NAD(P)H-dependent superoxide production in platelets: the role of angiotensin II and protein kinase C.

OBJECTIVES: Vascular NAD(P)H oxidase represents a major source for excessive superoxide production in hypertension. Angiotensin II (AngII) can activate NAD(P)H oxidase via the angiotensin II type 1 (AT1) receptor and protein kinase C (PKC). Platelets possess AT1 receptors and all the components of the NAD(P)H oxidase system. We employed this tissue model to explore mechanisms involved in AngII-mediated superoxide production. DESIGN AND METHODS: Platelet suspensions from hypertensive patients' blood were activated with AngII or phorbol 12-myristate 13-acetate (PMA). Inhibitors of NAD(P)H oxidase, PKC, and the AT1 receptor were employed to study their effects on superoxide production. RESULTS: Superoxide production was stimulated by AngII and PMA and attenuated by AT1 receptor antagonists (mean percentage reduction 80.2%, P<0.01) and inhibitors of PKC (mean reduction 94.8%, P<0.001) and NAD(P)H oxidase (mean reduction 100%, P< 0.001). CONCLUSIONS: AngII stimulates platelet superoxide production through activation of vascular NAD(P)H oxidase via the AT1 receptor and PKC.

Functional consequences of endothelial nitric oxide synthase uncoupling in congestive cardiac failure.

BACKGROUND: Impaired endothelium-mediated vasodilatation (EMVD) in congestive cardiac failure (CCF) has been 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, endothelial nitric oxide synthase (eNOS) produces O2*-. We studied the functional consequences of eNOS uncoupling in relation to EMVD in patients with CCF. METHODS AND RESULTS: We employed the platelet as a compartmentalized ex-vivo model to examine O2*- and NO production. When eNOS is functioning normally, incorporation of Nomega-Nitro-L-Arginine methyl ester (L-NAME, 1 mmol/L), results in increased O2*- detection, as inhibition of NO production prevents NO scavenging of O2*-. This was observed in controls and 9 of the CCF patients, in whom O2*- detection increased by 63% and 101%, respectively. In the remaining 9 CCF patients, incorporation of L-NAME reduced O2*- production by 39%, indicating O2*- production by eNOS uncoupling. Detection of platelet-derived NO was significantly greater in eNOS-coupled platelets compared with the uncoupled group (2.8+/-1.4 versus 0.9+/-0.4 pmol/108 platelets, P=0.04). Endothelium-dependent and -independent vasodilator responses to acetylcholine and sodium nitroprusside recorded using venous occlusion plethysmography were significantly impaired in patients exhibiting eNOS uncoupling. CONCLUSIONS: This study provides first evidence that platelet eNOS can become uncoupled in human CCF. Impaired endothelium-dependent and -independent vasodilator responses and diminished platelet-derived NO production occurred in association with enzyme uncoupling.