Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide q Matthew Whiteman a, * , Ling Li b , Iouri Kostetski c , Siew Hwa Chu a , Jia Ling Siau a , Madhav Bhatia b , Philip K. Moore b a Cardiovascular Biology Research Group, Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore 117597 b Cardiovascular Biology Research Group, Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore 117597 c Division of Bioengineering, Faculty of Engineering, National University of Singapore, Republic of Singapore 119260 Received 23 February 2006 Available online 6 March 2006 Abstract The gaseous mediators hydrogen sulphide (H 2 S) and nitric oxide ( Å NO) are synthesised in the body from L-cysteine and L-arginine, respectively. In the cardiovascular system, Å NO is an important regulator of vascular tone and its over- or under-production has been linked to a variety of diseases. The physiological significance of H 2 S is not yet clear but, like Å NO, it exhibits vasodilator activity and may play a part in septic and haemorrhagic shock, hypertension, regulation of cardiac contractility, and in inflammation. To date, there have been no reports of a chemical interaction between H 2 S and Å NO. Here we show that incubation of the H 2 S donor, sodium hydro- sulphide, with a range of Å NO donors and Å NO gas in vitro leads to the formation of a nitrosothiol molecule as determined by a com- bination of techniques; electron paramagnetic resonance, amperometry, and measurement of nitrite. We further show that this nitrosothiol did not induce cGMP accumulation in cultured RAW264.7 cells unless Å NO was released with Cu 2+ . Finally, using liver homogenates from LPS treated rats we present evidence for the endogenous formation of this nitrosothiol. These findings provide the first evidence for the formation of a novel nitrosothiol generated by reaction between H 2 S and Å NO. We propose that generation of this nitrosothiol in the body may regulate the physiological effects of both Å NO and H 2 S. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Reactive oxygen species; Hydrogen sulphide; Nitric oxide; Peroxynitrite; Cysthionine-b-synthase; Cystathionine-c-synthase H 2 S is a well-known and pungent toxic gas and its toxicol- ogy has been extensively studied (thoroughly reviewed in [1]). It is produced endogenously in mammalian tissues from the amino acids cysteine and homocysteine by pyridoxal-5 0 - phosphate-dependent enzymes such as cystathionine-c-lyase (CSE; E.C. 4.4.1.1) and cystathionine-b-synthetase (CBS; E.C. 4.2.1.22) (reviewed in [2,3]). CBS converts homocyste- ine to cystathionine and hydrolyses cysteine to equimolar amounts of serine and H 2 S whereas CSE converts cystathio- nine to cysteine yielding pyruvate, NH 3 , and H 2 S. H 2 S may also be formed in vivo from the enzymatic desulphura- tion of b-mercaptopyruvate derived from cysteine transam- ination [4]. Since the active form of H 2 S (H 2 S, HS  or S 2 ) in vivo has yet to be determined, the term ‘hydrogen sulphide’ has been used to encompass these three species. In sharp contrast to other gaseous mediators (i.e., nitric oxide and carbon monoxide; reviewed [5,6]), very little 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.02.154 q Abbreviations: CBS, cysthionine-b-synthase; CSE, cystathionine-c-syn- thase; cGMP, cyclic guanosine monophosphate; DMEM, Dulbecco’s modified Eagle’s medium; DD1, 3-bromo-3,4,4-trimethyl-3,4-dihydrodiazete 1,2-dioxide; DEA-NONOate, 2-(N,N-diethylamino)-diazenolate-2-oxide- diethylammonium salt; DETA-NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2- ammonioethyl)amino]diazen-1-ium-1,2-diolate; EPR, electron paramagnetic resonance; H 2 S, hydrogen sulphide; HgCl 2 , mercuric chloride; LPS, lipopolysaccharide; Å NO, nitric oxide; NO 2  , nitrite; ONOO  , peroxyni- trite; NaHS, sodium hydrosulphide; SNP, sodium nitroprusside; SIN-1, 3-morpholinosydnonimine. * Corresponding author. E-mail address: bchwml@nus.edu.sg (M. Whiteman). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 343 (2006) 303–310 BBRC