Chronic L-NAME Treatment Research Articles

6-NitroDopamine is an endogenous modulator of rat heart chronotropism

6-Nitrodopamine (6-ND) is released by rat vas deferens and exerts a potent contractile response that is antagonized by tricyclic antidepressants and α1-, β1- and β1/β2-adrenoceptor antagonists. The release of 6-ND, noradrenaline, adrenaline and dopamine from rat isolated right atria was assessed by tandem mass spectrometry. The effects of the catecholamines were evaluated in both rat isolated right atria and in anaesthetized rats. 6-ND was the major catecholamine released from the isolated atria and the release was significantly reduced in nitric oxide synthase inhibitor L-NAME pre-treated atria or in atria obtained from L-NAME chronically treated animals, but unaffected by tetrodotoxin. 6-ND (1 pM) significantly increased the atrial frequency, being 100 times more potent than noradrenaline and adrenaline. Selective β1-blockers reduced the atrial frequency only at concentrations that prevented the increases in atrial frequency induced by 6-ND 1pM. Conversely, β1-blockade did not affect dopamine (10 nM), noradrenaline (100 pM) or adrenaline (100 pM) effect. The reductions in atrial frequency induced by the β1-adrenoceptor antagonists were absent in L-NAME pre-treated atria and in atria obtained from chronic L-NAME-treated animals. Tetrodotoxin did not prevent the reduction in atrial frequency induced by L-NAME or by β1-blockers treated preparations. In anaesthetized rats, at 1 pmol/kg, only 6-ND caused a significant increase in heart rate. Inhibition of 6-ND synthesis by chronic L-NAME treatment reduced both atrial frequency and heart rate. The results indicate that 6-ND is a major modulator of rat heart chronotropism and the reduction in heart rate caused by β1-blockers are due to selective blockade of 6-ND receptor.

Anti-inflammatory, anti-oxidant and cardio-protective properties of novel fluorophenyl benzimidazole in L-NAME-induced hypertensive rats

BackgroundChronic inflammation and oxidative stress play important role in development of hypertension. Recently, we have reported novel fluorophenyl benzimidazole (FPD) for vasorelaxation and antihypertensive activity in SHRs. The present study envisaged the anti-inflammatory, anti-oxidant and cardio-protective properties of FPD in L-NAME model of hypertension with special emphasis on reversal of vascular remodeling, gene expression and restoration of hemodynamic. MethodsAntihypertensive activity of FPD was evaluated in L-NAME treated Wistar rats, and the parameters studied were anti-inflammatory activity, histomorphological changes, gene expression profile and anti-oxidant properties. ResultsFPD at 50 and 100 mg kg−1 once daily for 15 days significantly reduced SBP, DBP and MAP in L-NAME treated rats and the values were well comparable to vehicle control group. Further, FPD treatment showed a significant increase in hepatic GSH content, SOD, catalase activity, decreased MDA level and restoration of pro and anti-inflammatory cytokine levels. The mRNA expression profile of genes associated with regulation of vascular tone, remodeling and inflammation showed a significant level of alteration by chronic L-NAME treatment and was dose-dependently restored upon treatment with FPD. Further, FPD treatment restored serum lipid profile, CK, CK-MB and LDH level and also reversed the histomorphological changes like intimal wall thickening, hyperplasia of cardiomyocytes and ventricular wall thickening. ConclusionsTaken together, FPD produced potent antihypertensive activity in L-NAME model through vasorelaxation, anti-oxidative and anti-inflammatory properties leading to restoration of serum lipid profile, cardiac biomarker, expression profile of target genes and reversal of histomorphological changes.

Effects of chronic nitric oxide synthase inhibition on V’O2max and exercise capacity in mice

Acute inhibition of NOS by L-NAME (Nω-nitro-L-arginine methyl ester) is known to decrease maximal oxygen consumption (V'O2max) and impair maximal exercise capacity, whereas the effects of chronic L-NAME treatment on V'O2max and exercise performance have not been studied so far. In this study, we analysed the effect of L-NAME treatment, (LN2 and LN12, respectively) on V'O2max and exercise capacity (in maximal incremental running and prolonged sub-maximal incremental running tests), systemic NO bioavailability (plasma nitrite (NO2-) and nitrate (NO3-)) and prostacyclin (PGI2) production in C57BL6/J mice. Mice treated with L-NAME for 2weeks (LN2) displayed higher V'O2max and better running capacity than age-matched control mice. In LN2 mice, NO bioavailability was preserved, as evidenced by maintained NO2- plasma concentration. PGI2 production was activated (increased 6-keto-PGF1α plasma concentration) and the number of circulating erythrocytes (RBC) and haemoglobin concentration were increased. In mice treated with L-NAME for 12weeks (LN12), NO bioavailability was decreased (lower NO2- plasma concentration), and 6-keto-PGF1α plasma concentration and RBC number were not elevated compared to age-matched control mice. However, LN12 mice still performed better during the maximal incremental running test despite having lower V'O2max. Interestingly, the LN12 mice showed poorer running capacity during the prolonged sub-maximal incremental running test. To conclude, short-term (2weeks) but not long-term (12weeks) treatment with L-NAME activated robust compensatory mechanisms involving preservation of NO2- plasma concentration, overproduction of PGI2 and increased number of RBCs, which might explain the fully preserved exercise capacity despite the inhibition of NOS.

The effects of sildenafil after chronic L-NAME administration in male rat sexual behavior

Ferraz MMD, Quintella SL, Parcial ALN, Ferraz MR. The effects of sildenafil citrate and L-NAME on male rat sexual behaviour. PHARMACOL BIOCHEM BEHAV. Erectile dysfunction (ED) affects up to 50% of men between 40 and 70years of age. Significant advances in the pharmacological treatment of ED occurred in recent years, most notably the introduction of the first oral selective phosphodiesterase type-5 inhibitor, sildenafil. This study investigated the effectiveness of chronic oral treatment with L-NAME in rats as an experimental model of erectile dysfunction to evaluate new pharmacological agents that affect the sexual response. The effects of chronic oral L-NAME treatment, separately or in combination with sildenafil, on the sexual behaviour of male rats were evaluated. Filtered water was used as a control. Acute administration of L-NAME did not alter the sexual response compared with control, but sildenafil administration facilitated sexual behaviour after acute and chronic administration. Chronic L-NAME treatment inhibited motivational and consummatory measures of male rat sexual behaviour. Sildenafil prevented the inhibitory effects of L-NAME. The present results confirm that chronic oral treatment with a nitric oxide synthase inhibitor may be a relevant peripheral ED model to evaluate the effects of drugs on erectile function of male rats.

Nox2‐derived superoxide does not contribute to oxidative stress‐induced vascular upregulation of T‐type channel function (893.6)

Cardiovascular disease is characterised by reduced nitric oxide (NO) and increased oxidative stress. We have shown that these conditions increase the expression and contribution of T‐type channels to vascular tone through increased bioavailability of reactive oxygen species produced from NADPH oxidase (Nox). As Nox2‐generated superoxide has been causally implicated in several vascular disease models, we investigated whether Nox2 was also responsible for upregulation of T‐type channel function in cremaster arterioles in vivo, using wildtype (WT) and Nox2‐deficient mice (Nox2KO). We first validated a novel T‐type channel antagonist, TTA‐A2 (3µM; provided by Merck and Co, Inc) which demonstrated no cross reactivity with L‐type channel function in these arterioles. In both WT and Nox2KO mice, acute inhibition of NO with L‐NAME (10µM) significantly increased the T‐type contribution to vascular tone. Chronic L‐NAME treatment (40mg/kg/day) for 2 weeks significantly increased blood pressure of both WT and Nox2KO mice, as well as the T‐type channel component of arteriolar tone. Pharmacological inhibition of Nox1 (10µM ML171) and Nox4 (10µM VAS2870) together reversed the effect of chronic L‐NAME on T‐type channel function in Nox2KO mice. We conclude that Nox2‐generated superoxide is not responsible for the upregulation of T‐type calcium channels elicited by oxidative stress.Grant Funding Source: Supported by the Heart Foundation of Australia

Abstract 384: Tumor Necrosis Factor-alpha Antagonism Attenuates Hypertension and Renal Injury Induced by Chronic Blockade of Nitric Oxide and High Salt Intake in Mice

Earlier, we demonstrated that systemic blockade of nitric oxide (NO) generation by acute infusion of nitro-L-arginine methyl ester (L-NAME) in mice increases plasma and renal levels of tumor necrosis factor-alpha (TNF-α). In the present study, we examined the role of TNF-α in mediating hypertension and renal injury responses to chronic NO blockade by evaluating the responses to chronic administration of L-NAME (0.05 μg/g/min; by osmotic mini-pump) for 3 wks in mice (C57BL6) treated with or without TNF-α receptor antagonist, etanercept (ETN; 5 mg, ip, every alternate day). These mice were given either normal (NS; 0.03% NaCl) or high salt (HS; 4% NaCl) containing diets. Systemic blood pressure (BP) was measured by tail-cuff plethysmography. At the end of the experimental period, mice were sacrificed to collect plasma and renal tissue samples to measure TNF-α level by ELISA and to determine the extent of tissue injury such as glomerulosclerosis (GS; PAS staining) and renal interstitial fibrosis (RIF; Trichrome staining). L-NAME treatment caused increase in BP which was greater in HS (104±3 to 139±3 mmHg; n=6) than in NS group (96±6 to 124±6 mmHg; n=6). ETN treatment significantly attenuated this increase in BP in both L-NAME+HS (119±11 mmHg; n=6) and in L-NAME+NS (115±8 mmHg, n=6) groups. Renal tissue level of TNF-α was higher in L-NAME+NS (664±100 vs 325±73 pg/mg protein) and in L-NAME+HS (391±38 vs 114±17 pg/mg protein) groups compared to the levels in groups given NS (n=5) or HS (n=5) diet alone. However, the changes in plasma level of TNF-α due to chronic L-NAME treatment were minimal. ETN treatment lowered renal TNF-α level in L-NAME+NS (356±60 pg/mg protein) and in L-NAME+HS (259±65 pg/mg protein) groups. L-NAME treatment significantly increased GS (7.1±1.0 vs 17.9±2.3 % sclerosis area) and RIF, (1.5±0.1 vs 5.8±0.6 % fibrotic area) in HS group. However, these changes were minimal in NS group (GS, 10.3±1.0 vs 13.2±1.5 % and RIF, 1.3±0.1 vs 2.4±0.3 %). ETN treatment markedly attenuated the GS (11.8±0.4% and 11.9±0.6 %) and RIF (2.6±0.3% and 1.9±0.3%) both in L-NAME+HS and in L-NAME+NS groups. These data demonstrate that an enhancement in TNF-α activity is involved in mediating hypertension and renal injury responses to chronic NO blockade and HS intake.

Nitric oxide deficit elicits oxidative stress and augments T‐type calcium channel contribution to vascular tone

Nitric oxide (NO) bioavailability is reduced in cardiovascular disease however the impact on the mechanisms controlling vascular tone of pressurised arteries is unknown. Acute inhibition of NO with L‐NAME (10μM) significantly increased the T‐type (3μM NNC 55–0396) but not L‐type (1μM nifedipine) channel contribution to mouse cremaster arteriolar tone in vivo; an effect reversed by superfusion with the NO donor, SNP (100nM). Smooth muscle expression of Cav3.1 and Cav3.2 T‐type channels was also increased. In pressurised mesenteric arteries of Cav3.1ko and Cav3.2ko mice, acutely treated with L‐NAME, the contribution of T‐type channels was significantly less than in arteries from untreated mice. Chronic L‐NAME treatment (40mg/kg/day 2 weeks) increased vascular superoxide and the contribution of T‐type channels to cremaster tone. This was reversed by acute treatment with tempol (1mM), or inhibition of NADPH oxidase (500μM apocynin; 5μM DPI). Exogenous superoxide (xanthine oxidase, 3mU/ml) mimicked the effect of NO inhibition. We conclude that NO deficit significantly increases the contribution of Cav3.1and Cav3.2 T‐type calcium channels to vascular tone by increasing superoxide produced by NADPH oxidase.

95 REDUCED NITRIC OXIDE BIOAVAILABILTY AND INCREASED REACTIVE OXYGEN SPECIES INCREASE THE CONTRIBUITON OF T-TYPE CALCIUM CHANNELS TO VASCULAR TONE

Background: Hypertension is characterised by endothelial dysfunction and reduced nitric oxide (NO) bioavailability. Approximately 30% of hypertensive patients are unresponsive to current clinical treatments. We hypothesised that this is due to changes in the calcium channels mediating vascular tone. Methods: Male C57BL/6 mice (8-12weeks) were treated with the NO inhibitor L-NAME (0.5 mg/ml) for two weeks. Blood pressure was measured by tail-cuff plethysmography. L- (1 μmol/L nifedipine) and T- (3 μmol/L NNC 55-0396) type calcium channel function was measured in cremaster muscle arterioles of anaesthetised (fentanyl 0.002, medetomidine 0.02, midazolam 0.2 mg/hr, i.v.) mice, before and after acute inhibition of reactive oxygen species (ROS) with the NADPH oxidase inhibitors, apocynin (500 μmol/L) or DPI (5 μmol/L), or the superoxide dismutase mimetic, tempol (1mmol/L). Results: Chronic L-NAME treatment significantly elevated blood pressure (120 ± 1mmHg), compared to control (104 ± 2mmHg, n = 5-6, P<0.05). Although vascular tone of cremaster muscle arterioles in vivo was not significantly altered (L-NAME: 45 ± 2%, n = 23 vs. Control: 51 ± 4% maximal diameter, n = 8, P>0.05). Chronic L-NAME treatment resulted in a significant increase in the contribution of T-type calcium channels (37 ± 2% vs. 18 ± 2%, P<0.05). Acute treatment of arterioles of L-NAME treated mice with apocynin, DPI or tempol significantly reduced vascular tone (18 ± 2%, 12 ± 6% and 24 ± 4% maximal diameter, respectively, n = 5-7, P<0.05) and the contribution of T-type calcium channels (12 ± 3%, 15 ± 4% and 7 ± 2%, respectively, P<0.05). Conclusions: T-type calcium channels contribute minimally to tone under physiological conditions in vivo, however chronic reduction in NO bioavailability, as occurs during cardiovascular disease, significantly increases T-type channel function and this is reversible by inhibition of ROS.

Differential effects of Rho-kinase inhibitor and angiotensin II type-1 receptor antagonist on the vascular function in hypertensive rats induced by chronic l-NAME treatment

Little attention has been paid to the effect of Rho-kinase inhibitor on the vascular dysfunction of nitric oxide-deficient hypertension. We aimed to investigate whether the Rho-kinase inhibitor fasudil showed beneficial effect on the vascular dysfunction of the NG-nitro-l-arginine methyl ester (l-NAME) treated rat, as well as to compare the differential effects of fasudil and angiotensin II receptor antagonist valsartan on vascular function. In the present study, both valsartan and fasudil exerted antihypertensive action on the l-NAME-treated rats, while only valsartan attenuated the cardiac hypertrophy. Treatment with valsartan showed improvement on vascular reactivity to norepinephrine, KCl and CaCl2, whereas fasudil therapy showed little effect on vasoconstriction. Endothelium-dependent vasodilation to acetylcholine was reduced in the NO-deficient group but was normalized by the fasudil therapy. The increased expression of RhoA and Rho-kinase (ROCK) in the vasculature was corrected well to normal level by either valsartan or fasudil administration, which seemed to be at least partially responsible for the beneficial effect of the drug infusion. These findings suggest that the angiotensin II receptor antagonist interferes more with the contractile response than Rho-kinase inhibitor, whereas inhibition of Rho-kinase activity exhibits a better improvement on vasorelaxation than blockade of angiotensin II receptor.

Abstract 390: Cardiovascular Risk Factors and Endothelial Dysfunction Cause Rarefaction of the Collateral Circulation and Impaired Recovery from Ischemia

Aging and cardiovascular risk factors (CVRF) such as diabetes, hypertension, dyslipidemia and metabolic syndrome have been associated with collateral insufficiency in animals and humans. Long-standing CVRF as well as collateral insufficiency associate with poorer outcomes following acute ischemic stroke or myocardial infarction. Evidence suggests that endothelial dysfunction (decreased eNOS activity and NO bioavailability) is caused by and contributes to CVRF, and promotes atherosclerosis and thrombosis in patients with CVRF. Aging and eNOS deficiency have been shown to cause rarefaction of native (pre-existing) cerebral and hindlimb collaterals in mice and to reduce collateral remodeling in response to arterial occlusion. To determine whether endothelial dysfunction and CVRF cause collateral rarefaction and/or impair remodeling, we examined recovery of perfusion following femoral artery ligation (FAL) and native cerebral pial collateral extent (density and diameter) in the following mouse models (all 7-8 mos-age): chronic L-NAME treatment (endothelial dysfunction), ob/ob (obesity), LDLR -/- on high-fat diet (hyperlipidemia), LDLR -/- ; ob/ob (metabolic syndrome), RenTgMK +/- (hypertension), and Akita (type-1 diabetes). Hindlimb perfusion immediately after FAL (dependent on native collateral extent) and recovery over 21 days (dependent on collateral remodeling) were impaired by chronic endothelial dysfunction, hypertension, metabolic syndrome, and diabetes (p&lt;0.05) but not by obesity and hyperlipidemia. Similarly, native pial collateral extent was significantly reduced in models of endothelial dysfunction, hypertension, metabolic syndrome and diabetes. The functional significance of CVRF-induced collateral rarefaction was evident as more severe ischemic injury and use impairment in hindlimb and larger infarction in brain after middle cerebral artery occlusion. Investigations into the mechanisms of CVRF-induced collateral rarefaction and treatments to prevent it are ongoing. These data show that CVRF not only impair collateral remodeling but also cause significant loss of native collaterals, resulting in worse ischemic tissue injury after acute obstruction and fewer collaterals available for remodeling.

Long‐term administration of BAY 41‐2272 prevents bladder dysfunction in nitric oxide‐deficient rats

Chronic blockade of nitric oxide (NO) synthesis leads to detrusor smooth muscle overactivity. This study aimed to evaluate the protective effects of BAY 41-2272, a soluble guanlylate cyclase activator, on changes in cystometric parameters in NO-deficient rats. Rats were divided into the following groups: (a) control, (b) DMSO, (c) N(ω)-nitro-L-arginine methyl ester hydrochrolide (L-NAME), (d) BAY 41-2272 alone, and (e) L-NAME + BAY 41-2272. The NO synthase blocker L-NAME (20 mg/rat/day) was giving in the drinking water concomitantly or not with BAY 41-2272 (10 mg/kg/day, given by gavage). Chronic L-NAME treatment markedly increased the mean arterial blood pressure (MABP), and co-treatment with BAY 41-2272 nearly reversed L-NAME-induced rise on MABP. Non-void contractions were significantly increased in L-NAME group (0.90 ± 0.1 number/min) compared with either DMSO or control group (0.49 ± 0.1 number/min), which were prevented by co-treatment with BAY 41-2271 (0.56 ± 025 number/min; P < 0.05). The threshold pressure and peak pressure increased by 70% and 44% after chronic L-NAME treatment, while co-treatment with BAY 41-2272 largely attenuated both of these effects (27% and 22% increase, respectively). The frequency of micturition cycles decreased by about of 50% in L-NAME-treated rats compared with control animals, and co-treatment with BAY 41-2272 normalized this parameter. Our data show that long-term oral administration of BAY 41-2272 counteracts the bladder dysfunction seen in NO-deficient rats, indicating that restoration of the NO-cGMP pathway by this compound may be of beneficial value to treat bladder symptoms.

Administration of BAY 41-2272 prevents bladder dysfunction in nitric-oxide deficient rats.

to evaluate the protective effects of BAY 41-2272, a soluble guanylate cyclase activator, on changes in cystometric parameters in rats deficient in nitric oxide (NO). Rats were divided into the following groups: (a) control; (b) DMSO; (c) L-NAME; (d) BAY 41-2272 alone; (e) L-NAME + BAY 41-2272. The NO synthase blocker L-NAME (20 mg/rat/day) was given in drinking water concomitantly or not with BAY 41-2272 (10 mg/kg/day, given by gavage). Chronic L-NAME treatment markedly increased the mean arterial blood pressure, and co-treatment with BAY 41-2272 nearly reversed L-NAME-induced rise on mean arterial blood pressure. Non-void contractions were significantly increased in L-NAME group (0.90 ± 0.1 number/minute) compared with either DMSO or control group (0.49 ± 0.1 number/minute), which were prevented by co-treatment with BAY 41-2272 (0.56 ± 025 number/minute; p < 0.05). The threshold and peak pressure increased by 70 and 44%, respectively, after chronic L-NAME treatment, while co-treatment with BAY 41-2272 largely attenuated both effects (27 and 22% increase, respectively). The frequency of micturition cycles decreased by about of 50% in L-NAME-treated rats compared with control animals, and co-treatment with BAY 41-2272 normalized this parameter. Our data show that long-term oral administration of BAY 41-2272 counteracts the bladder dysfunction seen in NO-deficient rats, indicating that restoration of the NO-cGMP pathway by this compound may be of beneficial value to treat bladder symptoms.

CONTINUOUS LIGHT EXPOSURE- AND L-NAME-INDUCED LEFT VENTRICULAR REMODELLING: INTERACTIONS WITH MELATONIN AND CAPTOPRIL: PP.29.155

Objective: Blood pressure enhancement induced by continuous light exposure represents an attractive but rarely investigated model of experimental hypertension. Design and Method: The aim of our study was to show whether the combination of continuous light (24 h/day) exposure and chronic L-NAME treatment induces remodelling of the left ventricle and whether captopril or melatonin are able to modify these potential alterations. Six groups (n = 8/group) of 3-month old Wistar rats were treated for six weeks: control (untreated rats) (c), L-NAME-treated (40 mg/kg/day) (L), exposed to continuous light (24), L-NAME treated and exposed to continous light (L24), light-exposed-L-NAME rats which were treated with either captopril 100 mg/kg/day (L24C), or melatonin (10 mg/kg/24 h) (L24CM). Systolic blood pressure (SBP) was measured by tail-cuff method. Relative weight of the LV, endothelial nitric oxide synthase (eNOS) and angiotensin converting enzyme (ACE) expression in tissues, malondialdehyd (MD) concentration in plasma, and the level of hydroxyproline in collagenous protein fractions were measured. Results: SBP increase was prevented by both drugs investigated but more prominently by captopril in L24. Expression of eNOS was decreased in the aorta of L24 compared to controls and neither drug has improved it. Expression of ACE was increased in L24 and both drugs prevented this increase. Increased oxidative load, estimated by concentration of MD in plasma, was reduced by both drugs. Only captopril reduced LV hypertrophy in L24, while both captopril and melatonin reduced hydroxyproline concentration(captopril in soluble collagenous fraction and melatonin in insoluble collagenous fraction) of the LV. Conclusion: It is concluded that although melatonin - in comparison to captopril - did not prevented the increase of LV weight, both captopril and melatonin prevented LV fibrosis development. This protective effect of both drugs may be due to their antioxidative effect and by reducing the ACE expression.

Chronic Inhibition of Nitric-Oxide Synthase Potentiates Endothelium-Dependent Contractions in the Rat Aorta by Augmenting the Expression of Cyclooxygenase-2

Acute inhibition of nitric-oxide synthase (NOS) unmasks the release of endothelium-derived contracting factors (EDCFs). The present study investigated whether chronic inhibition of NOS modulates endothelium-dependent contractions. Eighteen-week-old male Sprague-Dawley rats were treated by daily gavage for 6 weeks with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (60 mg/kg) or vehicle (distilled water; 1 ml/kg). Chronic treatment with L-NAME increased arterial blood pressure. Isometric tension was measured in aortic rings with or without endothelium. Endothelium-dependent relaxations to acetylcholine and the calcium ionophore 5-(methylamino)-2-[(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187) were reduced in preparations from L-NAME-treated rats. The reduction in relaxation to A23187 was partially reversed by L-arginine (1 mM). In quiescent aortic rings, A23187 caused contractions in the presence of L-NAME and intact endothelium. The A23187-induced contractions were greater in rings from the L-NAME-treated rats than in those from the control group. These contractions were abolished by the cyclooxygenase (COX)-2 inhibitor N-[2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS-398) and the thromboxane-prostanoid (TP) receptor antagonist 3-((6R)-6-{[(4-chlorophenyl)sulfonyl]amido}-2-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)propanoate (S18886), but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560). Chronic L-NAME treatment reduced the level of nitric oxide in the plasma but increased COX activity in the aortic rings. Western blotting and immunohistochemical staining showed that endothelial NOS expression was reduced in the aortae of the chronic L-NAME-treated group. COX-1 expression was augmented slightly, whereas COX-2 expression was up-regulated markedly. The TP receptor expression was comparable with control. These experiments demonstrate that chronic NOS inhibition increases endothelium-dependent contractions of the rat aorta by inducing COX-2 expression and augmenting the production of EDCF.

Melatonin interactions with blood pressure and vascular function during l-NAME-induced hypertension

The mechanisms responsible for the antihypertensive effect of melatonin are not completely understood. To elucidate the possible role of the nitric oxide (NO) pathway in the hemodynamic actions of melatonin, the effects of this indolamine on vascular function during hypertension induced by the NO-synthase (NOS) inhibitor, N(omega)-nitro-L-arginine-methyl ester (L-NAME) were investigated. Four groups of male adult Wistar rats were employed: control, L-NAME (40 mg/kg), melatonin (10 mg/kg) and L-NAME + melatonin for 5 wks. Systolic and diastolic blood pressure were measured invasively in the carotid artery. Conjugated dienes concentration (an oxidative load marker), NOS RNA expression and its activity and RNA expression of cyclooxygenase-(COX)-1 and COX-2 were determined in the aorta. Acetylcholine-induced responses and their NO-mediated component were evaluated in femoral and mesenteric artery. Moreover, endothelium-derived constricting factor (EDCF)-dependent vasoconstriction and inner diameter were determined in the femoral artery. Chronic L-NAME treatment induced hypertension, elevated the oxidative load and inhibited NOS activity. Moreover, impaired NO-dependent relaxation, augmented EDCF-constriction, increased COX-2 expression and reduced arterial inner diameter were observed. Melatonin added to L-NAME treatment completely prevented elevation of the oxidative load in the aorta. However, melatonin was not able to prevent NOS activity decline, elevation of COX-2 expression or the impairment of vascular responses (except moderate improvement in relaxation of small mesenteric arteries) and it exerted only slight antihypertensive effect. In conclusion, in addition to the reduction of the oxidative load, the restoration of the NO pathway seems to play an important role in the antihypertensive effect of melatonin.

Chronic L‐NAME Treatment in Rats Resulted in A Significant Increase in the Systemic and Right Ventricular Pressure, But the Pulmonary Arterial Pressure was not Altered.

The important role of NO in the modulation of vascular reactivity and pressure development is well established. We recently found that an acute and short‐term (1 to 2 weeks) treatment with L‐NAME resulted in a marked systemic hypertension, while the right ventricular (RV) and pulmonary pressure (PP) was not affected, suggesting a marked regional difference in NO vasomodulatory action. Continued L‐NAME treatment for 3 to 4 weeks resulted in a marked left ventricular hypertrophy (LVH), a decreased LV ejection fraction as well as an increase in the RV pressure, while the PP remains protected, suggesting a presence of pressure gradient between the RV and the pulmonary circulation. Using 2‐dimension, M‐mode and Doppler echocardiography we found that chronic NO inhibition resulted in a thickened RV wall, a turbulent RV flow pattern, a decreased RV ejection fraction, and a narrowed area in the RV outflow tract. Further histomorphological studies identified that impediment to RV outflow tract was due to massive hypertrophy of the crista supraventricularis. A similar clinical syndrome of subpulmonary stenosis induced RV outflow tract obstruction has been reported in certain patients with pulmonary disorder. Our findings suggest that this animal model of chronic L‐NAME treatment may be useful to investigate the development of hypertrophic obstructive cardiomyopathy and its clinical management.

Nitric oxide synthases are crucially involved in the development of the severe cardiomyopathy of caveolin-1 knockout mice

Targeted ablation of caveolin-1 (cav-1) results in a severe cardiomyopathy. How the loss of cav-1 mediates these abnormalities is currently under investigation. Mounting evidence indicates that cav-1 acts as a negative regulator of endothelial nitric oxide synthase resulting in a constitutive hyperactivation of the nitric oxide (NO)-pathway in cav-1 knockout mice (cav-1 ko). In this context we hypothesized that disturbed NO signalling is implicated in these changes. To explore this question cav-1 ko were compared with knockout counterparts experiencing 2 month postnatal NO synthase inhibition by N G-nitro- l-arginine methyl ester ( l-NAME) treatment. Chronic l-NAME treatment resulted in significant improvements in heart function and exercise capacity in cav-1 ko. Furthermore, we found evidence for an enhanced radical stress in hearts of cav-1 ko which was markedly reduced by l-NAME treatment. Collectively, these findings suggest that NO synthases play a crucial role in the evolution of heart failure evident in cav-1 ko.

Ambivalent effect of chronic L-NAME treatment in the heart and brain

The goal of our study was to analyse a time course of NG-nitro-l-arginine methyl ester (L-NAME) effect on NOS particular isoforms and NF-κB protein expressions, total NOS activity, and blood pressure (BP) in rats. Adult 12-week-old male Wistar rats were subjected to treatment with L-NAME (40 mg/kg/day) for 4 and 7 weeks. Both 4- and 7-week-L-NAME treatments increased BP in comparison with age-matched controls. NOS activity was decreased after 4 weeks of L-NAME treatment in both heart and brain. However, prolonging the treatment to 7 weeks increased NOS activity in the heart, while NOS activity in the brain decreased more significantly.

Effects of Chronic Nitric Oxide Synthase Inhibition on Endothelium-Dependent and -Independent Relaxation in Arteries that Perfuse Skeletal Muscle of Swine

The purpose of this investigation was to test the hypothesis that chronic N(G)-nitro-l-arginine methyl ester (l-NAME) treatment produces differential effects on conduit artery and resistance arteriole relaxation responses to endothelium-dependent and -independent vasodilators in arteries that perfuse skeletal muscle of swine. To test this hypothesis, conduit skeletal muscle arteries and second-order skeletal muscle (2A) arterioles were harvested from 14 Yucatan swine that were chronically administered l-NAME and from 16 controls. In vitro assessments of vasorelaxation to increasing doses of acetylcholine (ACH), bradykinin (BK), and sodium nitroprusside (SNP) were performed in both conduit and 2A arterioles. l-NAME treatment produced a significant reduction in both BK and ACH relaxation responses in the conduit arteries. In contrast, the relaxation response and/or sensitivity to SNP were significantly greater in the intact, but not denuded, conduit arterial rings from chronically l-NAME-treated swine. There were no significant effects of chronic l-NAME treatment on vasodilation of skeletal muscle arterioles. These findings suggest (1) that unlike arterioles, skeletal muscle conduit arteries do not functionally compensate for a lack of NO through the upregulation of alternative vasodilator pathways; (2) that the greater relaxation response in conduit arteries of chronically l-NAME-treated swine to SNP can be explained by alterations to the endothelium.

Chronic-low-dose L-NAME treatment increases nitric oxide production and vasorelaxation in normotensive rats

N(G)-nitro-L-arginine methyl ester (L-NAME) is a non-specific nitric oxide (NO) synthase inhibitor, commonly used for the induction of NO-deficient hypertension. The aim of this study was to investigate the effect of chronic low-dose administration of L-NAME on NO production, vascular function and structure of the heart and selected arteries of rats. Adult male Wistar rats were treated with L-NAME in the dose of approximately 1.5 mg/kg/day in drinking water for 8 weeks. Basal blood pressure (BP) of rats (determined by tail-cuff) was 112+/-3 mm Hg. The low-dose administration of L-NAME significantly elevated BP measured on the third and sixth week of treatment vs. controls by approximately 9 % and 12 %, respectively. After this period, BP of L-NAME-treated rats returned to the control values. The relative left ventricular mass, heart fibrosis and collagen III/collagen I ratio were not affected by L-NAME. Similarly, there were no alterations in the cross-sectional area and wall thickness/diameter ratio of the aorta and the femoral artery of L-NAME-treated rats. NO synthase activity (determined by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline) was not altered in the hypothalamus of L-NAME-treated rats. Interestingly, chronic low-dose L-NAME treatment significantly elevated NO synthase activity in the left ventricle and aorta, increased endothelium-dependent acetylcholine-induced vasorelaxation and reduced serotonin-induced vasoconstriction of the femoral artery. The data suggest that chronic low-dose L-NAME treatment can increase NO production and vasorelaxation in normotensive rats without negative structural changes in the cardiovascular system.