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 diabetes and that the decrease of NO may be related to the pathogenesis of diabetic endothelial damage. NO synthase (NOS) catalyses the conversion of L-arginine to L-citrulline in the presence of oxygen and NADPH-diaphorase (NADPH-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 NADPH-d co-enzyme was visualized by a histochemical method and the endothelial isoform of NOS was localized by immunohistochemistry. In addition, eNOS gene expression was estimated by rt-PCR, and eNOS protein level was detected by Western blot analysis. The eNOS visualization, which involved immunoprecipitation, and the NADPH-d visualization, which involved histochemical staining, were both diminished in endothelial cells of the vascular wall of diabetic hearts, compared to non-diabetic hearts. The eNOS protein level, evaluated by Western blotting, was evident as an intense band in cardiac homogenates of non-diabetic and diabetic rats. The expression of mRNA for eNOS did not differ significantly between the two groups. These findings indicate that, in this rat heart model, diabetes does not influence the overall eNOS protein level or its mRNA level. However, there a diminution in the deposition of eNOS in cardiac endothelial cells of diabetic rats, versus non-diabetic controls, suggesting a relation between eNOS and the loss of vasodilatory response that is observed in diabetes.
Felaco
M
M
Department of Biomorphology, Faculty of Medicine and Surgery, Università degli Studi G. d'Annunzio-Chieti, Via dei Vestini 31, Chieti 66100, Italy. mfelaco@unich.it
Grilli
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De Lutiis
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Patruno
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Libertini
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Taccardi
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Di Napoli
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Di Giulio
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Barbacane
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Conti
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D000818:Animals; D015153:Blotting, Western; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology*; D006651:Histocytochemistry; D007150:Immunohistochemistry; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D009252:NADPH Dehydrogenase / Q000032:analysis; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011913:Rats, Inbred BB; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D009252:NADPH Dehydrogenase