Vasorelaxant and antiplatelet activity of 4,7-dimethyl-1,2, 5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide: role of soluble guanylate cyclase, nitric oxide and thiols

Abstract

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 significantly less potent vasodilator (pIC(50)=5.19+/-0.14). The vasorelaxant activity of FPTO and FPDO was inhibited by ODQ. oxyHb significantly 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 microM, respectively). The antiplatelet activity of FPTO (but not FPDO) was partially suppressed by oxyHb. The antiaggregatory effects of FPTO and FPDO were only partially blocked by sGC inhibitors. 6. FPTO and FPDO (10 - 20 microM) significantly 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 microM. On the contrary, inhibition of platelet aggregation is only partially related to sGC activation.

Figure 1

Structures of FPTO (4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide), FPDO (4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 5,6-dioxide) and DCF (3,4-dicyano-1,2,5-oxadiazole 2-oxide).

Figure 2

Effect of cysteine and glutathione on generation of nitrite from FPTO (n=5).

Figure 3

Effect of exogenous DTT on activation of crude rat lung soluble guanylate cyclase preparation by FPTO, FPDO (A) and spontaneous NO donor DEA/NO and basal activity (B) (n=6). Extract was prepared in the absence of thiols and total endogenous thiol concentration in the incubation medium was 16 μM (2.8 μM low molecular weight thiols). The latter value was added to the final DTT concentrations. Spontaneous NO donors (0.1 mM SIN-1 and 0.1 mM SNP) did not significantly activate the enzyme at DTT concentration up to 200 μM but the enzyme was efficiently stimulated in the presence of 10 mM DTT (58±4 and 114±17 fold, respectively) or 5 mM cysteine. Note the difference in ordinate scales between plots A and B.

Figure 4

Concentration-dependent relaxation of NA-precontracted aortic rings with FPTO and FPDO. For details see text. Data are mean±95% confidence limits (n=9–13).

Figure 5

Characterization of vasorelaxant activity of FPTO (A,C,E) and FPDO (B,D,F) in aortic rings precontracted with 0.5 μM NA. Endothelium-denuded (A,B) or intact rings (A–F) were pretreated 10 min before NA addition with 0.1 mM L-NOARG (A,B), 10 μM oxyHb or metHb (C,D), 1 μM ODQ or 10 U ml⁻¹ SOD (E,F) and cumulative concentration-response curves for FPTO or FPDO were recorded. The pIC₅₀ and R_max values for FPTO are given in Table 5. Values of FPDO activity were determined by fitting the data to a 3- or 4-parameter logistic equation and fitting quality was always satisfactory (P<0.05; Student's t-test). R_max shown are mean±s.e.mean and pIC₅₀ values were weighted versus standard error (mean±95% confidence intervals). Control: pIC₅₀=5.19±0.14, R_max=105.7±12.1% (n=9); without endothelium: pIC₅₀=5.58±0.05 (P<0.05 versus control), R_max=98.4±9.5 (n=4); with endothelium: 0.1 mM L-NOARG: pIC₅₀ 5.20±0.21, R_max=103.2±2.2 (n=4); 10 μM oxyHb: pIC₅₀ 4.92±0.16, R_max=54.7±16.5 (n=4); 10 μM metHb: pIC₅₀ 5.53±0.10, R_max=101.8±14.3 (n=4); 10 U ml⁻¹ SOD: pIC₅₀=5.66±0.17 (P<0.05 versus control), R_max=106.0±3.3% (n=4); 1 μM ODQ: pIC₅₀=4.87±0.20 (P<0.05 versus control), R_max=87.9±12.4 (n=4). Asterisks correspond to the points significantly different from control (P<0.05).

Figure 6

Effect of 1 mM cysteine on vasorelaxant activity of FPTO. Typical recording representative of five similar experiments is shown. Arrows correspond to addition of 1 mM cysteine (Cys) or 0.5 μM NA. Numbers from 1 to 6 correspond to cumulative concentrations of FPTO: 0.1 nM (1), 1 nM (2), 10 nM (3), 100 nM (4), 1 μM (5), and 10 μM (6). Control recording without cysteine (A) indicates that NA-dependent contraction is developing faster in the presence of cysteine (B) and vascular tone was stable during the experiment (C).

Figure 7

Inhibition of ADP-induced human platelet aggregation (A) and desaggregation of reversibly aggregated platelets (B) by FPTO and FPDO (n=4). SNP is shown as the reference.