Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats

Abstract

Experiments were performed to investigate whether nitric-oxide synthase (NOS) activity can be detected in vascular smooth muscle (VSM) from 12- to 14-week streptozotocin (STZ)-diabetic rats. Concentration-response curves to norepinephrine (NE) of superior mesenteric arteries from diabetic and age- and gender-matched control rats were obtained in the presence of dexamethasone (0.1 microM) to prevent in vitro induction of iNOS. Incubation of endothelium-intact arteries from diabetic rats with the nonselective NOS inhibitor, N(5)-(1-iminoethyl)L-ornithine (L-NIO) (300 microM), increased the NE sensitivity (expressed as the pD2 or -log EC50) from 6.58 +/- 0.05 to 8.39 +/- 0.12 (mean +/- S.E.M., n = 8). L-NIO produced a significantly smaller increase in the NE pD2 value in endothelium-intact arteries from control rats (from 6.51 +/- 0.03 to 7.08 +/- 0.03, p < 0.05). On endothelium removal, L-NIO still increased the NE pD2 value in diabetic arteries, from 7.48 +/- 0.03 to 8.38 +/- 0.15 (p < 0.05), but had no effect in control arteries. The selective iNOS inhibitor S-ethylisothiourea (EIT), but not the selective nNOS inhibitor 7-nitroindazole (7-NINA), produced an increase in the NE pD2 value in endothelium-denuded mesenteric arteries from diabetic but not control rats. Immunohistochemical staining indicated the presence of iNOS (but not eNOS or nNOS) in the medial and adventitial layers of mesenteric arteries from diabetic but not control rats. Quantitative measurement of cytosolic NOS activity indicated no significant calcium-dependent (nNOS) activity in control or diabetic arteries, or calcium-independent (iNOS) activity in control arteries. However, significant calcium-independent (iNOS) activity was detected in diabetic arteries. These data suggest that iNOS is functionally expressed in VSM of arteries from 12- to 14-week STZ-diabetic rats. The possible causes and consequences of the iNOS induction are discussed.