methods. 1983 Dec 16;65(1-2):55-63
6606682
J Biol Chem. 1987 Sep 5;262(25):12120-6
2442157
J Biol Chem. 1997 Apr 11;272(15):10135-43
9092559
Pharmacol Rev. 1991 Jun;43(2):109-42
1852778
J Biol Chem. 1995 Nov 17;270(46):27489-94
7499206
J Biol Chem. 2001 Aug 10;276(32):30392-8
11402048
Diabet Med. 2003 Oct;20(10):838-45
14510866
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7686-9
7644477
Mol Cell Biol. 1999 Oct;19(10):6765-74
10490615
FEBS Lett. 1994 Feb 28;340(1-2):124-8
8119395
J Biol Chem. 2001 Apr 13;276(15):12420-6
11278407
FASEB J. 2002 Oct;16(12 ):1584-94
12374781
Cell Signal. 1999 Aug;11(8):563-74
10433517
Anal Biochem. 1976 May 7;72:248-54
942051
J Biol Chem. 2002 Jan 18;277(3):1794-9
11707433
Am J Physiol Cell Physiol. 2001 Sep;281(3):C886-98
11502566
Nitric Oxide. 2003 Jun;8(4):253-61
12895435
Annu Rev Nutr. 1997;17:487-99
9240937