Mark D. Travis

Hemodynamic Effects of l- and d-S-Nitrosocysteine in the Rat Stereoselective S-Nitrosothiol Recognition Sites

The vasorelaxant effects of the endothelium-derived relaxing factor S-nitrosocysteine (SNC) may not be simply due to its decomposition to NO. The biological actions of SNC may also involve the transnitrosation of amino acids in the blood and in plasma membranes. The possibility that the SNC moiety possesses biological activity prompted us to examine whether the hemodynamic effects of this S-nitrosothiol involves the activation of stereoselective S-nitrosothiol receptors within the cardiovascular system. We examined (1) the hemodynamic effects produced by intravenous injections of the L and D isomers of SNC (L- and D-SNC, respectively; 100 to 800 nmol/kg), the L and D isomers of the parent thiols (L- and D-cysteine, respectively; 100 to 800 nmol/kg), the oxidized thiol L-cystine (100 to 800 nmol/kg), and the NO donor sodium nitroprusside (SNP, 1 to 36 micrograms/kg) in conscious freely moving rats, (2) the baroreceptor reflex-mediated changes in heart rate elicited in response to the falls in arterial pressure produced by L- and D-SNC and SNP in conscious rats, and (3) the relative decomposition of L- and D-SNC to NO upon addition to heparinized rat blood or upon direct application to cultured porcine aortic smooth muscle (PASM) cells. We now report that (1) L-SNC is a more potent hypotensive and vasodilator agent within the mesenteric bed and sympathetically intact and sympathetically denervated hindlimb beds of conscious rats than is D-SNC, (2) L- and D-SNC markedly inhibit baroreceptor reflex-mediated tachycardia in conscious rats and D-SNC is considerably more effective than L-SNC, (3) the intravenous injections of L- and D-cysteine or L-cystine do not affect arterial blood pressure or vascular resistances, and (4) L- and D-SNC decompose equally to NO upon application to rat blood or cultured PASM cells. These results suggest that the hemodynamic effects of endogenous SNC may involve its interaction with stereoselective S-nitrosothiol recognition sites within the vasculature and the baroreflex arc. These findings provide tentative evidence that membrane-bound S-nitrosothiol receptors may exist within the cardiovascular system.

l- and d-S-nitroso-β,β-dimethylcysteine differentially increase cGMP in cultured vascular smooth muscle cells

Abstract We examined the effects of the l - and d -isomers of S -nitroso- β , β -dimethylcysteine ( l - and d - S -nitrosopenicillamine, 10 −7 –10 −5 M) on the guanosine 3′,5′-cyclic monophosphate (cGMP) content of cultured porcine aortic smooth muscle cells and the decomposition of these stereoisomers to nitric oxide (NO). l - S -nitrosopenicillamine was a more potent generator of cGMP than d - S -nitrosopenicillamine although both stereoisomers equally decomposed to NO. The 10 −7 M concentration of l - or d - S -nitrosopenicillamine did not generate detectable amounts of NO although 10 −7 M l - S -nitrosopenicillamine but not d - S -nitrosopenicillamine generated significant amounts of cGMP. This study shows that the stereoisomeric configuration of S -nitrosopenicillamine is an important factor in its biological potency. The data suggest that the extracellular or intracellular generation of NO is not the only mechanism by which this S -nitrosothiol generates cGMP in vascular smooth muscle.

Blockade of voltage-sensitive Ca2+-channels markedly diminishes nitric oxide- but not l-S-nitrosocysteine- or endothelium-dependent vasodilation in vivo

The aim of this study was to determine the hemodynamic responses elicited by systemic injections of (i) the nitric oxide (NO)-donors, sodium nitroprusside (10 nmol/kg, i.v.) and (Z)-1-(N-methyl-N-(6(N-methylammoniohexyl)amino))diazen-1-ium-1, 2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.), (ii) the endothelium-derived S-nitrosothiol, L-S-nitrosocysteine (100 nmol/kg, i.v.), and (iii) the endothelium-dependent agonist, acetylcholine (1.0 microg/kg, i.v.), in anesthetized rats, before and after injection of the voltage-sensitive Ca(2+)-channel (Ca(VS)(2+)-channel) blocker, nifedipine (500 nmol/kg, i.v.). Before injection of nifedipine, the agents produced similar falls in mean arterial blood pressure, and in hindquarter and mesenteric vascular resistances. The depressor and vasodilator responses elicited by sodium nitroprusside and MAHMA NONOate were markedly attenuated by nifedipine. The falls in mean arterial blood pressure and mesenteric resistance elicited by L-S-nitrosocysteine and acetylcholine were not attenuated but the falls in hindquarter resistance were slightly attenuated by nifedipine. The cyclooxygenase inhibitor, indomethacin (10 mg/kg, i.v.), did not affect the actions of sodium nitroprusside, MAHMA NONOate, L-S-nitrosocysteine or acetylcholine or the effects of nifedipine on the hemodynamic actions of these compounds. The decomposition of sodium nitroprusside (0.2 nmol/ml), MAHMA NONOate (0.5 nmol/ml) and L-S-nitrosocysteine (2 nmol/ml) to NO upon addition to rat blood was not affected by nifedipine (10 microM). These findings suggest that (i) exogenously applied NO relaxes resistance arteries in vivo by inhibition of Ca(VS)(2+)-channels whereas L-S-nitrosocysteine and the non-prostanoid endothelium-derived relaxing factor (EDRF) released by acetylcholine acts by additional mechanisms, and (ii) this EDRF may be an S-nitrosothiol which acts independently of its decomposition to NO.

Stereoselective S-nitrosocysteine recognition sites in rat brain

We examined the effects of intracisternal (i.c.) injections (10-250 nmol) of the L- and D-isomers of S-nitrosocysteine (L- and D-S-nitrosocysteine) on the mean arterial blood pressure and heart rate of conscious rats, and the decomposition of L- and D-S-nitrosocysteine to nitric oxide (NO) upon addition to brain homogenates. The i.c. injection of L-S-nitrosocysteine produced initial falls in mean arterial blood pressure and heart rate which were followed by increases in these parameters. The i.c. injection of D-S-nitrosocysteine did not produce initial falls in mean arterial blood pressure or heart rate but produced the subsequent increases in these parameters. L- and D-S-nitrosocysteine decomposed equally to NO. These results suggest that the initial effects of L-S-nitrosocysteine may be due to the activation of stereoselective recognition sites on brain neurons.

Heterologous desensitization of β-adrenoceptor signal transduction in vivo

Abstract The hemodynamic actions of pituitary adenylate cyclase-activating polypeptide (PACAP-27) rapidly diminish upon repeated i.v. injection in rats treated with the nitric oxide synthase inhibitor, N G -nitro- l -arginine methyl ester ( l -NAME). We now report that the β-adrenoceptor agonist isoproterenol (0.5 μg/kg, i.v.) produces pronounced hypotensive and vasodilator effects in anesthetized rats pretreated with l -NAME (100 μmol/kg, i.v.). However, the hypotensive and vasodilator actions of isoproterenol were markedly diminished in l -NAME-treated rats in which tachyphylaxis to PACAP was induced immediately prior to the injection of the β-adrenoceptor agonist. This suggests that a reduction in tissue concentrations of nitric oxide-containing factors allows tachyphylaxis to PACAP-27-mediated vasodilation to occur in vivo and that this process leads to the heterologous desensitization of β-adrenoceptor-mediated responses.

Apparent association of MK-801-sensitive ion channels with l-S-nitrosocysteine recognition sites in the hindlimb vasculature of the rat

This study demonstrates that the decreases in hindquarter vascular resistance produced by the putative endothelium-derived S-nitrosothiol, L-S-nitrosocysteine, in urethane-anesthetized rats, were attenuated by a lower dose of the N-methyl-D-aspartate (NMDA) receptor ion-channel blocker, dizocilpine (MK-801, 200 microg/kg, i.v.), whereas they were augmented by a higher dose of dizocilpine (500 microg/kg, i.v.). In contrast, L-S-nitrosocysteine-induced decreases in mesenteric vascular resistance were not affected by either dose of dizocilpine. The vasodilator actions of L-S-nitrosocysteine in these beds were not affected by the competitive NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (2-AP5). The vasodilator actions of the nitric oxide (NO) donor, (Z)-1-mid R:N-methyl-N-[6(N-methylammoniohexyl)amino]mid R:diazen-1-ium-1,2-diolate (MAHMA NONOate), in these beds were not affected by dizocilpine or by 2-AP5. These findings suggest that L-S-nitrosocysteine recognition sites in hindquarter but not mesenteric beds may be associated with dizocilpine-sensitive ion-channels similar to those in NMDA receptors.