Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide?

Abstract

Background and purpose: The aim of these experiments was to evaluate the significance of the chemical reaction between hydrogen sulphide (H2S) and nitric oxide (NO) for the control of vascular tone.

Experimental approach: The effect of sodium hydrosulphide (NaHS; H2S donor) and a range of NO donors, such as sodium nitroprusside (SNP), either alone or together, was determined using phenylephrine (PE)-precontracted rat aortic rings and on the blood pressure of anaesthetised rats.

Key results: Mixing NaHS with NO donors inhibited the vasorelaxant effect of NO both in vitro and in vivo. Low concentrations of NaHS or H2S gas in solution reversed the relaxant effect of acetylcholine (ACh, 400 nM) and histamine (100 microM) but not isoprenaline (400 nM). The effect of NaHS on the ACh response was antagonized by CuSO(4) (200 nM) but was unaffected by glibenclamide (10 microM). In contrast, high concentrations of NaHS (200-1600 microM) relaxed aortic rings directly, an effect reduced by glibenclamide but unaffected by CuSO4. Intravenous infusion of a low concentration of NaHS (10 micromol kg(-1) min(-1)) into the anaesthetized rat significantly increased mean arterial blood pressure. L-NAME (25 mg kg(-1), i.v.) pretreatment reduced this effect.

Conclusions and implications: These results suggest that H2S and NO react together to form a molecule (possibly a nitrosothiol) which exhibits little or no vasorelaxant activity either in vitro or in vivo. We propose that a crucial, and hitherto unappreciated, role of H2S in the vascular system is the regulation of the availability of NO.

Figure 1

Representative traces showing the effect of NaHS (50–1600 μM) on rat aortic rings precontracted with PE (200 nM). NaHS injection at the peak of the PE-induced contraction evoked an additional contraction in endothelium-intact (first trace) but not in endothelium-denuded (second trace) rings. NaHS did not affect nonprecontracted rings (third trace) or precontracted rings treated with CuSO₄ (200 nM, fourth trace). Vertical bar indicates tension scale (g). Horizontal bar shows time (min). Drugs were injected at the arrows as indicated.

Figure 2

Effect of NaHS (100 μM) and NO donors administered alone or after mixing (1 min) with approximately EC₇₀s of either SNP (a; 0.8 nM), SIN-1 (b; 6.4 nM) or SNAP (c; 0.8 nM) on tone of the PE-precontracted rat aorta. Deionized water was used vehicle control. Results are expressed as % relaxation of PE-induced tone and are mean±s.e.m., n=6, ^*P<0.05.

Figure 3

Representative traces (a) and the concentration-related effect of NaHS (b; 50–1600 μM) on PE-precontracted aortic rings, which were partially relaxed by addition of acetylcholine (400 nM). Vertical bar indicates tension scale (g). Horizontal bar shows time (min). Drugs were injected at the arrows as indicated. Glibenclamide (b, 10 μM) was added to the organ bath 3 min before addition of NaHS. Deionized water was used as vehicle control and did not affect the ACh-mediated relaxation (P>0.05). Results show % contraction (reversal of ACh-mediated relaxation) and are mean±s.e.m., n=4–14, ^*P<0.05.

Figure 4

Concentration-related effect of H₂S gas in solution (a; 10–400 μM) on PE-precontracted aortic rings, which were partially relaxed by addition of acetylcholine (400 nM). Vertical bar indicates tension scale (g). Horizontal bar shows time (min). Drugs were injected at the arrows as indicated. Effect of CuSO₄ on concentration-related effect of NaHS (b; 50–1600 μM) on PE-precontracted aortic rings, which were partially relaxed by addition of acetylcholine (400 nM). CuSO₄ (b, 200 nM) was added to the organ bath 3 min before addition of NaHS. Deionized water was used as vehicle control and did not affect the ACh-mediated relaxation (P>0.05). Results show % contraction (reversal of ACh-mediated relaxation) and are mean±s.e.m., n=4–14, ^*P<0.05.

Figure 5

(a) Concentration-related effect of NaHS (a; 10–1600 μM) on PE-precontracted aortic rings, which were partially relaxed by addition of histamine (100 μM). Results show % contraction (reversal of histamine-mediated relaxation) and are mean±s.e.m., n=7. (b) Upper trace: representative trace showing the effect of NaHS (50–1600 μM) on PE-precontracted aortic rings, which were partially relaxed by addition of isoprenaline (400 nM). Vertical bar indicates tension scale (g). Horizontal bar shows time (min). Drugs were injected at the arrows as indicated. Lower graph: effect of NaHS (a; 50–1600 μM) on rat aortic rings, which had been precontracted with PE (200 nM) and then partially relaxed by addition of isoprenaline (400 nM). Deionized water was used a vehicle control. Results show % contraction (reversal of isoprenaline-mediated relaxation) and are mean±s.e.m., n=12.

Figure 6

(a) Upper graph: effect of SNP (3.3–16.5 nmol kg⁻¹, i.v.) and NaHS (5–20 μmol kg⁻¹, i.v.) on mean arterial blood pressure (MAP) of the anaesthetized rat. Lower graph: effect of a mixture of SNP (16.5 nmol kg⁻¹) and NaHS (5 μmol kg⁻¹) on blood pressure and subsequent time-related recovery of the response to SNP administered alone. For comparison the response to SNP (16.5 nmol kg⁻¹) injected before administration of the mixture is also shown. Results show reduction in MAP (mm Hg) % and are mean±s.e.m, n=4–6, ^*P<0.05 (cf. SNP alone). (b) Time-dependent effect of an i.v. infusion (10 min) of either saline, NaHS (10 μmol kg⁻¹ min⁻¹) or NaHS (10 μmol kg⁻¹ min⁻¹ administered 3 min after i.v. injection of L-NAME, 25 mg kg⁻¹) on mean arterial pressure (MAP) of the anaesthetized rat. The effect of the NaHS infusion was inhibited by pretreatment with L-NAME (P<0.05). Results show reduction in MAP (mm Hg) % and are mean±s.e.m., n=4–6.