Endothelial nitric oxide synthase (eNOS) expression and localization in healthy and diabetic rat hearts

Several studies suggest that nitric oxide (NO) production is reduced in dia betes and that the decrease of NO may be related to the pathogenesis of dia betic endothelial damage. NO synthase (NOS) catalyses the conversion of L-a rginine to L-citrulline in the presence of oxygen and NADPH-diaphorase (NAD PH-d). In this study, we evaluated the expression of endothelial NOS (eNOS) enzyme and its co-enzyme in diabetic rat hearts. Male Wistar rats (n = 20, 4 mo old) and 20 male Bio Breeding Wistar (BB/W) rats of the same age were used; the Wistar rats represent the control non-diabetic rats while the BB /W rats represent the diabetic group. After the hearts were excised, the NA DPH-d co-enzyme was visualized by a histochemical method and the endothelia l isoform of NOS was localized by immunohistochemistry. In addition, eNOS g ene expression was estimated by rt-PCR, and eNOS protein level was detected by Western blot analysis. The eNOS visualization, which involved immunopre cipitation, and the NADPH-d visualization, which involved histochemical sta ining, were both diminished in endothelial cells of the vascular wall of di abetic hearts, compared to non-diabetic hearts. The eNOS protein level, eva luated by Western blotting, was evident as an intense band in cardiac homog enates of non-diabetic and diabetic rats. The expression of mRNA for eNOS d id not differ significantly between the two groups. These findings indicate that, in this rat heart model, diabetes does not influence the overall eNO S protein level or its mRNA level. However, there is a diminution in the de position of eNOS in cardiac endothelial cells of diabetic rats, versus non- diabetic controls, suggesting a relation between eNOS and the loss of vasod ilatory response that is observed in diabetes. (received 7 November 2000, a ccepted 29 January 2001).