Increased Superoxide Production in Hypertensive Patients With Diabetes Mellitus: Role of Nitric Oxide Synthase

Background Hypertension and diabetes are important independent risk factors for increased oxidative stress and increased cardiovascular risk. The combination of hypertension and diabetes results in a dramatic increase in cardiovascular risk. Enhanced oxidative stress in hypertension and diabetes is linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O2.−), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, NO synthase III (NOS III) can also produce O2.−. We studied platelet superoxide production in patients with hypertension alone and in patients with coexistent diabetes mellitus, investigating the contribution of NOS III uncoupling to platelet superoxide production. Methods and Results Gel-filtered platelets were obtained and were stimulated with Phorbol 12-myristate 13-acetate, and O2.− production was detected using lucigenin-enhanced chemiluminescence. Superoxide production was significantly higher in patients with diabetes and hypertension (6.4 ± 1.6 pmol/min/108 platelets) than in patients with hypertension (1.6 ± 0.6 pmol/min/108 platelets) (P< .04). After incorporation of Nω-nitro-l-arginine methyl ester (L-NAME, 1 mmol/L), O2.− detection increased in 40% of patients with diabetes and hypertension and in 87% of patients with hypertension. This expected response results from L-NAME inhibition of NO production preventing NO scavenging of O2.−. A reduction in O2.− production in response to L-NAME occurred in the remaining patients and indicates O2.− production by the uncoupled NOS III enzyme. Conclusions This study provides first published evidence that NOS III can reside in the uncoupled state in patients with hypertension and, to a greater extent, in patients with coexisting hypertension and diabetes, and that it contributes significantly to increased superoxide production in these disease states.