Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion

Abstract

1. Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO*; NO gas solution) and nitroxyl ion (NO(-); from Angeli's salt). 2. The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 microM), concentration-dependently inhibited responses to all agents. 10 microM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3. The NO* scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 100 microM) and hydroxocobalamin (100 microM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4. The NO(-) inhibitor, L-cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO* and NO(-). Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Figure 1

Mean concentration-response curves to acetylcholine (a, d and g; n=4 – 7), nitric oxide gas solution (b, e and h; n=4) and Angeli's salt, (c, f and i; n=4) on mouse aortae pre-contracted with phenylephrine in the absence (control; closed symbols) and then in the presence (open symbols) of ODQ (0.3 μM, a, b and c; 1 μM, d, e and f; 10 μM, g, h and i; pre-incubation 30 min). The broken line represents the time control values for Angeli's salt (i.e second curve in the absence of ODQ; n=4). Relaxation responses are expressed as per cent reversal of the phenylephrine-induced contraction. Points are mean values with s.e.mean shown by vertical bars except when smaller than the size of the symbols. **0.01>P>0.001, ***P<0.001 Response significantly less than corresponding response in the absence of ODQ. ## 0.01>P>0.001, ### P<0.001 Significant parallel shift in curve based on a decrease in negative log IC₅₀ (see ‘log unit shifts' in Table 1).

Figure 2

Mean concentration-response curves to glyceryl trinitrate (a, d and g; n=4 – 7), sodium nitroprusside (b, e and h; n=4) and spermine NONOate (c, f and i; n=4) on mouse aortae pre-contracted with phenylephrine in the absence (control; closed symbols) and then in the presence (open symbols) of ODQ (0.3 μM, a, b and c; 1 μM, d, e and f; 10 μM, g, h and i; pre-incubation 30 min). Relaxation responses are expressed as per cent reversal of the phenylephrine-induced contraction. Points are mean values with s.e.mean shown by vertical bars except when smaller than the size of the symbols. **0.01>P>0.001, ***P<0.001 Response significantly less than corresponding response in the absence of ODQ. ## 0.01>P>0.001, ### P<0.001 Significant parallel shift in curve based on a decrease in negative log IC₅₀ (see ‘log unit shifts' in Table 1).

Figure 3

Mean responses to (a) nitric oxide gas solution (NO; 1 μM; n=4 – 6), (b) Angeli's salt (1 μM; n=4), (c) acetylcholine (ACh; 0.3 μM n=4), (d) sodium nitroprusside (SNP; 0.03 μM; n=4), (e) glyceryl trinitrate (GTN; 0.1 μM; n=4 – 6 (f) spermine NONOate (SperNO; 1 μM; n=3 – 4) on mouse aortae in the absence (control) and presence (treated) of carboxy-PTIO (c-PTIO; 100 μM), hydroxocobalamin (HC; 100 μM) or L-cysteine (3 mM); pre-incubation time 3 min. Relaxation responses are expressed as per cent reversal of the phenylephrine-induced contraction. Responses to Angeli's salt in the presence of inhibitors have been corrected for the time-related reduction in responses to this agent seen in the absence of inhibitors, using the correction factor referred to in the Methods. Points are mean values with s.e.mean shown by vertical bars. *0.05>P>0.01, **0.01>P>0.001, ***P<0.001 when compared with corresponding control value.