Pharmacology of Nitrovasodilators

Platelets have a crucial role in the maintenance of normal haemostasis, and perturbations of this system can lead to pathological thrombus formation and vascular occlusion, resulting in stroke, myocardial infarction and unstable angina. ADP released from damaged vessels and red blood cells induces platelet aggregation through activation of the integrin GPIIb-IIIa and subsequent binding of fibrinogen. ADP is also secreted from platelets on activation, providing positive feedback that potentiates the actions of many platelet activators. ADP mediates platelet aggregation through its action on two G-protein-coupled receptor subtypes. The P2Y1 receptor couples to Gq and mobilizes intracellular calcium ions to mediate platelet shape change and aggregation. The second ADP receptor required for aggregation (variously called P2Y(ADP), P2Y(AC), P2Ycyc or P2T(AC)) is coupled to the inhibition of adenylyl cyclase through Gi. The molecular identity of the Gi-linked receptor is still elusive, even though it is the target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel and AR-C66096 (ref. 9). Here we describe the cloning of this receptor, designated P2Y12, and provide evidence that a patient with a bleeding disorder has a defect in this gene. Cloning of the P2Y12 receptor should facilitate the development of better antiplatelet agents to treat cardiovascular diseases.

Nitroglycerin (glyceryl trinitrate, GTN), originally manufactured by Alfred Nobel, has been used to treat angina and heart failure for over 130 years. However, the molecular mechanism of GTN biotransformation has remained a mystery and it is not well understood why "tolerance" (i.e., loss of clinical efficacy) manifests over time. Here we purify a nitrate reductase that specifically catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from GTN, leading to production of cGMP and relaxation of vascular smooth muscle both in vitro and in vivo, and we identify it as mitochondrial aldehyde dehydrogenase (mtALDH). We also show that mtALDH is inhibited in blood vessels made tolerant by GTN. These results demonstrate that the biotransformation of GTN occurs predominantly in mitochondria through a novel reductase action of mtALDH and suggest that nitrite is an obligate intermediate in generation of NO bioactivity. The data also indicate that attenuated biotransformation of GTN by mtALDH underlies the induction of nitrate tolerance. More generally, our studies provide new insights into subcellular processing of NO metabolites and suggest new approaches to generating NO bioactivity and overcoming nitrate tolerance.

We sought to examine mechanisms underlying nitroglycerin (NTG) tolerance and "cross-tolerance" to other nitrovasodilators. Rabbits were treated for 3 d with NTG patches (0.4 mg/h) and their aortic segments studied in organ chambers. Relaxations were examined after preconstriction with phenylephrine. In NTG tolerant rabbit aorta, relaxations to cGMP-dependent vasodilators such as NTG (45 +/- 6%), SIN-1 (69 +/- 7%), and acetylcholine (ACh, 64 +/- 5%) were attenuated vs. controls, (90 +/- 2, 94 +/- 3, and 89 +/- 2% respectively, P < 0.05 for all), while responses to the cAMP-dependent vasodilator forskolin remained unchanged. In tolerant aorta, endothelial removal markedly enhanced relaxations to NTG and SIN-1 (82 +/- 4 and 95 +/- 3%, respectively). Other studies were performed to determine how the endothelium enhances tolerance. Vascular steady state .-O2 levels (assessed by lucigenin chemiluminescence) was increased twofold in tolerant vs. control vessels with endothelium (0.31 +/- 0.01 vs. 0.61 +/- 0.01 nmol/mg per minute). This difference was less in vessels after denudation of the endothelium. Diphenylene iodonium, an inhibitor of flavoprotein containing oxidases, and Tiron a direct .-O2 scavenger normalized .-O2 levels. In contrast, oxypurinol (1 mM) an inhibitor of xanthine oxidase, rotenone (50 microM) an inhibitor of mitochondrial electron transport and NG-nitro-L-arginine (100 microM) an inhibitor of nitric oxide synthase did not affect the chemiluminescence signals from NTG-tolerant aortas. Pretreatment of tolerant aorta with liposome-entrapped, pH sensitive superoxide dismutase (600 U/ml) significantly enhanced maximal relaxation in response to NTG, SIN-1, and ACh, and effectively reduced chemiluminescence signals. These studies show that continuous NTG treatment is associated with increased vascular .-O2-production and consequent inhibition of NO. mediated vasorelaxation produced by both exogenous and endogenous nitrovasodilators.