Systemic Nitric Oxide Synthase Inhibition Research Articles

Systemic NOS inhibition reduces contracting muscle oxygenation more in intact female than male rats

Females respond to baroreceptor stimulation with enhanced modulation of heart rate (HR) to regulate blood pressure and also express greater reliance on nitric oxide (NO) for vascular control compared to males. Sex differences in muscle oxygenation consequent to central hemodynamic challenge induced by systemic NO synthase (NOS) inhibition are unknown. We tested the hypotheses that systemic NOS inhibition would induce lower contracting skeletal muscle oxygenation in females compared to males. The spinotrapezius of Sprague-Dawley rats (females (♀) = 9, males (♂) = 9) was surgically exposed and contracted by electrical stimulation (180s, 1 Hz, ~6 V) under pentobarbital sodium anesthesia. Oxyphor G4 was injected into the muscle and phosphorescence quenching was used to measure the interstitial PO2 (PO2is, determined by O2 delivery-to-utilization matching) under control (Krebs-Henseleit solution) and after intra-arterial infusion of nitro-l-arginine methyl ester (l-NAME; NOS blockade; 10 mg kg−1). At rest, females showed a greater PO2is increase (ΔPO2is/ΔMAP) and HR (ΔHR/ΔMAP) reduction than males in response to the elevated MAP induced by systemic NOS inhibition (both p < 0.05). Following l-NAME, during the contracting steady-state, females exhibited lower PO2is than males (♂: 17.1 ± 1.4 vs ♀: 10.8 ± 1.4 mmHg, p < 0.05). The rate pressure product was lower in females than males (♂: 482 ± 14 vs ♀: 392 ± 29, p < 0.05) and correlated with the steady-state PO2is (r = 0.66, p < 0.05). These results support that females express greater reductions in HR than males in response to l-NAME-induced elevation of MAP via the baroreceptor reflex and provide new insights on how central hemodynamics affect skeletal muscle oxygenation in a sex-specific manner.

Renal Soluble Guanylate Cyclase Is Downregulated in Sunitinib-Induced Hypertension.

BackgroundThe tyrosine kinase inhibitor sunitinib causes hypertension associated with reduced nitric oxide (NO) availability, elevated renal vascular resistance, and decreased fractional sodium excretion. We tested whether (1) nitrate supplementation mitigates sunitinib‐induced hypertension and NO contributes less to renal vascular resistance as well as fractional sodium excretion regulation in sunitinib‐treated rats than in controls; and (2) renal soluble guanylate cyclase (sGC) is downregulated and sGC activation lowers arterial pressure in rats with sunitinib‐induced hypertension.Methods and ResultsArterial pressure responses to nitrate supplementation and the effects of systemic and intrarenal NO synthase (NOS) inhibition on renal hemodynamics and fractional sodium excretion were assessed in sunitinib‐treated rats and controls. Renal NOS and sGC mRNA as well as protein abundances were determined by quantitative polymerase chain reaction and Western blot. The effect of the sGC activator cinaciguat on arterial pressure was investigated in sunitinib‐treated rats. Nitrate supplementation did not mitigate sunitinib‐induced hypertension. Endothelium‐dependent reductions in renal vascular resistance were similar in control and sunitinib‐treated animals without and with systemic NOS inhibition. Selective intrarenal NOS inhibition lowered renal medullary blood flow in control but not in sunitinib‐treated rats without significant effects on fractional sodium excretion. Renal cortical sGC mRNA and sGC α1‐subunit protein abundance were less in sunitinib‐treated rats than in controls, and cinaciguat effectively lowered arterial pressure by 15‐20 mm Hg in sunitinib‐treated rats.ConclusionsRenal cortical sGC is downregulated in the presence of intact endothelium‐dependent renal vascular resistance regulation in developing sunitinib‐induced hypertension. This suggests that sGC downregulation occurs outside the renal vasculature, increases renal sodium retention, and contributes to nitrate resistance of sunitinib‐induced hypertension.

Effect of nebivolol on renal nitric oxide availability and tubular function in patients with essential hypertension.

Nebivolol is a selective β1 -receptor antagonist with vasodilating properties. In patients with essential hypertension, we tested the hypothesis that nebivolol increases systemic and renal nitric oxide (NO) availability using L-N(G) -monomethyl arginine (L-NMMA) as an inhibitor of NO production. In a randomized, placebo-controlled, crossover study, patients with essential hypertension were treated with nebivolol for five days, along with a standardized diet and fluid intake. We examined the acute effects of systemic NO synthase inhibition with L-NMMA on brachial blood pressure (bBP), pulse wave velocity (PWV) and central blood pressure (cBP) estimated by applanation tonometry, glomerular filtration rate (GFR), fractional excretion of sodium (FENa ), urinary excretion of both aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ ), and plasma concentrations of nitrate/nitrite (p-NOx ) and vasoactive hormones after five days' treatment with placebo and nebivolol. Nebivolol significantly reduced PWV, bBP, cBP and plasma renin, angiotensin II and aldosterone concentrations. The renal parameters, p-NOx and plasma arginine vasopressin concentration were not changed by nebivolol. There was no difference between nebivolol and placebo in the response to L-NMMA, with LMMA inducing a similar increase in PWV, bBP and cBP and a similar decrease in GFR, uAQP2 and u-ENaCγ and FENa [mean change -0.62% (95% confidence interval {CI} -0.40 to -0.84) during placebo vs. -0.57% (95% CI -0.46 to -0.68; P = 0.564) during nebivolol treatment]. Vasoactive hormones were changed to a similar extend by L-NMMA during administration of nebivolol and placebo. Nebivolol did not change p-NOx , and inhibition of NO synthesis induced the same response in blood pressure, GFR, renal tubular function and vasoactive hormones during nebivolol and placebo. Thus, the data did not support the hypothesis that nebivolol changes vascular and renal NO availability in patients with essential hypertension.

Chronic NOS inhibition accelerates NAFLD progression in an obese rat model.

The progression in nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis is a serious health concern, but the underlying mechanisms remain unclear. We hypothesized that chronic inhibition of nitric oxide (NO) synthase (NOS) via N(ω)-nitro-L-arginine methyl ester (L-NAME) would intensify liver injury in a rat model of obesity, insulin resistance, and NAFLD. Obese Otsuka Long-Evans Tokushima fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats received control or L-NAME (65-70 mg·kg(-1)·day(-1))-containing drinking water for 4 wk. L-NAME treatment significantly (P < 0.05) reduced serum NO metabolites and food intake in both groups. Remarkably, despite no increase in body weight, L-NAME treatment increased hepatic triacylglycerol content (+40%, P < 0.05) vs. control OLETF rats. This increase was associated with impaired (P < 0.05) hepatic mitochondrial state 3 respiration. Interestingly, the opposite effect was found in LETO rats, where L-NAME increased (P < 0.05) hepatic mitochondrial state 3 respiration. In addition, L-NAME induced a shift toward proinflammatory M1 macrophage polarity, as indicated by elevated hepatic CD11c (P < 0.05) and IL-1β (P = 0.07) mRNA in OLETF rats and reduced expression of the anti-inflammatory M2 markers CD163 and CD206 (P < 0.05) in LETO rats. Markers of total macrophage content (CD68 and F4/80) mRNA were unaffected by L-NAME in either group. In conclusion, systemic NOS inhibition in the obese OLETF rats reduced hepatic mitochondrial respiration, increased hepatic triacylglycerol accumulation, and increased hepatic inflammation. These findings suggest an important role for proper NO metabolism in the hepatic adaptation to obesity.

Role of atrial natriuretic peptide in mediating the blood pressure-independent natriuresis elicited by systemic inhibition of nitric oxide.

While it is clearly recognized that increased intrarenal nitric oxide (NO) levels elicit natriuresis, confounding data showing that systemic nitric oxide synthase inhibition (NOSi) also increases sodium excretion (UNaV) poses a conundrum. This response has been attributed to the associated increases in arterial pressure (AP); however, the increases in AP and in UNaV are temporally dissociated. The changes in regional renal haemodynamics induced by NOSi could also contribute to the alterations of UNaV. To evaluate the roles of AP and non-AP mechanisms mediating the natriuresis, N ω-nitro-l-arginine methyl ester hydrochloride (L-NAME) was infused i.v. at doses ranging from 5 to 50 μg/kg/min in anaesthetized rats. UNaV, perfusion of the cortex (cortical blood flow, CBF) and medulla (medullary blood flow, MBF) with laser-Doppler flowmetry and glomerular filtration rate (GFR) were measured. UNaV increased from 0.6 ± 0.2 to 1.6 ± 0.1 μmol/kg/min (P < 0.05) with the lower nonpressor doses. With the higher doses, AP increased from 116 ± 4 to 122 ± 4 mmHg and UNaV increased from 1.1 ± 0.3 to 3.3 ± 0.7 μmol/min/g (P < 0.002). UNaV increased similarly in a group where renal AP was maintained at baseline levels. The associated reductions in CBF (17 ± 5 and 38 ± 5 %) and MBF (27 ± 6 and 52 ± 6 %) would be expected to attenuate rather than contribute to the natriuresis. Plasma atrial natriuretic peptide (ANP) concentrations increased significantly following NOSi. Anantin, a natriuretic peptide receptor-A blocker, prevented or reversed the L-NAME-induced natriuresis without altering the L-NAME-induced changes in AP or CBF. The results indicate that increased ANP and related natriuretic peptides mediate the AP-independent natriuresis, at least partly, elicited by systemic L-NAME infusion and help resolve the conundrum of natriuresis during systemic NOSi.

Lack of changes in carotid artery compliance with systemic nitric oxide synthase inhibition

Proximal large elastic arteries (ascending aorta and carotid artery) have an important role in buffering the pulsatile pressure generated from the left ventricle, which forwards continuous peripheral blood flow and protects the brain microcirculation from end-organ damage. Although compliance of distal conduit arteries (extremities' arteries) is attenuated by the nitric oxide synthase (NOS) inhibition, it is yet unknown whether compliance of proximal elastic arteries changes by the systemic NOS inhibition. To address this question, we measured central artery compliance in 17 young adults (26±1 years) who underwent intravenous infusions of N(G)-monomethyl-L-arginine (L-NMMA) or saline (placebo) on separate days. Following the systemic NOS inhibition, the mean arterial pressure (MAP), total peripheral resistance and aortic augmentation index were significantly increased. However, carotid artery compliance was not affected significantly (from 0.10±0.01 to 0.11±0.01 mm2) per mmHg) and the β-stiffness index (an index of arterial compliance adjusted for the distending pressure) tended to decrease (from 6.63±0.35 to 6.06±0.42 a.u., P=0.07). These parameters were not altered with saline infusion. Changes in the β-stiffness index tended to correlate negatively with the corresponding changes in MAP (r = -0.31, P=0.07). These results suggest that carotid artery compliance remains unchanged during the systemic NOS inhibition in spite of systemic vasoconstriction.

Altered Nitric Oxide Bioavailability Contributes to Diesel Exhaust Inhalation‐Induced Cardiovascular Dysfunction in Man

BackgroundDiesel exhaust inhalation causes cardiovascular dysfunction including impaired vascular reactivity, increased blood pressure, and arterial stiffness. We investigated the role of nitric oxide (NO) bioavailability in mediating these effects.Methods and ResultsIn 2 randomized double‐blind crossover studies, healthy nonsmokers were exposed to diesel exhaust or filtered air. Study 1: Bilateral forearm blood flow was measured during intrabrachial infusions of acetylcholine (ACh; 5 to 20 μg/min) and sodium nitroprusside (SNP; 2 to 8 μg/min) in the presence of the NO clamp (NO synthase inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA) 8 μg/min coinfused with the NO donor SNP at 90 to 540 ng/min to restore basal blood flow). Study 2: Blood pressure, arterial stiffness, and cardiac output were measured during systemic NO synthase inhibition with intravenous l‐NMMA (3 mg/kg). Following diesel exhaust inhalation, plasma nitrite concentrations were increased (68±48 versus 41±32 nmol/L; P=0.006) despite similar l‐NMMA–induced reductions in basal blood flow (−20.6±14.7% versus −21.1±14.6%; P=0.559) compared to air. In the presence of the NO clamp, ACh and SNP caused dose‐dependent vasodilatation that was not affected by diesel exhaust inhalation (P>0.05 for both). Following exposure to diesel exhaust, l‐NMMA caused a greater increase in blood pressure (P=0.048) and central arterial stiffness (P=0.007), but reductions in cardiac output and increases in systemic vascular resistance (P>0.05 for both) were similar to those seen with filtered air.ConclusionsDiesel exhaust inhalation disturbs normal vascular homeostasis with enhanced NO generation unable to compensate for excess consumption. We suggest the adverse cardiovascular effects of air pollution are, in part, mediated through reduced NO bioavailability.Clinical Trial RegistrationURL: http://www.ClinicalTrials.gov. Unique identifiers: NCT00845767 and NCT01060930.

Effect of Systemic Nitric Oxide Synthase Inhibition on Optic Disc Oxygen Partial Pressure in Normoxia and in Hypercapnia

To investigate the effect of systemic nitric oxide synthase (NOS) inhibition on optic disc oxygen partial pressure (PO(2)) in normoxia and hypercapnia. Intervascular optic disc PO(2) was measured in 12 anesthetized minipigs by using oxygen-sensitive microelectrodes placed <50 microm from the optic disc. PO(2) was measured continuously during 10 minutes under normoxia, hyperoxia (100% O(2)), carbogen breathing (95% O(2), 5% CO(2)), and hypercapnia (increased inhaled CO(2)). Measurements were repeated after intravenous injection of N(omega)-nitro-L-arginine methyl ester (L-NAME) 100 mg/kg. Intravenous L-arginine 100 mg/kg was subsequently given to three animals. Before L-NAME injection, an increase was observed in optic disc PO(2) during hypercapnia (DeltaPO(2) = 3.2 +/- 1.7 mm Hg; 18%; P = 0.001) and carbogen breathing (DeltaPO(2) = 12.8 +/- 5.1 mm Hg; 69%; P < 0.001). Optic disc PO(2) in normoxia remained stable for 30 minutes after L-NAME injection (4% decrease from baseline; P > 0.1), despite a 21% increase of mean arterial pressure. Optic disc PO(2) increase under hypercapnia was blunted after L-NAME injection (DeltaPO(2) = 0.6 +/- 1.1 mm Hg; 3%; P > 0.1), and this effect was reversible by L-arginine. Moreover, L-NAME reduced the response to carbogen by 29% (DeltaPO(2) = 9.1 +/- 4.4 mm Hg; 49%; P = 0.01 versus before L-NAME). The response to hyperoxia was not affected. Whereas systemic NOS inhibition did not affect optic disc PO(2) in normoxia, a blunting effect was noted on the CO(2)-induced optic disc PO(2) increase. Nitric oxide appears to mediate the hypercapnic optic disc PO(2) increase.

Reduction in α-adrenergic receptor-mediated vascular tone contributes to improved arterial compliance with endurance training

Background Regular aerobic exercise improves large artery compliance in middle-aged and older humans. However, the underlying mechanisms are unknown. We tested the hypothesis that the improved central arterial compliance with endurance training is mediated by decreased α-adrenergic tone and/or increased endothelial function. Methods Seven sedentary healthy adults (60 ± 3 years) underwent systemic α-adrenergic blockade (phentolamine) and nitric oxide synthase (NOS) inhibition using N G-monomethyl- l-arginine in sequence before and after a 3-month moderate endurance training (walk/jog, 4–5 days/week). Phentolamine was given first to isolate the contribution of nitric oxide to arterial compliance by minimizing reflex suppression of sympathetic tone resulting from systemic NOS inhibition as well as to assess the α-adrenergic receptor-mediated modulation of arterial compliance. Results Baseline arterial compliance (via simultaneous ultrasound and applanation tonometry on the carotid artery) increased 34 ± 12% after exercise training ( P < 0.01). When α-adrenergic blockade was performed, arterial compliance increased 37 ± 6% ( P < 0.01) before the exercise training but did not change significantly after the training. Decreases in arterial compliance from the α-adrenergic blockade to the subsequent additional NOS blockade were not different before and after exercise training. Conclusion Our results suggest that the reduction in α-adrenergic receptor-mediated vascular tone contributes to the improved central arterial compliance with endurance training.

Reduction in Alpha-Adrenergic Receptor-Mediated Vascular Tone Contributes to Improved Arterial Compliance with Endurance Training

PURPOSE: Regular aerobic exercise improves large artery compliance in middleaged and older humans. However, the underlying mechanisms are unknown. We tested the hypothesis that the improved central arterial compliance with endurance training is mediated by decreased α-adrenergic tone and/or increased endothelial function. METHODS: Seven sedentary healthy adults (61±2 yr) underwent systemic α-adrenergic blockade (phentolamine) and nitric oxide synthase (NOS) inhibition using NG-monomethyl-L-arginine in sequence before and after a 3-month moderate endurance training (walk/jog, 4-5 days/week). Phentolamine was given first to isolate the contribution of nitric oxide to arterial compliance by minimizing reflex suppression of sympathetic tone resulting from systemic NOS inhibition as well as to assess the α-adrenergic receptor-mediated modulation of arterial compliance. RESULTS: Baseline arterial compliance (via simultaneous ultrasound and applanation tonometry on the carotid artery) increased 34±12% after exercise training (P<0.01). When α-adrenergic blockade was performed, arterial compliance increased 37±6% (P<0.01) before the exercise training but did not change significantly after the training. Decreases in arterial compliance from the α-adrenergic blockade to the subsequent additional NOS blockade were not different before and after exercise training. CONCLUSIONS: Our results suggest that the reduction in α-adrenergic receptormediated vascular tone contributes to the improved central arterial compliance with endurance training.

Carotid artery compliance and systemic nitric oxide synthase inhibition in young healthy adults

Decreased arterial compliance (increased arterial stiffness) is associated with cardiovascular events. Nitric oxide regulates vascular tone, which can influence arterial compliance. We previously investigated the effects of systemic NOS inhibition on arterial compliance under the systemic α‐adrenergic receptor blocking. In the present study, we investigated the effect of systemic NOS inhibition alone on central arterial compliance. Sixteen apparently healthy young adults (26±1 years) underwent intravenous infusions of NG‐monomethyl‐L‐arginine (L‐NMMA) or placebo (saline) on separate days. In the placebo control condition, no significant changes were observed in mean arterial pressure (68±2 vs. 71±1 mmHg), carotid artery compliance (0.10±0.01 vs. 0.10±0.01 mm2/mmHg), and β‐stiffness index (7.3±0.3 vs. 7.1±0.4 AU). After the administration of L‐NMMA, mean arterial pressure increased significantly (70±2 vs. 79±2 mmHg). However, there were no significant changes in carotid artery compliance (0.09±0.01 vs. 0.10±0.01 mm2/mmHg) or carotid β‐stiffness index (7.3±0.3 vs. 6.7±0.4 AU), an index adjusted for distending pressure. Our findings indicate that systemic NO inhibition does not modulate central arterial compliance in young healthy adults.

Systemic nitric oxide synthase inhibition improves coronary flow reserve to adenosine in patients with significant stenoses

We studied the impact of systemic infusion of the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on coronary flow reserve (CFR) in patients with coronary artery disease (CAD). We have previously demonstrated that CFR to adenosine was significantly increased after systemic infusion of L-NMMA in normal volunteers but not in recently transplanted denervated hearts. At baseline, myocardial blood flow (MBF; ml x min(-1) x g(-1)) was measured at rest and during intravenous administration of adenosine (140 microg x kg(-1) x min(-1)) in 10 controls (47 +/- 5 yr) and 10 CAD patients (58 +/- 8 yr; P < 0.01 vs. controls) using positron emission tomography and (15)O-labeled water. Both MBF measurements were repeated during intravenous infusion of 10 mg/kg L-NMMA. CFR was calculated as the ratio of MBF during adenosine to MBF at rest. CFR was significantly higher in healthy volunteers than in CAD patients and increased significantly after L-NMMA in controls (4.00 +/- 1.10 to 6.15 +/- 1.35; P < 0.0001) and in patients, both in territories subtended by stenotic coronary arteries (>70% luminal diameter; 2.06 +/- 1.13 to 3.21 +/- 1.07; P < 0.01) and in remote segments (3.20 +/- 1.23 to 3.92 +/- 1.62; P < 0.05). In conclusion, CFR can be significantly increased in CAD by a systemic infusion of L-NMMA. Similarly to our previous findings in normal volunteers, this suggests that adenosine-induced hyperemia in CAD patients is constrained by a mechanism that can be relieved by systemic NOS inhibition with L-NMMA.

Altered Nitric Oxide System in Patients With Open-Angle Glaucoma

To investigate the ocular blood flow response to systemic nitric oxide synthase inhibition in patients with primary open-angle glaucoma. In 12 patients with glaucoma and 12 age-matched control subjects, subfoveal choroidal blood flow, optic nerve head blood flow, ocular fundus pulsation amplitude, intraocular pressure, and systemic hemodynamic parameters were measured at baseline and after inhibition of nitric oxide synthase by intravenous administration of NG-monomethyl-L-arginine. The increase in blood pressure in response to NG-monomethyl-L-arginine was comparable between the 2 study cohorts. In patients with glaucoma, the decrease of optic nerve head blood flow (P = .03) and fundus pulsation amplitude (P<.001) during nitric oxide synthase inhibition was significantly less pronounced than in healthy control subjects. A tendency toward a reduced response in choroidal blood flow was seen (P = .051 between groups) in patients with glaucoma. This is the first in vivo study providing evidence for an altered ocular L-arginine/nitric oxide system in patients with glaucoma. Normalization of the ocular nitric oxide production may be beneficial in terms of normalization of ocular blood flow and neuroprotection of retinal ganglion cells.

Effect of Systemic Nitric Oxide Synthase Inhibition on Arterial Stiffness in Humans

Stiffening of large elastic arteries impairs the buffering function of the arterial system and contributes to cardiovascular disease. The aim of this study was to determine whether endothelium-derived nitric oxide (NO) modulates the stiffness of large elastic arteries in humans. Seven apparently healthy adults (60+/-3 years, 2 males and 5 females) underwent systemic alpha-adrenergic blockade (phentolamine) and systemic NO synthase inhibition using NG-monomethyl-L-arginine (L-NMMA) in sequence. Phentolamine was given first to isolate contribution of NO to arterial stiffness by preventing reflex changes in sympathetic tone that result from systemic NO synthase inhibition, and also to compare arterial stiffness at a similar mean arterial pressure. Mean arterial blood pressure decreased (p<0.05) after phentolamine infusion but returned to baseline levels after L-NMMA infusion. The carotid beta-stiffness index (via simultaneous ultrasound and applanation tonometry on the common carotid artery) did not change after the restraint of systemic alpha-adrenergic nerve activity (9.8+/-1.2 vs. 9.1+/-1.1 U) but increased (p<0.05) after NO synthase inhibition (12.6+/-2.0 U). These results suggest that NO appears to modulate central arterial stiffness in humans.

Effect of Systemic Nitric Oxide Synthase Inhibition on Arterial Stiffness in Humans

Stiffening of large elastic arteries impairs the buffering function of the arterial system and contributes to cardiovascular disease. Vascular tone is an important determinant of arterial stiffness. PURPOSE: To determine whether endothelium-derived nitric oxide modulates stiffness of large elastic artery in humans. METHODS: Seven apparently healthy adults (60±3 yr, 2 males and 5 females) underwent systemic alpha-adrenergic blockade (phentolamine) and systemic nitric oxide synthase inhibition using NG-monomethyl-L-arginine (L-NMMA) in sequence. Phentolamine was given first to isolate the contribution of nitric oxide to arterial stiffness by preventing reflex changes in sympathetic tone that result from systemic nitric oxide synthase inhibition as well as to test the extent to which sympathetic nervous system activity modulates arterial stiffness. RESULTS: Mean arterial pressure decreased (P<0.05) after phentolamine infusion, but returned to baseline levels after L-NMMA infusion. Carotid β -stiffness index (via simultaneous B-mode ultrasound and applanation tonometry on the common carotid artery) did not change after the restraint of systemic alpha-adrenergic nerve activity (9.8±1.2 vs. 9.1±1.1 U), but increased (P<0.05) after NO synthase inhibition (12.6±2.0 U). Similarly, aortic pulse wave velocity (PWV) did not change after phentolamine infusion (862±49 vs. 821±51 cm/sec), but increased (P<0.05) after L-NMMA infusion (982±69 cm/sec). Femoral artery β -stiffness index and leg PWV, measures of peripheral arterial stiffness, did not change significantly throughout the experiments. CONCLUSION: Nitric oxide appears to modulate central arterial stiffness in humans.

Blunted response to systemic nitric oxide synthase inhibition in the cerebral circulation of patients with Type 2 diabetes

Diabetes is a major risk factor for stroke, but the mechanisms that impart the excess risk are unclear. Endothelial dysfunction, which has been demonstrated in the coronary and peripheral vasculature of diabetic patients, is an important early marker of vascular disease. However, the effect of diabetes on cerebrovascular endothelium has not been examined. We sought to investigate the effect of diabetes on basal cerebrovascular endothelial function as assessed by response to the nitric oxide synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA). Fourteen men with Type 2 diabetes and 15 age-matched male control subjects were recruited. The participants had no clinically evident vascular disease and were taking no vasoactive or lipid-lowering medication. Each received a single 15-min intravenous infusion of L-NMMA (0.8 mol/kg/min). Cerebral blood flow was assessed by colour Doppler imaging of the internal carotid artery (ICA) at 10-min intervals for 20 min prior to and following the infusion. Middle cerebral artery velocity (MCAv) was assessed by transtemporal Doppler ultrasound at the same time points. L-NMMA produced a mean reduction in ICA flow area under curve (AUC) in the control group of 12.8 +/- 17.8% compared with a 2.1 +/- 21.7% reduction in the group with diabetes (P < 0.05), indicating blunted basal cerebrovascular response to NOS inhibition in the diabetic group. There was no significant change in MCAv following L-NMMA in either group. Mean +/- sd MAP rose 6.4 +/- 4.2 mmHg in the control group vs. 8.8 +/- 3.5 mmHg in the diabetic group [P = not significant (NS)]. No adverse event or symptom was reported. Response to NOS inhibition is impaired in the cerebral circulation of patients with diabetes. This observation is consistent with the elevated cerebrovascular risk reported in this population, and may represent a future therapeutic target in stroke prevention.

Modulation by NO of renal myogenic autoregulation: TGF dependency

Blood flow to the kidney is unique in that it is regulated to serve the urine-producing function of the kidney and is far in excess of any conceivable metabolic demand. As renal blood flow (RBF) is so high, and resistance thus so low, the capillaries in the glomerulus are potentially exposed to high transmural pressures. The need to stabilize glomerular capillary pressure plus the need for stable tubular loads to permit regulation of salt excretion has focused attention on the mechanisms by which RBF is stabilized when blood pressure fluctuates. Renal autoregulation, i.e. regulation by the kidney of its own blood flow, effectively stabilizes RBF over a wide range of blood pressure. In turn, autoregulation is the product of two mechanisms: a myogenic mechanism located entirely within the vascular smooth muscle of the preglomerular microcirculation, and tubuloglomerular feedback (TGF) by which information about the distal tubular load is transmitted through the macula densa and the juxtaglomerular apparatus to the afferent arteriole. Both these mechanisms are extensively modulated by a variety of paracrine and endocrine agents. In the 15 years since Tolins et al. (1990) showed that the nitro-arginine derivative Nω-monomethyl-l-arginine (l-NMMA) causes profound renal vasoconstriction, a great number of studies have been performed to characterize and understand the contribution of nitric oxide to the regulation of RBF. These studies have revealed that the renal circulation is perhaps the most sensitive in the body to non-selective inhibition of nitric oxide synthase (NOS) with complete inhibition causing at least a twofold increase in renal vascular resistance that is independent of the pronounced pressor response seen after systemic NOS inhibition. Early studies did not detect any effect of NOS inhibition on renal autoregulation. More recent studies using the classic steady-state, pressure ramp approach have shown that the lower pressure limit of autoregulation is extended to still lower pressures. At the same time, time series analysis has shown distinct augmentation of the myogenic mechanism after non-selective NOS inhibition. These effects have largely been attributed to inhibition of endothelial NOS although the kidney also contains neuronal NOS (nNOS), located largely at and around the macula densa. While selective inhibition of nNOS has been problematic, a number of studies have combined to demonstrate that inhibition of nNOS at the macula densa causes a stronger TGF response (e.g. Ichihara et al. 1998 and other references found in Just & Arendshorst, 2005). In this issue of The Journal of Physiology, Just & Arendshorst (2005) report studies that bear on two aspects of how NO affects renal autoregulation. As non-selective NOS inhibition causes such strong renal vasoconstriction, it is often considered that the enhancement of autoregulation is a consequence of the vasoconstriction. However, blood pressure fluctuates both upward and downward and NOS inhibition improves the stabilization of RBF. The major point made by Just and Arendshorst is the demonstration, which was logically necessary, that non-selective NOS inhibition augments both pressure-induced vasoconstriction and pressure-induced vasodilatation in the kidney. This finding is supported by the demonstration that equivalent vasoconstriction caused by Ang II does not duplicate the autoregulatory result, showing that the vasoconstriction is not sufficient to explain the change in autoregulation. In addition, they show in the same rats that non-selective NOS inhibition does not alter myogenic autoregulation in the hindquarters, indicating regional specificity. The authors further show that the augmentation of renal myogenic autoregulation induced by non-selective NOS inhibition is abrogated in the presence of furosemide. Furosemide of course is a loop diuretic which, at the dose used, blocks NaCl reabsorption from the thick ascending limb and effectively clamps TGF off. The strength of this interaction is somewhat surprising, given that both sources of nitric oxide within the renal cortex were inhibited, only one of which is under TGF control, and that nitric oxide is highly diffusive. However, this is undoubtedly a very interesting result that indicates regulation of the myogenic mechanism by tubular elements. Thus, a control system, TGF, that plays an important role linking renal inputs (RBF and GFR) to output (tightly regulated salt excretion) has now been linked mechanistically to the other autoregulatory system that had been presumed to be independent of tubular function.

Effect of intravenous L‐NMMA on nitric oxide production in collagenous colitis

Background: Nitric oxide (NO) is greatly increased in the colon of patients with inflammatory bowel disease of different aetiology, including collagenous colitis. In man, NO can be generated from NO synthases as well as from non‐enzymatic sources. The source of the NO found in luminal intestinal gas has not been exactly pinpointed. We investigated the effect of the NO synthase inhibitor L‐NMMA on intestinal concentrations of NO in patients with clinically active collagenous colitis. In addition, we measured NO levels from the respiratory tract. Methods: Ten patients with active collagenous colitis were studied. NO levels were measured from the respiratory tract and in the rectum before and after i.v. administration of L‐NMMA (7.5 mg/kg) using a chemiluminescence method. Results: Airway NO release decreased markedly, and mean arterial blood pressure increased in all patients following L‐NMMA treatment. Basal rectal NO levels were high (median >10 000 ppb) and decreased markedly in 5 patients after L‐NMMA. In contrast, NO levels remained completely unchanged or even increased in the other 5 patients. Conclusion: Rectal levels of NO are greatly increased in patients with symptomatic collagenous colitis. Intravenous administration of an NO synthase inhibitor reduced rectal NO only in half of the patients despite clear evidence of effective systemic NO synthase inhibition. This could indicate alternative NOS‐independent sources of intestinal NO in this disease.

Hypertension induced by nitric oxide synthase inhibition activates the atrial natriuretic peptide (ANP) system

We assessed the effect of nitric oxide (NO) synthase inhibition on plasma atrial natriuretic peptide (ANP) concentration and content in some brain structures [neurohypophysis (NH), adenohypophysis (AH), medial basal hypothalamus (MHB) and olfactory bulb (OB)] in rats before and after blood volume expansion (BVE). Male Wistar rats were injected i.p. with N ϖ-nitro- l-arginine ( l-NNA), 25 mg/kg of body weight, 40 min before the experiment (acute treatment) or l-NNA at a dose of 25 mg/kg body weight, twice a day, for 4 days (chronic treatment). The acute treatment caused an increase in the blood pressure and plasma ANP concentration in rats under basal conditions and after BVE. A decrease in ANP content was observed in the OB and NH, whereas no significant changes were found in the AH or MBH. In chronically treated rats, we also found an increase in blood pressure and in plasma ANP concentration under basal conditions and after BVE. The ANP content increased in the OB, NH and AH. These results indicate that systemic NO synthase inhibition increases ANP concentration in plasma and in areas of the central nervous system. We hypothesize that ANP participates in the hypertension-induced by NO synthesis blockade acting by baroreceptors input to the brain to stimulate ANP release and synthesis that reduces NO prival hypertension.

Cardiac nerves affect myocardial stunning through reactive oxygen and nitric oxide mechanisms.

The goal of this study was to investigate the role of cardiac nerves on the response to 90-minute coronary artery stenosis (CAS), which reduced coronary blood flow by 40% for 90 minutes, and subsequent myocardial stunning after reperfusion in chronically instrumented conscious pigs. In pigs with regional cardiac denervation (CD), myocardial stunning was intensified, ie, at 12 hours reperfusion wall thickening (WT) was depressed more, P<0.05, in CD (-46+/-5%) as compared with intact pigs (-31+/-3%) and remained depressed in CD at 24 hours reperfusion (-45+/-6%). Although the TTC technique was negative for infarct, histopathological analysis revealed patchy necrosis present in 11+/-2% of the area at risk. In intact pigs, WT had essentially recovered at 24 hours without infarct. In CD pigs treated with either an antioxidant, N-2-mercaptopropionyl glycine (MPG, 100 mg/kg per hour) or systemic nitric oxide synthase inhibition using N(omega)-nitro-L-arginine (L-NA, 30 mg/kg for 3 days), recovery of wall thickening was similar to that in pigs with intact nerves and without evidence of infarct. Immunohistochemistry analysis for 3-nitrotyrosine in tissue after CAS and 1 hour reperfusion demonstrated enhanced peroxynitrite-related protein nitration in pigs with regional CD compared with pigs with intact cardiac nerves, and pigs with regional CD and MPG or L-NA. Thus, reperfusion after myocardial ischemia in the setting of CD results in enhanced stunning and development of infarct. The underlying mechanism appears to involve nitric oxide and reactive oxygen species.