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BAY 41-8543 is a nitric oxide (NO)-independent stimulator of soluble guanylyl cyclase (sGC). Responses to intravenous injections of BAY 41-8543 were investigated under baseline and elevated tone conditions and when NO synthase (NOS) was inhibited with [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME). Under baseline conditions, intravenous injections of BAY 41-8543 caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and increases in cardiac output. When pulmonary arterial pressure was increased to ~30 mmHg with an intravenous infusion of U-46619, intravenous injections of BAY 41-8543 produced larger dose-dependent decreases in pulmonary arterial pressure, and the relative decreases in pulmonary and systemic arterial pressure in response to the sGC stimulator were similar. Treatment with L-NAME markedly decreased responses to BAY 41-8543 when pulmonary arterial pressure was increased to similar values (~30 mmHg) in U-46619-infused and in U-46619-infused plus L-NAME-treated animals. The intravenous injection of a small dose of sodium nitroprusside (SNP) when combined with BAY 41-8543 enhanced pulmonary and systemic vasodilator responses to the sGC stimulator in L-NAME-treated animals. The present results indicate that BAY 41-8543 has similar vasodilator activity in the systemic and pulmonary vascular beds when pulmonary vasoconstrictor tone is increased with U-46619. These results demonstrate that pulmonary and systemic vasodilator responses to BAY 41-8543 are significantly attenuated when NOS is inhibited by L-NAME and show that vasodilator responses to BAY 41-8543 are enhanced when combined with a small dose of SNP in L-NAME-treated animals. The present results are consistent with the concept that pulmonary and systemic vasodilator responses to the sGC stimulator are NO-independent; however, the vasodilator activity of the compound is greatly diminished when endogenous NO production is inhibited with L-NAME. These data show that BAY 41-8543 has similar vasodilator activity in the pulmonary and systemic vascular beds in the rat. soluble guanylyl cyclase stimulator; pulmonary and systemic vascular beds; U-46619; [N.sup.[omega]]-nitro-L-arginine methyl ester; sodium nitroprusside doi: 10.1152/ajpheart.01101.2009.

This study determined the role of nitric oxide (NO) in neurogenic vasodilation in mesenteric resistance arteries of the toad Bufo marinus. NO synthase (NOS) was anatomically demonstrated in perivascular nerves, but not in the endothelium. ACh and nicotine caused TTX-sensitive neurogenic vasodilation of mesenteric arteries. The ACh-induced vasodilation was endothelium-independent and was mediated by the NO/ soluble guanylyl cyclase signaling pathway, inasmuch as the vasodilation was blocked by the soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the NOS inhibitors [N.sup.[omega]]-nitro-L-arginine methyl ester and [N.sup.[omega]]-nitro-L-arginine. Furthermore, the ACh-induced vasodilation was significantly decreased by the more selective neural NOS inhibitor [N.sup.5]-(1-imino-3-butenyl)-L-ornithine. The nicotine-induced vasodilation was endothelium-independent and mediated by NO and calcitonin gene-related peptide (CGRP), inasmuch as pretreatment of mesenteric arteries with a combination of [N.sup.[omega]]-nitro-L-arginine and the CGRP receptor antagonist CGRP-(8-37) blocked the vasodilation. Clotrimazole significantly decreased the ACh-induced response, providing evidence that a component of the NO vasodilation involved [Ca.sup.2+]-activated [K.sup.+] or voltage-gated [K.sup.+] channels. These data show that NO control of mesenteric resistance arteries of toad is provided by nitrergic nerves, rather than the endothelium, and implicate NO as a potentially important regulator of gut blood flow and peripheral blood pressure. amphibian; autonomic nervous system; nitric oxide synthase; endothelium doi:10.1152/ajpregu.00148.2009

The transient receptor potential vallinoid type 4 (TRPV4) channel has been implicated in the endothelial shear response and flow-mediated dilation, although the precise functions of this channel remain poorly understood. In the present study, we investigated the role of TRPV4 in shear stress-induced endothelial Ca2+ entry and the potential link between this signaling response and relaxation of small resistance arteries. Using immunohistochemical analysis and RT-PCR, we detected strong expression of TRPV4 protein and mRNA in the endothelium in situ and endothelial cells freshly isolated from mouse small mesenteric arteries. The selective TRPV4 agonist GSK1016790A increased endothelial Ca2+ and induced potent relaxation of small mesenteric arteries from wild-type (WT) but not [TRPV4.sup.-/-] mice. Luminal flow elicited endothelium-dependent relaxations that involved both nitric oxide and EDHFs. Both nitric oxide and EDHF components of flow-mediated relaxation were markedly reduced in TRPV4 /- mice compared with WT controls. Using a fura-2/[Mn.sup.2+] quenching assay, shear was observed to produce rapid [Ca.sup.2+] influx in endothelial cells, which was markedly inhibited by the TRPV4 channel blocker ruthenium red and TRPV4-specific short interfering RNA. Flow elicited a similar TRPV4-mediated [Ca2.sup.+] entry in HEK-293 cells transfected with TRPV4 channels but not in nontransfected cells. Collectively, these data indicate that TRPV4 may be a potential candidate of mechanosensitive channels in endothelial cells through which the shear stimulus is transduced into [Ca.sup.2+] signaling, leading to the release of endothelial relaxing factors and flow-mediated dilation of small resistance arteries. transient receptor potential; endothelium; endothelium-derived factors; calcium signaling doi: 10.1152/ajpheart.00854.2009

LeBianc A J, Reyes R, Kang LS, Dailey RA, Stallone JN, Moningka NC, Muller-Delp JM. Estrogen replacement restores fiow-induced vasodilation in coronary arterioles of aged and ovariectomized rats. Am J Physiol Regul Integr Comp Physiol 297: R1713-R1723, 2009. First published October 7, 2009; doi: 10.1152/ajpregu.00178.2009.--The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flow-induced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized + estrogen replaced (OVE) female Fischer-344 rats to assess flow-induced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 [+ or -] 4 vs. old: 34 [+ or -] 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by ~160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with [N.sup.g]-nitro-L-arginine methyl ester (L-NAME), declined with age. L-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, L-NAME reduced flow-induced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flow-induced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age. endothelial nitric oxide synthase; Akt; nitric oxide; ovariectomy doi: 10.1152/ajpregu.00178.2009

It has been suggested that nitric oxide (NO) stimulates the cyclooxygenase (COX)-dependent mechanisms in the ocular vasculature; however, the importance of the pathway in regulating retinal circulation in vivo remains to be elucidated. Therefore, we investigated the role of COX-dependent mechanisms in NO-induced vasodilation of retinal blood vessels in thiobutabarbital-anesthetized rats with and without neuronal blockade (tetrodotoxin treatment). Fundus images were captured with a digital camera that was equipped with a special objective lens. The retinal vascular response was assessed by measuring changes in diameter of the retinal blood vessel. The localization of COX and soluble guanylyl cyclase in rat retina was examined using immunohistochemistry. The NO donors (sodium nitroprusside and NOR3) increased the diameter of the retinal blood vessels but decreased systemic blood pressure in a dose-dependent manner. Treatment of rats with indomethacin, a nonselective COX inhibitor, or SC-560, a selective COX-1 inhibitor, markedly attenuated the vasodilation of retinal arterioles, but not the depressor response, to the NO donors. However, both the vascular responses to NO donors were unaffected by the selective COX-2 inhibitors NS-398 and nimesulide. Indomethacin did not change the retinal vascular and depressor responses to hydralazine, 8-(4-chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (a membrane-permeable cGMP analog) and 8-(4-chlorophenylthio)-adenosine-3', 5'-cyclic monophosphate (a membrane-permeable cAMP analog). Treatment with SQ 22536, an adenylyl cyclase inhibitor, but not ODQ, a soluble guanylyl cyclase inhibitor, significantly attenuated the NOR3-induced vasodilation of retinal arterioles. The COX-1 immunoreactivity was found in retinal blood vessels. The retinal blood vessel was faintly stained for soluble guanylyl cyclase, although the apparent immunoreactivities on mesenteric and choroidal blood vessels were observed. These results suggest that NO exerts a substantial part of its dilatory effect via a mechanism that involves COX-1-dependent pathway in rat retinal vasculature. cyclooxygenases; prostaglandins; retinal blood vessel doi: 10.1152/ajpregu.91005.2008.

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation. nitrite reduction; nitric oxide; anerobic glycolysis; hypoxia; red blood cell; adenosine 5'-triphosphate doi: 10.1152/ajpheart.01233.2008

In animal models, administration of the adenosine analog AICA-riboside has shown beneficial effects on ischemia-reperfusion injury and glucose homeostasis. The vascular and/or metabolic effects of AICA-riboside administration in humans remain to be established. AICA-riboside was infused intra-arterially in four different dosages up to 8 mg x [min.sup.-1] x [dl.sup.-1] in 24 healthy subjects. Forearm blood flow (FBF) and glucose uptake and plasma glucose, free fatty acid, and AICA-riboside concentrations were assessed. We also combined AICA-fiboside infusion (2 mg x [min.sup.-1] x [dl.sup.-1]) with the intra-arterial administration of the adenosine receptor antagonist caffeine (90 [micaro]g x [min.sup.-1 x [dl.sup.-1]; n = 6) and with the endothelial NO synthase inhibitor L-NMMA (0.4 mg x [min.sup.-1] x [dl.sup.-1] = 6). Additional in vitro experiments were performed to explain our in vivo effects of AICA-riboside in humans. AICA-riboside increased FBF dose dependently from 2.0 [+ or -] 0.2 to 13.2 [+ or -] 1.9 ml x [min.sup.-1] x [dl.sup.-1] maximally (P < 0.05 for all dosages). The latter was not reduced by caffeine administration but was significantly attenuated by L-NMMA infusion. Despite high plasma AICA-riboside concentrations, forearm glucose uptake did not change. In vitro experiments showed rapid uptake of AICA-riboside by the equilibratire nucleoside transporter in erythrocytes and subsequent phosphorylation to AICA-ribotide. We conclude that AICA-riboside induces a potent vasodilator response in humans that is mediated by NO. Despite high local plasma concentrations, AICA-riboside does not increase skeletal muscle glucose uptake. 5-aminoimidazole-4-carboxamide; nitric oxide; forearm blood flow; forearm glucose uptake

Impaired nitric oxide-cGMP signaling contributes to severe pulmonary hypertension after birth, which may in part be due to decreased soluble gnanylate cyclase (sGC) activity. Cinacignat (BAY 58-2667) is a novel sGC activator that causes vasodilation, even in the presence of oxidized heine or heine-free sGC, but its hemodynamic effects have not been studied in the perinatal lung. We performed surgery on eight fetal (126 [+ or -] 2 days gestation) lambs (full term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery to measure blood flow, and a catheter was placed in the left pulmonary artery for drug infusion. Cinaciguat (0.1-100 [micro]g over 10 rain) caused dose-related increases in pulmonary blood flow greater than fourfold above baseline and reduced pulmonary vascular resistance by 80%. Treatment with IH-[l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ), an sGC-oxidizing inhibitor, enhanced cinacignat-induced pulmonary vasodilation by > 120%. The pulmonary vasodilator effect of cinacignat was prolonged, decreasing pulmonary vascular resistance for > 1.5 h after brief infusion. In vitro stimulation of ovine fetal pulmonary artery smooth muscle cells with cinacignat after ODQ treatment resulted in a 14-fold increase in cGMP compared with non-ODQ-treated cells. We conclude that cinaciguat causes potent and sustained fetal pulmonary vasodilation that is augmented in the presence of oxidized sGC and speculate that cinacignat may have therapeutic potential for severe neonatal pulmonary hypertension. BAY 58-2667; cGMP; nitric oxide; persistent pulmonary hypertension of the newborn; pulmonary hypertension

The free radical form of nitric oxide (N[O.sup.x]) is a well-known mediator of vascular tone. What is not so well recognized is that N[O.sup.x] exists in several different redox forms. There is considerable evidence that N[O.sup.x] and its one-electron reduction product, nitroxyl (HNO), have pharmacologically distinct actions that extend into the regulation of the vasculature. The aim of this study was to compare the vasorelaxation mechanisms of HNO and N[O.sup.x], including an examination of the ability of these redox variants to hyperpolarize and repolarize vascular smooth muscle cells from rat mesenteric arteries. The HNO donor Angeli's salt (0.1 nM-10 [micro]M) caused a concentration-dependent hyperpolarization of vessels at resting tone and a simultaneous, concentration-dependent vasorelaxation and repolarization of vessels precontracted and depolarized with methoxamine. Both vasorelaxation and repolarization responses to Angeli's salt were significantly attenuated by both the HNO scavenger L-cysteine (3 mM) and the voltage-dependent [K.sup.+] ([K.sub.v]) channel inhibitor 4-aminopyridine (4-AP; 1 mM) and virtually abolished by the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 [micro]M) or 30 mM [K.sup.+]. In contrast, N[O.sup..] (0.01-1 [micro]M) repolarized arteries to a lesser extent than HNO, and these responses were resistant to inhibition by ODQ (10 [micro]M) and 4-AP (1 mM). Blockade of [K.sub.v] channels (1 mM 4-AP) also significantly inhibited the repolarization response to YC-1 (0.l-10 [micro]M), confirming a role for sGC/cGMP in the activation of [K.sub.v] channels in this preparation. We conclude that HNO causes vasorelaxation via a cGMP-dependent activation of [K.sub.v] channels and that there are different profiles of vasorelaxant activity for the redox siblings HNO and N[O.sup.x]. nitroxyl; hyperpolarization; soluble guanylate cyclase; [K.sup.+] channels; nitric oxide

Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species, thereby reducing nitric oxide (NO*) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO., endothelial nitric oxide synthase (eNOS), also contributes. It is also unclear whether resistance vessels that utilize both NO* and non-NO* vasodilatory mechanisms, undergo alteration of non-NO*mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hey: 89.2 [+ or -] 49.0 [micro]M) compared with age-matched controls (Hey: 6.6 [+ or -] 1.9 [micro]M), using first-order mesenteric arteries. Dilation to ACh ([10.sup.-9]-[10.sup.-4] M) was measured in isolated, cannulated, and pressurized (75 mmHg) arteries with and without [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) ([10.sup.-4] M) and/or indomethacin ([10.sup.-5] M) to test endothelium-dependent dilation and non-NO*-dependent dilation, respectively. The time course of dilation to ACh ([10.sup.-4] M) was examined to compare the initial transient dilation due to non-NO*, non-prostacyclin mechanism and the sustained dilation due to NO'. These experiments indicated that endothelium-dependent dilation was attenuated (P < 0.05) in HHcy arteries due to downregulation of only NO*-dependent dilation. Western blot analysis indicated significantly less (P < 0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no difference in phospho-T495-eNOS/eNOS. S 1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic dietinduced HHcy in mice impairs eNOS protein expression and phos- phorylation at S 1179, coincident with impaired NO*-dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation and microvascular disease that is common with HHcy. homocysteine; endothelial nitric oxide synthase; S1179; vasodilation

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and mis-identification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures. red blood cell vasodilation | S-nitrosohemoglobin | S-nitrosylation

Microvascular pathophysiology associated with type 2 diabetes mellitus (T2DM) contributes to several aspects of the morbidity associated with the disease. We quantified the contribution of nitric oxide (NO) to the cutaneous vasodilator response to nonpainful local warming in subjects with T2DM (average duration of diabetes mellitus 7 [+ or -] 1 yr) and in age-matched control subjects. We measured skin blood flow in conjunction with intradermal microdialysis of [N.sup.G]-nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor) or vehicle during 35 min of local warming to 42[degrees]C. Microdialysis of sodium nitroprusside (SNP) was used for assessment of maximum cutaneous vascular conductance (CVC). Resting CVC was higher in T2DM subjects at vehicle sites (T2DM: 19 [+ or -] 2 vs. control: 11 [+ or -] 3%maxCVC; P < 0.05); this difference was abolished by L-NAME (T2DM: 10 [+ or -] 1 vs. control: 8 [+ or -] 1%maxCVC; P > 0.05). The relative contribution of NO to the vasodilator response to local warming was not different between groups (T2DM: 46 [+ or -] 4 vs. control: 44 [+ or -] 6%maxCVC; P > 0.05). However, absolute CVC during local warming was ~25% lower in T2DM subjects (T2DM: 1.79 [+ or -] 0.15 AU/mmHg; controls: 2.42 [+ or -] 0.20 AU/mmHg; P < 0.01), and absolute CVC during SNP was ~20% lower (T2DM: 1.91 [+ or -] 0.12 vs. control: 2.38 [+ or -] 0.13 AU/mmHg; P < 0.01). We conclude that the relative contribution of NO to vasodilation during local warming is similar between subjects with T2DM and control subjects, although T2DM was associated with a lower absolute maximum vasodilation. skin blood flow; vasodilation; local warming; thermoregulation

One of the most intriguing areas of research in erythrocyte physiology is the interaction of hemoglobin with nitric oxide (NO). These two molecules independently fulfill diverse and complex physiological roles, while together they subtly modulate microvascular perfusion in response to second-by-second changes in local metabolic demand, contributing to hypoxic vasodilation. It is through an appreciation of the temporal and structural constraints of the microcirculation that the principal requirements of the physiological interplay between NO and hemoglobin are revealed, elucidating the role of the erythrocyte in hypoxic vasodilation. Among the candidate molecular mechanisms, only S-nitrosohemoglobin (SNO-hemoglobin) directly fulfills the physiological requirements. Thus, NO is transported by red blood cells to microvascular sites of action in protected form as an S-nitrosothiol on the highly conserved hemoglobin [beta]-93 Cys residue, invariant in birds and mammals. SNO-hemoglobin dispenses NO bioactivity to microvascular cells on the release of oxygen, physiologically coupling hemoglobin deoxygenation to vasodilation. SNO-hemoglobin is the archetype for the role of S-nitrosylation in a newly identified class of biological signals, and disturbances in SNO-hemoglobin activity are associated with the pathogenesis of several important vascular diseases. nitrite; nitrite reduction; S-nitrosylation; S-nitrosohemoglobin

Red blood cells (RBCs) act as [O.sub.2]-responsive transducers of vasodilator and vasoconstrictor activity in lungs and tissues by regulating the availability of nitric oxide (NO). Vasodilation by RBCs is impaired in diseases characterized by hypoxemia. We have proposed that the extent to which RBCs constrict vs. dilate vessels is, at least partly, controlled by a partitioning between NO bound to heme iron and to Cys[beta]193 thiol of hemoglobin (Hb). Heroes sequester NO, whereas thiols deploy NO bioactivity. In recent work, we have suggested that specific micropopulations of NO-liganded Hb could support the chemistry of S-nitrosohemoglobin (SNO-Hb) formation. Here, by using nitrite as the source of NO, we demonstrate that a (T state) micropopulation of a heme-NO species, with spectral and chemical properties of Fe(III)NO, acts as a precursor to SNO-Hb formation, accompanying the allosteric transition of Hb to the R state. We also show that at physiological concentrations of nitrite and deoxyHb, a S-nitrosothiol precursor is formed within seconds and produces SNO-Hb in high yield upon its prompt exposure to [O.sub.2] or CO. Deoxygenation/reoxygenation cycling of oxyHb in the presence of physiological amounts of nitrite also efficiently produces SNO-Hb. In contrast, high amounts of nitrite or delays in reoxygenation inhibit the production of SNO-Hb. Collectively, our data provide evidence for a physiological S-nitrosothiol synthase activity of tetrameric Hb that depends on NO-Hb micropopulations and suggest that dysfunction of this activity may contribute to the pathophysiology of cardiopulmonary and blood disorders. hypoxic vasodilation | nitric oxide | S-nitrosohemoglobin | S-nitrosylation

To study the role of nitric oxide (NO) in regulating oxygen consumption by vessel walls, the oxygen consumption rate of arteriolar walls in rat cremaster muscle was measured in vivo during flow-induced vasodilation and after inhibiting NO synthesis. The oxygen consumption rate of arteriolar walls was calculated based on the intra- and perivascular P[o.sub.2] values measured by phosphorescence quenching laser microscopy. The perivascular P.sub.[o.sub.2] value of the arterioles during vasodilation was significantly higher than under control conditions, although the intravascular P[o.sub.2] values under both conditions were approximately the same. Inhibition of NO synthesis, on the other hand, caused a significant increase in arterial blood pressure and a significant decrease in arteriolar diameter. Inhibition of NO synthesis also caused a significant decrease in both the intra- and perivascular P[o.sub.2] values of the arterioles. Inhibition of NO synthesis increased the oxygen consumption rate of the vessel walls by 42%, whereas enhancement of flow-induced NO release decreased it by 34%. These results suggest that NO plays an important role not only as a regulator of peripheral vascular tone but also as a modulator of tissue oxygenation by reducing oxygen consumption by vessel walls. In addition, enhancement of NO release during exercise may facilitate efficient oxygen supply to the surrounding high metabolic tissue. flow-induced vasodilation; [[N.sup.[omega]]-nitro-L-arginine methyl ester; oxygen transport; vascular tone; vascular endothelial cells

The mechanism by which hypoxia [low partial pressure of [O.sub.2] (p[O.sub.2])] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heine iron-nitrosyl (FeNO) species that are deficient in p[O.sub.2]-governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). S-nitrosothiol (SNO)-deficient RBCs produce impaired vasodilator responses in vitro and exaggerated pulmonary vasoconstrictor responses in vivo and are defective in oxygenating the blood. RBCs from hypoxemic patients with elevated pulmonary arterial pressure (PAP) exhibit a similar FeNO/SNO imbalance and are thus deficient in p[O.sub.2]-coupled vasoregulation. Chemical restoration of SNO-Hb levels in both animals and patients restores the vasodilator activity of RBCs, and this activity is associated with improved oxygenation and lower PAPs. hemoglobin | red blood cell vasodilation | S-nitrosylation

The identity of the cellular mechanisms through which nitroglycerin (glyceryl trinitrate, GTN) elicits nitric oxide (NO)-based signaling to dilate blood vessels remains one of the longest standing foci of investigation and sources of controversy in cardiovascular biology. Recent evidence suggests an unexpected role for mitochondria. We show here that bioconversion by mitochondria of clinically relevant concentrations of GTN results in activation of guanylate cyclase, production of cGMP, vasodilation in vitro, and lowered blood pressure in vivo, which are eliminated by genetic deletion of the mitochondrial aldehyde dehydrogenase (mtALDH). In contrast, generation of vasoactivity from alternative nitro(so)-vasodilators is unaffected. In mtALD[H.sup.-/-] mice and their isolated vascular tissue, GTN bioactivity can still be generated, but only at substantially higher concentrations of GTN and by a mechanism that does not exhibit tolerance. Thus, mtALDH is necessary and sufficient for vasoactivity derived from therapeutic levels of GTN, and, more generally, mitochondria can serve as a source of NO-based cellular signals that may originate independently of NO synthase activity. nitric oxide | nitrite | S-nitrosothiol | nitrate tolerance

Decreased nitric oxide (NO) bioavailability is associated with a number of pathological conditions. Administration of a supplemental source of NO can counter the pathological effects arising from decreased NO bioavailability. A class of NO-nucleophile adducts that spontaneously release NO (NONOates) has been developed, and its members show promise as therapeutic sources of NO. Because the NONOates release NO spontaneously, a significant portion of the NO may be consumed by the myriad of NO reactive species present in the body. Here we develop a model to analyze the efficacy of NO delivery, by membrane-impermeable NONOates, in the resistance arterioles. Our model identifies three features of blood vessels that will enhance NONOate efficacy: 1) the amount of NO delivered to the abluminal region increases with lumen radius: 2) the presence of a flow-induced red blood cell-free zone will augment NO delivery: and 3) extravasation of the NONOate into the interstitial space will increase abluminal NO delivery. These results suggest that NONOates may be more effective in larger vessels and that NONOate efficacy can be altered by modifying permeability to the interstitial space. vasodilation; NONOate; microcirculation; extravasation

No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/ cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown [via antisense oligodeoxynucleotide (ODN) applications] of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (~50% reduction in SNAP [EC.sub.50]). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis. nitric oxide; multidrug resistance protein 5; phosphodiesterase 5

Research was conducted to examine the hypothesis that vasoactive compounds, which includes nitric oxide and hypertonic sodium, may diffuse from venous endothelial cells and blood to the arterial wall during intestinal absorption. The perivascular NO was measured using NO-sensitive recessed-tip microelectrodes. Results reveal that venous to arterial communication transpired through diffusion of endothelium-dependent and -independent dilator substances from the vein to the artery.

The relationship between conformational movement and catalytic action in the reductase domain of neuronal NO synthase (nNOS) is analyzed by alteration of the interface between the FAD and FMN domains. It is observed that the R1229E mutation disrupts the FAD-FMN domain interface in the isolated reductase domain (nNOSrd) such that there is no regulatory interaction between the two domains.