Insulin-stimulated NAD(P)H oxidase activity increases migration of cultured vascular smooth muscle cells.
BACKGROUND
We reported that insulin stimulates NAD(P)H oxidase activity but not migration of cultured rat vascular smooth muscle cells (VSMCs). Because angiotensin II (Ang II) increases NAD(P)H oxidase activity in these cells, we wished to determine whether insulin stimulates migration of Ang II-treated VSMCs by synergistically stimulating enzyme activity.


METHODS
Cultured rat VSMC superoxide anion (O2-) production, cyclic GMP production, and migration were measured by lucigenin luminescence, immunoassay, and wound closure rate, respectively. Nitric oxide (NO) scavenging was measured by inhibition of NO-induced fluorescence of 4-5-diaminofluorescin.


RESULTS
Insulin (1 nmol/L) did not affect and Ang II (100 nmol/L) stimulated VSMC migration by 65% (P < .05), but together stimulated it by 150% (P < .05 versus Ang II) by a mechanism inhibited by the NAD(P)H oxidase inhibitors, diphenyleneiodonium (DPI) or gp91ds-tat. Insulin and Ang II stimulated O2- production by 34% and 35%, respectively (both P < .05), but together synergistically stimulated it by 143% (P < .05 versus insulin or Ang II) in a DPI or gp91ds-tat-sensitive manner. Neither insulin nor Ang II measurably affected NO scavenging, but together reduced NO availability by 46% in a DPI-sensitive manner (P < .05) and significantly inhibited NO-stimulated cyclic GMP production.


CONCLUSIONS
Insulin synergestically stimulates NAD(P)H oxidase activity in Ang II-treated cultured rat VSMCs causing increased migration.
Yang
Ming
M
Division of Renal Diseases and Hypertension, Department of Internal Medicine, University of Texas Medical School, Houston, Texas 77030, USA.
Foster
Estrella
E
Kahn
Andrew M
AM
D000634:Aminoquinolines / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009861:Onium Compounds / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D000634:Aminoquinolines; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D007328:Insulin; D020030:Nitric Oxide Donors; D009861:Onium Compounds; C110881:S-nitro-N-acetylpenicillamine; D014662:Vasoconstrictor Agents; C473031:gp91ds-tat protein, chimeric; D013481:Superoxides; D000804:Angiotensin II; C007517:diphenyleneiodonium; C041715:6-anilino-5,8-quinolinedione; D019255:NADPH Oxidases; D006162:Guanylate Cyclase; D010396:Penicillamine; D006152:Cyclic GMP