L-NAME In Rats Research Articles

BPC 157 as a Therapy for Retinal Ischemia Induced by Retrobulbar Application of L-NAME in Rats.

Providing NO-system importance, we suggest that one single application of the NOS-blocker L-NAME may induce retinal ischemia in rats, and that the stable pentadecapeptide BPC 157 may be the therapy, since it may interact with the NO-system and may counteract various adverse effects of L-NAME application. A rat retinal ischemia study was conducted throughout 4 weeks, including fundoscopy, behavior presentation, tonometry, and histology assessment. Retrobulbar L-NAME application (5 mg/kg; 0.5 mg/0.1 ml saline/each eye) in rats immediately produced moderate generalized irregularity in the diameter of blood vessels with moderate atrophy of the optic disc and faint presentation of the choroidal blood vessels, and these lesions rapidly progressed to the severe stage. The specific L-NAME–induced vascular failure points to normal intraocular pressure (except to very transitory increase upon drug retrobulbar administration). When BPC 157 (10 μg; 10 ng/kg, as retrobulbar application, 1 μg; 1 ng/0.1 ml saline/each eye) is given at either 20 min after L-NAME or, lately, at 48 h after L-NAME, the regular retrobulbar L-NAME injection findings disappear. Instead, fundoscopy demonstrated only discrete generalized vessel caliber irregularity with mild atrophy of the optic disc, and then, quite rapidly, normal eye background and choroidal blood vessels, which remain in all of the subsequent periods. Also, histology assessment at 1, 2, and 4 weeks shows that BPC 157 counteracted the damaged inner plexiform layer and inner nuclear layer, and revealed normal retinal thickness. The poor behavioral presentation was also rescued. Thus, while further studies will be done, BPC 157 counteracted L-NAME–induced rat retinal ischemia.

Butterfly Pea Flower (Clitoria ternatea Linn.) Extract Ameliorates Cardiovascular Dysfunction and Oxidative Stress in Nitric Oxide-Deficient Hypertensive Rats.

In this study, we examine whether Clitoria ternatea Linn. (CT) can prevent Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced cardiac and vascular dysfunction in rats. Male Sprague Dawley rats were given L-NAME (40 mg/kg, drinking water) and orally administered with CT extract (300 mg/kg/day) or lisinopril (2.5 mg/kg/day) for 5 weeks. The main phytochemical components of the CT extract were found to be flavonoids. The CT extract alleviated the high blood pressure in rats receiving L-NAME. Decreased vasorelaxation responses to acetylcholine and enhanced contractile responses to sympathetic nerve stimulation in aortic rings and mesenteric vascular beds of L-NAME treated rats were ameliorated by CT extract supplementation. Left ventricular hypertrophy and dysfunction were developed in L-NAME rats, which were partially prevented by CT extract treatment. The CT extract alleviated upregulated endothelial nitric oxide synthase expression, decreased plasma nitrate/nitrite levels, and increased oxidative stress in L-NAME rats. It suppressed high levels of serum angiotensin-converting enzyme activity, plasma angiotensin II, and cardiac angiotensin II type 1 receptor, NADPH oxidases 2, nuclear factor-kappa B, and tumor necrosis factor-alpha expression. The CT extract, therefore, partially prevented L-NAME-induced hypertension and cardiovascular alterations in rats. These effects might be related to a reduction in the oxidative stress and renin–angiotensin system activation due to L-NAME in rats.

Therapy of the rat hemorrhagic cystitis induced by cyclophosphamide. Stable gastric pentadecapeptide BPC 157, L-arginine, L-NAME

We focused on the cyclophosphamide-induced hemorrhagic cystitis (100 mg/kg/day intraperitoneally throughout three days) as a particular NO-system disturbance, and therapy possibilities. We demonstrated that it may be attenuated by subsequent administration of the NOS substrate L-arginine (100 mg/kg/day intraperitoneally), aggravated by NOS-blocker L-NAME (5 mg/kg/day intraperitoneally), all influenced by the stable gastric pentadecapeptide BPC 157 (10 μg/kg/day, 10 ng/kg/day, intraperitoneally or perorally, in drinking water). Regularly, cyclophosphamide dose- and time-dependently induced severe hemorrhagic cystitis lesions, gross lesions, and corresponding urothelial necrosis, vesical edema, erosion, hemorrhage, inflammation, and ulceration, microscopically. The bladder wet weight dramatically increased. Functionally, already after first cyclophosphamide administration, there is an increased leak point pressure. Until the second cyclophosphamide administration, L-arginine consistently attenuated regular cyclophosphamide-induced severe hemorrhagic cystitis lesions, grossly and microscopically, but not functionally. L-NAME aggravated these lesions and eradicated beneficial effect of L-arginine when combined. BPC 157 administration after cyclophosphamide, given in either dose or in either regimen markedly attenuated all cyclophosphamide lesions, grossly, microscopically. The increase of the bladder wet weight was consistently attenuated. Functionally, increased leak point pressure was reversed to the values noted in normal rats. The similar findings were noted in rats that received BPC 157 together with L-NAME or L-arginine, given alone or combined. Thus, the lesions are NO-related based on the administration of L-NAME as well as administration of L-arginine, and their mutual interaction, and counteraction by BPC 157 application. Likewise, we reveal new therapeutic possibilities, emphasizing stable gastric pentadecapeptide BPC 157 and L-arginine, versus L-NAME in rats underwent cyclophosphamide-induced cystitis.

Class side effects: decreased pressure in the lower oesophageal and the pyloric sphincters after the administration of dopamine antagonists, neuroleptics, anti-emetics, L-NAME, pentadecapeptide BPC 157 and L-arginine.

The ulcerogenic potential of dopamine antagonists and L-NAME in rats provides unresolved issues of anti-emetic neuroleptic application in both patients and experimental studies. Therefore, in a 1-week study, we examined the pressures within the lower oesophageal and the pyloric sphincters in rats [assessed manometrically (cm H2O)] after dopamine neuroleptics/prokinetics, L-NAME, L-arginine and stable gastric pentadecapeptide BPC 157 were administered alone and/or in combination. Medication (/kg) was given once daily intraperitoneally throughout the 7days, with the last dose at 24h before pressure assessment. Given as individual agents to healthy rats, all dopamine antagonists (central [haloperidol (6.25mg, 16mg, 25mg), fluphenazine (5mg), levomepromazine (50mg), chlorpromazine (10mg), quetiapine (10mg), olanzapine (5mg), clozapine (100mg), sulpiride (160mg), metoclopramide (25mg)) and peripheral(domperidone (10mg)], L-NAME (5mg) and L-arginine (100mg) decreased the pressure within both sphincters. As a common effect, this decreased pressure was rescued, dose-dependently, by BPC 157 (10µg, 10ng) (also note that L-arginine and L-NAME given together antagonized each other's responses). With haloperidol, L-NAME worsened both the lower oesophageal and the pyloric sphincter pressure, while L-arginine ameliorated lower oesophageal sphincter but not pyloric sphincter pressure, and antagonized L-NAME effect. With domperidone, L-arginine originally had no effect, while L-NAME worsened pyloric sphincter pressure. This effect was opposed by L-arginine. All these effects were further reversed towards a stronger beneficial effect, close to normal pressure values, by the addition of BPC 157. In addition, NO level was determined in plasma, sphincters and brain tissue. Thiobarbituric acid reactive substances (TBARS) were also assessed. Haloperidol increased NO levels (in both sphincters, the plasma and brain), consistently producing increased TBARS levels in the plasma, sphincters and brain tissues. These effects were all counteracted by BPC 157 administration. In conclusion, we revealed that BPC 157 counteracts the anti-emetic neuroleptic class side effect of decreased pressure in sphincters and the dopamine/NO-system/BPC 157 relationship.

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.

0042 : Chronic increase in blood flow in rat mesenteric resistance arteries improved endothelium (NO)-mediated dilation: essential role of estrogens

Resistance arteries (RA) have a key role in the control of local blood flow. They undergo outward expansive remodeling in response to a chronic increase in blood flow as seen in post-ischemic collateral arteries growth. This remodeling is associated with improved endothelium-dependent dilation. We hypothesized that estrogens play a role in flow-mediated improvement of endothelium-dependent dilation. Local increase in blood flow in one mesenteric artery was obtained local surgery in three-month old ovariectomized female rats treated or not with 17-b-estradiol (E2). Changes in arterial function and structure were measured after 2 weeks. After 2 weeks, arterial diameter increased in high flow (HF) compared to normal flow (NF) arteries in ovariectomized rats treated with E2, not in untreated rats. Acetylcholine-mediated relaxation was higher in HF than in NF arteries in control and OVX rats treated with E2. In untreated rats, the relaxation was lower in HF than in NF vessels. eNOS expression level was higher in HF than in NF vessels in E2-treated rats only. Acetylcholine-mediated relaxation was fully dependent on the production of NO in E2-treated rats (total inhibition by the NO-synthesis blocker L-NAME). In untreated OVX rats L-NAME blocked only partly the relaxation. Endothelium-independent relaxation (sodium nitroprusside) was not affect by OVX or by E2. Serotoninand phenylephrine-mediated contraction was higher in HF than in NF arteries in both treated and untreated OVX rats. Thus, we demonstrated the essential role of endogenous E2 in flowmediated improvement of endothelium (NO)-mediated dilation in mesenteric resistance arteries.

The influence of trait anxiety on the elevation of arterial pressure induced by l-NAME in rats

Due to the high prevalence of anxiety disorders and hypertension comorbidity in the general population, the establishment of anxiety as a risk factor for elevated blood pressure, or the reverse, is of great relevance. In this context, animal models can be of great scientific value, as they permit the control of several variables. Bearing this in mind, the influence of anxiety, not as a state, but as a personality trait (trait anxiety), on blood pressure elevation and vice versa were investigated for the first time in rats, using the free-exploratory paradigm (FEP). Sixty adult male Wistar rats were evaluated on FEP and categorized according to their levels of anxiety. From this sample, 24 animals with high (n=12) and low (n=12) trait anxiety were allocated to two treatment groups: (1) l-NAME (NG-nitro-l-arginine methyl ester, 20mg/kg, p.o., for 7 days to increase blood pressure; n=6/anxiety category); (2) CTRL (tap water, p.o., for 7 days; n=6/anxiety category). During treatment, measurements of systolic blood pressure (SBP) were taken daily. After treatment, the animals were again tested on FEP. SBP and trait anxiety levels were compared pre- and post-treatment. Additionally, correlations between trait anxiety levels and SBP increases (l-NAME group) were analyzed. The results showed that l-NAME was able to induce significant SBP elevation, but only for the high-anxious animals, while SBP elevation did not significantly interfere with anxiety levels. A significant correlation between anxiety levels and SBP peaks in response to l-NAME was also shown. No differences were observed between the levels of anxiety before and after treatment. These findings suggest that individuals with high trait anxiety are more susceptible to increases in blood pressure, but that high blood pressure does not affect the levels of trait anxiety.

Involvement of inducible nitric oxide synthase and dimethylarginine dimethylaminohydrolase in Nω‐Nitro‐L‐arginine methyl ester (L‐NAME)‐induced hypertension (LB676)

Chronic administration of L‐NAME in rats is an excellent model to study the induction and progression of a NO deficiency‐induced endothelial dysfunctional state. This study was performed to determine how different vascular beds compensate for the loss in nitric oxide. Male Wistar rats received L‐NAME (50mg/kg/day in drinking water) or no drug for 6 weeks. Aorta, pulmonary and renal arteries were isolated for determination of basal cGMP production, PDE5 activity, dimethylarginine dimethylaminohydrolase (DDAH) and hemeoxygenase (HO) activity. mRNA expression studies were done by real time PCR. Plasma IL‐1β level was measured by ELISA. Results: L‐NAME‐induced hypertension was associated with an increase in mean arterial pressure and plasma IL‐1β. The treatment had varied effects on mRNA expression levels of eNOS, soluble guanylyl cyclase, PDE5 and HO1 but showed an increased iNOS and decreased DDAH2 expression in all three tissues studied. Basal cGMP levels were decreased in all tissues, but PDE5 activity was increased only in renal and pulmonary artery. DDAH activity was significantly reduced, whereas HO activity was significantly increased in all the tissues. This study showed that different vascular beds do not react similarly to chronic NO inhibition and that vascular inflammation triggered by an increase in iNOS and indirectly by a decrease in DDAH activity might be the causative factors for the progression of vascular dysfunction in this model.

Renoprotective effect of sitagliptin against hypertensive nephropathy induced by chronic administration of L-NAME in rats: Role of GLP-1 and GLP-1 receptor

The present study was undertaken to assess the possible protective effects of sitagliptin, a dipeptidyl peptidase 4-inhibitor (DPP4), against Nω-nitro-L-arginine methyl ester (L-NAME) induced hypertensive nephropathy in rats. Hypertension was induced in adult rats by administration of L-NAME for 6 weeks. Rats were treated with sitagliptin (10mg/kg/day or 30mg/kg/day) for six weeks. Chronic L-NAME administration resulted in depletion of serum nitric oxide (NO) associated with elevation in the mean arterial pressure. When compared with the control group; serum urea, serum creatinine, albuminuria, urinary N-acetyl-ß-d-glucosaminidase (NAG) level and renal tissue malondialdhyde (MDA) content were significantly elevated, while creatinine clearance, serum level of glucagon like peptide-1 (GLP-1) as well as renal tissue superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were signifcantly decreased in L-NAME treated group. Renal expression of mRNA for eNOS and GLP-1 receptors were reduced in the L-NAME treated group as compared with the control group. Treatment with sitagliptin (10mg/kg or 30mg/kg) successfully ameliorated the deleterious effects of L-NAME on the all tested parameters. Our study indicates a novel protective effect of sitagliptin against L-NAME induced hypertensive nephropathy. An effect which is mediated through, increasing serum level of GLP-1, upregulation of GLP-1 receptors, which in turn, lead to induction of expression eNOS, increased serum NO level, tandem with decreased lipid perodixation and restore the antioxidant defense mechanisms. It is worth mentioning that the effects produced by sitaglipin (30mg/kg) were superior to the effects obtained by the lower dose.

Hepatoprotective and anti-inflammatory effects of silibinin on experimental preeclampsia induced by l-NAME in rats

AimsInhibition of nitric oxide synthase with N-omega-nitro-l-arginine methyl ester (l-NAME) has been employed as an experimental model of human preeclampsia. This study determined the protective effect of silibinin, a flavonoid with anti-inflammatory and hepatoprotective properties on the deleterious effects observed in experimentally induced preeclampsia in rats. Main methodsPregnant Wistar rats were treated during gestation (days 10–20) with l-NAME (70–80mg/kg/day) in drinking water or with l-NAME plus silibinin (100mg/kg/day, orally) starting at day 0, day 7 or day 14 of pregnancy. Systolic blood pressure was recorded from gestation days 0 to 21. A control group of pregnant non-treated rats was analyzed similarly. On day 21 the rats were euthanized and the following parameters were evaluated: proteinuria, platelet count, liver histopathology and reproductive outcome. Tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1β), IL-6, IL-10 and interferon-gamma (IFN-γ) were determined in liver homogenate by enzyme immunoassay. Key findingsIn comparison with the l-NAME group the silibinin treatment reduced the values of systolic blood pressure, proteinuria, TNF-α, IL-1β and IFN-γ in liver, normalized the platelet count and improved fetal outcomes. Histopathological lesions in liver of the l-NAME group showed intense mononuclear inflammatory infiltrate and thickening of muscle tunica of arterial vessel, mainly in the periportal area. Silibinin treatment induced attenuation of periportal inflammatory infiltrate, showing an association between inflammatory infiltrate and TNF-α, IL-1β and IFN-γ levels in liver homogenate. SignificanceSilibinin administration to l-NAME-treated rats displays anti-inflammatory and immunomodulatory actions that may contribute to its hepatoprotective effects and improve reproductive outcomes in experimental preeclampsia.

P27 Hepatoprotective effects of silibinin on experimental preeclampsia induced by L-NAME in rats is associated with reduction in inflammatory cytokines levels

P27 Hepatoprotective effects of silibinin on experimental preeclampsia induced by L-NAME in rats is associated with reduction in inflammatory cytokines levels

Modulation by Cytochrome P450-4A ω-Hydroxylase Enzymes of Adrenergic Vasoconstriction and Response to Reduced PO2 in Mesenteric Resistance Arteries of Dahl Salt-Sensitive Rats

This study evaluated the contribution of the 20-HETE/cytochrome P450-4A ω-hydroxylase (CYP4A) system to the early development of salt-induced vascular changes in Dahl salt-sensitive (SS) rats. CYP4A expression and 20-HETE production were evaluated and responses to norepinephrine, endothelin, and reduced PO₂ were determined by video microscopy in isolated mesenteric resistance arteries from SS rats fed high salt (HS; 4% NaCl) diet for three days vs. low salt (LS; 0.4% NaCl) controls. CYP4A enzyme inhibition with dibromododecenyl methylsulfimide (DDMS) selectively reduced norepinephrine sensitivity and restored impaired vasodilation in response to reduced PO₂ in SS rats fed HS diet. In the presence of DDMS, vasodilatation to reduced PO₂ was eliminated by indomethacin and unaffected by l-NAME in rats fed LS diet, and eliminated by l-NAME and unaffected by indomethacin in rats fed HS diet. The 20-HETE agonist WIT003 restored norepinephrine sensitivity in DDMS-treated arteries of HS-fed rats. HS diet increased vascular 20-HETE production and CYP4A protein levels by ∼24% and ∼31%, respectively, although these differences were not significant. These findings support the hypothesis that the 20-HETE/CYP4A system modulates vessel responses to norepinephrine and vascular relaxation to reduced PO₂ in mesenteric resistance arteries of SS rats fed HS diet.

Is Nitric Oxide a Mediator of the Effects of Low-Intensity Electrical Stimulation on Bone in Ovariectomized Rats?

Low-intensity electrical stimulation (LIES) may counteract the effects of ovariectomy (OVX) on nitric oxide synthase (NOS) expression, osteocyte viability, bone structure, and microarchitecture in rats (Lirani-Galvão et al., Calcif Tissue Int 84:502-509, 2009). The aim of the present study was to investigate if these effects of LIES could be mediated by NO. We analyzed the effects of NO blockage (by L-NAME) in the response to LIES on osteocyte viability, bone structure, and microarchitecture in OVX rats. Sixty rats (200-220 g) were divided into six groups: sham, sham-L-NAME (6 mg/kg/day), OVX, OVX-L-NAME, OVX-LIES, and OVX-LIES-L-NAME. After 12 weeks, rats were killed and tibiae collected for histomorphometric analysis and immunohistochemical detection of endothelial NOS (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). In the presence of L-NAME, LIES did not counteract the OVX-induced effects on bone volume and trabecular number (as on OVX-LIES). L-NAME blocked the stimulatory effects of LIES on iNOS and eNOS expression of OVX rats. Both L-NAME and LIES decreased osteocyte apoptosis. Our results showed that in OVX rats L-NAME partially blocks the effects of LIES on bone structure, turnover, and expression of iNOS and eNOS, suggesting that NO may be a mediator of some positive effects of LIES on bone.

EFFECT OF AEROBIC EXERCISE ON THE AUTONOMIC CONTROL OF HEART RATE IN RATS SUBMITTED TO L‐NAME HYPERTENSION

Hypertension induced by chronic blockade of nitric oxide synthesis with L-NAME is characterized by autonomic imbalance with sympathetic predominance. On the other hand, physical exercise leads to cardiac autonomic imbalance with vagal predominance. The aim of this study was evaluate autonomic control of heart rate (HR) in hypertension induced by L-NAME in rats submitted to aerobic exercise. Rats were splited into 4 groups: control, treated with L-NAME (70 mg/kg/day, 7 days), trained by swimming during 12 weeks and trained by swimming and treated with L-NAME in the last week. L-NAME group presented tachycardia and hypertension as compared with other groups. Sympathovagal balance evaluated by pharmacological autonomic receptors blockade with methylatropine and propranolol also was found altered in L-NAME group, characterizing sympathetic predominance. HR variability studied by spectral analysis showed decrease in low frequency (LF: 0.2–0.75 Hz) in L-NAME group as compared with other groups. Sympathovagal balance was found similar between trained group as compared with control rats. However, basal HR and intrinsic heart rate was found decreased in trained rats. Exercise training did not prevent, but attenuated hypertension levels induced by L-NAME. Moreover, L-NAME trained group presented lower basal HR as compared with trained group, however with similar intrinsic heart rate. Support: FAPESP, FAEPA

The intrarenal blood flow distribution and role of nitric oxide in diabetic rats

A few attempts have so far been made to determine the regional renal blood flow distribution in experimental diabetic rats. In the present experiment, 3 weeks after successful streptozotocin injection in diabetic rats (n = 8), the blood flows in the renal superficial and deep cortexes and outer medulla with implanted fibers were measured by laser-Doppler techniques. Renal blood flow was measured by an ultrasonic flow probe placed around the renal artery. Studies were performed at the baseline condition, during the administration of nonselective nitric oxide synthesis inhibitor, nitro- l-arginine methyl-ester ( l-NAME), and during the postinfusion period. The results showed that superficial cortical blood flow and deep cortical blood flow were significantly greater ( P < .05) in diabetic rats compared with control rats (n = 8) (superficial cortical blood flow, 2.18 ± 0.22 vs 1.55 ± 0.21 V; deep cortical blood flow, 1.32 ± 0.13 vs 0.99 ± 0.14 V) with the significant increase in renal blood flow (18.1 ± 3.3 vs 14.5 ± 2.7 mL/min). Furthermore, it was shown that in diabetic rats the intravenous infusion of a low dose of l-NAME, which did not alter medullary blood flow, decreased cortical blood flow (CBF) ( P < .05), whereas in control rats l-NAME did not affect CBF but a high dose of l-NAME decreased medullary blood flow ( P < .05). We conclude that in early diabetic nephropathy the blood flow is increased in both the superficial and deep cortexes, and nitric oxide plays an important role in regulating the CBF during the development of diabetic nephropathy.

Decrease in Cochlear Blood Flow with Infusion of Nitric Oxide Synthase Inhibitor and its Recovery with L-arginine Infusion: Comparison with Abdominal Blood Flow and Auricular Blood Flow

We observed changes in cochlear blood flow (CoBF), abdominal blood flow (AbBF) and auricular blood flow (AuBF) in rats after administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine-methyl ester (L-NAME). Ten min after i.v. infusion of L-NAME, L-arginine, a substrate of NO, was infused (100 mg/kg) intravenously. Using a laser Doppler flowmeter, changes in blood flow were recorded from the basal turn of the right cochlea, abdominal wall or right auricle, and systemic blood pressure (BP) was recorded simultaneously from the left femoral artery. As another parameter of vascular response, vascular conductance (VC) was calculated from blood flow/mean BP. I.v. infusion of L-NAME produced a dose-dependent depression of cochlear VC at concentrations of 0.2 (-18.9%), 1 (-37.9%), 5 (-45.8%) and 10 mg/kg (-48.3%). Infusion of L-arginine partially reversed the decrease of CoBF caused by L-NAME. The group that received a 0.2 mg/kg infusion of L-NAME showed the largest degree of recovery (23.8%) with L-arginine, while the 10 mg/kg group showed the smallest degree of recovery (10.1%). AbBF also decreased after infusion of L-NAME (5 mg/kg) but to a lesser degree (-41.1% depression of VC) and with no significance compared to the decrease in CoBF (5 mg/kg L-NAME). Likewise, AuBF showed a decrease (-44.0% depression of VC) after infusion of L-NAME (10 mg/kg), the decrease being less than that of CoBF (10 mg/kg L-NAME). Recoveries from these decreased levels of VC in the AbBF and AuBF groups appeared to be smaller than those in the CoBF groups at the same dose of L-NAME (5.5% vs 17.3% in abdominal VC; 5.3% vs 10.1% in auricular VC). In a previous study comparing the CoBF changes caused by i.v. infusion and round window application of L-NAME, we proposed that i.v. infusion of L-NAME in rats primarily affects the precapillary arteriole of the spiral modiolar artery, which effectively regulates cochlear microcirculation as a resistance artery. Thus we assume that there may exist an active pathway of the NO/soluble guanylate cyclase/cyclic guanosine monophosphate system in the above vessels. With regard to the finding of a smaller recovery of VC with L-arginine in both the AbBF and AuBF groups, we consider that differences in L-arginine availability or uptake, and in the synthesis of NO, may exist between the cochlear and cutaneous vasculatures.

Hypertension Plus Diabetes Mimics the Cardiomyopathy Induced by Nitric Oxide Inhibition in Rats

We compared the myocardial lesions caused by the long-term inhibition of nitric oxide (NO) biosynthesis with those associated with renovascular hypertension (two-kidney, one-clip model [2K-1C]) and superimposed streptozotocin-induced diabetes mellitus (DM). Prospective trial. University laboratory. Male Wistar rats were classified into the following groups: (1) a control group; (2) the L-NAME group (treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester [L-NAME], 75 micro mol per rat per day, orally); (3) the 2K-1C group (renovascular hypertension); (4) the DM group (treatment with streptozotocin, 60 mg/kg via intraperitoneal route); and (5) the 2K-1C plus DM group (renovascular hypertension and streptozotocin-induced DM). Arterial BP was measured by a tail-cuff method for 3 weeks, after which histologic and stereological analysis of the heart was done and cardiac NO synthase type 3 (NOS3) levels were assessed by Western blotting. The circulating levels of nitrates/nitrites and thromboxane B(2) (TXB(2), the stable metabolite of thromboxane A(2)) were also measured. In DM and 2K-1C rats, the myocardial lesions consisted mainly of recent myocardial infarcts, which were more severe in the 2K-1C plus DM group. In L-NAME-treated rats, multiple foci of reparative fibrosis and fresh myocardial necrosis resembled the severe lesions found in the 2K-1C plus DM group. Although NOS3 protein expression increased (19 to 44%; p < 0.05) in all treated groups, serum nitrate/nitrite levels decreased only in the L-NAME group and the 2K-1C plus DM group. These two groups also showed a more pronounced increase in TXB(2) concentrations. These results indicate that the association of hypertension and DM mimics the alterations induced by L-NAME in rats, which suggests a role for NO in the pathophysiology of hypertensive-diabetic cardiomyopathy.

Decrease in Cochlear Blood Flow with Infusion of Nitric Oxide Synthase Inhibitor and its Recovery with L-arginine Infusion: Comparison with Abdominal Blood Flow and Auricular Blood Flow

We observed changes in cochlear blood flow (CoBF), abdominal blood flow (AbBF) and auricular blood flow (AuBF) in rats after administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine-methyl ester (L-NAME). Ten min after i.v. infusion of L-NAME, L-arginine, a substrate of NO, was infused (100 mg/kg) intravenously. Using a laser Doppler flowmeter, changes in blood flow were recorded from the basal turn of the right cochlea, abdominal wall or right auricle, and systemic blood pressure (BP) was recorded simultaneously from the left femoral artery. As another parameter of vascular response, vascular conductance (VC) was calculated from blood flow/mean BP. I.v. infusion of L-NAME produced a dose-dependent depression of cochlear VC at concentrations of 0.2 (-18.9%), 1 (-37.9%), 5 (-45.8%) and 10 mg/kg (-48.3%). Infusion of L-arginine partially reversed the decrease of CoBF caused by L-NAME. The group that received a 0.2 mg/kg infusion of L-NAME showed the largest degree of recovery (23.8%) with L-arginine, while the 10 mg/kg group showed the smallest degree of recovery (10.1%). AbBF also decreased after infusion of L-NAME (5 mg/kg) but to a lesser degree (-41.1% depression of VC) and with no significance compared to the decrease in CoBF (5 mg/kg L-NAME). Likewise, AuBF showed a decrease (-44.0% depression of VC) after infusion of L-NAME (10 mg/kg), the decrease being less than that of CoBF (10 mg/kg L-NAME). Recoveries from these decreased levels of VC in the AbBF and AuBF groups appeared to be smaller than those in the CoBF groups at the same dose of L-NAME (5.5% vs 17.3% in abdominal VC; 5.3% vs 10.1% in auricular VC). In a previous study comparing the CoBF changes caused by i.v. infusion and round window application of L-NAME, we proposed that i.v. infusion of L-NAME in rats primarily affects the precapillary arteriole of the spiral modiolar artery, which effectively regulates cochlear microcirculation as a resistance artery. Thus we assume that there may exist an active pathway of the NO/soluble guanylate cyclase/cyclic guanosine monophosphate system in the above vessels. With regard to the finding of a smaller recovery of VC with L-arginine in both the AbBF and AuBF groups, we consider that differences in L-arginine availability or uptake, and in the synthesis of NO, may exist between the cochlear and cutaneous vasculatures.

Inhibition of nitric oxide synthesis by L-name exacerbates acute lung injury induced by hepatic ischemia-reperfusion.

Hepatic Kupffer cells and pulmonary alveolar macrophages together constitute a macrophage-axis involved in the regulation of regional and systemic inflammatory responses. Systemic inflammatory response syndrome induced by overproduced pro-inflammatory mediators is the major cause of adult respiratory distress syndrome. In the present study, we examined the anti-inflammatory role of nitric oxide (NO) in a rat model of acute lung injury induced by hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia by clamping the relevant branches of hepatic artery and portal vein, followed by a 4-h reperfusion achieved by removal of the vascular clamp. Four groups of animals were studied: sham control + saline; sham control + N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v., 10 min before reperfusion); HI/R + saline; HI/R + L-NAME. Results show that (1) administration of L-NAME to rats subjected to HI/R decreased plasma NO levels; however, the attenuation of NO increased plasma alanine aminotransferase (ALT) activity and superoxide generation in the ischemic lobes of liver, compared to HI/R alone. (2) Inhibition of NO synthesis with L-NAME in rats subjected to HI/R also enhanced systemic inflammatory response as assessed by the increase in the number of circulating leukocytes and levels of plasma tumor necrosis factor-alpha (TNFalpha) and interleukin 1-beta (IL-1beta). (3) The overwhelming systemic inflammatory response induced by administration of L-NAME in rats subjected to HI/R also augmented pulmonary vascular permeability and superoxide generation in the lung tissue. (4) Pulmonary alveolar macrophages isolated from rats subjected to HI/R + L-NAME produced higher levels of TNFalpha and IL-1beta in the supernatant of culture medium than that of rats subjected to HI/R alone. (5) There were no differences between the groups of sham + saline and sham + L-NAME in terms of plasma NO levels and ALT activity, circulating leukocytes, superoxide generation in the liver and lung, lavage protein levels, and TNFalpha and IL-1beta levels in plasma and bronchoalveolar lavage fluid. Our results suggest that inhibition of NO synthesis by L-NAME in rats subjected to HI/R not only augments ischemic liver injury, but also enhances the systemic inflammatory response and exacerbates remote lung injury. The increase in TNFalpha and IL-1beta production by alveolar macrophages may, in part, account for L-NAME-induced enhancement of acute lung injury.

Role of nitric oxide in renal function in rats with short and prolonged periods of streptozotocin-induced diabetes.

Nitric oxide (NO) has been proposed to play a significant role in renal function. In addition, NO production has been found to increase in diabetes mellitus. The present study aimed to clarify the mechanism responsible for NO action in renal function in rats with short (10 days) or prolonged periods (8 weeks) of diabetic induction. Male Wistar rats were induced to develop diabetes mellitus by intraperitoneal injection of streptozotocin (STZ) (65 mg/kg b.w.), whereas the age-matched control rats were given normal saline. After diabetic induction for 10 days or 8 weeks, the experiment was begun. Three consecutive periods of 30 min each, were designed consisting of one control period, the first and the second period of L-arginine or L-NAME or insulin infusion. Mean arterial pressure (MAP) was determined every 15 min. Arterial blood and urine samples were collected to determine the plasma glucose level (PG), glomerular filtration rate (GFR), effective renal plasma flow (ERPF), urine flow rate (V), urinary protein excretion (Upro), fractional excretion of glucose (FEG) and fractional excretion of sodium (FENa) in each period. No significant differences of MAP were apparent between control rats and rats with diabetic induction. L-arginine infusion had no effect whereas L-NAME markedly increased MAP in normal rats and rats after the short period of diabetes induction. Pressor response to L-NAME in rats exposed to the prolonged period of diabetes induction was lower than that of age-matched control rats. During L-NAME infusion, the PG level significantly declined from 394.9+/-13.1 - 338.0+/-14.1 mg/dl and from 399.9+/-7.9 - 354.3+/-18.8 mg/dl in rats after short and prolonged periods of diabetic induction, respectively. GFR significantly increased whereas ERPF slightly increased in diabetic rats. The elevation of GFR could be reversed by L-NAME or insulin infusion but it increased again after simultaneous infusion of insulin and glucose. Increases in V, the Upro and FEG without changes of FENa, were apparent in diabetic rats. Either L-arginine or L-NAME infusion could not reverse elevations of V, Upro and FEG. The rise of both V and Upro was reversed along with the attenuation of high FEG during insulin infusion, and it rose again close to the diabetic level during simultaneous infusion of insulin and glucose. Elevation of GFR, V and Upro appeared along with a rise of the PG level by approximately 300-350 mg/dl in diabetic rats. Both NO and hyperglycaemia are involved in modulating renal hyperfiltration in diabetic rats. The elevations of urine flow rate and urinary excretion of both protein and glucose would be expected to represent the reduction of renal tubular reabsorption rather than renal hyperfiltration in diabetic rats. NO does not participate in the change of renal tubular function in diabetic rats. There was a parallel change of urine flow rate and urinary excretion of protein in diabetic rats. The rise of the PG level itself would account for the increases of GFR, V, Upro and FEG in diabetic rats. Glomerular hyperfiltration, diuresis and proteinuria in diabetic rats are not exhibited until the PG level rises to = 300-350 mg/dl.