Carbon monoxide (CO), a vasodilator, has been implicated as an activator of soluble guanylyl cyclase (sGC) to effect smooth muscle relaxation; however, this idea has not received universal support. The purpose of this study was to examine the effects of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) on relaxation of rabbit aortic rings (RARs) induced by CO. Administration of 10 microM ODQ completely abolished relaxation of RARs by CO (30 microM), whereas only a partial attenuation of NO-induced relaxation was achieved by the same concentration of ODQ. The results of this study suggest that CO-mediated relaxation of RARs is mediated by sGC and indicate that ODQ may serve as a useful tool in the investigation of the actions of CO. Furthermore, these observations support the idea that ODQ is less potent in inhibiting relaxations by NO, thereby implicating a component of NO-induced relaxation that is independent of sGC/cGMP.

No abstract

Previously, we reported on the antagonism by pyocyanin (PYO) of the relaxant effects of nitrovasodilators such as glyceryl trinitrate, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1). The purpose of the present study was to elucidate the mechanism of this action of PYO by examining its effect on the steps considered to be necessary for nitrovasodilator-induced relaxation of blood vessels. PYO (10 microM) attenuated the accumulation of guanosine-3',5'-cyclic monophosphate (cGMP) in rabbit aorta induced by nitrovasodilators SIN-1, SNAP, and GTN, 65, 81, and 67%, respectively. Additionally, PYO (1 or 10 microM) interfered with in vitro activation of soluble guanylyl cyclase. PYO did not inhibit vascular relaxation induced by 8-bromo-cyclic guanosine monophosphate. PYO (10 microM) also decreased the quantity of nitric oxide measured in the headspace above intact vascular tissue incubated with glyceryl trinitrate in the presence of oxygen. These observations are consistent with the interpretation that PYO interfered with the nitrovasodilator action of glyceryl trinitrate by inactivation of NO or by inhibition of enzymatic biotransformation of GTN; this would result in decreased guanylyl cyclase activation and thus lowering cellular levels of cGMP. NO chemiluminescence studies with SIN-1 (10 microM) revealed that this NO donor produced NO in a time-dependent manner and PYO (10 microM) caused no inhibition of NO production, but in fact, potentiated NO release after 10 min of incubation (1395 +/- 179 pmol NO compared with 1088 +/- 154 pmol NO). NO production from 10 microM SNAP was similarly potentiated by PYO after 0.5, 2, 5, and 10 min of incubation. Therefore, it is likely that PYO acts as an inhibitor of guanylyl cyclase with respect to NO donors, SIN-1 and SNAP, but it also appears that PYO can exert additional inhibitory effects in the case of vascular relaxation by GTN. Such differences in relaxant effects may reflect inhibition of enzymatic biotransformation that is unique to GTN or that PYO may complex with an alternative redox form of NO (perhaps NO+) that is generated by vascular metabolic activation of GTN.

1 The purpose of the present study was to assay NO formation from GTN biotransformation by the rabbit aortic strip (RAS) at times concurrent with its vasorelaxation. Such an assay is an important test of the prodrug hypothesis where it is postulated that glyceryl trinitrate (GTN) is biotransformed to nitric oxide (NO), the active species that initiates vascular smooth muscle relaxation. To test such a hypothesis, we propose that a sample of smooth muscle, poorly responsive to GTN, yet sensitive to the effects of NO could be used to detect RAS production of NO from GTN. 2 Muscle strips of rabbit taenia coli (RTCS) and RAS in close apposition, were mounted in tissue baths, and muscle relaxation was recorded with isometric force transducers. Tissues were submaximally precontracted with 30-35mM K+ depolarizing solution and exposed to increasing concentrations of GTN (0.1 nM-l0IM).3 EC25 for GTN-induced relaxation of RTCS in the presence of RAS was significantly decreased to that for RTCS in the absence of RAS (5.9 ± 3.0 X 10-8 M and 5.5 ± 3.7 X 10-6 M, respectively). Mean maximal levels of GTN-induced relaxation of similarly precontracted RTCS also differed in the presence and absence of RAS, viz., 80.8 ± 2.1% and 29.8 ± 8.3% respectively. 4 RTCS was found to relax upon administration of NO gas bubbled through the incubation medium. Analysis of tissue bath medium revealed that the NO concentration to which RTCS was exposed attained a maximum of 33 nM. Relaxation of RTCS by NO gas was inhibited by 1 gM reduced haemoglobin. 5 For GTN-incubation with intestinal and vascular smooth muscle preparations, NO formation was greater with RAS compared to RTCS. Thus, in the two-issue bioassay, the RAS was the predominant source of NO formation from GTN. 6 Reduced deoxyhaemoglobin (1 pM), a potent extracellular NO scavenger, was found to decrease the augmented GTN-induced relaxation in the RTCS-RAS sandwich preparation from 17.3 ± 1.8% to 8.0 ± 0.8%. The . This proposed prodrug hypothesis of GTN-induced vasorelaxation involves denitration of GTN to glyceryl-1,2-dinitrate and /or glyceryl-1,3-dinitrate with the attendant formation of the free radical NO. It is believed that NO activates soluble guanylyl cyclase (Diamond & Holmes, 1975;Katsuki et al., 1977;Murad et al., 1978;Gruetter et al., 1981), thereby elevating guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels and lowering intracellular free Ca2" concentration, which results in smooth muscle relaxa-'Author for Correspondence. tion. Although the actual bioactivation pathway of GTN to NO remains uncertain, several hypotheses have been put forward. These include enzymatic metabolism by cytosolic glutathione-S-transferases (Needleham et al., 1969;Hill et al., 1992), non-enzymatic formation of NO by thiol-containing compounds such as cysteine (Feelisch & Noack, 1987) and haemoprotein-mediated GTN biotransformation Marks et al., 1989).Because of the uncertainty surrounding metabolic activation of GTN, substantial research has been conducted to test the original prodrug...

In the present study, the role of thiols on glyceryl trinitrate (GTN) induced relaxation of rabbit taenia coli strips (RTCS) was investigated. This study was designed to test the hypothesis that a deficiency in thiols is responsible for RTCS insensitivity to GTN, and thus thiols play a key role in the enzymatic activation of GTN. Isolated RTCS bathed in normothermic, oxygenated Krebs solution were pretreated with the thiols L-cysteine (5 mM) and N-acetyl-L-cysteine (NAC, 5 mM) for 30 min and washed. The effects of GTN were determined by changes in isometric tension of K(+)-precontracted RTCS. Both L-cysteine and NAC resulted in increased relaxations to GTN (0.1 nM-10 microM) as the GTN relaxation EC50 decreased compared with that of the untreated RTCS (L-cysteine, 0.06 +/- 0.12 microM and NAC, 0.08 +/- 0.03 microM versus control 0.25 +/- 0.08 microM, n = 5, p < 0.05). In contrast, 5 mM D-cysteine had no significant effects on the RTCS GTN relaxation EC50 (0.16 +/- 0.13 microM, n = 5). Similarly, the thiol donor L-methionine significantly increased RTCS sensitivity to GTN, as the relaxation EC50 decreased from the control value of 0.25 +/- 0.08 microM to 10 +/- 4 nM (n = 5, p < 0.001), whereas the D-isomer did not. These results are consistent with the idea that thiols play a key stereospecific role in the metabolic activation of GTN in RTCS. However, RTCS treated with amino acids were still less sensitive to GTN compared with vascular tissue, and this suggests that RTCS may be deficient in some other enzyme(s) relative to vascular tissue that is (are) responsible for the activation of GTN.