Long-term Nitric Oxide Synthase Research Articles

Chronic Inhibition of Nitric Oxide Synthases Impairs Spatiotemporal Learning and Memory to a Similar Extent in C57BL/6 and hAPP23+/− Mice

Due to global population growth, age-related disorders like cardiovascular disease and dementia are anticipated to increase. Recent data suggests a connection between cardiovascular disease and neurodegeneration, especially focusing on arterial stiffness (AS) and Alzheimer’s disease (AD). In light of this, we conducted a study to explore the impact of long-term nitric oxide synthase (NOS) isoform inhibition, which leads to AS, on neurobehavioral performance. We also compared these effects in an AD model and control mice. C57BL/6 and hAPP23+/− mice (an established AD model) were given 0.5 mg/mL N(G)-Nitro-L-Arginine Methyl Ester (L-NAME) in their drinking water for 16 weeks. Our findings indicate that chronic non-selective NOS inhibition increased AS and reduced spatiotemporal learning and memory in both C57BL/6 and hAPP23+/− mice. These effects were consistent across both groups, emphasizing the role of neuronal NOS (nNOS) in cognitive aging, regardless of genetic predisposition to AD.

Long-term nitric oxide synthase inhibition prevents 17β-estradiol-induced suppression of cyclooxygenase-dependent contractions and enhancement of endothelium-dependent hyperpolarization-like relaxation in mesenteric arteries of ovariectomized rats

Endothelial dysfunction is associated with a reduced bioavailability of nitric oxide (NO). In this study, the effects of 17β-estradiol supplement on endothelial function were examined in ovariectomized (OVX) rats following long-term inhibition of NO synthases with L-NAME. Female Sprague Dawley rats were ovariectomized at 12 weeks old. They were supplemented with 17β-estradiol (25 μg/kg/day, intramuscularly) or its vehicle (olive oil) until they were killed. At 18 weeks old, they were administered daily with NO synthase inhibitor L-NAME (60 mg/kg, by gavage) or its vehicle (distilled water) for 6 weeks. Rats were then anesthetized for blood pressure measurement and for isolation of mesenteric arteries and aortae for isometric tension measurement. Long-term L-NAME-treatment, without or with 17β-estradiol supplement, resulted in reduced plasma nitrite/nitrate level without causing an increase in blood pressure in OVX rats. Acute inhibition of cyclooxygenase (COX) with indomethacin improved relaxations of mesenteric arteries to the calcium ionophore A23187 in OVX rats, and in those with long-term L-NAME-treatment without or with 17β-estradiol supplement, but not in those with female hormone supplement only. 17β-estradiol supplement or long-term L-NAME-treatment resulted in a greater endothelium-dependent hyperpolarization-like relaxation in mesenteric arteries. In the quiescent aorta, 17β-estradiol supplement or long-term L-NAME-treatment unmasked the COX-dependent components of A23187-induced contractions, but prevented that of the smooth muscle contractions to U46619 in OVX rats. In summary, long-term 17β-estradiol-supplement results in differential effects in different blood vessel types, and its beneficial vascular effects are masked under the conditions with NO synthase inhibition.

Long-term l-NAME treatment potentiates the blood–brain barrier disruption during pentylenetetrazole-induced seizures in rats

We investigated whether the severity of blood–brain barrier disruption caused by pentylenetetrazole-induced seizures is modified by long-term nitric oxide synthase inhibition in rats. Rats were given N-omega-nitro- l-arginine methyl ester ( l-NAME), a nitric oxide synthase inhibitor, in drinking water for 4 weeks, and then treated with pentylenetetrazole to induce seizures. Damage to the blood–brain barrier was investigated using Evans blue dye extravasation. Serum nitric oxide concentration was decreased in l-NAME-treated rats ( P < 0.01). l-NAME and/or pentylenetetrazole treatments elevated systolic blood pressure of animals ( P < 0.01). l-NAME caused an increase in the mortality rate after pentylenetetrazole injection leading to the death of animals at about 15 min after the onset of the seizure. Pentylenetetrazole-induced seizures in rats treated with l-NAME caused a significant increase in Evans blue dye extravasation into cerebral cortex, diencephalon and cerebellum, as compared with seizures evoked by pentylenetetrazole injection to l-NAME-untreated rats ( P < 0.01). Data presented here suggest that the degree of blood–brain barrier disruption induced by seizures is more pronounced in long-term nitric oxide deficiency.

Pivotal role of PAI-1 in a murine model of hepatic vein thrombosis

Hepatic veno-occlusive disease (VOD) is a common complication of high-dose chemotherapy associated with bone marrow transplantation. While the pathogenesis of VOD is uncertain, plasminogen activator inhibitor-1 (PAI-1) has emerged as a diagnostic marker and predictor of VOD in humans. In this study, we investigated the role of PAI-1 in a murine model of VOD produced by long-term nitric oxide synthase inhibition using L-NAME. After 6 weeks, wild-type (WT) mice developed extensive fibrinoid hepatic venous thrombi and biochemical evidence of hepatic injury and dysfunction. In contrast, PAI-1-deficient mice were largely protected from the development of hepatic vein thrombosis. Furthermore, WT mice that received tiplaxtinin, an antagonist of PAI-1, were effectively protected from L-NAME-induced thrombosis. Taken together, these data indicate that NO and PAI-1 play pivotal and antagonistic roles in hepatic vein thrombosis and that PAI-1 is a potential target in the prevention and treatment of VOD in humans.

Potential Roles of Plasminogen Activator System in Coronary Vascular Remodeling Induced by Long-term Nitric Oxide Synthase Inhibition

K. Kaikita, J. A. Schoenhard, C. A. Painter, R. T. Ripley, N. J. Brown, A. B. Fogo and D. E. Vaughan. Potential Roles of Plasminogen Activator System in Coronary Vascular Remodeling Induced by Long-term Nitric Oxide Synthase Inhibition. Journal of Molecular and Cellular Cardiology (2002) 34, 617–627. Recent studies have indicated that a number of factors contribute to the pathophysiology in response to nitric oxide synthase (NOS) inhibition. We previously demonstrated that plasminogen activator inhibitor-1 deficient (PAI-1−/−) mice are protected against hypertension and perivascular fibrosis induced by relatively short-term NOS inhibition. In this study, we compared the temporal changes in systolic blood pressure and coronary perivascular fibrosis induced by long-term treatment withNω -nitro- L -arginine methyl ester (L -NAME) in wild type (WT), PAI-1−/− and tissue-type plasminogen activator deficient (t-PA−/−) mice. After initiating L -NAME, systolic blood pressure increased in all groups at 2 weeks. Over a 16 week study period, systolic blood pressure increased to 143±3mmHg (mean±SEM) in WT animals, 139±2 in t-PA−/− mice vs 129±2 in PAI-1−/− mice (P < 0.01). Coronary perivascular fibrosis increased in L -NAME-treated WT and t-PA−/− mice compared to each control group (P<0.01 in WT, P<0.05 in t-PA−/−), while PAI-1−/− mice were protected against fibrosis induced by L -NAME. t-PA deficiency did not accentuate the vascular pathology or the changes in blood pressure. In situ zymography demonstrated augmented gelatinolytic activity in PAI-1−/− mice at baseline, suggesting that PAI-1 deficiency prevents the increase of collagen deposition by promoting matrix degradation. Plasma TGF-β1 levels increased in L -NAME-treated WT and PAI-1−/− mice (P < 0.01), but not in L -NAME-treated t-PA−/− mice. These findings support the hypothesis that the plasminogen activator system protects against the structural vascular changes induced by long-term NOS inhibition. While PAI-1 deficiency protects against L -NAME-induced hypertension and perivascular fibrosis, t-PA deficiency does not exacerbate the vascular pathology or hypertension.

Beneficial effect of pentaerythrityl tetranitrate on functional and morphological changes in the rat thoracic aorta evoked by long-term nitric oxide synthase inhibition

The present study examined whether pentaerythrityl tetranitrate (PETN), a tolerance-devoid exogenous donor of nitric oxide (NO), could attenuate functional and morphological changes in the rat thoracic aorta evoked by 6-week NO synthase inhibition by N G -nitro- l-arginine methyl ester ( l-NAME). Systolic blood pressure in l-NAME+PETN-treated rats (163±1 mm Hg) was significantly lower than in l-NAME-treated rats (172±2 mm Hg) but was still higher than in age-matched controls (126±2 mm Hg). Six weeks of treatment of rats with l-NAME significantly inhibited endothelium-dependent relaxation of the isolated thoracic aorta induced by acetylcholine. The inhibitory effect of l-NAME was entirely reversed by the simultaneous treatment with PETN. The enhancing effect of l-NAME on noradrenaline-induced contraction was antagonised by long-term treatment with PETN. Wall thickness, cross-sectional area and wall/diameter ratio of the thoracic aorta in l-NAME-treated rats were markedly increased. In the l-NAME+PETN-treated rats, the increment of these parameters was significantly lower. The results suggest that PETN administered to rats during development of NO-deficient hypertension prevented functional impairment and at the same time reduced structural changes in the thoracic aorta induced by long-term inhibition of NO synthase.

Plasminogen activator inhibitor-1 deficiency prevents hypertension and vascular fibrosis in response to long-term nitric oxide synthase inhibition.

Long-term inhibition of nitric oxide synthase (NOS) is known to induce hypertension and perivascular fibrosis. Recent evidence also suggests that long-term NOS inhibition induces expression of plasminogen activator inhibitor-1 (PAI-1) in vascular tissues and that PAI-1 may contribute to the development of fibrosis after chemical or ionizing injury. On the basis of these observations, we hypothesized that PAI-1 may influence the vascular response to long-term NOS inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME). We compared the temporal changes in systolic blood pressure and coronary perivascular fibrosis in PAI-1-deficient (PAI-1(-/-)) and wild-type (WT) male mice (N=6 per group). At baseline, there were no significant differences in blood pressure between groups. After initiation of L-NAME, systolic blood pressure increased in both groups at 2 weeks. Over an 8-week study period, systolic blood pressure increased to 141+/-3 mm Hg in WT animals versus 112+/-4 mm Hg in PAI-1(-/-) mice (P<0.0001). The extent of coronary perivascular fibrosis increased significantly in L-NAME-treated WT mice (P<0.01 versus PAI-1(-/-) mice). Cardiac type I collagen mRNA expression was greater in control (P<0.01) and L-NAME-treated PAI-1(-/-) (P<0.05) groups than in control WT mice, indicating that PAI-1 deficiency prevents the increase of collagen deposition by promoting matrix degradation. These findings suggest that PAI-1 deficiency alone is sufficient to protect against the structural vascular changes that accompany hypertension in the setting of long-term NOS inhibition. Direct inhibition of vascular PAI-1 activity may provide a new therapeutic strategy for the prevention of arteriosclerotic cardiovascular disease.

Marked variation in responses to long-term nitric oxide inhibition during pregnancy in outbred rats from two different colonies

Objective: Some but not all studies have shown that long-term nitric oxide synthase inhibition during pregnancy induces symptoms similar to those of preeclampsia that include hypertension, proteinuria, and intrauterine growth restriction. This study was undertaken to compare the effects of long-term nitric oxide synthase inhibition during pregnancy on blood pressure and fetal weight between Sprague-Dawley rats from outbred colonies of two different suppliers. Study Design: Osmotic minipumps were inserted on day 10 or day 17 of pregnancy in Sprague-Dawley rats obtained from Charles River Laboratories, Inc, Wilmington, Mass, or Harlan Sprague Dawley, Inc, Indianapolis, Ind. The pumps were set to deliver vehicle only (control group) or N ω-nitro-L -arginine methyl ester (a nitric oxide synthase inhibitor) at a rate of 50 mg/d until postpartum day 7. Systolic blood pressures were measured daily with the tail-cuff method. Neonatal weights and survival were recorded. Results:N ω-nitro-L -arginine methyl ester infusion initiated on gestational day 10 increased blood pressure relative to control levels in all rats studied. Harlan rats were much more sensitive to the hypertensive effect of N ω-nitro-L -arginine methyl ester. When N ω-nitro-L -arginine methyl ester infusion was initiated on gestational day 17, blood pressure increased only in Harlan rats. Pups born to Harlan rats treated with N ω-nitro-L -arginine methyl ester had lower birth weights and a higher stillbirth rate than did pups of Charles River rats. The degree of hypertension was significantly correlated with the deleterious effects of N ω-nitro-L -arginine methyl ester on the fetuses. Conclusion: Within the same strain of rats the effects of long-term nitric oxide synthase inhibition on blood pressure and fetal outcome depended on the original animal colony, with animals from Harlan Sprague Dawley being more sensitive than those from Charles River Laboratories. This difference in response between animals from different suppliers is most likely caused by genetic differences inbred into the strain. In addition to explaining some of the reported inconsistencies between laboratories, these results may also provide insights into the genetic basis of preeclampsia. (Am J Obstet Gynecol 2001;184:686-93.)

Differential Effects of Endothelin A and B Receptor Antagonism on Fetal Growth in Normal and Nitric Oxide-Deficient Rats

To determine the role of endothelin (ET) in fetal and placental growth in rats with and without long-term nitric oxide synthase (NOS) inhibition. Pregnant rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME) or saline and with one of three ET receptor antagonists or vehicle. The antagonists included A-182086 (nonselective) as well as A-127722 and FR-139317 (both ET(A) selective). Treatment was begun on day 14 of gestation. On gestational day 21, a hysterotomy was done. Litter size was recorded, and viability and fetal and placental weights were determined. Results were analyzed by analysis of variance or by a Kruskal-Wallis nonparametric analysis. In the absence of L-NAME, fetal and placental weights were not affected by ET(A)-selective antagonism but were significantly decreased by nonselective receptor antagonism (P <.001 and P <.05 for fetal and placental weights, respectively). Infusion of L-NAME resulted in fetal and placental growth restriction (P <.001). In the setting of L-NAME infusion, fetal and placental weights were increased by the ET(A)-selective antagonists (P <.01) but not by the nonselective antagonist, compared with weights from animals treated with L-NAME alone. There were more fetal deaths with L-NAME treatment (P <.05), but their occurrence was not significantly affected by any of the ET receptor antagonists. Endothelin-A antagonism alone did not affect fetal or placental growth, whereas combined ET(A) plus ET(B) antagonism produced fetal and placental growth restriction. In the setting of long-term NOS inhibition, ET(A)-selective antagonism improved fetal and placental growth, whereas antagonism of both ET(A) and ET(B) receptors did not. Endothelin contributes to NOS inhibition-induced growth restriction acting through the ET(A) receptor.

Long-term nitric oxide synthase inhibition in rat pregnancy reduces renal kallikrein.

This study was performed to test the hypothesis that long-term nitric oxide synthase (NOS) inhibition during pregnancy may alter the predominance of the vasodilator kallikrein system. Sprague-Dawley rats were treated with the competitive inhibitor of NOS N(omega)-nitro-L-arginine (L-NNA, 50 mg. kg(-1). d(-1), dissolved in water) from days 7 to 21 of pregnancy. Rats were studied before treatment (day 5), at days 11, 17, and 21 of pregnancy (during treatment), and at postpartum days 7 and 21 (after the drug was withdrawn at delivery). Each group (n=5 to 8) had its corresponding control group (C) that received only vehicle. Additional rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME) alone or with an excess of L-arginine. At each study day, we measured blood pressure, collected urine overnight, obtained blood samples, and processed the kidneys for conventional histology and immunohistochemistry. In L-NNA rats, fetal and placental weights were reduced at days 17 and 21. Blood pressure was higher at days 17 and 21, returning to normal after L-NNA was removed. Urinary kallikrein activity was lower at days 11 and 17 (L-NNA=1147+/-213 and C=2317+/-146 nmol/16 h, P<0.001). Plasma renin activity was reduced at day 21 (L-NNA=9.6+/-2.1 and C=25.9+/-5 ng x mL(-1) x h(-1), P<0.05) and remained lower at postpartum day 7 x L-NNA rats exhibited glomerular lesions and tubular atrophy, particularly of connecting tubules that displayed reduced kallikrein staining. Tubulointerstitial infiltrating macrophages (ED1+) were also observed. Renal lesions were present as early as day 11 and persisted at day 7 postpartum. L-NAME rats exhibited similar alterations that were attenuated with an excess of L-arginine. We postulate that the reduction in renal kallikrein may contribute to the hemodynamic alterations described in this model.

Long-term nitric oxide synthase inhibition in ontogenesis: Neurogenic and endothelial control of arteries

Long-term nitric oxide synthase inhibition in ontogenesis: Neurogenic and endothelial control of arteries

Long-term nitric oxide blockade in the pregnant rat: Effects on blood pressure and plasma levels of endothelin-1

OBJECTIVE: Abnormalities in the production of nitric oxide and endothelin-1 have been implicated in the development of preeclampsia. We postulated that long-term nitric oxide synthase inhibition with L-nitro-arginine methyl ester would induce sustained hypertension, a rise in plasma levels of endothelin-1, and fetal growth restriction. STUDY DESIGN: Conscious virgin and pregnant Sprague-Dawley rats received infusions of vehicle or L-nitro-arginine methyl ester (2.5 mg/kg/hr) for 11 days. Mean arterial pressure was assessed serially. On day 21 of gestation (or equivalent in virgin rats) plasma was collected for endothelin-1 levels; pup weight and litter size were determined. Data were analyzed with analysis of variance and regression techniques. RESULTS: Mean arterial pressure was constant in virgin control rats ( n = 7) but declined in pregnant control rats ( n = 11) as gestation advanced. Nitric oxide synthase inhibition in virgin ( n = 10) and pregnant ( n = 11) rats caused sustained elevations in mean arterial pressure (165 ± 7 vs 100 ± 3 mm Hg, L-nitro-arginine methyl ester vs control virgin rats, p < 0.0001; 149 ± 5 vs 91 ± 2 mm Hg, L-nitro-arginine methyl ester vs control pregnant rats, p < 0.0001). L-nitro-arginine methyl ester induced a rise in plasma endothelin-1 levels in virgin (4.4 ± 0.1 vs 3.5 ± 0.1 pg/ml, L-nitro-arginine methyl ester vs control, p < 0.0001) and pregnant rats (3.0 ± 0.1 vs 2.6 ± 0.1 pg/ml, L-nitro-arginine methyl ester vs control, p < 0.0001). Pregnant rats had lower endothelin-1 levels than did virgin rats ( p < 0.0001). Mean arterial pressure and endothelin-1 were significantly correlated in pregnant rats. L-nitro-arginine methyl ester decreased pup weight (2.4 ± 0.4 vs 3.7 ± 0.2 gm/pup/litter, L-nitro-arginine methyl ester vs control, p < 0.01) and litter size (6.6 ± 1.3 vs 10.2 ± 0.9 pups/litter, L-nitro-arginine methyl ester vs control, p < 0.05). CONCLUSIONS: Long-term nitric oxide synthase blockade causes sustained hypertension, elevated levels of endothelin-1, and fetal growth restriction. Although the endocrine and pressor effects are not unique to pregnancy, this model clearly induces some of the changes seen in preeclampsia and may be useful for studying specific interventions. (Am J Obstet Gynecol 1996;175:484-8.)

Role of endothelin-1 in hypertension induced by long-term inhibition of nitric oxide synthase

We examined the effect of long-term nitric oxide (NO) synthase inhibition on vascular and renal endothelin-1 levels and evaluated the antihypertensive effect of endothelin ET A receptor antagonist FR139317 (( R)2-[( R)-2-[( S)-2-[[1-(hexahydro-1 H-azepinyl)]-carbonyl]amino-4-methyl-pentanoyl]amino-3-[3-(1-methyl-1 H-indolyl)]propionyl]amino rats in which NO synthase was blocked. Chronic NO blockade was produced by oral administration of the NO synthase inhibitor N G-nitro- l-arginine for 4 weeks, which produced sustained hypertension. At the end of this time, there were no significant changes in aortic and renal immunoreactive-endothelin levels between N G-nitro- l-arginine-treated hypertensive rats and normotensive control rats. Intravenous injection of FR139317 (10 mg/kg), which had a sufficient hypotensive effect on deoxycorticosterone acetate-salt hypertensive rats, to N G-nitro- l-arginine-treated hypertensive rats produced only a moderate hypotensive effect, to the same degree as seen in normotensive rats. The results indicate that long-term NO synthase inhibition did not affect vascular and renal endothelin-1 levels in these rats. It seems likely that endothelin-1 and ET A receptors do not contribute to the sustained hypertension induced by NO synthesis blockade.

Long-term nitric oxide synthase inhibition and distensibility of carotid artery in intact rats.

The goal of the present study was to evaluate the effect of long-term nitric oxide synthase inhibition by NG-nitro-L-arginine-methyl ester (L-NAME) on the morphology and viscoelastic properties of the carotid arteries in rats. Twelve-week-old Wistar-Kyoto rats were treated for 6 weeks with either the nitric oxide synthase inhibitor L-NAME (0.4 g/L in drinking water; L-NAME rats, n = 13) or tap water (control rats, n = 13). Age-matched spontaneously hypertensive rats (SHR, n = 14) received tap water for the same period. The internal diameter of the common carotid artery was measured continuously with an echo-tracking device with the rats under anesthesia with halothane. Intra-arterial pressure was monitored on the contralateral side. L-NAME rats exhibited arterial pressures similar to those of SHR. The distensibility pressure-curve determined in L-NAME rats was a direct continuation of that obtained in control rats. In contrast the distensibility in SHR was increased (P < .01, SHR versus L-NAME rats). Carotid artery cross-sectional area and left ventricular weight index were increased similarly in SHR and L-NAME rats compared with control rats. Thus the hypertension caused by long-term nitric oxide synthesis inhibition was not associated with the increased arterial distensibility observed in SHR despite similar blood pressure elevations, similar arterial hypertrophy, and consequently similar wall stress. This suggests a role for nitric oxide in regulating the mechanical behavior of arteries exposed to high blood pressure.

Angiotensin blockade reverses hypertension during long-term nitric oxide synthase inhibition.

Blockade of the renin-angiotensin system was studied in male Sprague-Dawley rats during long-term inhibition of nitric oxide synthase. Nitro-L-arginine-methyl ester (L-NAME) was placed in the drinking water for 4 weeks (approximately 100 mg/kg per day). Separate groups of rats were coadministered the angiotensin II antagonist A-81988 in the drinking water ranging from approximately 0.001 to 1 mg/kg per day. Control groups received only tap water or A-81988 alone. Each week, rats were placed in metabolic cages, and tail-cuff blood pressures and blood samples were taken. L-NAME produced a sustained elevation in tail-cuff pressure that was completely prevented by A-81988. No changes in creatinine clearance, sodium excretion, plasma creatinine concentration, or blood urea nitrogen were observed. Food and water intakes were identical in all groups. Water excretion was significantly increased in L-NAME-treated animals regardless of additional inhibitor treatment, suggesting a possible role for nitric oxide synthase in the control of water excretion; this effect was independent of blood pressure. Although less potent than A-81988, the angiotensin II antagonist losartan and the angiotensin converting enzyme inhibitor enalapril also blocked L-NAME-induced hypertension. In a separate series of experiments, rats were not given A-81988 until 2 weeks after hypertension had fully developed in L-NAME-treated rats. Within 1 week of treatment with the angiotensin II antagonist, tail-cuff pressure returned to normal. We conclude from these studies that long-term inhibition of endogenous nitric oxide production produces an angiotensin II-dependent form of hypertension.