- Email: [email protected]
Synergism between hydrogen sulfide (H2S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom
Life Sciences 80 (2007) 1664 – 1668 www.elsevier.com/locate/lifescie
Synergism between hydrogen sulfide (H2S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom H.C. Liew a,c , H.E. Khoo a , P.K. Moore b , M. Bhatia b , J. Lu c , S.M. Moochhala b,c,⁎ a
Department of Biochemistry, National University of Singapore, Blk MD4A, 10 Kent Ridge Crescent, 119260, Singapore b Department of Pharmacology, National University of Singapore, Blk MD2, 18 Medical Drive, 119260, Singapore c DMERI, DSO National Laboratories, 27 Medical Drive, 117510, Singapore Received 7 July 2006; accepted 15 December 2006
Abstract Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10–320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-NG-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and β-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-γ-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H2S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H2S and NO act synergistically in effecting SNTX-induced vasorelaxation. © 2007 Elsevier Inc. All rights reserved. Keywords: Stonustoxin (SNTX); Synanceja horrida; Vasorelaxation; Hydrogen sulfide (H2S); Nitric oxide (NO)
Introduction Stonustoxin (Stonefish, National University of Singapore, SNTX) is a 148 kDa, dimeric, lethal protein factor isolated from the venom of the stonefish Synanceja horrida (Poh et al., 1991). It possesses a number of biological activities including speciesspecific haemolysis and platelet aggregation, edema-induction, hypotension, endothelium-dependent vasorelaxation, and inhibition of neuromuscular function in the mouse hemidiaphragm and chick biventer cervicis muscles (Low et al., 1993; Khoo et al., 1995; Chen et al., 1997; Sung et al., 2002). Lethality arising from SNTX is due to the marked hypotension that results from a potent vasorelaxant effect (Low et al., 1993). Abbreviations: L-NAME, L-N G-nitro arginine methyl ester; PAG, D, Lproparglyglycine; BCA, β-cyano-L-alanine; PE, L-phenylephrine; CSE, Cystathionine-γ-lyase; NOS, Nitric Oxide Synthase; ACh, Acetylcholine. ⁎ Corresponding author. DMERI, DSO National Laboratories, 27 Medical Drive, 117510, Singapore. E-mail address: [email protected] (S.M. Moochhala). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.058
Hydrogen sulfide (H2S) is a colorless and flammable gas with a strong odor of rotten eggs. It occurs naturally in crude petroleum, natural gas and volcanic gases and is produced from the bacterial breakdown of organic matter and from industrial activities associated with food processing, coke ovens, kraft paper mills, tanneries and petroleum refineries (US Environmental Protection Agency — Public health statement for H2S, 1999). Although H2S is associated with many industrial fatalities (Burnett et al., 1977; Guidotti, 1994), it is produced endogenously in mammalian cells during L-cysteine metabolism (Stipanuk and Beck, 1982; Stipanuk, 2004; Kamoun, 2004). In rat serum, circulating H2S concentration is ∼46 μM (Zhao et al., 2001). Relatively high levels of H2S (50–160 μM) have also been measured in brains of rat, human and cow (Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990). Rat ileum as well as different vascular tissues such as rat thoracic aorta and portal vein also generate measurable amounts of H2S (Zhao et al., 2001; Hosoki et al., 1997). In addition, H2S-generating enzymes have also been
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
detected in liver, kidney and heart (Stipanuk and Beck, 1982; De La Rosa et al., 1987; Nagahara et al., 1998). In this paper, we were interested to determine whether H2S may be involved in SNTX-induced vasorelaxation. Hosoki et al. (1997) first described the possible physiological role of H2S as a smooth muscle relaxant in the thoracic aorta. Since SNTX causes vasorelaxation, we investigated whether H2S may be a possible mediator of the effect of SNTX. In addition, since Hosoki et al. (1997) also reported that H2S acts synergistically with NO to cause vascular smooth muscle relaxation; we aimed to determine if H2S is involved in SNTX-induced vasorelaxation, and whether H2S acts synergistically with NO in this regard. Materials and methods
1665
The tension of the aortic rings was monitored with a force displacement transducer (ADInstruments model: MLT0201). The basal tone of the aortic ring was set at 1.0–1.5 g and rings were allowed to stabilize for 1 h prior to experimentation. The organ bath was maintained at 37 ± 1 °C throughout the entire experiment. Vasorelaxation studies After equilibration, aortic rings were exposed to 0.32 μM PE (EC85 from preliminary experiments) followed by 2.56 μM ACh (EC85 from preliminary experiments). 0.32 μM PEprecontracted aortic rings that exhibit greater or equal to 70% vasorelaxation with 2.56 μM ACh were termed as endotheliumintact aortic rings and used for inhibitor studies involving SNTX.
Purification of SNTX Data analysis SNTX was purified from crude S. horrida venom according to the method of Poh et al. (1991). Briefly, stonefish venom was extracted from the venom sac of the dorsal fin, lyophilized and stored at − 80 °C prior to purification. During purification, 253.6 mg lyophilized venom was reconstituted in 1.0 ml of 0.05 M sodium phosphate buffer, pH 7.4 (buffer A) and centrifuged at 12,000 g for 15 min to remove insoluble material. The supernatant was applied to a Sephacryl S-200 HR gel filtration column (1.6 × 100 cm) previously equilibrated with buffer A. Elution was carried out at a constant flow rate of 1 ml/ min and 3 ml fractions were collected. The absorbance of the fractions was monitored at OD280. Peak 1 fractions from Sephacryl S-200 HR gel filtration was pooled together and loaded onto a DEAE Bio-Gel A (100–200 mesh) gel column (1.2 × 8.8 cm) that was pre-equilibrated with 0.05 M sodium phosphate buffer, pH 7.4 (buffer B). The DEAE column was washed with 60 ml of buffer B before a linear sodium chloride gradient (0 to 0.15 M sodium chloride in 0.05 M sodium phosphate buffer pH 7.4, 150 ml) was initiated. 2.5 ml fractions were collected and their absorbance monitored at OD280. Peak 4 fractions from DEAE gel filtration containing SNTX were analyzed for protein concentrations before storage at − 80 °C. All protein-handling operations described were carried out at 4 °C.
The results are expressed as mean ± SE from which EC50 values were calculated. Student's one-tailed, unpaired t-test was
Tissue preparation for organ bath studies All experiments were undertaken in accordance with the Singaporean National Advisory Committee for Laboratory Animal Research (NACLAR) Guidelines on the responsible care and use of laboratory animals. Male Sprague–Dawley rats (250–300 g) were killed by cervical dislocation. The thoracic aorta was extracted and cut into rings of approximately 2 mm in length. Care was taken not to damage the endothelium during the entire process. Aortic rings were gently hooked to a string and a metal hook, and transferred to a 2.5 ml organ bath containing Krebs solution [(in mM): NaCl 118; KCl 4.8; KH2PO4 1.2; CaCl2 2.5; NaHCO3 25; MgSO4 2.4; D-(+)-glucose 11.0] aerated with 5% CO2:95% O2.
Fig. 1. Dose–response curves of PE and ACh on 2 mm rat thoracic aortic rings. (a) Vasoconstriction of 2 mm rat thoracic aortic rings by cumulative concentrations of PE. (b) Vasorelaxation of 0.32 μM PE-precontracted thoracic aortic rings to cumulative concentrations of ACh.
1666
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
used to compare the mean difference between control and tested groups.
Involvement of hydrogen sulfide (H2S) in SNTX-induced vasorelaxation
Results
SNTX induced concentration-dependent vasorelaxation of endothelium-intact, PE-precontracted thoracic aortic rings (logEC50 = 2.02 ± 0.004 ng/ml). Pretreatment of aortic rings with PAG (1 mM) for 1 h prior to vasorelaxation with SNTX, shifted the logEC50 of SNTX to the right i.e. logEC50 = 2.04 ± 0.003 ng/ml (p b 0.05) (Fig. 3a,c). Similar results were obtained with BCA (1 mM), with logEC50 of stonustoxin shifted to the right, i.e. 2.14 ± 0.003 ng/ml (p b 0.05) (Fig. 3b,c). In addition, the maximum vasorelaxation induced by SNTX was decreased from 49.1 ± 0.3% to 39.0 ± 0.1% in the presence of 1 mM PAG ( p b 0.05) (Table 1). In the presence of 1 mM BCA, the maximum vasorelaxation induced by SNTX was decreased to 38.8 ± 0.1% ( p b 0.05).
Organ bath studies Preliminary experiments were undertaken to show the effect of L-phenylephrine (PE) and acetylcholine (ACh) on 2 mm rat thoracic aortic rings (Fig. 1). Cumulative addition of PE led to progressive vasoconstriction (EC50 = 0.039 μM; EC85 = 0.32 μM, n = 12) (Fig. 1a). Addition of ACh cumulatively to PE-precontracted aortic rings resulted in progressive vasorelaxation (EC50 = 0.082 μM; EC85 = 2.56 μM, n = 12) (Fig. 1b). Involvement of NO in SNTX-induced vasorelaxation 10–320 ng/ml SNTX caused concentration-dependent vasorelaxation of PE-precontracted aortic rings (logEC50 = 2.02 ± 0.004 ng/ml; maximum vasorelaxation = 49.1 ± 0.3%, n = 9) (Fig. 2a). Preincubation of aortic rings with L-NAME (10 μM) for 1 h completely abolished the vasorelaxant effect of SNTX (Fig. 2b).
Synergy between H2S and NO in SNTX-induced vasorelaxation SNTX induced concentration-dependent vasorelaxation of endothelium-intact, PE-precontracted thoracic aortic rings (logEC50 = 2.02 ± 0.004 ng/ml). In the presence of L-NAME (1 μM), the logEC50 of SNTX was shifted to the right to
Fig. 2. Nitric oxide involvement in SNTX-induced vasorelaxation. (a) Dose–response curve of 0.32 μM PE-precontracted thoracic aortic rings to cumulative concentration of SNTX. (b) Experimental profiles showing the effect of saline control or 10 μM L-NAME on SNTX-induced vasorelaxation.
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
1667
Table 1 Effect of various enzyme inhibitors on the logEC 50 and maximum vasorelaxation induced by SNTX on endothelium-intact, 0.32 μM PEprecontracted thoracic aortic rings Experimental group
logEC50 (ng/ml)
Maximum vasorelaxation (%)
SNTX + Ctl SNTX + 1 μM L-NAME SNTX + 1 mM PAG SNTX + 1 mM PAG + 1 μM L-NAME SNTX + 1 mM BCA SNTX + 1 mM BCA + 1 μM L-NAME
2.022 ± 0.004342 2.255 ± 0.01073 2.041 ± 0.003128 NA 2.136 ± 0.003100 2.379 ± 0.006519
49.05 ± 0.2564 31.38 ± 0.5588 38.93 ± 0.1312 NA 38.81 ± 0.1333 25.62 ± 0.3006
logEC50 of SNTX was decreased to 2.38 ± 0.006 ng/ml (Fig. 4b) and the maximum vasorelaxation decreased to 25.6 ± 0.3% (Table 1). Discussion Purified SNTX (10–320 ng/ml) added cumulatively to endothelium-intact, PE-precontracted rat thoracic aortic rings
Fig. 3. Effect of PAG and BCA on SNTX-induced vasorelaxation. (a) Effect of 1 mM PAG on 0.32 μM PE-precontracted thoracic aortic rings. (b) Effect of 1 mM BCA on 0.32 μM PE-precontracted thoracic aortic rings. (c) Comparison of the effect of 1 mM PGA and 1 mM BCA on the vasorelaxation of precontracted aortic rings. Data are represented as mean ± SEM. ⁎p b 0.05 vs. saline control.
logEC50 = 2.26 ± 0.01 ng/ml (p b 0.05) (Fig. 4a) and the maximum vasorelaxation was decreased from 49.1 ± 0.26% to 31.4 ± 0.6% (p b 0.05) (Table 1). In the presence of PAG (1 mM) and L-NAME (1 μM), the vasorelaxant effect of SNTX on PEprecontracted aortic strips was completely abolished (Fig. 4a). In the presence of BCA (1 mM) and L-NAME (1 μM), the
Fig. 4. Synergism between H2S and NO in SNTX-induced vasorelaxation. (a) Synergistic inhibition by PAG and L-NAME on SNTX-induced vasorelaxation of endothelium-intact, 0.32 μM PE-precontracted thoracic aortic rings. (b) Synergistic inhibition by BCA and L-NAME on SNTX-induced vasorelaxation of endothelium-intact, 0.32 μM PE-precontracted thoracic aortic rings. Data are represented as mean ± SEM.
1668
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
progressively causes vasorelaxation, whose effect was inhibited by L-NAME indicating that nitric oxide (NO) is likely to be a mediator of this effect. This data is consistent with the findings of Low et al. (1993). To determine if H2S, a novel endogenous gasotransmitter, plays a role in vasorelaxation by SNTX, D, L-proparglyglycine (PAG) and β-cyano-L-alanine (BCA) were used in organ bath studies to inhibit cystathionineγ-lyase (CSE; EC 4.4.1.1) — the only H2S-generating enzyme that has been identified in vascular tissues to date (Hosoki et al., 1997; Zhao et al., 2001). PAG is an irreversible inhibitor of CSE (IC50 = 10− 4 M, Stipanuk and Beck, 1982; Uren et al., 1978) while BCA, a structural analogue of PAG, is a competitive inhibitor of CSE (IC50 = 10− 5 M, Uren et al., 1978; Pfeffer and Ressler, 1967). Preincubation of endothelium-intact, PEprecontracted thoracic aortic rings with PAG (1 mM) or BCA (1 mM) for 1 h prior to SNTX vasorelaxation of PE-precontracted aortic tissues resulted in inhibition of the vasorelaxant effect of SNTX and suggested that H2S is involved in SNTX-induced vasorelaxation. Since H2S is possibly a mediator of the vasorelaxant effect of SNTX, we were interested to determine if it works together with NO to bring about vasorelaxation. Both a CSE-inhibitor and nitric oxide synthase inhibitor were co-applied and the results showed that preincubation of the aortic rings with L-NAME in conjunction with either PAG or BCA led to an inhibition of SNTX-induced vasorelaxation. The inhibitory effect is much greater than when either inhibitor was used individually and suggests that H2S and NO may well work together to bring about SNTX-mediated vasorelaxation of precontracted aortic rings. Conclusion Stonustoxin (SNTX), a toxin purified from S. horrida venom causes vasorelaxation of precontracted rats’ aorta via the L-arginine–nitric oxide synthase pathway (Low et al., 1993; Poh et al., 1991). Hydrogen sulfide (H2S), a well known toxic gas associated with many industrial fatalities (Burnett et al., 1977; Guidotti, 1994) was found recently to be an endogenously generated smooth muscle relaxant that works in synergy with NO (Hosoki et al., 1997). In this paper, we show that H2S is an endogenous mediator of SNTX-induced vasorelaxation and that it appears to act synergistically with NO. To date, this is the first report which shows H2S working in concert with NO to mediate the biological effect of a toxin. References Burnett, W.W., King, E.G., Grace, M., Hall, W.F., 1977. Hydrogen sulfide poisoning: review of 5 years’ experience. Canadian Medical Association Journal 117 (11), 1277–1280.
Chen, D., Kini, R.M., Yuen, R., Khoo, H.E., 1997. Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues. Biochemistry Journal 325, 685–691. De La Rosa, J., Drake, M.R., Stipanuk, M.H., 1987. Metabolism of cysteine and cysteine sulfinate in rat and cat hepatocytes. Journal of Nutrition 117, 549–558. Goodwin, L.R., Francom, D., Dieken, F.P., Taylor, J.D., Warenycia, M.W., Reiffenstein, R.J., Dowling, G., 1989. Determination of sulfide in brain tissue by gas dialysis/ion chromatography: postmortem studies and two case reports. Journal of Analytical Toxicology 13, 105–109. Guidotti, T.L., 1994. Occupational exposure to hydrogen sulfide in the sour gas industry: some unresolved issues. International Archives of Occupational and Environmental Health 66 (3), 153–160. Hosoki, R., Matsiki, N., Kimura, H., 1997. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochemical and Biophysical Research Communications 237, 527–531. Kamoun, P., 2004. Endogenous production of hydrogen sulfide in mammals. Amino acids 26, 243–254. Khoo, H.E., Hon, W.M., Lee, S.H., Yuen, R., 1995. Effects of stonustoxin (lethal factor from Synanceja horrida venom) on platelet aggregation. Toxicon 33 (8), 1033–1041 Aug. Low, K.S.Y., Gwee, M.C.E., Yuen, R., Gopalakrishnakone, P., Khoo, H.E., 1993. Stonustoxin: a highly potent endothelium-dependent vasorelaxant in the rat. Toxicon 31, 1471–1478. Nagahara, N., Ito, T., Kitamura, H., Nishino, T., 1998. Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis. Histochemistry and Cell Biology 110, 243–250. Pfeffer, M., Ressler, C., 1967. Beta-cyanoalanine, an inhibitor of rat liver cystathionase. Biochemical Pharmacology 242, 2299–2308. Poh, C.H., Yuen, R., Khoo, H.E., Chung, M., Gwee, M., Gopalakrishnakone, P., 1991. Purification and partial characterization of stonustoxin from Synanceja horrida venom. Computational Biochemical Physiology 99B (4), 793–798. Savage, J.C., Gould, D.H., 1990. Determination of sulfide in brain tissue and rumen fluid by ion interaction, reversed phase high-performance liquid chromatography. Journal of Chromatography 526, 540–545. Stipanuk, M.H., 2004. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annual Review of Nutrition 24, 539–577. Stipanuk, M.H., Beck, P.W., 1982. Characterization of the enzymic capacity for cysteine desulphydration in liver and kidney of the rat. Biochemical Journal 206, 267–277. Sung, J.M.L., Low, K.S.Y., Khoo, H.E., 2002. Characterization of the mechanism underlying stonustoxin-mediated relaxant response in the rat aorta in vitro. Biochemical Pharmacology 7149, 1–6. Uren, J.R., Ragin, R., Chaykovsky, Y.M., 1978. Modulation of cysteine metabolism in mice — effects of proparglyglycine and L-cysteine-degrading enzymes. Biochemical Pharmacology 27, 2807–2814. Warenycia, M.W., Goodwin, L.R., Benishin, C.G., Reiffenstein, R.J., Francom, D.M., Taylor, J.D., Dieken, F.P., 1989. Acute hydrogen sulfide poisoning: demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels. Biochemical Pharmacology 38, 973–981. Zhao, W., Zhang, J., Lu, Y., Wang, R., 2001. The vasorelaxant effect of H2S as a novel endogenous KATP channel opener. European Molecular Biology Organization Journal 20, 6008–6016.
Synergism between hydrogen sulfide (H2S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom H.C. Liew a,c , H.E. Khoo a , P.K. Moore b , M. Bhatia b , J. Lu c , S.M. Moochhala b,c,⁎ a
Department of Biochemistry, National University of Singapore, Blk MD4A, 10 Kent Ridge Crescent, 119260, Singapore b Department of Pharmacology, National University of Singapore, Blk MD2, 18 Medical Drive, 119260, Singapore c DMERI, DSO National Laboratories, 27 Medical Drive, 117510, Singapore Received 7 July 2006; accepted 15 December 2006
Abstract Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10–320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-NG-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and β-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-γ-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H2S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H2S and NO act synergistically in effecting SNTX-induced vasorelaxation. © 2007 Elsevier Inc. All rights reserved. Keywords: Stonustoxin (SNTX); Synanceja horrida; Vasorelaxation; Hydrogen sulfide (H2S); Nitric oxide (NO)
Introduction Stonustoxin (Stonefish, National University of Singapore, SNTX) is a 148 kDa, dimeric, lethal protein factor isolated from the venom of the stonefish Synanceja horrida (Poh et al., 1991). It possesses a number of biological activities including speciesspecific haemolysis and platelet aggregation, edema-induction, hypotension, endothelium-dependent vasorelaxation, and inhibition of neuromuscular function in the mouse hemidiaphragm and chick biventer cervicis muscles (Low et al., 1993; Khoo et al., 1995; Chen et al., 1997; Sung et al., 2002). Lethality arising from SNTX is due to the marked hypotension that results from a potent vasorelaxant effect (Low et al., 1993). Abbreviations: L-NAME, L-N G-nitro arginine methyl ester; PAG, D, Lproparglyglycine; BCA, β-cyano-L-alanine; PE, L-phenylephrine; CSE, Cystathionine-γ-lyase; NOS, Nitric Oxide Synthase; ACh, Acetylcholine. ⁎ Corresponding author. DMERI, DSO National Laboratories, 27 Medical Drive, 117510, Singapore. E-mail address: [email protected] (S.M. Moochhala). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.058
Hydrogen sulfide (H2S) is a colorless and flammable gas with a strong odor of rotten eggs. It occurs naturally in crude petroleum, natural gas and volcanic gases and is produced from the bacterial breakdown of organic matter and from industrial activities associated with food processing, coke ovens, kraft paper mills, tanneries and petroleum refineries (US Environmental Protection Agency — Public health statement for H2S, 1999). Although H2S is associated with many industrial fatalities (Burnett et al., 1977; Guidotti, 1994), it is produced endogenously in mammalian cells during L-cysteine metabolism (Stipanuk and Beck, 1982; Stipanuk, 2004; Kamoun, 2004). In rat serum, circulating H2S concentration is ∼46 μM (Zhao et al., 2001). Relatively high levels of H2S (50–160 μM) have also been measured in brains of rat, human and cow (Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990). Rat ileum as well as different vascular tissues such as rat thoracic aorta and portal vein also generate measurable amounts of H2S (Zhao et al., 2001; Hosoki et al., 1997). In addition, H2S-generating enzymes have also been
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
detected in liver, kidney and heart (Stipanuk and Beck, 1982; De La Rosa et al., 1987; Nagahara et al., 1998). In this paper, we were interested to determine whether H2S may be involved in SNTX-induced vasorelaxation. Hosoki et al. (1997) first described the possible physiological role of H2S as a smooth muscle relaxant in the thoracic aorta. Since SNTX causes vasorelaxation, we investigated whether H2S may be a possible mediator of the effect of SNTX. In addition, since Hosoki et al. (1997) also reported that H2S acts synergistically with NO to cause vascular smooth muscle relaxation; we aimed to determine if H2S is involved in SNTX-induced vasorelaxation, and whether H2S acts synergistically with NO in this regard. Materials and methods
1665
The tension of the aortic rings was monitored with a force displacement transducer (ADInstruments model: MLT0201). The basal tone of the aortic ring was set at 1.0–1.5 g and rings were allowed to stabilize for 1 h prior to experimentation. The organ bath was maintained at 37 ± 1 °C throughout the entire experiment. Vasorelaxation studies After equilibration, aortic rings were exposed to 0.32 μM PE (EC85 from preliminary experiments) followed by 2.56 μM ACh (EC85 from preliminary experiments). 0.32 μM PEprecontracted aortic rings that exhibit greater or equal to 70% vasorelaxation with 2.56 μM ACh were termed as endotheliumintact aortic rings and used for inhibitor studies involving SNTX.
Purification of SNTX Data analysis SNTX was purified from crude S. horrida venom according to the method of Poh et al. (1991). Briefly, stonefish venom was extracted from the venom sac of the dorsal fin, lyophilized and stored at − 80 °C prior to purification. During purification, 253.6 mg lyophilized venom was reconstituted in 1.0 ml of 0.05 M sodium phosphate buffer, pH 7.4 (buffer A) and centrifuged at 12,000 g for 15 min to remove insoluble material. The supernatant was applied to a Sephacryl S-200 HR gel filtration column (1.6 × 100 cm) previously equilibrated with buffer A. Elution was carried out at a constant flow rate of 1 ml/ min and 3 ml fractions were collected. The absorbance of the fractions was monitored at OD280. Peak 1 fractions from Sephacryl S-200 HR gel filtration was pooled together and loaded onto a DEAE Bio-Gel A (100–200 mesh) gel column (1.2 × 8.8 cm) that was pre-equilibrated with 0.05 M sodium phosphate buffer, pH 7.4 (buffer B). The DEAE column was washed with 60 ml of buffer B before a linear sodium chloride gradient (0 to 0.15 M sodium chloride in 0.05 M sodium phosphate buffer pH 7.4, 150 ml) was initiated. 2.5 ml fractions were collected and their absorbance monitored at OD280. Peak 4 fractions from DEAE gel filtration containing SNTX were analyzed for protein concentrations before storage at − 80 °C. All protein-handling operations described were carried out at 4 °C.
The results are expressed as mean ± SE from which EC50 values were calculated. Student's one-tailed, unpaired t-test was
Tissue preparation for organ bath studies All experiments were undertaken in accordance with the Singaporean National Advisory Committee for Laboratory Animal Research (NACLAR) Guidelines on the responsible care and use of laboratory animals. Male Sprague–Dawley rats (250–300 g) were killed by cervical dislocation. The thoracic aorta was extracted and cut into rings of approximately 2 mm in length. Care was taken not to damage the endothelium during the entire process. Aortic rings were gently hooked to a string and a metal hook, and transferred to a 2.5 ml organ bath containing Krebs solution [(in mM): NaCl 118; KCl 4.8; KH2PO4 1.2; CaCl2 2.5; NaHCO3 25; MgSO4 2.4; D-(+)-glucose 11.0] aerated with 5% CO2:95% O2.
Fig. 1. Dose–response curves of PE and ACh on 2 mm rat thoracic aortic rings. (a) Vasoconstriction of 2 mm rat thoracic aortic rings by cumulative concentrations of PE. (b) Vasorelaxation of 0.32 μM PE-precontracted thoracic aortic rings to cumulative concentrations of ACh.
1666
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
used to compare the mean difference between control and tested groups.
Involvement of hydrogen sulfide (H2S) in SNTX-induced vasorelaxation
Results
SNTX induced concentration-dependent vasorelaxation of endothelium-intact, PE-precontracted thoracic aortic rings (logEC50 = 2.02 ± 0.004 ng/ml). Pretreatment of aortic rings with PAG (1 mM) for 1 h prior to vasorelaxation with SNTX, shifted the logEC50 of SNTX to the right i.e. logEC50 = 2.04 ± 0.003 ng/ml (p b 0.05) (Fig. 3a,c). Similar results were obtained with BCA (1 mM), with logEC50 of stonustoxin shifted to the right, i.e. 2.14 ± 0.003 ng/ml (p b 0.05) (Fig. 3b,c). In addition, the maximum vasorelaxation induced by SNTX was decreased from 49.1 ± 0.3% to 39.0 ± 0.1% in the presence of 1 mM PAG ( p b 0.05) (Table 1). In the presence of 1 mM BCA, the maximum vasorelaxation induced by SNTX was decreased to 38.8 ± 0.1% ( p b 0.05).
Organ bath studies Preliminary experiments were undertaken to show the effect of L-phenylephrine (PE) and acetylcholine (ACh) on 2 mm rat thoracic aortic rings (Fig. 1). Cumulative addition of PE led to progressive vasoconstriction (EC50 = 0.039 μM; EC85 = 0.32 μM, n = 12) (Fig. 1a). Addition of ACh cumulatively to PE-precontracted aortic rings resulted in progressive vasorelaxation (EC50 = 0.082 μM; EC85 = 2.56 μM, n = 12) (Fig. 1b). Involvement of NO in SNTX-induced vasorelaxation 10–320 ng/ml SNTX caused concentration-dependent vasorelaxation of PE-precontracted aortic rings (logEC50 = 2.02 ± 0.004 ng/ml; maximum vasorelaxation = 49.1 ± 0.3%, n = 9) (Fig. 2a). Preincubation of aortic rings with L-NAME (10 μM) for 1 h completely abolished the vasorelaxant effect of SNTX (Fig. 2b).
Synergy between H2S and NO in SNTX-induced vasorelaxation SNTX induced concentration-dependent vasorelaxation of endothelium-intact, PE-precontracted thoracic aortic rings (logEC50 = 2.02 ± 0.004 ng/ml). In the presence of L-NAME (1 μM), the logEC50 of SNTX was shifted to the right to
Fig. 2. Nitric oxide involvement in SNTX-induced vasorelaxation. (a) Dose–response curve of 0.32 μM PE-precontracted thoracic aortic rings to cumulative concentration of SNTX. (b) Experimental profiles showing the effect of saline control or 10 μM L-NAME on SNTX-induced vasorelaxation.
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
1667
Table 1 Effect of various enzyme inhibitors on the logEC 50 and maximum vasorelaxation induced by SNTX on endothelium-intact, 0.32 μM PEprecontracted thoracic aortic rings Experimental group
logEC50 (ng/ml)
Maximum vasorelaxation (%)
SNTX + Ctl SNTX + 1 μM L-NAME SNTX + 1 mM PAG SNTX + 1 mM PAG + 1 μM L-NAME SNTX + 1 mM BCA SNTX + 1 mM BCA + 1 μM L-NAME
2.022 ± 0.004342 2.255 ± 0.01073 2.041 ± 0.003128 NA 2.136 ± 0.003100 2.379 ± 0.006519
49.05 ± 0.2564 31.38 ± 0.5588 38.93 ± 0.1312 NA 38.81 ± 0.1333 25.62 ± 0.3006
logEC50 of SNTX was decreased to 2.38 ± 0.006 ng/ml (Fig. 4b) and the maximum vasorelaxation decreased to 25.6 ± 0.3% (Table 1). Discussion Purified SNTX (10–320 ng/ml) added cumulatively to endothelium-intact, PE-precontracted rat thoracic aortic rings
Fig. 3. Effect of PAG and BCA on SNTX-induced vasorelaxation. (a) Effect of 1 mM PAG on 0.32 μM PE-precontracted thoracic aortic rings. (b) Effect of 1 mM BCA on 0.32 μM PE-precontracted thoracic aortic rings. (c) Comparison of the effect of 1 mM PGA and 1 mM BCA on the vasorelaxation of precontracted aortic rings. Data are represented as mean ± SEM. ⁎p b 0.05 vs. saline control.
logEC50 = 2.26 ± 0.01 ng/ml (p b 0.05) (Fig. 4a) and the maximum vasorelaxation was decreased from 49.1 ± 0.26% to 31.4 ± 0.6% (p b 0.05) (Table 1). In the presence of PAG (1 mM) and L-NAME (1 μM), the vasorelaxant effect of SNTX on PEprecontracted aortic strips was completely abolished (Fig. 4a). In the presence of BCA (1 mM) and L-NAME (1 μM), the
Fig. 4. Synergism between H2S and NO in SNTX-induced vasorelaxation. (a) Synergistic inhibition by PAG and L-NAME on SNTX-induced vasorelaxation of endothelium-intact, 0.32 μM PE-precontracted thoracic aortic rings. (b) Synergistic inhibition by BCA and L-NAME on SNTX-induced vasorelaxation of endothelium-intact, 0.32 μM PE-precontracted thoracic aortic rings. Data are represented as mean ± SEM.
1668
H.C. Liew et al. / Life Sciences 80 (2007) 1664–1668
progressively causes vasorelaxation, whose effect was inhibited by L-NAME indicating that nitric oxide (NO) is likely to be a mediator of this effect. This data is consistent with the findings of Low et al. (1993). To determine if H2S, a novel endogenous gasotransmitter, plays a role in vasorelaxation by SNTX, D, L-proparglyglycine (PAG) and β-cyano-L-alanine (BCA) were used in organ bath studies to inhibit cystathionineγ-lyase (CSE; EC 4.4.1.1) — the only H2S-generating enzyme that has been identified in vascular tissues to date (Hosoki et al., 1997; Zhao et al., 2001). PAG is an irreversible inhibitor of CSE (IC50 = 10− 4 M, Stipanuk and Beck, 1982; Uren et al., 1978) while BCA, a structural analogue of PAG, is a competitive inhibitor of CSE (IC50 = 10− 5 M, Uren et al., 1978; Pfeffer and Ressler, 1967). Preincubation of endothelium-intact, PEprecontracted thoracic aortic rings with PAG (1 mM) or BCA (1 mM) for 1 h prior to SNTX vasorelaxation of PE-precontracted aortic tissues resulted in inhibition of the vasorelaxant effect of SNTX and suggested that H2S is involved in SNTX-induced vasorelaxation. Since H2S is possibly a mediator of the vasorelaxant effect of SNTX, we were interested to determine if it works together with NO to bring about vasorelaxation. Both a CSE-inhibitor and nitric oxide synthase inhibitor were co-applied and the results showed that preincubation of the aortic rings with L-NAME in conjunction with either PAG or BCA led to an inhibition of SNTX-induced vasorelaxation. The inhibitory effect is much greater than when either inhibitor was used individually and suggests that H2S and NO may well work together to bring about SNTX-mediated vasorelaxation of precontracted aortic rings. Conclusion Stonustoxin (SNTX), a toxin purified from S. horrida venom causes vasorelaxation of precontracted rats’ aorta via the L-arginine–nitric oxide synthase pathway (Low et al., 1993; Poh et al., 1991). Hydrogen sulfide (H2S), a well known toxic gas associated with many industrial fatalities (Burnett et al., 1977; Guidotti, 1994) was found recently to be an endogenously generated smooth muscle relaxant that works in synergy with NO (Hosoki et al., 1997). In this paper, we show that H2S is an endogenous mediator of SNTX-induced vasorelaxation and that it appears to act synergistically with NO. To date, this is the first report which shows H2S working in concert with NO to mediate the biological effect of a toxin. References Burnett, W.W., King, E.G., Grace, M., Hall, W.F., 1977. Hydrogen sulfide poisoning: review of 5 years’ experience. Canadian Medical Association Journal 117 (11), 1277–1280.
Chen, D., Kini, R.M., Yuen, R., Khoo, H.E., 1997. Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues. Biochemistry Journal 325, 685–691. De La Rosa, J., Drake, M.R., Stipanuk, M.H., 1987. Metabolism of cysteine and cysteine sulfinate in rat and cat hepatocytes. Journal of Nutrition 117, 549–558. Goodwin, L.R., Francom, D., Dieken, F.P., Taylor, J.D., Warenycia, M.W., Reiffenstein, R.J., Dowling, G., 1989. Determination of sulfide in brain tissue by gas dialysis/ion chromatography: postmortem studies and two case reports. Journal of Analytical Toxicology 13, 105–109. Guidotti, T.L., 1994. Occupational exposure to hydrogen sulfide in the sour gas industry: some unresolved issues. International Archives of Occupational and Environmental Health 66 (3), 153–160. Hosoki, R., Matsiki, N., Kimura, H., 1997. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochemical and Biophysical Research Communications 237, 527–531. Kamoun, P., 2004. Endogenous production of hydrogen sulfide in mammals. Amino acids 26, 243–254. Khoo, H.E., Hon, W.M., Lee, S.H., Yuen, R., 1995. Effects of stonustoxin (lethal factor from Synanceja horrida venom) on platelet aggregation. Toxicon 33 (8), 1033–1041 Aug. Low, K.S.Y., Gwee, M.C.E., Yuen, R., Gopalakrishnakone, P., Khoo, H.E., 1993. Stonustoxin: a highly potent endothelium-dependent vasorelaxant in the rat. Toxicon 31, 1471–1478. Nagahara, N., Ito, T., Kitamura, H., Nishino, T., 1998. Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis. Histochemistry and Cell Biology 110, 243–250. Pfeffer, M., Ressler, C., 1967. Beta-cyanoalanine, an inhibitor of rat liver cystathionase. Biochemical Pharmacology 242, 2299–2308. Poh, C.H., Yuen, R., Khoo, H.E., Chung, M., Gwee, M., Gopalakrishnakone, P., 1991. Purification and partial characterization of stonustoxin from Synanceja horrida venom. Computational Biochemical Physiology 99B (4), 793–798. Savage, J.C., Gould, D.H., 1990. Determination of sulfide in brain tissue and rumen fluid by ion interaction, reversed phase high-performance liquid chromatography. Journal of Chromatography 526, 540–545. Stipanuk, M.H., 2004. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annual Review of Nutrition 24, 539–577. Stipanuk, M.H., Beck, P.W., 1982. Characterization of the enzymic capacity for cysteine desulphydration in liver and kidney of the rat. Biochemical Journal 206, 267–277. Sung, J.M.L., Low, K.S.Y., Khoo, H.E., 2002. Characterization of the mechanism underlying stonustoxin-mediated relaxant response in the rat aorta in vitro. Biochemical Pharmacology 7149, 1–6. Uren, J.R., Ragin, R., Chaykovsky, Y.M., 1978. Modulation of cysteine metabolism in mice — effects of proparglyglycine and L-cysteine-degrading enzymes. Biochemical Pharmacology 27, 2807–2814. Warenycia, M.W., Goodwin, L.R., Benishin, C.G., Reiffenstein, R.J., Francom, D.M., Taylor, J.D., Dieken, F.P., 1989. Acute hydrogen sulfide poisoning: demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels. Biochemical Pharmacology 38, 973–981. Zhao, W., Zhang, J., Lu, Y., Wang, R., 2001. The vasorelaxant effect of H2S as a novel endogenous KATP channel opener. European Molecular Biology Organization Journal 20, 6008–6016.