Chronic Nitric Oxide Synthase Research Articles

Hypotensive effect of endothelin-1 in nitric oxide-deprived, hypertensive pregnant rats

Both nitric oxide (NO) and endothelin-1 (ET-1) are important mediators in the regulation of vascular tone during pregnancy and preeclampsia. This study was designed to investigate the ET-1-induced hypotensive effect in late pregnant rats (P) and in NO-deprived hypertensive pregnant rats (TP), a model of preeclampsia. From day 13 of pregnancy Wistar rats were fed a control or an N ω-nitro-L-arginine-enriched diet. On gestational day 20, mean arterial pressure (MAP ± SEM, in mm Hg) and heart rate (HR) were measured with a carotid catheter in anesthetized rats after a bolus intravenous injection of several agonists and antagonists. After 7 days of chronic NO synthase inhibition, there was a significant increase in MAP (+45 ± 3.9, P < .01) and 24-h urinary nitrate excretion was significantly decreased ( P < .05). ET-1 bolus injection (0.1 nmol/kg) was rapidly followed by a significant decrease in MAP and a slight delayed increase, with no change in HR. The magnitude of the decrease had significantly dropped off in P (−30 ± 2.2) as compared to that in TP (−46 ± 5.1) and in virgin rats (−51 ± 6.3) ( P < .05). In P and TP, in vivo depressor effect was also obtained with sarafotoxin S6c, a specific ET B agonist, and blocked by the specific ET B antagonist BQ-788. After inhibition of cyclooxygenase with acetylsalicylic acid, the ET-1-induced hypotension was not modified either in P or in TP. In conclusion, the present data highlight an enhanced ET B receptor mediated hypotensive effect of ET-1 in anesthetized TP as compared to P. The magnitude of the hypotensive effect of ET-1 observed in TP is of the same order as that in virgin rats and neither NO nor vasodilator prostaglandins seem to be involved in TP. The enhanced hypotensive effect of ET-1 could be a beneficial counterbalancing mechanism in this rat model of preeclamptic pathology where an increased sensitivity to vasoconstrictor agents is generally described.

Reversal of cardiovascular remodelling with candesartan.

Cardiovascular remodelling, defined as ventricular and vascular hypertrophy together with fibrosis, characterises hypertension following inhibition of the production of the endogenous vasodilator, nitric oxide (NO). This study has determined whether the cardiovascular remodelling following chronic NO synthase inhibition can be reversed by administration of the selective angiotensin II AT1-receptor antagonist, candesartan. Male Wistar rats were treated with L-nitroarginine methyl ester (L-NAME, 400 mg/l in drinking water) for eight weeks and with candesartan cilexetil (2 mg/kg/day by oral gavage) for the last four weeks. L-NAME-treated rats became hypertensive with systolic blood pressure increasing from 110±4 mmHg (control) to 170±10 mmHg. Rats developed left ventricular hypertrophy (control 1.70±0.06; L-NAME 2.10±0.04 mg/kg body wt) with markedly increased deposition of perivascular and interstitial collagen. Candesartan returned blood pressure, left ventricular weights and collagen deposition to control values. Echocardiographic assessment showed concentric hypertrophy with an increased fractional shortening; this was reversed by candesartan treatment. Heart failure was not evident. In the isolated Langendorff heart, diastolic stiffness increased in L-NAME-treated rats while the rate of increase in pressure (+dP/dt) increased after eight weeks only; candesartan reduced collagen deposition and normalised +dP/dt. In isolated left ventricular papillary muscles, the potency (negative log EC50) of noradrenaline as a positive inotropic compound was unchanged, (control 6.56±0.14); maximal increase in force before ectopic beats was reduced from 5.0±0.4 mN to 2.0±0.2 mN. Noradrenaline potency as a vasoconstrictor in thoracic aortic rings was unchanged, but maximal contraction was markedly reduced from 25.2±2.0 mN to 3.0±0.3 mN; this was partially reversed by candesartan treatment. Thus, chronic inhibition of NO production with L-NAME induces hypertension, hypertrophy and fibrosis with increased toxicity and significant decreases in vascular responses to noradrenaline. These changes were at least partially reversible by treatment with candesartan, implying a significant role of AT1-receptors in L-NAME-induced cardiovascular changes.

Role of nitric oxide in regional blood flow in angiotensin II-induced hypertensive rats.

The present study was designed to evaluate the contribution of nitric oxide (NO) to regional hemodynamics during the early phase of angiotensin II (Ang II)-induced hypertension. The responses of regional blood flow to chronic NO synthase inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME) were assessed using radioactive microspheres in conscious Ang II-infused hypertensive rats. Ang II-infused rats (270 ng/kg/min, subcutaneously for 12 days: n=11) showed higher mean arterial pressure (MAP: 153+/-4 mmHg) and total peripheral resistance (TPR: 1.61+/-0.06 mmHg/min/ml), and lower cardiac output (CO: 102+/-3 ml/min) than vehicle-infused normotensive rats (115+/-2 mmHg, 0.96+/-0.05 mmHg/min/ml and 130+/-7 ml/min, n=11, respectively). The blood flow rates in the brain, spleen, large intestine and skin were significantly reduced in Ang III-infused rats compared with vehicle-infused rats, while those in the lung, heart, liver, kidney, adrenal gland, small intestine, and skeletal muscle were similar. Treating Ang II-infused rats with L-NAME (75 mg/l in drinking water for 10 days, n=11) resulted in higher MAP (166+/-6 mmHg) and TPR (1.89+/-0.18 mmHg/min/ml) and lower CO (87+/-7 m/min) than untreated Ang II-infused rats. L-NAME-treated Ang II-infused rats showed widespread increases in regional vascular resistance and reduced blood flow rates in the kidney (3.81+/-0.27 ml/min/g) and skeletal muscle (0.20+/-0.03 ml/min/g) compared with untreated Ang II-infused rats (6.88+/-0.27 and 0.33+/-0.04 ml/min/g, respectively). However, there were no significant differences in the flow rates of other organs investigated between these animals. An NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409: 30 microg/kg/min, i.v.), significantly decreased MAP (110+/-6 mmHg) and TPR (1.23+/-0.18 mmHg/min/ml) without significant changes in CO (89+/-9 ml/min) in L-NAME-treated Ang II-infused rats. Furthermore, FK409 partially reversed blood flow rates in the kidney (4.72+/-0.40 ml/min/g) and skeletal muscle (0.25+/-0.02 ml/min/g)in these animals. These results suggest that NO counteracts, at least in part, the vasoconstrictor effects of elevated Ang II levels in renal and skeletal muscle vascular beds, and is an important modulator in the regulation of blood flow to these organs during the development of Ang II-induced hypertension.

Mycophenolate Mofetil Reduces Renal Injury in the Chronic Nitric Oxide Synthase Inhibition Model.

-We and others have recently shown that mycophenolate mofetil (MMF) reduces renal inflammation and glomerular and interstitial injury in the 5/6 renal ablation model. In the present study, we investigated whether MMF limits renal injury in a model of chronic nitric oxide (NO) inhibition associated with a high-salt diet and characterized by progressive systemic hypertension, albuminuria, glomerular sclerosis and ischemia, interstitial expansion, and progressive macrophage infiltration. Adult male Münich-Wistar rats were distributed among 3 groups: HS, rats receiving a high-salt diet (3.2% Na); HS+N, HS rats orally treated with the NO inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), 25 mg. kg(-1). d(-1); and HS+N+MMF, HS+N rats orally treated with MMF, 10 mg. kg(-1). d(-1). Renal hemodynamics were studied after 15 days of treatment; histological and immunohistochemical studies were conducted after 30 days of treatment. MMF treatment did not reverse the hemodynamic alterations characteristic of this model. Renal injury in the HS+N group was associated with macrophage and lymphocyte infiltration. Treatment with MMF reduced glomerular and interstitial injury and limited macrophage and lymphocyte infiltration. These results suggest that renal inflammation is a strong independent factor in the pathogenesis of the nephropathy associated with the HS+N model.

Predisposition of spontaneously hypertensive rats to develop renal injury during nitric oxide synthase inhibition

Chronic nitric oxide (NO) synthase (NOS) inhibition results in renal injury. Hypertension is an important risk factor for renal injury. We studied the influence of preexistent hypertension on the sensitivity for renal injury induced by chronic NOS inhibition in rats. Spontaneously hypertensive (SHR) and normotensive Wistar–Kyoto (WKY) rats were treated with 3, 10, 30 and 100 mg/l Nω-nitro-l-arginine (l-NNA) until death. Systolic blood pressure and proteinuria were measured regularly and compared with time-control measurements in untreated SHR and WKY. In WKY, 3 and 10 mg/l l-NNA did not affect systolic blood pressure, while 30 and 100 mg/l l-NNA resulted in an increase in systolic blood pressure after 12 and 4 weeks, respectively. In contrast in SHR, every dose of l-NNA resulted in an increase in systolic blood pressure after 2 weeks. In WKY, 3 and 10 mg/l l-NNA did not affect proteinuria or survival, while 30 and 100 mg/l l-NNA resulted in an increase in proteinuria after 30 and 9 weeks, and a median survival of 36 and 12 weeks, respectively. In SHR, 3, 10, 30 and 100 mg/l l-NNA resulted in an increase in proteinuria after 30, 12, 3 and 3 weeks, and a median survival of 41, 20, 5 and 3 weeks, respectively. Thus, at every dose of the inhibitor, chronic NOS inhibition resulted in far earlier increases in systolic blood pressure and proteinuria and a marked increase in mortality in SHR as compared to WKY. Indeed, a very low dosage of l-NNA that caused no harm in WKY was followed by marked increases in proteinuria and blood pressure and decreased survival in SHR. Hypertension strongly increases the vulnerability to cardiovascular risk factors that compromise the NO-system.

7-Nitroindazole reduces cerebral blood flow following chronic nitric oxide synthase inhibition

Blood flow and glucose utilization were measured in rat brain after chronic l-NAME treatment followed by acute 7-nitroindazole. Following chronic l-NAME, blood flow was not significantly different from control. Treatment with acute 7-nitroindazole reduced blood flow to the same extent in both chronic saline and l-NAME groups. Glucose utilization was unaffected. These results suggest that residual NOS activity in brain is sufficient to provide tonic, NO-dependent cerebrovascular dilator tone.

High-calcium diet enhances vasorelaxation in nitric oxide-deficient hypertension.

Because the effects of calcium supplementation on arterial tone in nitric oxide-deficient hypertension are unknown, we investigated the influence of elevating dietary calcium from 1.1 to 3.0% in Wistar rats treated with N(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg. kg(-1). day(-1)) for 8 wk. A high-calcium diet attenuated the development of hypertension induced by L-NAME and abrogated the associated impairments of endothelium-independent mesenteric arterial relaxations to nitroprusside, isoproterenol, and cromakalim. Endothelium-dependent relaxations to acetylcholine during nitric oxide synthase inhibition in vitro were decreased in L-NAME rats and improved by calcium supplementation. The inhibition of cyclooxygenase by diclofenac augmented the responses to acetylcholine in L-NAME rats but not in calcium + L-NAME rats. When hyperpolarization of smooth muscle was prevented by KCl precontraction, the responses to acetylcholine during combined nitric oxide synthase and cyclooxygenase inhibition were similar in all groups. Furthermore, superoxide dismutase enhanced the acetylcholine-induced relaxations in L-NAME rats but not in calcium + L-NAME rats. In conclusion, calcium supplementation reduced blood pressure during chronic nitric oxide synthase inhibition and abrogated the associated impairments in endothelium-dependent and -independent arterial relaxation. The augmented vasorelaxation after increased calcium intake in L-NAME hypertension may be explained by enhanced hyperpolarization and increased sensitivity to nitric oxide in arterial smooth muscle and decreased vascular production of superoxide and vasoconstrictor prostanoids.

Angiotensin in the nucleus tractus solitarii contributes to neurogenic hypertension caused by chronic nitric oxide synthase inhibition.

Activation of the sympathetic nervous system and renin-angiotensin system has been suggested to contribute to the hypertension caused by chronic nitric oxide synthase inhibition. The aim of the present study was to determine whether angiotensin within the nucleus tractus solitarii (NTS) plays a role in activation of the sympathetic nervous system in this model. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg. kg(-1). d(-1) in drinking water) for 2 weeks. Experiments were performed on anesthetized rats with denervated arterial and cardiopulmonary baroreceptors. Arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA) were measured. Microinjection of an angiotensin II type 1 (AT(1)) receptor antagonist (CV11974) or an angiotensin II type 2 (AT(2)) receptor antagonist (PD123319) into the depressor region within the NTS (identified by prior injection of L-glutamate) was performed. Microinjection of CV11974, but not of PD123319, produced greater decreases in arterial pressure, heart rate, and RSNA in L-NAME-treated rats than in control rats. The administration of hexamethonium resulted in a larger fall in arterial pressure in L-NAME-treated rats than in control rats. The ACE mRNA level in the brain stem was greater in L-NAME-treated rats than in control rats. These results suggest that increased sympathetic nerve activity plays a role in hypertension caused by chronic nitric oxide synthase inhibition and that activation of the renin-angiotensin system in the NTS is involved at least in part in this increased sympathetic nerve activity via AT(1) receptors.

Cardiovascular effects of chronic nitric oxide synthase inhibition in genetically hypertensive rats.

1. The possible role of an endothelial defect in the hypertension of the New Zealand genetically hypertensive (GH) rat strain was assessed by examining cardiovascular responses to the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) and the endothelium-dependent depressor agent acetylcholine (ACh). The vascular sensitivity of the hindquarter to nitric oxide (NO) was examined using the NO donor sodium nitroprusside (SNP). 2. NG-Nitro-L-arginine methyl ester (10 mg/kg per day in drinking water) was given to GH and normotensive (N) rats from age 7-9 weeks, with GH and N untreated control groups. Systolic blood pressure (tail-cuff) was monitored weekly from age 5-9 weeks. At age 9 weeks, pressure responses to various vasoactive agents were measured in vivo and in the rat isolated hindquarter. Left ventricular (LV) mass was measured at the time of death. 3. NG-Nitro-L-arginine methyl ester induced a greater hypertensive effect in GH (P < 0.001) compared with N (P < 0.05) rats and caused a significant increase in hindquarter perfusion pressure in GH rats only (P < 0.01). 4. Genetically hypertensive rats had LV hypertrophy that was exacerbated by L-NAME (P < 0.01). Left ventricular hypertrophy was not induced by L-NAME in N rats. 5. The normalized response to ACh did not differ between GH and N control rats and was unaffected by L-NAME treatment in vivo and in vitro except at the highest ACh dose (3 micrograms/kg) in GH hindquarters (P < 0.01). The response to SNP was similar in GH and N hindquarters and enhanced by L-NAME in GH (0.1 microgram; P < 0.05) and N rats (0.01 microgram, P < 0.01; 0.01 microgram, P < 0.001). 6. These results suggest that the L-arginine/NO system is not deficient in GH rats and that endothelial function in the GH hindquarter is preserved. They confirm that NO is involved in mediating blood pressure in GH and N rats and raise the possibility that a non-NO-mediated mechanism may underlie ACh-induced vasodilation in GH and N.

Unchanged cardiac angiotensin II levels accompany losartan-sensitive cardiac injury due to nitric oxide synthase inhibition

Chronic nitric oxide synthase (NOS) inhibition results in hypertension and myocardial injury. In a rapid and severe model of chronic NOS inhibition, we determined the role of angiotensin II in these effects by using angiotensin II receptor blockade and by measuring cardiac angiotensin II concentrations before and during development of cardiac damage. Rats received either no treatment, the NOS inhibitor Nω-nitro- l-arginine ( l-NNA; 500 mg/l), the angiotensin AT 1 receptor antagonist losartan (400 mg/kg chow), or l-NNA plus losartan for 21 days. In the second protocol, five groups of rats received l-NNA (500 mg/l) for 0, 4, 7, 14 and 21 days, respectively. l-NNA increased systolic blood pressure (SBP) (227±8 versus 143±6 mm Hg; P<0.01), heart weight index (0.44±0.02 versus 0.32±0.01; P<0.01) and induced coronary vasculitis and myocardial necrosis. Co-treatment with losartan prevented all changes. l-NNA during 4 days decreased cardiac angiotensin II (23±4 versus 61±15 fmol/g; P<0.05). Although after 7 days, fresh infarcts and after 14 days organized infarcts were present, cardiac angiotensin II was only slightly increased after 21 days (100±10 fmol/g; P<0.05). In conclusion, losartan-sensitive cardiac damage due to chronic NOS inhibition is not associated with primary increase of cardiac angiotensin II, suggesting that chronic NOS inhibition increases cardiac sensitivity for angiotensin II.

THE EFFECTS OF CHRONIC [formula omitted]-NAME AND [formula omitted]-ARGININE ADMINISTRATION ON β-ADRENERGIC RESPONSIVENESS OF STZ-DIABETIC RAT ATRIA

Recent studies have shown that NO acts as a negative inotrope and chronotrop in cardiac muscle. In the present study, we investigated the effects of the chronic administration of l -NAME and l -arginine on 14-week streptozotocin (STZ)-diabetic rat atria. To study these effects, we compared the alterations of inotropic and chronotropic responses to isoprenaline (ISO) on electrically-driven left atria and spontaneously beating right atria. In addition, we compared the blood pressures of rats in all groups. The chronic administration of L-arginine resulted in a significant reduction in blood pressure of the diabetic rats. On the other hand, the chronic nitric oxide synthase inhibition resulted in a significant increase in blood pressure of diabetic animals. To our findings, maximum positive inotropic responses of ISO diminished in STZ-diabetic, L-arginine and L-NAME treated diabetic groups relative to controls but neither the basal contractility of the left atria nor the pD2values were altered significantly in all groups. The basal atrial rate and maximum positive chronotropic responses to ISO were found to be significantly lower in treated and untreated diabetic groups, no significant changes were observed in pD2values. Our results demonstrate that the changes in inotropic and chronotropic responses in diabetic rat atria were not influenced by the chronic administration of L-arginine and L-NAME treatments.

The effects of chronic nitric oxide synthase suppression on glioma pathophysiology

Nitric oxide synthase (NOS) is strongly expressed in glioma and has an important role in tumour blood flow (TBF) regulation. Whether manipulation of NOS function within a tumour can have any therapeutic effect is unknown. This study therefore evaluated the pathophysiological effects of chronic systemic NOS inhibition on experimental rodent glioma blood flow, growth and necrosis.To determine the duration and pathophysiological effects of systemic NOS inhibition, Ng-nitroL-arginine methyl ester (L-NAME) was given to rats bearing C6 glioma acutely (single dose i.v., 30 mg kg) or for either 4 or 7 days (i.p. 75 mg kg day) prior to study.TBF and local cerebral blood flow (LCBF) were measured using C14-iodoantipyrine quantitative autoradiography. Tumour volume, tumoural necrosis and tumoural NOS were measured using conventional neuropathology and immunocytochemistry.Acute and 4-day L-NAME administration produced significant TBF reductions (‘;48 and ‘;39%, respectively) with less marked changes in LCBF (‘;35 and ‘;15%, respectively). Seven-day L-NAME administration reduced tumour volume ( p =0.12), increased tumoural necrosis ( p <0.05), but immunohistochemistry showed no difference in tumoural NOS expression. These results confirm that NOS has a significant role in the pathophysiology of experimental glioma, and that in this glioma model the effects of chronic systemic NOS inhibition are, for the period under study, predominately anti-tumoural. Whether chronic NOS inhibition is useful as an adjunct in glioma therapy or provides the opportunity for novel therapeutic approaches requires further study.

CHRONIC NITRIC OXIDE INHIBITION AGGRAVATES HYPERTENSION IN ERYTHROPOIETIN-TREATED RENAL FAILURE RATS

Alterations in nitric oxide (NO) and endothelin-1 (ET-1) production have recently been reported in erythropoietin (r-HuEPO)-induced hypertension in renal failure rats. The present study was designed to evaluate the effect of NO synthase inhibition with the L-arginine analog NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP) and ET-1 production in control and in uremic rats treated or not treated with r-HuEPO. Renal failure was induced by a two-stage 5/6 nephrectomy. Control and uremic rats were studied separately and subdivided into four groups: vehicle, r-HuEPO, L-NAME + vehicle and L-NAME + r-HuEPO. L-NAME (100 mg/kg/day), r-HuEPO (100 U/kg, subcutaneously, three times per week), the vehicle or both were administered during 4 weeks in control rats and during 2 weeks in uremic rats. Systolic BP was recorded before and after the onset of treatment at weeks 2 and 4 in control rats and at weeks 1 and 2 in uremic rats. Hematocrit, serum creatinine, plasma, blood vessel (thoracic aorta and mesenteric artery bed) and renal cortex immunoreactive (ir) ET-1 concentrations were measured at the end of the protocol. L-NAME enhanced BP in control and uremic rats and the increase was significantly higher in uremic rats under r-HuEPO therapy (222 ± 7 mmHg vs 198 ± 6 mmHg, p<0.05). L-NAME induced an increase in thoracic aorta ir-ET-1 concentrations in control and uremic rats. In contrast, ir-ET-1 concentrations were unchanged in the mesenteric arterial bed and the renal cortex of control and uremic animals. R-HuEPO increased thoracic aorta ir-ET-1 contents in L-NAME treated control and uremic rats. These results underline the important role of NO release in opposing the action of vasopressors on blood vessel tone which appears more important in uremic rats treated with r-HuEPO. L-NAME treatment increased large vessel, but not small resistance artery ir-ET-1 concentrations, suggesting differential regulation of ET-1 production in different vascular beds under chronic NO synthase inhibition.

Tissue and blood levels of zinc, copper, and magnesium in nitric oxide synthase blockade-induced hypertension.

The aim of this study was to determine the levels of tissue and blood zinc (Zn), copper (Cu), magnesium (Mg) in nitric oxide (NO) synthase blockade-induced hypertension. A group of albino rats received a NO synthase inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME, 60 mg/kg/d) in their drinking water for 21 d. L-NAME intake caused a progressive rise in this group's resting mean arterial blood pressure compared to a control group (p < 0.01). There were no differences between the groups with regard to tissue and blood levels of Zn or Cu; however, Mg concentrations were significantly lower in the hypertensive rats' erythrocytes (20.2% reduction from control levels), cerebral cortex (17.0%), heart (9.1%), renal cortex (12%), renal medulla (16.7%), and in the tissues of the caval vein (23.7%), mesenteric artery (29.8%), renal artery (18.4%), and renal vein (22.1%). There were no significant Mg concentration changes in the hypertensive group's plasma, cerebellum, liver, duodenum, or aortal tissue. These findings suggest that Mg depletion may play a role in the blood pressure rise that occurs in the model of chronic NO synthase inhibition-induced hypertension.

Calcium antagonist inhibits glomerular cell apoptosis and injuries of L-NAME exacerbated nephrosclerosis in SHR.

Increased apoptosis of glomerular cells, with progression of glomerulosclerosis, overactivity of the renin-angiotensin system and elevation of glomerular pressure, follows chronic nitric oxide synthase (NOS) inhibition in spontaneously hypertensive rats (SHR). To gain insight into the regulation of glomerular cell apoptosis in severe nephrosclerosis, we investigated apoptosis, the expression of proliferative cell nuclear antigen (PCNA) in glomeruli, and glomerular morphometric changes in 20-week-old SHR, SHR treated with NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 80 mg/l in drinking water), and SHR treated with L-NAME and the calcium antagonist, efonidipine (20 mg/kg per day), for 3 weeks. Apoptosis in non-sclerotic glomeruli was quantified by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. The increase in systolic blood pressure and the severe proteinuria with severe nephrosclerosis induced by chronic NOS inhibition were completely prevented by efonidipine. Furthermore, the glomerular area and capillary tuft area were markedly increased in rats treated with efonidipine compared with both control rats (+30 and +42%, respectively, p<0.01) and rats treated with L-NAME (+35 and +56%, respectively, p<0.01)-treated rats. This calcium antagonist also significantly inhibited the both increases of the glomerular cell apoptosis index (-72%) and the PCNA index (+44%), therefore the alteration between apoptosis and proliferation slightly increased the number of glomerular cells (subcapsular, +22%, p<0.01; juxtamedullary, +2%, not significant). Thus, the calcium antagonist efonidipine seems to play an important role in the regulation of apoptosis and proliferation of glomerular cells and may be effective in preventing nephrosclerosis exacerbated by NOS inhibition.

Nitric oxide and the cerebral-blood-flow response to somatosensory activation following deafferentation.

The single-vibrissa stimulation model in the rat was utilized to study the microvascular coupling between functional activation and local cerebral blood flow (LCBF) in both normal cortex and in cortex that had been peripherally deafferented. In addition, the role of chronic nitric oxide synthase (NOS) inhibition on the LCBF response to vibrissa stimulation was examined. One-day-old rats underwent deafferentation of all vibrissae on one side of the face, sparing C3, and received daily administration of either saline or N omega-nitro-L-arginine (L-NA). After seven weeks of treatment, LCBF was measured autoradiographically in conscious rats with [14C]N-isopropyl-p-iodoamphetamine while C3 was stimulated bilaterally. Stimulation produced a greater increase in LCBF in the deafferented cortex of both the saline (30.4%) and L-NA treated (25.7%) animals than in the intact cortex (19.9% and 16%, respectively). The area of activation of LCBF (0.176 mm2) was comparable to the area metabolically activated (0.149 mm2), and the increase in area of LCBF activation following deafferentation (169%) was smaller than the increase in area that was metabolically activated (287%). Chronic inhibition of NOS did not alter the spatial extent of the blood-flow response.

Micropuncture analysis of tubuloglomerular feedback regulation in transgenic mice.

Micropuncture methods have been used widely as a means to define the function of single tubules and study the functional connection between tubules and afferent arterioles (so-called tubuloglomerular feedback [TGF]). Transgenic mouse strains have become a new research tool with the potential of shedding new light on the role of specific gene products in renal tubular and vascular function. The micropuncture approach has therefore been adapted to studies in the mouse kidney. Although the data presented here support the feasibility of using this technique in the mouse, technical improvements are desirable in the areas of anesthesia, ureteral urine collections, blood collections, volume replacement, and functional stability for extended time periods. During ketamine/inactin anesthesia, TGF responses could regularly be elicited in wild-type mice. In contrast, changes in loop flow did not alter stop-flow pressure in angiotensin II type 1A receptor and angiotensin-converting enzyme knockout mice. Infusion of angiotensin II in subpressor doses partially restored TGF responsiveness in angiotensin-converting enzyme knockout animals. Normal TGF responses compared to wild type were found in nitric oxide synthase I and thromboxane receptor knockout mice. Using free-flow micropuncture techniques, the proximal-distal single-nephron GFR difference was found to be augmented in aquaporin-1 and Na/H exchanger-3 knockout mice, suggesting TGF activation in these strains of mice. These results support an essential role of angiotensin II in TGF regulation mediated through the angiotensin II type 1A receptor. Chronic nitric oxide synthase I and thromboxane receptor deficiency did not change TGF responsiveness. Aquaporin-1 and Na/H exchanger-3 deficiency enhances TGF suppression of TGF probably by volume depletion-mediated TGF sensitization. The use of micropuncture methodology in transgenic mice combines old and new research tools in a way that promises to yield important new insights into single-nephron function in physiologic and pathophysiologic conditions.

Losartan-sensitive renal damage caused by chronic NOS inhibition does not involve increased renal angiotensin II concentrations

Chronic nitric oxide synthase (NOS) inhibition results in hypertension, proteinuria, and renal morphological changes. Continuous angiotensin II (Ang II) blockade prevents these effects, suggesting an essential role of Ang II. However, it is not known whether renal Ang II concentrations are primarily increased or whether the scarcity of NO allows normal concentrations of Ang II to cause these detrimental effects. Therefore, we measured renal Ang II concentrations before and during the development of renal damage. Group 1 served as controls. Groups 2 through 5 received the NOS inhibitor Nomega-nitro-L-arginine (L-NNA; 40 mg/kg/day) for 4, 7, 14, and 21 days, respectively. Systolic blood pressure (SBP), proteinuria, glomerular filtration rate (GFR), and renal and blood Ang II were measured. In a separate experiment, rats were treated with L-NNA + the Ang II AT1 receptor blocker losartan to determine the functional effects of endogenous Ang II during chronic NOS inhibition. L-NNA treatment resulted in an increase in SBP from day 4 (161 +/- 4 vs. 135 +/- 4 mm Hg in control, P < 0.05) to day 21 (230 +/- 9 mm Hg). GFR was decreased from day 4 (1.9 +/- 0.2 vs. 2.5 +/- 0.2 ml/min in control, P < 0.05) to day 21 (1.2 +/- 0.2 ml/min). Proteinuria was increased from day 14 (85 +/- 14 vs. 6 +/- 1 mg/day in control, P < 0.05) to day 21 (226 +/- 30 mg/day). L-NNA treatment during four days resulted in a significant decrease in renal Ang II (183 +/- 32 vs. 454 +/- 40 fmol/g in control, P < 0.05). On day 7, 14, and 21, renal Ang II was not significantly different from the control. Blood Ang II was not significantly different from the control on days 4, 7, and 14 but was significantly increased after 21 days of L-NNA treatment (215 +/- 35 vs. 78 +/- 13 fmol/ml in control, P < 0.05). Ang II type-1 (AT1) receptor blockade prevented the severe renal injury and hypertension induced by chronic NOS inhibition. Losartan-sensitive renal damage caused by chronic NOS inhibition does not involve increased renal Ang II concentrations. This suggests that the detrimental effects of endogenous Ang II are increased during chronic NOS inhibition. Thus, when NO levels are low, normal Ang II concentrations can cause renal injury and hypertension.

The effect of chronic nitric oxide synthase inhibition on blood pressure and heart rate in unrestrained pregnant rats as recorded by radiotelemetry

Objectives: The objective of this study was to determine the effect of chronic nitric oxide synthase inhibition on heart rate and intravascular blood pressure in unrestrained pregnant rats as recorded by radiotelemetry. Study Design: Heart rate and systolic and diastolic blood pressures were monitored beginning with day 6 of pregnancy and until 1 week post partum with a radiotelemetric device. On day 10 of pregnancy osmotic minipumps were implanted subcutaneously and loaded to continuously deliver N G-nitro- L -arginine methyl ester (50 mg/d per rat, n = 6 animals) or vehicle (control group, n = 6 animals). Results: Blood pressure in the animals treated with N G-nitro- L -arginine methyl ester significantly increased compared with that in the control group and heart rate significantly decreased immediately after nitric oxide synthase blockade. Blood pressure then trended downward as gestation progressed, until the difference between the control group and the group treated with N G-nitro- L -arginine methyl ester became nonsignificant after day 17. Refractoriness to nitric oxide synthase blockade was especially evident in the diastolic pressure. Systolic, diastolic, and mean blood pressures in the rats treated with N G-nitro- L -arginine methyl ester were again significantly higher than those in the control group immediately after delivery and remained so despite a lower heart rate until the experiment was ended on postpartum day 6. Conclusions: Radiotelemetry can be used to monitor heart rate and intra-arterial blood pressure in unstressed, unrestrained animals. Chronic inhibition of nitric oxide does not cause sustained hypertension throughout pregnancy. Nitric oxide does not appear to be the only factor responsible for the vascular changes in pregnancy. The factors responsible for the refractoriness to nitric oxide synthase blockade are specific to pregnancy and disappear immediately after delivery. (Am J Obstet Gynecol 1999;181:159-64.)

Chronic studies on the interaction between nitric oxide and endothelin in cardiovascular and renal function.

1. Chronic inhibition of nitric oxide synthase (NOS) results in a persistent hypertension, while chronic blockade of endothelin ETA receptors has little effect on arterial pressure. These findings indicate that nitric oxide (NO) plays a more significant role than ET-1 in the long-term maintenance of arterial pressure. 2. Although endothelin (ET) appears to contribute to the hypertension in the early stages of NOS inhibition, blockade of either ETA or both ETA and ETB receptors has only a minor effect on the hypertension beyond the initial 2 weeks of NOS inhibition. 3. Endothelin may play a role in vascular lesion development associated with NOS inhibition, at least within the kidney, which may be related to angiotensin II activity. 4. The processes involved in the hypertension associated with chronic NOS inhibition appear to be dynamic and may include an evolution of ET-1 action. Variability in results from different laboratories may be related to genetic factors and choice of pharmacological agents.