Endothelial nitric-oxide synthase (NOS-III

Abstract-Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8Ϯ10.3% inhibition, nϭ8; PϽ0.05). Key Words: mechanotransduction Ⅲ nonselective cation channels Ⅲ cardiovascular physiology I n 1902, Bayliss made the seminal observation that resistance arteries possess an innate ability to constrict in response to elevations in intraluminal pressure. 1 He described this phenomenon as the myogenic response. Today, we understand that myogenic constriction is a major determinant of peripheral resistance and organ perfusion. 2,3 As such, it plays an important role in the maintenance of an appropriate level of perfusion to vascular beds, independent of systemic pressure, providing a mechanism whereby tissues are protected from variations in blood pressure. 2,3 Myogenic responses predominate in small resistance arteries (Ͻ500 m) 4 and are considered to be attributable to an increase in smooth muscle intracellular free calcium concentration ([Ca 2ϩ ] i ) after depolarization of these cells. 5 Many studies have attempted to elucidate the signaling mechanisms involved in the myogenic response and several possibilities have been proposed including activation of ion channels, ion exchangers/transporters, and enzyme systems/ second messengers. 2 In particular, there is compelling evidence supporting a role for stretch activated ion channels and the arachidonate metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). 2 However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear.Until recently the myogenic response was thought to be derived entirely from a direct effect of intraluminal pressure on the smooth muscle. 6 It is now clear that the endothelium,

Soluble guanylyl cyclase (sGC) is an important effector for nitric oxide (NO). It acts by increasing intracellular cyclic GMP (cGMP) levels to mediate numerous biological functions. Recently, 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) was identified as a novel and selective inhibitor of this enzyme. Therefore, ODQ may represent an important pharmacological tool for differentiating cGMP-mediated from cGMP-independent effects of NO. In the present study, we examined the inhibitory action of ODQ both functionally and biochemically. In phenylephrine-preconstricted, endothelium-intact, isolated aortic rings from the rat, ODQ, in a concentration-dependent manner, increased contractile tone and inhibited relaxations to authentic NO with maximal effects at 3 microM. Pretreatment of vascular rings with ODQ induced a parallel, 2-log-order shift to the right of the concentration-response curves (CRCs) to histamine, ATP, NO, the NO-donors S-nitrosoglutathione, S-nitroso-N-acetyl-D,L-penicillamine, and spermine NONOate [N-[4-[1-(3-amino propyl)-2-hydroxy-2-nitroso hydrazino]butyl]-1, 3-propane diamine], and the direct sGC-stimulant [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] YC-1 but did not affect relaxations induced by papaverine and atriopeptin II. Moreover, the rightward shift of the CRCs to Angeli's salt, peroxynitrite, and linsidomine was similar to that of NO. These results suggested that ODQ is specific for sGC. Furthermore, they indicate that NO can cause vasorelaxation independent of cGMP. Three interesting exceptions were observed to the otherwise rather uniform inhibitory effect of ODQ: the responses to acetylcholine, glycerol trinitrate, and sodium nitroprusside. The latter two agents are known to require metabolic activation, possibly by cytochrome P-450-type proteins. The 3- to 5-log-order rightward shift of their CRCs suggests that, in addition to sGC, ODQ may interfere with heme proteins involved in the bioactivation of these NO donors and the mechanism of vasorelaxation mediated by acetylcholine. In support of this notion, ODQ inhibited hepatic microsomal NO production from both glycerol trinitrate and sodium nitroprusside as well as NO synthase activity in aortic homogenates. The latter effect seemed to require biotransformation of ODQ. Collectively, these data reveal that ODQ interferes with various heme protein-dependent processes in vascular and hepatic tissue and lacks specificity for sGC.

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H 4 Bip, has been attributed to enzyme-associated H 4 Bip. To elucidate further H 4 Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H 4 Bip and inhibited H 4 Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of V max ) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H 4 Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H 4 Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H 4 Bip redox cycling in NOS. Rather, H 4 Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.