In vivo assessment of microvascular nitric oxide production and its relation with blood flow

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.     

Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion

Endothelin (ET) receptor antagonism protects from ischemia-reperfusion injury. We hypothesized that the cardioprotective effect is related to nitric oxide (NO) bioavailability. Buffer-perfused rat and mouse hearts were subjected to ischemia and reperfusion. At the onset of ischemia, the rat hearts received vehicle, the dual endothelin type A/type B (ETA/ETB) receptor antagonist bosentan (10 microM), the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 microM), the combination of bosentan and L-NMMA or the combination of bosentan, L-NMMA, and the NO substrate L-arginine (1 mM). Hearts from wild-type and endothelial NO synthase (eNOS)-deficient mice received either vehicle or bosentan. Myocardial performance, endothelial function, NO outflow, and eNOS expression were monitored. Bosentan significantly improved myocardial function during reperfusion in rats and in wild-type mice, but not in eNOS-deficient mice. The functional protection afforded by bosentan was inhibited by L-NMMA, whereas it was restored by L-arginine. Myocardial expression of eNOS (immunoblotting) increased significantly in bosentan-treated rat hearts compared with vehicle hearts. Recovery of NO outflow during reperfusion was enhanced in the bosentan-treated rat heart. The endothelium-dependent vasodilator adenosine diphosphate increased coronary flow by 18 +/- 9% at the end of reperfusion in the bosentan group, whereas it reduced coronary flow by 7 +/- 5% in the vehicle group (P < 0.001). The response to the endothelium-independent dilator sodium nitroprusside was not different between the two groups. In conclusion, the dual ETA/ETB receptor antagonist bosentan preserved endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.     

Heat shock protein 90 and tyrosine kinase regulate eNOS NO* generation but not NO* bioactivity

An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-*) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO* release from stimulated endothelial cell cultures and increased O(2)(-*) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-*) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.     

Impaired nitric oxide-mediated vasodilation in transgenic sickle mouse

Transgenic sickle mice expressing human beta(S)- and beta(S-Antilles)-globins show intravascular sickling, red blood cell adhesion, and attenuated arteriolar constriction in response to oxygen. We hypothesize that these abnormalities and the likely endothelial damage, also reported in sickle cell anemia, alter nitric oxide (NO)-mediated microvascular responses and hemodynamics in this mouse model. Transgenic mice showed a lower mean arterial pressure (MAP) compared with control groups (90 +/- 7 vs. 113 +/- 8 mmHg, P < 0.00001), accompanied by increased endothelial nitric oxide synthase (eNOS) expression. N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of NOS, caused an approximately 30% increase in MAP and approximately 40% decrease in the diameters of cremaster muscle arterioles (branching orders: A2 and A3) in both control and transgenic mice, confirming NOS activity; these changes were reversible after L-arginine administration. Aminoguanidine, an inhibitor of inducible NOS, had no effect. Transgenic mice showed a decreased (P < 0.02-0.01) arteriolar dilation in response to NO-mediated vasodilators, i.e., ACh and sodium nitroprusside (SNP). Indomethacin did not alter the responses to ACh and SNP. Forskolin, a cAMP-activating agent, caused a comparable dilation of A2 and A3 vessels ( approximately 44 and 70%) in both groups of mice. Thus in transgenic mice, an increased eNOS/NO activity results in lower blood pressure and diminished arteriolar responses to NO-mediated vasodilators. Although the increased NOS/NO activity may compensate for flow abnormalities, it may also cause pathophysiological alterations in vascular tone.     

Dexamethasone lacks effect on blood pressure in mice with a disrupted endothelial NO synthase gene.

Cushing's syndrome and systemic administration of glucocorticoids are associated with hypertension, but the underlying molecular mechanism is only partially understood. We have shown previously that dexamethasone downregulates the expression of the endothelial NO synthase (eNOS) gene in human endothelial cells and in the rat and that this may contribute to the blood pressure-raising effect of the steroid [Proc. Natl. Acad. Sci. USA 96 (1999) 13357]. In the current communication, we demonstrated that dexamethasone increased mean arterial blood pressure in wild-type C-57 Bl6 mice (eNOS+/+ mice), but had no effect on blood pressure in mice with a disrupted eNOS gene (eNOS-/- mice) derived from the same strain. The NOS inhibitor ethylisothiourea, used for control purposes, showed a hypertensive effect in eNOS+/+ mice, but no such effect in eNOS-/- mice. Serum NO2-/NO3- levels, an indicator of total body NO synthesis, decreased significantly when eNOS+/+ mice were treated with dexamethasone. eNOS-/- mice had lower serum NO2-/NO3- levels per se, which were not changed significantly by dexamethasone. Dexamethasone decreased the expression of eNOS in three major organs of the mouse investigated, namely the heart, the liver, and the kidney. We conclude that the expressional downregulation of eNOS and the ensuing reduction in vascular NO production contributes to the hypertension caused by glucocorticoids.     

L-Arginine uptake affects nitric oxide production and blood flow in the renal medulla

Experiments were performed to determine whether L-arginine transport regulates nitric oxide (NO) production and hemodynamics in the renal medulla. The effects of renal medullary interstitial infusion of cationic amino acids, which compete with L-arginine for cellular uptake, on NO levels and blood flow in the medulla were examined in anesthetized rats. NO concentration in the renal inner medulla, measured with a microdialysis-oxyhemoglobin trapping technique, was significantly decreased by 26-44% and renal medullary blood flow, measured by laser Doppler flowmetry, was significantly reduced by 20-24% during the acute renal medullary interstitial infusion of L-ornithine, L-lysine, and L-homoarginine (1 micromol.kg(-1).min(-1) each; n = 6-8/group). In contrast, intramedullary infusion of L-arginine increased NO concentration and medullary blood flow. Flow cytometry experiments with 4-amino-5-methylamino-2',7'-difluorescein diacetate, a fluorophore reactive to intracellular NO, demonstrated that L-ornithine, L-lysine, and L-homoarginine decreased NO by 54-57% of control, whereas L-arginine increased NO by 21% in freshly isolated inner medullary cells (1 mmol/l each, n > 1,000 cells/experiment). The mRNA for the cationic amino acid transporter-1 was predominantly expressed in the inner medulla, and cationic amino acid transporter-1 protein was localized by immunohistochemistry to the collecting ducts and vasa recta in the inner medulla. These results suggest that L-arginine transport by cationic amino acid transport mechanisms is important in the production of NO and maintenance of blood flow in the renal medulla.     

Induction of iNOS restricts functional activity of both eNOS and nNOS in pig cerebral artery

The aim of the study was to investigate the effect of iNOS expression on eNOS and nNOS functional activity in porcine cerebral arteries. iNOS was induced in pig basilar arteries using lipopolysaccharide (LPS). Arteries expressing iNOS generated NO and relaxed when challenged with L-arginine (30 microM), an effect that was reduced by treatment with dexamethasone (coincubated with LPS) and prevented by the iNOS inhibitor 1400 W (administered 10 min prior to precontraction). eNOS was activated by A23187 and was found to be impaired in arteries that had iNOS induced (A23187 1 microM relaxation: control 110+/-8%, LPS-treated 50+/-16% ; p<0.05, N=5-6). This was due mainly to reduced formation of NO by A23187 (NO concentration in response to A23187 1 microM: control 25+/-6 nM, LPS-treated 0.8+/-1.2 nM; p<0.001, N=5-6), in addition to a small reduction in the vasodilator response to the NO-donors NOC-22 and SIN-1. Cerebral vasodilation produced by stimulation of intramural nitrergic nerves was impaired in arteries that had iNOS induced, and this was reversed by 1400 W (control 23+/-4% relaxation, LPS-treated 11+/-1% relaxation, LPS plus 1400 W 10 microM treated 25+/-2% relaxation; p<0.01 for control versus LPS, N=6). It is concluded that the induction of iNOS in cerebral arteries reduces NO-mediated vasodilation initiated by eNOS and by nNOS, primarily by modulation of NO formation.     

Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS-/- mice

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.     

Roles of endogenous prostaglandins and nitric oxide in inhibitions of gastric emptying and accelerations of gastrointestinal transit by escins Ia, Ib, IIa, and IIb in mice

We reported previously that escins Ia, Ib, IIa, and IIb, isolated from horse chestnuts, inhibited the 30-min gastric emptying (GE) in mice. In this study, the effects of escins Ia-IIb on gastrointestinal transit (GIT), and the roles of endogenous prostaglandins (PGs) and nitric oxide (NO) in the effects of escins Ia--IIb on GE and GIT were investigated in fasted mice. Escins Ia-IIb (12.5-50 mg/kg, p.o.) dose-dependently accelerated GIT. Both GE inhibitions and GIT accelerations by escins Ia-IIb (25 mg/kg) were markedly attenuated by pretreatment with indomethacin (10 mg/kg, s.c., an inhibitor of PGs synthesis). Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.p., an inhibitor of constitutive and inducible NO synthase) attenuated the effects of escins Ia-IIb on GIT, but not on GE. The effect of L-NAME was reversed by L-arginine (600 mg/kg, i.p., a substrate of NO synthase), but not by D-arginine (900 mg/kg, i.p., the enantiomer of L-arginine). The GIT accelerations of escins Ia-IIb were not attenuated by pretreatment with D-NAME (10 mg/kg, i.p., the enantiomer of L-NAME) or dexamethasone (5 mg/kg, i.p., an inhibitor of inducible form of NO synthase). The results suggest that endogenous PGs play an important role in both GE inhibitions and GIT accelerations, and constitutive NO is involved in the GIT accelerations, by escins Ia--IIb in mice.     

Mice with targeted deletion of eNOS develop hyperdynamic circulation associated with portal hypertension

Systemic vasodilation is the initiating event of the hyperdynamic circulatory state, being most likely triggered by increased levels of vasodilators, primarily nitric oxide (NO). Endothelial NO synthase (eNOS) is responsible for this event. We tested the hypothesis that gene deletion of eNOS and inducible NOS (iNOS) may inhibit the development of the hyperdynamic circulatory state in portal hypertensive animals. To test this hypothesis, we used mice lacking eNOS (eNOS-/-) or eNOS/iNOS (eNOS/iNOS-/-) genes. A partial portal vein ligation (PVL) was used to induce portal hypertension. Sham-operated animals were used as a control. Hemodynamic characteristics were tested 2 wk after surgery. As opposed to our hypothesis, PVL also caused significant reduction in peripheral resistance in eNOS-/- compared with sham animals (0.33 +/- 0.02 vs. 0.41 +/- 0.03 mmHg. min x kg body wt x ml(-1); P = 0.04) and in eNOS/iNOS-/- animals with PVL compared with that of the sham-operated group (0.44 +/- 0.02 vs. 0.54 +/- 0.04; P = 0.03). This demonstrates that, despite gene deletion of eNOS, the knockout mice developed hyperdynamic circulation. Compensatory vasodilator molecule(s) are upregulated in place of NO in the systemic and splanchnic circulation in portal hypertensive animals.     

Advancing age produces sex differences in vasomotor kinetics during and after skeletal muscle contraction

Little is known of the vasomotor responses of skeletal muscle arterioles during and following muscle contraction. We hypothesized that aging leads to impaired arteriolar responses to muscle contraction and recovery. Nitric oxide (NO) availability, which is age dependent, has been implicated in components of these kinetics. Therefore, we also hypothesized that changes in the kinetics of vascular responses are associated with the NO pathway. Groups were young (3 mo), old (24 mo), endothelial NO synthase knockout (eNOS-/-), and N(G)-nitro-L-arginine (L-NA)-treated male and female C57BL/6 mice. The kinetics of vasodilation during and following 1 min of contractions of the gluteus maximus muscle were recorded in second-order (regional distribution) and third-order (local control) arterioles. Baseline, peak (during contraction), and maximal diameters (pharmacological) were not affected by age or sex. The kinetics of dilation and recovery were not different between males and females at the young age. There was a significant slowing of vasodilation at the onset of contractions (approximately 2-fold; P < 0.05) and a significant speeding of recovery ( approximately 5-fold; P < 0.05) in old males vs. old females and vs. young eNOS-/-, and L-NA did not affect the kinetics at the onset of muscle contraction. eNOS-/- mimicked the rapid recovery of old males in second-order arterioles; acute NO production (L-NA) explained approximately 50% of this effect. These data demonstrate fundamental age-related differences between the sexes in the dynamic function of skeletal muscle arterioles. Understanding how youthful function persists in females but not males may provide therapeutic insight into clinical interventions to maintain dynamic microvascular control of nutrient supply with age.     

Age impairs Flk-1 signaling and NO-mediated vasodilation in coronary arterioles

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (approximately 6 mo) and old (approximately 24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P相似文献   

Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.     

Nitroso-redox balance in control of coronary vasomotor tone

Reactive oxygen species (ROS) are essential in vascular homeostasis but may contribute to vascular dysfunction when excessively produced. Superoxide anion (O(2)(·-)) can directly affect vascular tone by reacting with K(+) channels and indirectly by reacting with nitric oxide (NO), thereby scavenging NO and causing nitroso-redox imbalance. After myocardial infarction (MI), oxidative stress increases, favoring the imbalance and resulting in coronary vasoconstriction. Consequently, we hypothesized that ROS scavenging results in coronary vasodilation, particularly after MI, and is enhanced after inhibition of NO production. Chronically instrumented swine were studied at rest and during exercise before and after scavenging of ROS with N-(2-mercaptoproprionyl)-glycine (MPG, 20 mg/kg iv) in the presence or absence of prior inhibition of endothelial NO synthase (eNOS) with N(ω)-nitro-L-arginine (L-NNA, 20 mg/kg iv). In normal swine, MPG resulted in coronary vasodilation as evidenced by an increased coronary venous O(2) tension, and trends toward increased coronary venous O(2) saturation and decreased myocardial O(2) extraction. These effects were not altered by prior inhibition of eNOS. In MI swine, MPG showed a significant vasodilator effect, which surprisingly was abolished by prior inhibition of eNOS. Moreover, eNOS dimer/monomer ratio was decreased after MI, reflecting eNOS uncoupling. In conclusion, ROS exert a small coronary vasoconstrictor influence in normal swine, which does not involve scavenging of NO. This vasoconstrictor influence of ROS is slightly enhanced after MI. Since inhibition of eNOS abolished rather than augmented the vasoconstrictor influence of ROS in swine with MI, while eNOS dimer/monomer ratio was decreased, our data imply that uncoupled eNOS may be a significant source of O(2)(·-) after MI.     

Chronic renal failure alters endothelial function in cerebral circulation in mice

We examined structure, composition, and endothelial function in cerebral arterioles after 4 wk of chronic renal failure (CRF) in a well-defined murine model (C57BL/6J and apolipoprotein E knockout female mice). We also determined quantitative expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (on serine 1177 and threonine 495), and caveolin-1; quantitative expression of markers of vascular inflammation or oxidative stress [Rock-1, Rock-2, VCAM-1, and peroxisome proliferator-activated receptor-γ (PPARγ)]; and the plasma concentration of L-arginine and asymmetric dimethylarginine (ADMA). Our hypothesis was that endothelial function would be impaired in cerebral arterioles during CRF following either a decrease in NO production (through alteration of eNOS expression or regulation) or an increase in NO degradation (due to oxidative stress or vascular inflammation). Endothelium-dependent relaxation was impaired during CRF, but endothelium-independent relaxation was not. CRF had no effect on cerebral arteriolar structure and composition. Quantitative expressions of eNOS, eNOS phosphorylated on serine 1177, caveolin-1, Rock-1, Rock-2, and VCAM-1 were similar in CRF and non-CRF mice. In contrast, quantitative expression of PPARγ (which exercises a protective role on blood vessels) was significantly lower in CRF mice, whereas quantitative expression of eNOS phosphorylated on the threonine 495 (the inactive form of eNOS) was significantly higher. Lastly, the plasma concentration of ADMA (a uremic toxin and an endogenous inhibitor of eNOS) was elevated and plasma concentration of L-arginine was low in CRF. In conclusion, endothelial function is impaired in a mouse model of early stage CRF. These alterations may be related (at least in part) to a decrease in NO production.     

Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation

Nitric oxide (NO) relaxes the internal anal sphincter (IAS), but its enzymatic source(s) remains unknown; neuronal (nNOS) and endothelial (eNOS) NO synthase (NOS) isoforms could be involved. Also, interstitial cells of Cajal (ICC) may be involved in IAS relaxation. We studied the relative roles of nNOS, eNOS, and c-Kit-expressing ICC for IAS relaxation using genetic murine models. The basal IAS tone and the rectoanal inhibitory reflex (RAIR) were assessed in vivo by a purpose-built solid-state manometric probe and by using wild-type, nNOS-deficient (nNOS-/-), eNOS-deficient (eNOS-/-), and W/W(v) mice (lacking certain c-Kit-expressing ICC) with or without L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME) treatment. Moreover, the basal tone and response to electrical field stimulation (EFS) were studied in organ bath using wild-type and mutant IAS. In vivo, the basal tone of eNOS-/- was higher and W/W(v) was lower than wild-type and nNOS-/- mice. L-arginine administered rectally, but not intravenously, decreased the basal tone in wild-type, nNOS-/-, and W/W(v) mice. However, neither L-arginine nor L-NAME affected basal tone in eNOS-/- mice. In vitro, L-arginine decreased basal tone in wild-type and nNOS-/- IAS but not in eNOS-/- or wild-type IAS without mucosa. The in vivo RAIR was intact in wild-type, eNOS-/-, and W/W(v) mice but absent in all nNOS-/- mice. EFS-induced IAS relaxation was also reduced in nNOS-/- IAS. Thus the basal IAS tone is largely controlled by eNOS in the mucosa, whereas the RAIR is controlled by nNOS. c-Kit-expressing ICC may not be essential for the RAIR.     

Resveratrol protects myocardial ischemia-reperfusion injury through both NO-dependent and NO-independent mechanisms

We previously showed that resveratrol (3,4',5-trihydroxystilbene) stimulates NO production and is cardioprotective in rat heart subjected to ischemia-reperfusion (I/R rat heart). We now show that in I/R rat heart, inducible nitric oxide synthase (iNOS) expression is markedly induced, while expression of endothelial nitric oxide synthase (eNOS) and nueronal nitric oxide synthase (nNOS) is unchanged. In animals preconditioned with resveratrol (0.5 to 1 mg/kg body wt), I/R-induced iNOS induction is abrogated; however, expression of eNOS and nNOS is greatly upregulated. The protective effects of resveratrol on I/R rat heart include reduced rhythm disturbances, reduced cardiac infarct size, and decreased plasma levels of lactate dehydrogenase (LDH) and creatine kinase (CK). Among these, the reductions in LDH/CK levels and infarct size are NO-dependent as the coadministration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg body wt) with resveratrol abolishes the resveratrol effect. In contrast, the reductions in the severity of ventricular arrhythmia and mortality rate are not affected by L-NAME coadministration, suggesting that a NO-independent mechanism is involved.     

Modulation by NO of acetylcholine release in the ileum of wild-type and NOS gene knockout mice

Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.     

Arginase 1 contributes to diminished coronary arteriolar dilation in patients with diabetes

Arginase 1, via competing with nitric oxide (NO) synthase for the substrate L-arginine, may interfere with NO-mediated vascular responses. We tested the hypothesis that arginase 1 contributes to coronary vasomotor dysfunction in patients with diabetes mellitus (DM). Coronary arterioles were dissected from the right atrial appendages of 41 consecutive patients with or without DM (the 2 groups suffered from similar comorbidities), and agonist-induced changes in diameter were measured with videomicroscopy. We found that the endothelium-dependent agonist ACh elicited a diminished vasodilation and caused constriction to the highest ACh concentration (0.1 μM) with a similar magnitude in patients with (18 ± 8%) and without (17 ± 9%) DM. Responses to ACh were not significantly affected by the inhibition of NO synthesis with N(G)-nitro-L-arginine methyl ester in either group. The NO donor sodium nitroprusside-dependent dilations were not different in patients with or without DM. Interestingly, we found that the presence of N(G)-hydroxy-L-arginine (10 μM), a selective inhibitor of arginase or application of L-arginine (3 mM), restored ACh-induced coronary dilations only in patients with DM (to 47 ± 6% and to 40 ± 19%, respectively) but not in subjects without DM. Correspondingly, the protein expression of arginase 1 was increased in coronary arterioles of patients with DM compared with subjects without diabetes. Moreover, using immunocytochemistry, we detected an abundant immunostaining of arginase 1 in coronary endothelial cells of patients with DM, which was colocalized with NO synthase. Collectively, we provided evidence for a distinct upregulation of arginase 1 in coronary arterioles of patients with DM, which contributes to a reduced NO production and consequently diminished vasodilation.