Acute Nitric Oxide Synthase Research Articles

Acute nitric oxide synthase inhibition induces greater increases in blood pressure in female versus male Wistar Kyoto rats.

Nitric oxide (NO) contributes to blood pressure (BP) regulation via its vasodilatory and anti-inflammatory properties. We and others previously reported sex differences in BP in normotensive and hypertensive rat models where females have lower BP than age-matched males. As females are known to have greater NO bioavailability than age-matched males, the current study was designed to test the hypothesis that anesthetized female normotensive Wistar Kyoto rats (WKY) are more responsive to acute NOS inhibition-induced increases in BP compared to male WKY. Twelve-week-old male and female WKY were randomized to infusion of the nonspecific NOS inhibitor NG -nitro-L-arginine methyl ester (L-NAME, 1 mg/kg/min) or selective NOS1 inhibition with vinyl-L-NIO (VNIO, 0.5 mg/kg/min) for 60 min. Mean arterial BP, glomerular filtration rate (GFR), urine volume, and electrolyte excretion were assessed before, and during L-NAME or VNIO infusion. L-NAME and VNIO significantly increased BP in both sexes; however, the increase in BP with L-NAME infusion was greater in females versus males compared to baseline BP values. Acute infusion of neither L-NAME nor VNIO for 60 min altered GFR in either sex. However, urine volume, sodium, chloride and potassium excretion levels increased comparably in male and female WKY with L-NAME and VNIO infusion. Our findings suggest sex differences in BP responses to acute non-isoform-specific NOS inhibition in WKY, with females being more responsive to L-NAME-induced elevations in BP relative to male WKY. However, sex differences in the BP response did not coincide with sex differences in renal hemodynamic responses to acute NOS inhibition.

Intravital assessment of precapillary pulmonary arterioles of type 1 diabetic mice shows oxidative damage and increased tone in response to NOS inhibition.

Microvascular dilation, important for peripheral tissue glucose distribution, also modulates alveolar perfusion and is inhibited by loss of bioavailable nitric oxide (NO) in diabetes mellitus (DM). We hypothesized that DM-induced oxidative stress decreases bioavailable NO and pulmonary precapillary arteriolar diameter, causing endothelial injury. We examined subpleural pulmonary arterioles after acute NO synthase (NOS) inhibition with NG-nitro-l-arginine methyl ester (l-NAME) in streptozotocin (STZ)- and saline (CTRL)-treated C57BL/6J mice. Microvascular changes were assessed by intravital microscopy in the right lung of anesthetized mice with open chest and ventilated lungs. Arteriolar tone in pulmonary arterioles (27.2-48.7 µm diameter) increased in CTRL mice (18.0 ± 11% constriction, P = 0.034, n = 5) but decreased in STZ mice (13.6 ± 7.5% dilation, P = 0.009, n = 5) after l-NAME. Lung tissue dihydroethidium (DHE) fluorescence (superoxide), inducible NOS expression, and protein nitrosylation (3-nitrotyrosine) increased in STZ mice and correlated with increased glucose levels (103.8 ± 8.8 mg/dL). Fluorescently labeled fibrinogen administration and fibrinogen immunostaining showed fibrinogen adhesion, indicating endothelial injury in STZ mice. In CTRL mice, vasoconstriction to l-NAME was likely due to the loss of bioavailable NO. Vasodilation in STZ mice may be due to decreased formation of a vasoconstrictor or emergence of a vasodilator. These findings provide novel evidence that DM targets the pulmonary microcirculation and that decreased NO bioavailability and increased precapillary arteriolar tone could potentially lead to ventilation-perfusion abnormalities, exacerbating systemic DM complications.NEW & NOTEWORTHY Diabetes pulmonary and microvascular consequences are well recognized but have not been characterized. We assessed lung microvascular changes in a live anesthetized mouse model of type 1 diabetes, using a novel intravital microscopy technique. Our results show new evidence that a diabetes-induced decrease in lung nitric oxide bioavailability underlies oxidative damage, enhanced platelet activation, and endothelial injury causing pulmonary microvascular dysfunction and altered vasoreactivity. These findings could provide novel strategies to prevent or reverse diabetes systemic consequences.

Subpleural Microvascular Dysfunction in the Intact Mouse Lung in Conjunction with Increased Blood Glucose Levels, Oxidative Stress and Altered Nitric Oxide Signaling in a Model of Type‐1 Diabetes

Microvascular dilation important for distributing the blood glucose load to peripheral tissues, such as skeletal muscle, is inhibited by the loss of bioavailable nitric oxide (NO) in diabetes. The lungs could also be a target since NO signaling modulates alveolar perfusion. We tested the hypothesis that diabetes‐induced oxidative stress with subsequent over‐expression of inducible NO synthase (iNOS) and increased protein nitrosylation, leads to decreased bioavailable NO and consequently to pulmonary microvascular dysfunction. Effects of acute NO synthase (NOS) inhibition with L‐NAME on sub‐pleural pulmonary arterioles and alveoli were examined in streptozotocin‐treated C57BL/6J mice (STZ) and saline‐treated controls (Control). Microvascular changes were assessed by intra‐vital microscopy of the intact right lung in anesthetized mice (7 to 11 months old) with open chest and ventilated lungs when the lungs were statically held inflated for brief periods. Pulmonary arteriolar diameters and alveoli were measured before and after acute administration of L‐NAME, a nitric oxide synthase (NOS) inhibitor. L‐NAME significantly increased arteriolar tone in control mice (n=5). In contrast, tone decreased in STZ mice (n=5). Fixed lung tissue sections from these experiments were stained for superoxide detection by fluorescence microscopy (DHE staining) and immuno‐stained for iNOS expression and protein nitrosylation (3‐nitrotyrosine, 3‐NT) by immunohistochemistry. Superoxide formation significantly increased in STZ (22.8 ± 15.0%, p = 0.02) compared to Control (3.8 ± 1.0%) and correlated with increased blood glucose levels in STZ (372.2 ± 43.3 mg/dL) compared to Control (103.8 ± 8.8 mg/dL). Expression of iNOS also increased significantly in STZ (4.7 ± 3.1%, p = 0.009) compared to Control (0.06 ± 0.07%) as did 3‐NT formation in STZ (5.4 ± 5.5%, p = 0.008 compared to Control (0.007 ± 0.01%). Our data suggest that diabetes‐induced systemic effects alter lung endothelial signaling. Control mice had a physiological vasoconstrictor response to L‐NAME likely due to the loss of the vasodilator influence of bioavailable NO. In contrast, NOS inhibition with L‐NAME appeared to have a vasodilator effect in STZ mice by decreasing formation of a vasoconstrictor, or unmasking a vasodilator. DHE staining and 3‐NT immunoreactivity indicated increased oxidative stress related to blood glucose levels. We conclude that diabetes may adversely affect lung microvasculature by decreasing NO bioavailability and inducing oxidative injury. Altered NO‐induced responses may cause pulmonary endothelial injury and microvascular dysfunction leading to ventilation‐perfusion abnormalities that exacerbate systemic complications of diabetes.Support or Funding InformationUniversity of Louisville, School of Medicine Collaborative Enhancement GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute Nitric Oxide Synthase Inhibition Accelerates Transendothelial Insulin Efflux In Vivo.

Before insulin can stimulate glucose uptake in muscle, it must be delivered to skeletal muscle (SkM) through the microvasculature. Insulin delivery is determined by SkM perfusion and the rate of movement of insulin across the capillary endothelium. The endothelium therefore plays a central role in regulating insulin access to SkM. Nitric oxide (NO) is a key regulator of endothelial function and stimulates arterial vasodilation, which increases SkM perfusion and the capillary surface area available for insulin exchange. The effects of NO on transendothelial insulin efflux (TIE), however, are unknown. We hypothesized that acute reduction of endothelial NO would reduce TIE. However, intravital imaging of TIE in mice revealed that reduction of NO by l-NG-nitro-l-arginine methyl ester (l-NAME) enhanced the rate of TIE by ∼30% and increased total extravascular insulin delivery. This accelerated TIE was associated with more rapid insulin-stimulated glucose lowering. Sodium nitroprusside, an NO donor, had no effect on TIE in mice. The effects of l-NAME on TIE were not due to changes in blood pressure alone, as a direct-acting vasoconstrictor (phenylephrine) did not affect TIE. These results demonstrate that acute NO synthase inhibition increases the permeability of capillaries to insulin, leading to an increase in delivery of insulin to SkM.

Increased Subpleural Pulmonary Arteriolar Tone Associated with Inhibition of Nitric Oxide Synthase in a Mouse Model of Type‐1 Diabetes

Type‐1 diabetes, characterized by elevation of blood glucose, inflammation and oxidative stress, results in endothelial injury that alters nitric oxide (NO) signaling and impairs microvascular regulation in a variety of vascular beds. NO signaling also plays a prominent role in the modulation of alveolar perfusion. Therefore, the lungs may be particularly vulnerable to diabetes‐induced endothelial changes. We hypothesized that diabetes‐induced oxidative injury, resulting in decreased NO bioavailability and enhanced fibrinogen‐endothelial interaction, induces endothelial injury and pulmonary microvascular dysfunction in the mouse lung. The effects of acute NO synthase (NOS) inhibition with L‐NAME on subpleural arterioles were examined in streptozotocin‐treated C57BL/6J mice (STZ) and their saline‐treated controls (Control). Microvascular changes were assessed by intravital microscopy of the right lung in anesthetized mice (7 to 11 months old) with open chest and ventilated lungs. Pulmonary arteriolar diameters (ranging from 27.2 – 48.7 μm) were measured before and after acute administration of the nitric oxide synthase (NOS) inhibitor, L‐NAME (0.1mg/g body wt). L‐NAME caused a 13.6 ± 7.5% constriction (n=5, p=0.034) in Control mice. In contrast, there was an 18.0 ± 11% dilation (p<0.009, n=5) in STZ mice. Intra‐arterial administration of fluorescently‐labeled fibrinogen in STZ mice, without L‐NAME treatment, resulted in adherence of fibrinogen in the microvascular network, indicating endothelial injury. These data suggest that diabetes‐induced systemic effects may affect lung endothelial function. Control mice displayed a physiological vasoconstriction response to L‐NAME resulting from a loss of the NO vasodilator influence. In contrast, while L‐NAME may have decreased bioavailable NO in the diabetic lung, inhibition of NOS with L‐NAME appeared to have a vasodilator effect by decreasing formation of reactive nitrogen species or by unmasking another vasodilator. We conclude that diabetes may adversely affect lung microvasculature by inducing inflammation and decreasing NO bioavailability. Altered NO‐induced responses may subsequently cause significant pulmonary endothelial injury and microvascular dysfunction that could lead to ventilation‐perfusion abnormalities and exacerbate systemic complications of diabetesSupport or Funding InformationUniversity of Louisville, School of Medicine Collaborative Enhancement GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute nitric oxide synthase inhibition enhances trans‐endothelial insulin efflux and muscle insulin sensitivity in vivo

Before insulin can stimulate muscle glucose uptake, it must first traverse the continuous endothelium of skeletal muscle (SkM) capillaries. Despite the importance of trans‐endothelial insulin transport to SkM insulin sensitivity, little is known about the regulation of this process in vivo. We previously developed a technique by which we can measure the rate of trans‐endothelial insulin efflux using intravital microscopy of a fully bioactive, fluorescent insulin probe (ins‐647). Using this technique, we showed that the movement of insulin across the endothelium in SkM occurs by a fluid‐phase transport mechanism which is not saturable and does not involve the insulin receptor. Furthermore, we have demonstrated that trans‐endothelial insulin efflux is impaired in obese, insulin resistant mice. Insulin resistance in humans and rodents is associated with endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability. Studies in cultured endothelial cells suggest that insulin transport by endothelial cells is positively regulated by NO. These findings led to the hypothesis that NO is a positive regulator of trans‐endothelial insulin efflux.To test this hypothesis, we treated male C57Bl/6 mice with an intravenous bolus of either 30 mg/kg L‐NG‐Nitroarginine methyl ester (L‐NAME; NO synthase inhibitor), or saline. L‐NAME treatment increased mean arterial blood pressure by ~30 mmHg, indicating that NO synthase was successfully inhibited. Intravital ins‐647 imaging demonstrated that L‐NAME enhanced the rate at which plasma insulin equilibrated with the interstitial space by ~25%, indicating enhanced trans‐endothelial insulin efflux. The more rapid trans‐endothelial movement of insulin was associated with a 25% increase in SkM insulin sensitivity as determined by the accumulation of 2[14C]2‐deoxyglucose (2DG) in the gastrocnemius. To test the effects of increasing NO availability on insulin efflux, mice were treated with 100 μg/kg/min sodium nitroprusside (SNP; NO donor) or saline. SNP had a small, but non‐significant blood pressure‐lowering effect. SNP had no effect on either trans‐endothelial insulin efflux kinetics or gastrocnemius 2DG accumulation. To test whether the effects of NO modulation were due to changes in blood pressure, we infused a primed, continuous infusion of either phenylephrine (300 μg/kg bolus; 300 μg/kg/min infusion) or hydralazine (1 mg/kg bolus; 1 mg/kg/min infusion) into mice. Phenylephrine and hydralazine are direct smooth‐muscle acting drugs which raise and lower blood pressure independent of NO, respectively. Intravital ins‐647 imaging demonstrated that neither phenylephrine nor hydralazine had any effect on trans‐endothelial insulin efflux. Taken together, these results suggest that, surprisingly, reduction of NO by L‐NAME treatment increases trans‐endothelial insulin efflux. The mechanism by which lowering NO enhances the movement of insulin may involve effects of L‐NAME on SkM hemodynamics or direct effects on the capillary endothelium.Support or Funding InformationThis work was supported by NIH grants R01DK054902 and U24 DK059637 to D.H.W.; grant F31DK109594 to I.M.W.; and grant T32DK007563 to I.M.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of nitric oxide synthase unmasks vigorous vasoconstriction in established pulmonary arterial hypertension.

It is widely accepted that impaired bioavailability of endothelial nitric oxide (NO) plays a critical role in the pathophysiology of pulmonary arterial hypertension (PAH). However, there are published data that show that relatively many PAH patients respond favorably to acetylcholine‐induced pulmonary vasodilation during their follow‐up period, when diverse stages of the disorder are included. We hypothesized that NO bioavailability varies depending on the progression of PAH. Adult rats were exposed to the VEGF receptor blocker Sugen5416 and 3 weeks of hypoxia followed by return to normoxia for various additional weeks. All rats developed increased right ventricular systolic pressure (RVSP) and occlusive lesion formation at 1, 3, 5, and 8 weeks after the Sugen5416 injection. Acute NO synthase blockade did not change the elevated RVSP at the 1‐week time point, while it further increased RVSP markedly at the 3‐, 5‐, and 8‐week time points, leading to death in all rats tested at 8 weeks. Acetylcholine caused significant reduction in RVSP at the 8‐week but not the 1‐week time point, whereas sodium nitroprusside decreased the pressure similarly at both time points. Increased NO‐mediated cGMP production was found in lungs from the 8‐week but not the 1‐week time point. In conclusion, despite its initial impairment, NO bioavailability is restored and endogenous NO plays a critical protective role by counteracting severe pulmonary vasoconstriction in established stages of PAH in the Sugen5416/hypoxia/normoxia‐exposed rats. Our results provide solid pharmacological evidence for a major contribution of a NO‐suppressed vasoconstrictor component in the pathophysiology of established PAH.

Calcium channel blockade blunts the renal effects of acute nitric oxide synthase inhibition in healthy humans.

Our aim was to investigate whether blockade of calcium channels (CCs) or angiotensin II type 1 receptors (AT1R) modulates renal responses to nitric oxide synthesis inhibition (NOSI) in humans. Fourteen sodium-replete, healthy volunteers underwent 90-min infusions of 3.0 μg·kg-1·min-1 NG-nitro-l-arginine methyl ester (l-NAME) on 3 occasions, preceded by 3 days of either placebo (PL), 10 mg of manidipine (MANI), or 50 mg of losartan (LOS). At each phase, mean arterial pressure (MAP), glomerular filtration rate (GFR; inulin), renal blood flow (RBF; p-aminohippurate), urinary sodium (UNaV), and 8-isoprostane (U8-iso-PGF2αV; an oxidative stress marker) were measured. With PL + l -NAME, the following changes were observed: +6% MAP (P < 0.005 vs. baseline), -10% GFR, -20% RBF, -49% UNaV (P < 0.001), and +120% U8-iso-PGF2αV (P < 0.01). In contrast, MAP did not increase during LOS + l-NAME or MANI + l-NAME (P > 0.05 vs. baseline), whereas renal changes were the same during LOS + l-NAME vs. PL + l-NAME (ANOVA, P > 0.05). However, during MANI + l-NAME, changes vs. baseline in GFR (-6%), RBF (-12%), and UNaV (-34%) were blunted vs. PL + l-NAME and LOS + l-NAME (P < 0.005), and the rise in U8-iso-PGF2αV was almost abolished (+37%, P > 0.05 vs. baseline; P < 0.01 vs. PL + l-NAME or LOS + l-NAME). We conclude that, since MANI blunted l-NAME-induced renal hemodynamic changes, CCs participate in the renal responses to NOSI in healthy, sodium-replete humans independent of changes in MAP and without the apparent contribution of the AT1R. Because the rise in U8-iso-PGF2αV was essentially prevented during MANI + l-NAME, CC blockade may oppose the renal effects of NOSI in part by counteracting oxidative stress responses to acutely impaired renal NO bioavailability.

Nitric oxide synthase inhibition causes acute increases in glomerular permeability in vivo, dependent upon reactive oxygen species.

There is increasing evidence that the permeability of the glomerular filtration barrier (GFB) is partly regulated by a balance between the bioavailability of nitric oxide (NO) and that of reactive oxygen species (ROS). It has been postulated that normal or moderately elevated NO levels protect the GFB from permeability increases, whereas ROS, through reducing the bioavailability of NO, have the opposite effect. We tested the tentative antagonism between NO and ROS on glomerular permeability in anaesthetized Wistar rats, in which the left ureter was cannulated for urine collection while simultaneously blood access was achieved. Rats were systemically infused with either l-NAME or l-NAME together with the superoxide scavenger Tempol, or together with l-arginine or the NO-donor DEA-NONOate, or the cGMP agonist 8-bromo-cGMP. To measure glomerular sieving coefficients (theta, θ) to Ficoll, rats were infused with FITC-Ficoll 70/400 (mol/radius 10-80 Å). Plasma and urine samples were analyzed by high-performance size-exclusion chromatography (HPSEC) for determination of θ for Ficoll repeatedly during up to 2 h. l-NAME increased θ for Ficoll70Å from 2.27 ± 1.30 × 10-5 to 8.46 ± 2.06 × 10-5 (n = 6, P < 0.001) in 15 min. Tempol abrogated these increases in glomerular permeability and an inhibition was also observed with l-arginine and with 8-bromo-cGMP. In conclusion, acute NO synthase inhibition in vivo by l-NAME caused rapid increases in glomerular permeability, which could be reversed by either an ROS antagonist or by activating the guanylyl cyclase-cGMP pathway. The data strongly suggest a protective effect of NO in maintaining normal glomerular permeability in vivo.

Oxidative stress‐induced changes in nitric oxide availability contribute to increased pulmonary microvascular tone in a mouse model of Alzheimer's disease (1082.2)

Inflammation and oxidative stress are well‐known components of Alzheimer's disease (AD) that cause cardio‐respiratory abnormalities. To determine if AD‐induced inflammation occurs in the lung and affects its function, we hypothesized that these processes of AD cause pulmonary microvascular dysfunction by decreasing lung nitric oxide (NO) bioavailability. We assessed pulmonary involvement in an established mouse transgenic AD model expressing the human APP gene 770 isoform with the Swedish, Dutch and Iowa mutations, and in wild type (WT) controls. Mice 7‐20 months old were used, since at these ages AD model mice have microvascular injuries. Effects of acute NO synthase (NOS) inhibition with L‐NAME on pulmonary microvascular diameter were examined in the right lung of anesthetized, open chest AD and WT mice by intravital microscopy while the lungs were statically inflated with O2. Arteriolar constriction to L‐NAME was blunted in AD mice compared to WT controls. Lung immunohistochemistry showed a significant increase in superoxide production and inducible NOS (iNOS) in the AD model mice. Our data suggest that despite iNOS induction, superoxide formation decreases lung NO bioavailability in AD, resulting in changes in pulmonary vascular tone. The loss of this vasodilator influence indicates cardiovascular dysfunction may be initiated in AD through a generalized inflammatory process.Grant Funding Source: Supported by AG017173 to RPF

Acute nitric oxide synthase inhibition and endothelin-1-dependent arterial pressure elevation.

Key evidence that endogenous nitric oxide (NO) inhibits the continuous, endothelin (ET)-1-mediated drive to elevate arterial pressure includes demonstrations that ET-1 mediates a significant component of the pressure elevated by acute exposure to NO synthase (NOS) inhibitors. This review examines the characteristics of this pressure elevation in order to elucidate potential mechanisms associated with the negative regulation of ET-1 by NO and, thereby, provide potential insight into the vascular pathophysiology underlying NO dysregulation. We surmise that the magnitude of the ET-1-dependent component of the NOS inhibitor-elevated pressure is (1) independent of underlying arterial pressure and other pressor pathways activated by the NOS inhibitors and (2) dependent on relatively higher NOS inhibitor dose, release of stored and de novo synthesized ET-1, and ETA receptor-mediated increased vascular resistance. Major implications of these conclusions include: (1) the marked variation of the ET-1-dependent component, i.e., from 0 to 100% of the pressure elevation, reflects the NO-ET-1 regulatory pathway. Thus, NOS inhibitor-mediated, ET-1-dependent pressure elevation in vascular pathophysiologies is an indicator of the level of compromised/enhanced function of this pathway; (2) NO is a more potent inhibitor of ET-1-mediated elevated arterial pressure than other pressor pathways, due in part to inhibition of intravascular pressure-independent release of ET-1. Thus, the ET-1-dependent component of pressure elevation in vascular pathophysiologies associated with NO dysregulation is of greater magnitude at higher levels of compromised NO.

Role of propolis on biochemical parameters in kidney and heart tissues against l-NAME induced oxidative injury in rats

Nitric oxide (NO), produced by endothelial NO synthase, is recognised as a central antiinflammatory and antiatherogenic principle in the vasculature. Epidemiological and clinical studies have demonstrated that a growing list of natural products, as components of the daily diet or phytomedical preparations, may improve vascular function by enhancing NO bioavailability. In this article, we investigated antioxidant effects of propolis on biochemical parameters in kidney and heart tissues of acute NO synthase inhibited rats by Nω-nitro-l-arginine methyl ester (l-NAME). There was increase (p < 0.001) in the activities of catalase and malondialdehyde levels in the l-NAME treatment groups when compared with control rats, but NO levels were decreased in both kidney and heart tissues. There were statistically significant changes (p < 0.001) in these parameters of l-NAME + propolis treated rats as compared with l-NAME-treated group. In summary, propolis may influence endothelial NO production.

Different contributions of the endothelin ET(A) receptor to hypertension induced by acute or chronic inhibition of nitric oxide synthesis.

The effects of FR139317((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carb onyl] amino-4-methyl-pentanoyl] amino-3-[3-(1-methyl-1H-indoyl)]propionyl]-amino-3-(2-pyridyl)prop ionic acid), an endothelin ET(A) receptor antagonist, on systemic and renal haemodynamic responses and excretory responses to chronic or acute nitric oxide (NO) synthase inhibition with NG-nitro-L-arginine (NOARG) have been examined. An intravenous bolus injection of FR139317 (10 mg kg(-1)) to chronic NO-deficient hypertensive rats (2.74 mM NOARG in drinking water for 4 weeks) elicited only a slight decrease in mean arterial pressure (MAP), to the same extent as seen in normotensive control rats. Injection of this drug induced no alteration of the renal haemodynamics of this chronic hypertensive model. Urine formation in control rats was significantly reduced by administration of FR139317. No significant decrease in urine formation was observed in the chronic NO-deficient rats. Acute intravenous injection of NOARG (5 mg kg(-1)) induced a gradual and significant increase in MAP, with a significant decrease in renal blood flow. A slight but insignificant diuretic effect was observed. In animals pretreated with FR139317 (10 mg kg(-1) i.v.) NOARG induced a significantly less potent increase in MAP, whereas similar renal haemodynamic responses to NOARG were observed. In contrast to the FR139317-untreated group, urine formation tended to decrease after administration of NOARG. These results suggest that endothelin, via the ET(A) receptor, contributes to the systemic pressor response to acute NO synthase inhibition, although renal vasoconstriction and functional changes induced by acute NO synthase inhibition are independent of ET(A) receptor-related effects. These results imply that action of endothelin via the ET(A) receptor is not involved in the maintenance of sustained hypertension induced by chronic NO synthase inhibition.

Knockout of Angiotensin 1–7 Receptor Mas Worsens the Course of Two-Kidney, One-Clip Goldblatt Hypertension: Roles of Nitric Oxide Deficiency and Enhanced Vascular Responsiveness to Angiotensin II

Aims: The present study was performed to evaluate the effects of target disruption of the G-protein-coupled receptor Mas for angiotensin 1–7 [Ang(1–7)] in knockout mice on the course of two-kidney, one-clip (2K1C) Goldblatt hypertension. Methods: Knockout and wild-type mice underwent clipping of one renal artery. Blood pressure (BP) was monitored by radiotelemetry. The mice were either untreated or chronically treated with the superoxide (O₂^–) scavenger tempol (400 mg/l) or the inhibitor of NADPH oxidase apocynin (1 g/l) administered in drinking water. Results: Knockout mice responded to clipping by accelerated increases in BP and the final BP was significantly higher than that in wild-type mice. Chronic treatment with tempol or apocynin elicited similar antihypertensive effects in 2K1C/knockout as in 2K1C/wild-type mice. Acute nitric oxide synthase inhibition caused greater BP increases in 2K1C/wild-type than in 2K1C/knockout mice. Conclusion: Our present findings support the notion that the angiotensin-converting enzyme 2-Ang(1–7)-Mas axis serves as an important endogenous physiological counterbalancing mechanism that partially attenuates the hypertensinogenic actions of the activated renin-angiotensin system. The impairment in this axis may contribute to the deterioration of the course of 2K1C Goldblatt hypertension.

The role of nitric oxide in the development of neurogenic pulmonary edema in spinal cord-injured rats: the effect of preventive interventions

Neurogenic pulmonary edema (NPE) is an acute life-threatening complication following an injury of the spinal cord or brain, which is associated with sympathetic hyperactivity. The role of nitric oxide (NO) in NPE development in rats subjected to balloon compression of the spinal cord has not yet been examined. We, therefore, pretreated Wistar rats with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) either acutely (just before the injury) or chronically (for 4 wk prior to the injury). Acute (but not chronic) L-NAME administration enhanced NPE severity in rats anesthetized with 1.5% isoflurane, leading to the death of 83% of the animals within 10 min after injury. Pretreatment with either the ganglionic blocker pentolinium (to reduce blood pressure rise) or the muscarinic receptor blocker atropine (to lessen heart rate decrease) prevented or attenuated NPE development in these rats. We did not observe any therapeutic effects of atropine administered 2 min after spinal cord compression. Our data indicate that NPE development is dependent upon a marked decrease of heart rate under the conditions of high blood pressure elicited by the activation of the sympathetic nervous system. These hemodynamic alterations are especially pronounced in rats subjected to acute NO synthase inhibition. In conclusion, nitric oxide has a partial protective effect on NPE development because it attenuates sympathetic vasoconstriction and consequent baroreflex-induced bradycardia following spinal cord injury.

Effects of Acute Nitric Oxide Synthase Inhibition on Lower Leg Vascular Function in Chronic Tetraplegia

Background/Objective: To improve our understanding of the lower-leg vascular responses of nitric oxide synthase inhibition in persons with tetraplegia.Participants: Six people with chronic tetraplegia and 6 age-matched controls.Methods: Lower-leg relative vascular resistance and venous volume variation were obtained by venous occlusion plethysmography and blood pressure by auscultation at baseline. Postintravenous infusion of the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl-ester (1 mg-kg-1) or placebo on separate days.Results: At baseline in the group with tetraplegia compared with controls, mean arterial pressure and relative vascular resistance of the leg were significantly lower. After nitric oxide synthase inhibition, mean arterial pressure and lower leg vascular resistance were significantly elevated in both groups. There were no group or intervention differences in venous volume variation.Conclusion: These preliminary results suggest that nitric oxide synthase inhibition with 1 mg-kg-1 NG- nitro-L-arginine-methyl-ester normalizes seated blood pressure and lower leg vascular resistance to control group baseline levels.

Pivotal role of angiotensin II receptor subtype 1A in the development of two-kidney, one-clip hypertension: study in angiotensin II receptor subtype 1A knockout mice

The present study was performed to examine in two-kidney, one-clip (2K1C) Goldblatt hypertensive mice: first, the relative contribution of angiotensin II receptor subtypes 1A (AT(1A)) and 1B (AT(1B)); second, the role of angiotensin II type 2 (AT(2)) receptors in the development of hypertension in wild-type (AT(1A)+/+) and AT(1A) receptor knockout (AT(1A)-/-) mice; and third, the role of increased nitric oxide synthase activity in counteracting the hypertensinogenic action of angiotensin II in this model. AT(1A)+/+ and AT(1A)-/- mice underwent clipping of one renal artery and were infused with either saline vehicle or selective AT(2) receptor agonist CGP-42112A (CGP). Blood pressure was monitored by radiotelemetry. Blood pressure responses to the nitric oxide synthase inhibitor nitro-L-arginine-methyl-ester were evaluated. AT(1A)+/+ mice responded to clipping by a rise in blood pressure that was not modified by CGP infusion. Clip placement caused a slight increase in blood pressure in AT(1A)-/- mice that remained significantly lower than in AT(1A)+/+ mice. Acute nitric oxide synthase inhibition caused greater increase in blood pressure in 2K1C/AT(1A)+/+ than in AT(1A)+/+ mice. The present data support the critical role of AT(1A) receptors in the development of 2K1C hypertension, whereas AT(1B) receptors play only a minor role in blood pressure regulation in this model of angiotensin II-dependent hypertension. Activation of AT(2) receptors does not play an antagonistic role in the AT(1) receptor-mediated hypertensinogenic actions of angiotensin II in this model. Finally, enhanced nitric oxide synthase activity plays a protective role by counteracting the vasoconstrictor influences of angiotensin II in 2K1C hypertensive mice.

Effect of nitric oxide on exercise-induced proteinuria in rats.

Temporary proteinuria occurring after exercise is a common finding, and it is explained predominantly by alterations in renal hemodynamics. In this study, we investigated whether nitric oxide (NO), which is known to have an effect on renal hemodynamics and to increase during exercise, has a role in postexercise proteinuria. In the first step of this study, the effect of acute NO synthase blockage on exercise proteinuria was evaluated. The urinary protein levels in animals that performed acute exhaustive treadmill running exercise were considerably elevated compared with the control animals. Significantly elevated urinary protein levels were also detected in animals that received Nomega-nitro-L-arginine methyl ester before exhaustion, compared with both control and exhausted groups, and mixed-type proteinuria was detected in electrophoresis, as in all exhausted animals. In the second step of the study, a NO donor (isosorbide mononitrate) was given to rats 1 h before exhaustive exercise. Mixed-type proteinuria and the elevation in urinary protein levels that occur as a consequence of exhaustive exercise were prevented by NO donor treatment. Finally, in the third step of our study, a calcium channel blocker (diltiazem), another vasodilator, was applied to the rats 1 h before exhaustive exercise. Urinary protein levels were not different in exhausted rats with or without calcium channel blocker treatment. On the other hand, in both groups, urinary protein levels were higher than in the control group. The tail-cuff blood pressure alterations caused by vasodilator drug applications before exercise were not different for NO donor and calcium channel blocker groups. These results suggest that endogenous NO might prevent the postexercise proteinuria from becoming more severe by affecting hemodynamic changes that occur during exercise.

Prostacyclin-mediated compensatory mechanism in the coronary circulation during acute NO synthase blockade

Nitric oxide (NO) in contrast to most prostanoids, plays a major role in the maintenance of coronary arterial tone under physiological conditions. However, in case of endothelial damage or other NO-depleting situations the importance of other vasodilating mechanisms may be increased. The aim of the present study was to investigate the crosstalk between the L-arginine - NO and the prostanoid systems in isolated rat hearts. Coronary flow and cardiac dynamics were measured in a standard Langendorff perfusion system. Application of indomethacin in the perfusion media failed to change coronary flow. Administration of L-NA, however, significantly decreased coronary flow by 24.8 ± 2.3% (p < 0.01 vs. untreated control). In the presence of indomethacin, L-NA decreased coronary flow to an even greater extent by 35.8 ± 5.2% (p < 0.05 vs. L-NA alone). Treatment of the preparations with L-NA or indomethacin failed to change cardiac work, coapplication of both drugs together, however, decreased cardiac work by 45 ± 11% (p < 0.05 vs. untreated control). Heart rate remained constant throughout the experimental period and did not differ significantly between the treatment groups. The prostacyclin content of the effluent from the L-NA treated hearts was significantly higher than that of controls. We conclude that in case of decreased NO levels in the coronary circulation, arterial tone is maintained by prostacyclin production.

Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats.

Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg) elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade) and complete autonomic blockade (38 +/- 3, 55 +/- 6, 54 +/- 5, 45 +/- 5 mmHg, respectively; N = 9, P = NS). L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 +/- 8, 38 +/- 5, 46 +/- 6 bpm, respectively; N = 9, P = NS). Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 3 bpm, P<0.05 vs intact animals, N = 9). Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9) caused a similar increase in blood pressure (45 +/- 5, 38 +/- 3, 44 +/- 9 mmHg, respectively; P = NS) and heart rate (31 +/- 4, 34 +/- 3, 35 +/- 4 bpm, respectively; P = NS). Addition of L-NAME (500 micro M) to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9). In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.