Role of hydrogen sulphide in haemorrhagic shock in the rat: protective effect of inhibitors of hydrogen sulphide biosynthesis

Abstract

Haemorrhagic shock (60 min) in the anaesthetized rat resulted in a prolonged fall in the mean arterial blood pressure (MAP) and heart rate (HR). Pre-treatment (30 min before shock) or post-treatment (60 min after shock) with inhibitors of cystathionine gamma lyase (CSE; converts cysteine into hydrogen sulphide (H(2)S)), dl-propargylglycine or beta-cyanoalanine (50 mg kg(-1), i.v.), or glibenclamide (40 mg kg(-1), i.p.), produced a rapid, partial restoration in MAP and HR. Neither saline nor DMSO affected MAP or HR. Plasma H(2)S concentration was elevated 60 min after blood withdrawal (37.5+/-1.3 microM, n=18 c.f. 28.9+/-1.4 microM, n=15, P<0.05). The conversion of cysteine to H(2)S by liver (but not kidney) homogenates prepared from animals killed 60 min after withdrawal of blood was significantly increased (52.1+/-1.6 c.f. 39.8+/-4.1 nmol mg protein(-1), n=8, P<0.05), as was liver CSE mRNA (2.7 x). Both PAG (IC(50), 55.0+/-3.2 microM) and BCA (IC(50), 6.5+/-1.2 microM) inhibited liver H(2)S synthesizing activity in vitro. Pre-treatment of animals with PAG or BCA (50 mg kg(-1), i.p.) but not glibenclamide (40 mg kg(-1), i.p., K(ATP) channel inhibitor) abolished the rise in plasma H(2)S in animals exposed to 60 min haemorrhagic shock and prevented the augmented biosynthesis of H(2)S from cysteine in liver. These results demonstrate that H(2)S plays a role in haemorrhagic shock in the rat. CSE inhibitors may provide a novel approach to the treatment of haemorrhagic shock.

Figures

Figure 1

Effect of haemorrhagic shock on (a) MAP and (b) HR in anaesthetized rats. Arrows indicate timing of blood withdrawal and saline (0.9% w v−1 NaCl, 1 ml kg−1) injection (60 min after withdrawal of blood). (c) Effect of PAG and BCA (50 mg kg−1, i.v.) in control (i.e. unshocked) anaesthetized rats. Results show MAP in mmHg, and are mean±s.e.m., n=8.

Figure 2

Time-dependent effect of pre-treatment (a; 30 min before blood withdrawal of blood) and post-treatment (b; 60 min after withdrawal of blood) with PAG (50 mg kg−1, i.v.) on MAP in anaesthetized rats subjected to haemorrhagic shock. Arrows indicate timing of drug injection. Results show MAP in mmHg, and are mean±s.e.m., n=8, +P<0.05 c.f. control MAP (−30 min), *P<0.05 c.f. haemorrhagic shock measured at 45 min.

Figure 3

Time-dependent effect of pre-treatment (a; 30 min before blood withdrawal of blood) and post-treatment (b; 60 min after withdrawal of blood) with BCA (50 mg kg−1, i.v.) on MAP in anaesthetized rats subjected to haemorrhagic shock. Arrows indicate timing of drug injection. Results show MAP in mmHg, and are mean±s.e.m., n=8, +P<0.05 c.f. control MAP (−30 min), *P<0.05 c.f. haemorrhagic shock measured at 45 min.

Figure 4

(a) Time-dependent effect of post-treatment (60 min after withdrawal of blood) with glibenclamide (40 mg kg−1, i.p.) or DMSO vehicle (0.15 ml kg−1, i.p.) on MAP in anaesthetized rats subjected to haemorrhagic shock. Arrows indicates timing of drug or vehicle injection. Results show MAP in mmHg, and are mean±s.e.m., n=8, +P<0.05 c.f. control MAP (−30 min), *P<0.05 c.f. haemorrhagic shock measured at 60 min. (b) Time-dependent effect of glibenclamide (40 mg kg−1, i.p.) on MAP in control (i.e. unshocked) anaesthetized rats. Results show MAP in mmHg and are mean±s.e.m., n=8.

Figure 5

Effect of post-treatment (60 min after withdrawal of blood) with BCA (50 mg kg−1, i.v.), glibenclamide (40 mg kg−1, i.p.) or both BCA and glibenclamide (at doses stated) on MAP of shocked rats determined 120 min after withdrawal of blood. Results show MAP in mmHg, and are mean±s.e.m., n=8, *P<0.05 c.f. MAP of shocked animals measured at 45 min.

Figure 6

(a) Formation of H2S from cysteine (10 mM) in the presence of pyridoxal 5′ phosphate (1 mm) following incubation (37°C, 30 min). Homogenates were prepared from livers removed from anaesthetized animals prior to (BS) or 60 or 120 min after haemorrhagic shock (AS). T=0 shows the concentration of H2S in control liver incubations in which the reaction was stopped at zero time by addition of 10% w v−1 trichloroacetic acid (250 μl) prior to addition of cysteine (10 mM) and incubation (37°C, 30 min), as described above. (b) Formation of H2S from cysteine (10 mM) in the presence of pyridoxal 5′ phosphate (1 mm) following incubation (37°C, 30 min) in animals pre-treated with either PAG or BCA (50 mg kg−1, i.v.). Results show H2S formation as nmol formed mg protein−1, and are mean±s.e.m., n=8, *P<0.05 c.f. T=0 controls, +P<0.05 c.f. H2S formation before haemorrhagic shock (BS).

Figure 7

Inhibition of rat liver H2S synthesis by PAG and BCA in vitro. Results show % inhibition of H2S formation (c.f. incubates containing an equivalent of water vehicle), and are mean±s.e.m., n=5–6.

Figure 8

(a) Representative blots from three livers, showing the presence of CSE (445 b.p., 29 cycles). M shows molecular weight markers. (b) Quantitation of blots shown in (a). Data indicate the relative intensities in arbitrary units, and are mean±s.e.m., n=3, *P<0.05.