Endothelial cells play a unique role in modulating vascular tone by releasing endothelium-derived relaxing factor (EDRF). Defects in EDRF may predispose the diabetic to a higher risk of hypertension, atherosclerosis, coronary artery vasospasm and sudden death. This proposal will examine the hypothesis that diabetes selectively uncouples relaxation to receptor-operated endothelium-dependent vasodilators (RO-EDV), but not receptor-independent endothelium vasodilators (RI-EDV). The specific role oxygen-derived free radicals (ODFR)in this defect will be investigated. Blood vessels (aorta, femoral and mesenteric artery) from streptozotocin-diabetic and genetically-diabetic BB rats will be used and compared to glucose- infused rats. Selectivity and specificity for impaired response to RO- EDV vs. RI-EDV and endothelium-independent vasodilators will be examined (Specific Aim #1). In vivo intervention (Specific Aim #2) using surgery (pancreatic transplantation) or therapeutic intervention (insulin, antioxidant, free radical scavengers, iron chelator) will be evaluated to test for prevention or reversal of endothelial dysfunction. The mechanism of impaired EDRF will be examined (Specific Aim #3). The bioassay technique will be utilized in which endothelial- perfusion of control or experimental donor segments are challenged with agonists which release EDRF and acts upon detector rings (without endothelium). This will answer: Does the diabetic endothelium release less EDRF?; Is the diabetic vascular smooth muscle less responsive to authentic EDRF and/or nitric oxide?; Do ODFR produced by the diabetic endothelium or diabetic vascular smooth muscle impair or inactivate EDRF activity? The influence of insulin on intracellular glucopenia or EDRF release or action will also be tested. Additional protocols will test for a potential endothelium-derived constricting factor or differential changes in cGMP (using radioimmunoassay). Superoxide dismutase, catalase and glutathione peroxidase activities will be examined in blood vessels to assess radical scavenging capacity. Production of ODFR by blood vessels (with or without endothelium) will be examined by spectrophotometric techniques and by state-of-the art electron spin resonance spectroscopy using loop-gap resonators with spin trapping techniques. Intracellular calcium signal transduction in response to RO-EDV and RI-EDV will be evaluated using fluorescence spectroscopy (Fura-2) in cultured bovine aortic endothelial cells subjects to conditions mimicking the diabetic environment (Specific Aim #4). Endothelial cells will be incubated with either glucose, fatty acids, ketone bodies or diabetic sera (including very low density lipoproteins) to examine selective uncoupling of intracellular calcium signal transduction in response to RO-EDV. The role of ODFR in this uncoupling will also be evaluated. Data obtained from this study will help define a mechanism for defective EDRF and understanding the role EDRF may play in the etiology of hypertension and other vascular complications associated with diabetes. It will also provide experimental evidence for the efficacy of pancreatic transplantation or therapeutic intervention to prevent or reverse endothelial dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL047072-03
Application #
2223385
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Project Start
1993-04-01
Project End
1997-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Pieper, G M; Lai, C S (1999) Biological evaluation of the nitric oxide-trapping agent, N-methyl-D-glucamine dithiocarbamate-Fe2+, as a probe of nitric oxide activity released from control and diabetic rat endothelium. Jpn J Pharmacol 80:359-70
Pieper, G M (1999) Enhanced, unaltered and impaired nitric oxide-mediated endothelium-dependent relaxation in experimental diabetes mellitus: importance of disease duration. Diabetologia 42:204-13
Pieper, G M; Dondlinger, L A (1998) Antioxidant pyrrolidine dithiocarbamate prevents defective bradykinin-stimulated calcium accumulation and nitric oxide activity following exposure of endothelial cells to elevated glucose concentration. Diabetologia 41:806-12
Cooper, M; Lindholm, P; Pieper, G et al. (1998) Myocardial nuclear factor-kappaB activity and nitric oxide production in rejecting cardiac allografts. Transplantation 66:838-44
Pieper, G M; Adams, M B; Roza, A M (1998) Pancreatic transplantation reverses endothelial dysfunction in experimental diabetes mellitus. Surgery 123:89-95
Dembny, K D; Roza, A M; Johnson, C et al. (1998) Heparin interferes with the determination of plasma nitric oxide by inhibition of enzymatic conversion of nitrate to nitrite by nitrate reductase. Clin Chim Acta 275:107-14
Pieper, G M (1998) Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension 31:1047-60
Pieper, G M; Dembny, K; Siebeneich, W (1998) Long-term treatment in vivo with NOX-101, a scavenger of nitric oxide, prevents diabetes-induced endothelial dysfunction. Diabetologia 41:1220-6
Pieper, G M; Siebeneich, W (1998) Oral administration of the antioxidant, N-acetylcysteine, abrogates diabetes-induced endothelial dysfunction. J Cardiovasc Pharmacol 32:101-5
Nakanishi, A L; Roza, A M; Adams, M B et al. (1998) Electron spin resonance analysis of heme-nitrosyl and reduced iron-sulfur centered complexes in allogeneic, heterotopic cardiac transplants: effects of treatment with pyrrolidine dithiocarbamate. Free Radic Biol Med 25:201-7

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