1 Certain heterocyclic N-oxides are vasodilators and inhibitors of platelet aggregation. The pharmacological activity of the furoxan derivative condensed with pyridazine di-N-oxide 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (FPTO) and the corresponding furazan (FPDO) was studied. 2 FPTO reacted with thiols generating nitrite (NO), S-nitrosoglutathione and hydroxylamine (nitroxyl) and converted oxyHb to metHb. FPDO did not generate detectable amounts of NO-like species but reacted with thiols and oxyHb. 3 FPTO and FPDO haem-dependently stimulated the activity of soluble guanylate cyclase (sGC) and this stimulation was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by 0.1 mM dithiothreitol. 4 FPTO relaxed noradrenaline-precontracted aortic rings and its concentration-response curve was biphasic (pIC 50 =9.03+0.13 and 5.85+0.06). FPDO was signi®cantly less potent vasodilator (pIC 50 =5.19+0.14). The vasorelaxant activity of FPTO and FPDO was inhibited by ODQ. oxyHb signi®cantly inhibited only FPTO-dependent relaxation. 5 FPTO and FPDO were equipotent inhibitors of ADP-induced platelet aggregation (IC 50 =0.63+0.15 and 0.49+0.05 mM, respectively). The antiplatelet activity of FPTO (but not FPDO) was partially suppressed by oxyHb. The antiaggregatory eects of FPTO and FPDO were only partially blocked by sGC inhibitors. 6 FPTO and FPDO (10 ± 20 mM) signi®cantly increased cyclic GMP levels in aortic rings and platelets and this increase was blocked by ODQ. 7 Thus, FPTO can generate NO and, like FPDO, reacts with thiols and haem. The vasorelaxant activity of FPTO and FPDO is sGC-dependent and a predominant role is played by NO at FPTO concentrations below 1 mM. On the contrary, inhibition of platelet aggregation is only partially related to sGC activation.

Exogenous endothelin-1 (ET-1) or high concentrations of the peptide in pathological conditions have marked effects on vascular reactivity. In order to evaluate the role of endogenous ET-1 we investigated responsiveness of conduit (aorta) and of resistant-like (tail artery) vessels in ET-1-deficient rats. Elimination of circulating ET-1 was achieved by active immunization of Wistar rats with a peptide-haemocyanin conjugate (anti-ET-1 group), leading to a marked reduction in plasma level of the peptide in comparison with that of vehicle-treated animals (control group): 1.9 fmol/ml vs 4.9 fmol/ml, respectively. The immunization was associated with a slight elevation of mean arterial pressure, whereas heart rate remained unchanged. In the anti-ET-1 group rings of isolated aorta displayed reduced sensitivity to ET-1: EC50 = 6.57 nM vs 2.95 nM in the control group. Tail arteries of the ET-1-deficient rats showed diminished responses to ET-1, the maximal rise in perfusion pressure: +5.2 mmHg vs +13.6 mmHg in the control group. After immunization, rings of isolated aorta displayed no changes in endothelium-dependent relaxation to acetylcholine (Ach, EC50 = 0.20 microM vs 0.35 microM in the control group), whereas experiments on perfused tail artery showed a twofold reduction in Ach effects. Thus, depletion of circulating ET-1 induces slight changes in haemodynamics associated with altered vessel responsiveness to vasoactive substances.

In the present work we evaluated the role of circulating endothelin-1 (ET-1) in haemodynamics and the adrenal medulla responses to stress using PD-142893, a mixed endothelin-A- and -B- (ET(A)/ET(B)) receptor antagonist. Male Wistar rats were chronically instrumented with arterial and venous catheters and a microdialysis probe placed into the adrenal medulla. Stress was induced by a 1 h period of immobilization. PD-142893 was infused (20 microg/kg/min) for 90 min starting 30 min before stress onset. Concentrations of norepinephrine and epinephrine in dialysate samples were measured by high-performance liquid chromatography (HPLC). At rest animals displayed a stable level of norepinephrine (85 +/- 34 pg/ml) and epinephrine (176 +/- 57 pg/ml) in adrenal perfusate and constant haemodynamic parameters. Stress increased adrenal secretion (norepinephrine 206 +/- 50%, epinephrine 202 +/- 45%) associated with hypertension (peak 141 +/- 3 mmHg) and tachycardia (peak 505 +/- 5 bpm). No significant changes in haemodynamics or of plasma catecholamine levels were observed during infusion of PD-142893. The antagonist did not significantly change the stress-induced increase in catecholamine secretion, tachycardia or hypertension. Thus, in Wistar rats, circulating ET-1 seems not to be essential for blood pressure control or adrenal catecholamine secretion during acute stress.

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The endothelin (ET) system was studied in August rats, with genetically determined high sensitivity to stress, and in control Wistar rats. Radioimmunoassay revealed a significant difference in plasma endothelin-1 (ET-1) levels of August vs Wistar rats (7.1 fmol/ml vs 50.0 fmol/ml). Immobilization of the animals increased these values up to 11.0 fmol/ml and 65.2 fmol/ml, respectively. Elevation of ET-1 was associated with an increase in blood pressure, which was similar in both strains, whereas the heart rate increase was diminished in the stress-sensitive rats. The mixed endothelin-A/endothelin-B- (ET(A)/ET(B)) receptor antagonist PD142893 suppressed stress-induced elevation of blood pressure in August, but not in Wistar rats. In both strains, heart rate responses to stress were insensitive to the ET receptor blockade. Aortic rings of August rats displayed diminished sensitivity to the vasoconstrictor action of ET-1 vs that of Wistar rats (EC50 = 22.1 nM vs 6.3 nM, respectively). In noradrenaline-precontracted tail arteries, 50 nM ET-1 elicited further constriction without any vasodilator effects. ET-1-induced increase in perfusion pressure was greater in tail arteries of Wistar rats. Thus, endogenous ET-1 can play a strain-dependent role in the stress-induced responses of haemodynamics and the alterations in endothelin-dependent regulation may be responsible for the differences in vascular reactivity of Wistar and August rats.