We designed experiments to characterize the role of superoxide anions in the mediation of endothelium-dependent contractions in isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% Oj-6% CO 2 (37°C, pH 7.4). Radioimmunoassay was used to determine the levels of cyclic GMP and cyclic AMP. Calcium ionophore A23187 (1(T 9 to 1(T 6 mol/L) caused concentration-dependent contractions. The removal of endothelium abolished the effect of A23187. Contractions to A23187 were reversed into relaxations in the presence of superoxide dismutase (150 U/mL) or the prostaglandin H 2 /thromboxane A 2 receptor antagonist SQ29548 (10~6 mol/L). Af G -nitro-Larginine methyl ester (3xlO~4 mol/L) augmented contractions to A23187. In rings with endothelium, A23187 (3 x 1(T 7 mol/L) significantly increased levels of both cyclic AMP and cyclic GMP. Indomethacin (10~5 mol/L) inhibited stimulatory effects of A23187 on cyclic AMP production. In contrast, indomethacin augmented A23187-induced production of cyclic GMP. Selective augmentation of cyclic GMP production by indomethacin appears to be due to protection of nitric oxide or a closely related molecule released following translocation of calcium into endothelial cells. Our findings suggest that (1) an increased concentration of calcium in endothelial cells may activate both cyclooxygenase and the L-arginine/nitric oxide pathway, (2) arachidonic acid metabolism via cyclooxygenase is a source of superoxide anions, and (3) superoxide anions may be responsible for impairment of balance between relaxing and contracting factors leading to contraction of underlying smooth muscle cells. (Hypertension. 1994^3:229-235.)

Experiments were designed to determine the role of the L-arginine pathway in endothelium-dependent relaxations to vasopressin. The effects of L-arginine analogues NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), and NG-monomethyl-L-arginine (L-NMMA) on basal and vasopressin-induced activity of nitric oxide synthase were studied in isolated canine basilar arteries. Rings with and without endothelium were suspended for isometric tension recording in Krebs-Ringer bicarbonate solution bubbled with 94% O2-6% CO2 (37 degrees C, pH 7.4). Radioimmunoassay was used to determine the level of guanosine 3',5'-cyclic monophosphate (cGMP). All experiments were performed in the presence of indomethacin, a cyclooxygenase inhibitor. L-NAME and L-NMMA caused endothelium-dependent contractions and inhibited basal production of cGMP. In contrast, L-NNA did not affect basal tone or basal production of cGMP. L-Arginine analogues inhibited relaxations to vasopressin but did not affect relaxations to a nitric oxide donor, molsidomine (SIN-1). The effects of L-NNA, L-NAME, and L-NMMA were reversed in the presence of L-arginine. The relaxations to vasopressin were associated with an increase of cGMP levels in the arterial wall. This effect of vasopressin was inhibited in the presence of L-NNA. These studies suggest that the relaxations to vasopressin are mediated by activation of the endothelial L-arginine pathway, leading to increased production of nitric oxide, with subsequent activation of guanylate cyclase in smooth muscle cells. In canine basilar artery, L-NAME and L-NMMA are nonselective inhibitors of both basal and stimulated production of nitric oxide, whereas L-NNA selectively inhibits vasopressin-induced activation of the L-arginine pathway.

This study concerned reactive oxygen species for their potential to activate human platelet GP IIb/IIIa receptors. All cells produce reactive oxygen species - radicals that can abstract electrons and hydrogen atoms from biological molecules to alter cell function. In many cells, radicals contribute to cellular signaling. In platelets, the predominant oxidant effect is platelet activation. Less is known concerning oxidants and GP IIb/IIIa receptor activation. The first aim of the current study was to confirm that although both H(2)O(2) and tert butyl hydroperoxide both predispose platelets to aggregation; neither directly activates GP IIb/IIIa receptors. The second aim was to demonstrate that even in the presence of extracellular redox iron; H(2)O(2) does not activate GP IIb/IIIa receptors. The third aim was to determine if extracellular superoxide anions evoke GP IIb/IIIa activation. Finally, a role for intra-platelet iron in GP IIb/IIIa activation was examined. Intracellular superoxide anions are produced in excess during platelet activation and curiously, they are uniquely able to increase intracellular free iron. This iron can, in a redox manner, generate radicals and these iron dependent species modulate signaling systems, including systems associated with adhesion receptor activation. In the current studies, platelets in suspension were exposed to H(2)O(2) and to tert butyl hydroperoxide, to H(2)O(2) plus ferrous or ferric chloride (+/- ascorbate to enhance iron redox cycling) and to xanthine plus xanthine oxidase to generate extra-platelet superoxide anions. Intra-platelet iron was increased with iron ionophore 8-hydroxyquinoline. During flow cytometry, intra-platelet oxidant state was assessed with the redox sensitive fluorescent indicator H2DCF, while GP IIb/IIIa activation was assessed using fluorescent antibody PAC-1. Results showed that although all the oxidizing systems examined increased intra-platelet oxidant state, GP IIb/IIIa receptors were not activated by H(2)O(2), by tert butyl hydroperoxide, by H(2)O(2) plus iron (+/- ascorbate) or by xanthine plus xanthine oxidase. In contrast, iron plus ionophore 8-hydroxyquinoline evoked GP IIb/IIIa activation. Platelet positivity for PAC-1 increased from 2 +/- 0.2 to 28 +/- 7% (P < 0.005). However this response, although vigorous, was less than 56 +/- 8% (P < 0.001) evoked by thrombin 0.1 milliunit/ml. In conclusion, the results indicated that oxidant systems external to platelets did not activate GP IIb/IIIa receptors while increased intra-platelet iron was associated with appearance of cytosolic oxidizing species and with GP IIb/IIIa receptor activation.

The fact that amrinone inhibited human platelet activation at the cellular level and protected against experimental coronary thrombosis in vivo in dogs suggests a potentially advantageous antithrombotic action for this inotropic and vasodilator drug.

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