An investigation of some S‐nitrosothiols, and of hydroxy‐arginine, on the mouse anococcygeus

Abstract

1 The effect of five S-nitrosothiols, and of the stereoisomers of N0-hydroxy-arginine (HOARG), were investigated on the mouse anococcygeus. 2 All five S-nitrosothiols produced concentration-related (0.1-100IM) relaxations of carbachol (50 LM)-induced tone; the order of potency was S-nitroso-L-cysteine (CYSNO)> S-nitroso-N-acetyl-D,Lpenicillamine (SNAP)>S-nitrosoglutathione (GSNO)> S-nitrosocoenzyme A (CoASNO)> S-nitroso-N-acetyl-L-cysteine (NACNO). The relaxations were unaffected by the nitric oxide synthase (NOS) inhibitor, L-NG-nitro-arginine (10 JIM) (L-NOARG).3 Cold-storage of the tissue for 72 h resulted in loss of sympathetic and non-adrenergic, noncholinergic (NANC) nerve function. NOS activity in the tissue was reduced by 97%. Despite this, relaxations induced by the S-nitrosothiols were unaffected. 4 Haemoglobin (50 jM) attenuated relaxations induced by NO and the S-nitrosothiols, although responses to 3-isobutyl-1-methyl-xanthine were unaffected. N-methyl-hydroxylamine (2 mM) which has been shown previously to produce selective inhibition of NANC and nitrovasodilator responses in this tissue, also reduced responses to all S-nitrosothiols.5 Hydroquinone (100 gM) greatly reduced relaxations to CYSNO (by 88%) but had no effect on those to SNAP, GSNO, CoASNO or NACNO. Since hydroquinone does not reduce responses to NANC stimulation, CYSNO is unlikely to be the NANC transmitter. 6 L-HOARG by itself (up to 100 piM) had no significant effect on carbachol-induced tone or on NANC (10 Hz; 10 s train every 100 s) relaxations. However, it produced reversal of the inhibitory effects of L-NOARG (10;pM), being only slightly less potent than L-arginine. D-HOARG was without effect. L-HOARG had no effect on relaxations induced by 1.51iM NO. 7 The results show that S-nitrosothiols are potent relaxants of the mouse anococcygeus; they act directly on the smooth muscle with a mechanism similar to NO and other nitrovasodilators. In addition, the results are consistent with L-HOARG being an intermediate in the biosynthesis of NO from L-arginine, although there is no evidence for it acting to stabilize NO extracellularly.

Inhibition of relaxations to nitrergic stimulation of the mouse anococcygeus by duroquinone

“…One of the objects of the study was to compare the effects of DQ with those of HQ, which has been shown previously to differentiate between NO-and field stimulation-induced relaxations of the mouse anococcygeus (Hobbs et al, 1991;Gibson et al, 1992). Unlike DQ, HQ still failed to inhibit nitrergic relaxations after DETCA treatment.…”

“…NO solutions were prepared as described previously (Gibson & Mirzazadeh, 1989). Drugs used were (from Sigma unless stated otherwise): 1,4-benzoquinone (Aldrich), 8-bromo-cyclic guanosine monophosphate, carbachol (BDH), diethyldithiocarbamic acid, duroquinone (Aldrich), guanethidine sulphate, hydroquinone, lucigenin (bis-methylacridinium nitrate), nitric oxide (99%, BDH), S-nitrosoglutathione (prepared as described previously, Gibson et al, 1992), phentolamine HC1, sodium nitroprusside, superoxide dismutase (copper/zinc containing enzyme from bovine erythrocytes), xanthine, xanthine oxidase (from buttermilk).…”

“…However, several aspects of this novel (nitrergic) neurotransmission system await full elucidation, including the observation that a number of drugs are potent inhibitors of relaxations induced by exogenous NO, but have little effect on relaxation due to nitrergic nerve stimulation; these drugs include hydroquinone (HQ, Hobbs et al, 1991;Gibson et al, 1992), hydroxocobalamin (Rand & Li, 1993), pyrogallol and LY83583 (Gillespie & Sheng, 1990;Barbier & Lefebvre, 1992;Knudsen et al, 1992;Liu et al, 1994). Such results have inspired a number of possible explanations (for reviews see Gibson et al, 1995;Rand & Li, 1995): these are: (1) the NO produced in the nerve terminal may be released in a modified form, perhaps attached to a carrier molecule; (2) the rapid rate of diffusion of NO released from a point source (Wood & Garthwaite, 1994) may render it resistant to the actions of scavenger molecules, at least over short distances; and (3) NO may indeed be released as a free radical, but may be protected by other substances which preferentially interact with potential NO-scavengers.…”

Photon pharmacology of an iron‐sulphur cluster nitrosyl compound acting on smooth muscle

“…Oxyhaemoglobin was freshly prepared by treatment of bovine haemoglobin with a ten fold excess of sodium dithionite followed by dialysis as described by Gibson et al (1992) and Gao & Vanhoutte (1993).…”

l-Arginine protects human heart cells from low-volume anoxia and reoxygenation

“…Confluent plates were used, and NO production was measured in extracellular fluid collected at the end of each of the stabilization and reoxygenation steps (at 30 and 150 min for the normoxic controls). NO production was not measured for SNAP treatment groups; it is well established that SNAP produces NO independently of cellular metabolism (6,12,14,29,36).…”