Relationship between muscle sympathetic nerve activity and systemic hemodynamics during nitric oxide synthase inhibition in humans

Large interindividual differences exist in resting sympathetic nerve activity (SNA) among normotensive humans with similar arterial pressure (AP). We recently showed inverse relationships of resting SNA with cardiac output (CO) and vascular adrenergic responsiveness that appear to balance the influence of differences in SNA on blood pressure. In the present study, we tested whether nitric oxide (NO)-mediated vasodilation has a role in this balance by evaluating hemodynamic responses to systemic NO synthase (NOS) inhibition in individuals with low and high resting muscle SNA (MSNA). We measured MSNA via peroneal microneurography, CO via acetylene uptake and AP directly, at baseline and during increasing systemic doses of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Baseline MSNA ranged from 9 to 38 bursts/min (13 to 68 bursts/100 heartbeats). L-NMMA caused dose-dependent increases in AP and total peripheral resistance and reflex decreases in CO and MSNA. Increases in AP with L-NMMA were greater in individuals with high baseline MSNA (PANOVA<0.05). For example, after 8.5 mg/kg of L-NMMA, in the low MSNA subgroup (n=6, 28+/-4 bursts/100 heartbeats), AP increased 9+/-1 mmHg, whereas in the high-MSNA subgroup (n=6, 58+/-3 bursts/100 heartbeats), AP increased 15+/-2 mmHg (P<0.01). The high-MSNA subgroup had lower baseline CO and smaller decreases in CO with L-NMMA, but changes in total peripheral resistance were not different between groups. We conclude that differences in CO among individuals with varying sympathetic traffic have important hemodynamic implications during disruption of NO-mediated vasodilation.     

Effect of systemic nitric oxide synthase inhibition on postexercise hypotension in humans.

An acute bout of aerobic exercise results in a reduced blood pressure that lasts several hours. Animal studies suggest this response is mediated by increased production of nitric oxide. We tested the extent to which systemic nitric oxide synthase inhibition [N(G)-monomethyl-L-arginine (L-NMMA)] can reverse the drop in blood pressure that occurs after exercise in humans. Eight healthy subjects underwent parallel experiments on 2 separate days. The order of the experiments was randomized between sham (60 min of seated upright rest) and exercise (60 min of upright cycling at 60% peak aerobic capacity). After both sham and exercise, subjects received, in sequence, systemic alpha-adrenergic blockade (phentolamine) and L-NMMA. Phentolamine was given first to isolate the contribution of nitric oxide to postexercise hypotension by preventing reflex changes in sympathetic tone that result from systemic nitric oxide synthase inhibition and to control for alterations in resting sympathetic activity after exercise. During each condition, systemic and regional hemodynamics were measured. Throughout the study, arterial pressure and vascular resistances remained lower postexercise vs. postsham despite nitric oxide synthase inhibition (e.g., mean arterial pressure after L-NMMA was 108.0+/-2.4 mmHg postsham vs. 102.1+/-3.3 mmHg postexercise; P<0.05). Thus it does not appear that postexercise hypotension is dependent on increased production of nitric oxide in humans.     

Effect of nitric oxide synthase inhibition on cardiorespiratory responses in the conscious rat

Gozal, David, José E. Torres, Yair M. Gozal, andSanford M. Littwin. Effect of nitric oxide synthase inhibition on cardiorespiratory responses in the conscious rat. J. Appl. Physiol. 81(5): 2068-2077, 1996.Nitricoxide synthase (NOS) blockade was used to test the cardioventilatoryresponses to hypercapnia and hypoxia in freely behaving animals.Chronically instrumented adult Sprague-Dawley rats were studied beforeand after intravenous administration of either 100 mg/kg ofN^G-nitro-L-arginine methylester (L-NAME), a nonspecificNOS blocker, or 10 mg/kg ofS-methyl-L-thiocitrulline(SMTC), a selective neural NOS inhibitor.L-NAME injection inducedsustained blood pressure (BP) elevation with transient tachycardia andincreased minute ventilation (E), whichreturned to baseline within minutes. SMTC elicited similar, althoughtransient, BP increases; however, heart rate andE decreased.L-NAME and SMTC did not modifyoverall steady-state hypercapnic responses. In controlconditions, hypoxia induced early Eincreases with further E enhancementsat 30 min. L-NAME increased theearly E response to 10%O₂ but induced lateE reductions in hypoxia. SMTC did notchange early E responses but inducedmarked reductions in the later Ehypoxic responses. In control animals, hypoxia induced a significantheart rate increase. This increase was absent during the early response after SMTC and was followed in bothL-NAME- and SMTC-treated animals by significant heart rate reductions to values below room air. Similarly, the sustained BP response to hypoxia in control animals wasabsent after administration of NOS inhibitors. These findings suggestthat NOS activity exerts excitatory influences on respiration andcardiac chronotropy and sustained vasomotor tone during hypoxia. Wespeculate that NOS-mediated mechanisms may play an important role inhypoxia-induced ventilatory roll-off during wakefulness.

     

Acetylcholine chloride and renal hemodynamics during postnatal maturation in conscious lambs.

To test the hypothesis that acetylcholine-induced relaxation of the renal artery decreases with postnatal age, we measured parameters of renal hemodynamics before and for 35 s after aortic suprarenal injection of acetylcholine in conscious, chronically instrumented lambs aged approximately 1 wk (n = 5) and approximately 6 wk (n = 5). Acetylcholine was administered in one of five doses ranging from 0 to 10 mg/kg body wt; doses were administered randomly, in the same volume. There were significant age- and dose-dependent changes in renal vascular resistance after acetylcholine administration, such that the response was greater in 1-wk-old lambs. After the highest dose tested, renal vascular resistance decreased by 13.6 +/- 7.3 (SD) mmHg. ml(-1). min. g kidney wt in 1-wk-old lambs and by 9.1 +/- 3.2 mmHg. ml(-1). min. g kidney wt in 6-wk-old lambs at 35 s. We also observed a transient renal vasoconstriction before the renal vasodilatation in 6-wk-old lambs but not in 1-wk-old animals. These data provide the first age- and dose-dependent effects of exogenous administration of acetylcholine on renal hemodynamics during maturation in conscious animals.     

Influence of renal denervation on renal effects of acute nitric oxide and ETA/ETB receptor inhibition in conscious normotensive rats.

The role of renal nerves in the effects of concomitant NO synthase and non-selective ET(A/)ET(B) receptor inhibition on renal function was investigated in conscious normotensive Wistar rats. NO synthase inhibition alone (10 mg/kg b. w. i.v. L-NAME) in sham-operated rats with intact renal nerves induced an increase in systolic, diastolic and mean arterial pressure, urine flow rate, sodium, chloride and calcium excretion (p<0.05). The effect of L-NAME was markedly reduced by bosentan (10 mg/kg b.w. i.v.) and the values of urine flow rate, sodium, chloride and calcium excretions returned to control level (p<0.05). L-NAME administration one week after a bilateral renal denervation increased blood pressure to a similar extent as in sham-operated rats but decreased urine flow rate (p<0.05) and did not change electrolyte excretion. ET(A/)ET(B) receptor inhibition with bosentan during NO synthase inhibition in the renal denervated rats did not produce changes in urine flow rate or electrolyte excretion. NO synthase inhibition as well as concurrent NO synthase and ET(A/)ET(B) receptor inhibition did not change clearance of inulin or paraaminohippuric acid in sham-operated or renal denervated rats. These results indicate that renal sympathetic nerves play an important modulatory role in NO and endothelin induced effects on renal excretory function.     

Direct and reflexive effects of nitric oxide synthase inhibition on blood pressure

Direct effects of vasoactive substances on blood pressure can be examined in individuals with tetraplegia due to disruption of descending spinal pathways to sympathetic preganglionic neurons, as cervical lesions interfere with baroreceptor reflex buffering of sympathetic outflow. In this study, we assessed effects of the nitric oxide synthase inhibitor nitro-L-arginine methyl ester (L-NAME) on mean arterial pressure, heart rate, and plasma norepinephrine concentrations in individuals with tetraplegia vs. effects shown in a neurologically intact control group. Seven individuals with tetraplegia and seven age-matched controls received, on separate visits and in the following order, placebo (30 ml normal saline) and 0.5, 1, 2, and 4 mg/kg L-NAME intravenously over 60 min. Supine hemodynamic data were collected, and blood was sampled at the end of each infusion and at 120, 180, and 240 min thereafter. L-NAME increased mean arterial pressure, and the relative increase was greater in the tetraplegia group than in the control group. Heart rate was reduced after L-NAME administration in both groups. L-NAME decreased plasma norepinephrine in the control group but not in the group with tetraplegia. These findings suggest that reflexive sympathoinhibition normally buffers the pressor response to nitric oxide synthase inhibition, an effect that is not evident in individuals with tetraplegia as a result of decentralized sympathetic vasomotor control.     

Photoperiod-dependent effects of neuronal nitric oxide synthase inhibition on aggression in Siberian hamsters

Many nontropical species undergo physiological and behavioral adaptations in response to seasonal changes in photoperiod, or day length. In most rodent species, short winter photoperiods reduce testosterone concentrations, which provoke gonadal regression and reduce testosterone-dependent behaviors such as mating and aggression. Seasonally-breeding Siberian hamsters, however, are paradoxically more aggressive in short-days, despite much reduced reproductive activity and testosterone concentrations. Nitric oxide (NO) signaling has been proposed as part of an alternate mechanism underlying this phenomenon. A reduction in neuronal nitric oxide synthase (nNOS), the enzyme responsible for synthesizing NO in the brain, is associated with increased aggression in male short-day hamsters. In the present study, we hypothesized that pharmacological inhibition of nNOS would increase aggressive behavior in long days, but not in short days because nNOS is already reduced. Adult male Siberian hamsters were housed in either long (LD 16:8h) or short (LD 8:16h) photoperiods for 8weeks, then treated with either the selective nNOS inhibitor, 3-bromo-7-nitroindazole (3BrN) or oil vehicle, and subsequently tested for aggression in a resident-intruder test. Treatment with 3BrN increased attack frequency and duration in long days, but had no effect in short days. Short days also reduced testosterone concentrations, without any effect of treatment. These data provide further evidence linking reduced nNOS to elevated short-day aggression and support a role for NO signaling in this phenomenon.     

Effects of renal denervation on cardiovascular and renal responses to ACE inhibition in conscious lambs

Smith, Francine G., Suzanne Chan, and Saskia N. De Wildt.Effects of renal denervation on cardiovascular and renal responsesto ACE inhibition in conscious lambs. J. Appl.Physiol. 83(2): 414-419, 1997.Cardiovascular andrenal effects of either the angiotensin-converting enzyme inhibitorcaptopril or vehicle were measured in chronically instrumented lambs inthe presence (intact; n = 6) andabsence of renal sympathetic nerves (denervated; n = 5) to determine whether there wasan interaction between the renin-angiotensin system and renalsympathetic nerves early in life. Captopril caused a similar decreasein mean arterial pressure (P < 0.001) in intact and denervated lambs, predominantly through a decreasein diastolic pressure. Heart rate was increased from 177 ± 34 to213 ± 22 (SD) beats/min during captopril compared with vehicleinfusion in intact lambs. In denervated lambs, basal heart rates wereelevated to 218 ± 33 beats/min; there was no further increase inheart rate during captopril compared with vehicle infusion. Captoprilinfusion caused a decrease in renal vascular resistance but only in theabsence of renal nerves. These findings provide evidence to suggestthat early in life there is an interaction between renal sympatheticnerves and the renin-angiotensin system in regulating renalhemodynamics and the baroreflex control of the heart.

     

Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth rat

Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) rat. We previously showed that the Wistar-Furth (WF) rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD rats, whereas low-dose NOSI caused progression of CKD in WF rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N(G)-nitro-l-arginine methyl ester (l-NAME) (150 and 300 mg/l for 6-10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic l-NAME. SD given 150 mg/l l-NAME for 6 wk developed proteinuria and renal injury, whereas WF rats receiving 150 mg/l l-NAME for 6-10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD rat. There was little impact on renal nitric oxide synthase expression with l-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF rats, whereas WF rats showed a much smaller increment in glomerular blood pressure compared with SD rats. In conclusion, WF rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD rat. This protection may be associated with protection from glomerular hypertension.     

The effects of cyclooxygenase and nitric oxide synthase inhibition on oxidative stress in isolated rat heart

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas still exist about potential impact of these drugs on cardiovascular system. The present study was aimed to estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA], and SC-560) on oxidative stress in isolated rat heart, with special focus on l-arginine/NO system. The hearts of male Wistar albino rats (total number n = 96, each group 12 rats, 8 weeks old, body mass 180–200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40–120 cmH₂O). After control experiments the hearts were perfused with the following drugs: 100 μmol/l ASA (Aspirin), alone or in combination with 30 μmol/l l-NAME, 0.3 μmol/l meloxicam (movalis) with or without 30 μmol/l l-NAME, 3 μmol/l meloxicam (alone or in combination with 30 μmol/l l-NAME), 30 μmol/l l-NAME, and administration of 0.25 μmol/l SC-560. In samples of coronary venous effluent the following oxidative stress markers were measured spectrophotometrically: index of lipid peroxidation (measured as thiobarbituric acid reactive substances [TBARS]), superoxide anion radical release (O₂ ^?), and hydrogen peroxide (H₂O₂). While ASA was found to have an adverse influence on redox balance in coronary circulation, and coronary perfusion, meloxicam and SC-560 do not negatively affect the intact model of the heart. Furthermore, all effects were modulated by NOS inhibition. It seems that interaction between COX and l-arginine/NO system truly exists in coronary circulation, and can be one of the possible causes for achieved effects. That means: those effects induced by different inhibitors of COX are modulated by subsequent inhibition of NOS.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.     

Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-l-arginine methyl ester

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [³H]l-arginine into [³H]l-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.     

Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase.

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.     

Effect of nitric oxide synthase inhibition on hypercapnia-induced hypothermia and hyperventilation

Hypercapnia elicits hypothermia in a numberof vertebrates, but the mechanisms involved are not well understood. Inthe present study, we assessed the participation of the nitric oxide(NO) pathway in hypercapnia-induced hypothermia and hyperventilation bymeans of NO synthase inhibition by usingN-nitro-L-arginine(L-NNA). Measurements ofventilation, body temperature, and oxygen consumption were performed inawake unrestrained rats before and afterL-NNA injection(intraperitoneally) and L-NNA injection followed by hypercapnia (5%CO₂). Control animals received saline injections. L-NNA alteredthe breathing pattern during the control situation but not duringhypercapnia. A significant (P < 0.05) drop in body temperature was measured after bothL-NNA (40 mg/kg) and 5%inspired CO₂, with a drop inoxygen consumption in the first situation but not in the second.Hypercapnia had no effect onL-NNA-induced hypothermia. Theventilatory response to hypercapnia was not changed byL-NNA, even thoughL-NNA caused a drop in bodytemperature. The present data indicate that the two responses elicitedby hypercapnia, i.e., hyperventilation and hypothermia, do not share NOas a common mediator. However, theL-arginine-NO pathwayparticipates, although in an unrelated way, in respiratory function andthermoregulation.

     

Evaluation of nitric oxide synthase activity and inhibition kinetics by chemiluminescence.

The binding affinity (K(I)) and inactivation rate (k(inact)) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.     

Acute intracerebroventricular insulin microinjection after nitric oxide synthase inhibition of renal sodium handling in rats

The role of the central nervous system (CNS) in the control of hydrosaline homeostasis has been strikingly demonstrated by several studies. Recent and growing evidence suggests that insulin or a nonapeptide-derived from the C-terminus of the insulin beta-chain may influence many brain functions. However, there is little information on the insulin-activated neural pathways regulating urinary sodium excretion. Also, we examined the influence of nitric oxide synthase activity by chronic oral administration of N(omega)-nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, after previous i.c.v. administration of insulin to unanesthetized, unrestrained rats that were randomly assigned to one of seven separated groups: (a) i.c.v. 0.15 M NaCl-injected (n = 11) and i.c.v. 126 ng (n = 11) insulin-injected rats; (b) i.c.v. insulin-injected in systemic L-NAME-treated (n = 10) and vehicle-treated insulin-injected rats (n = 10); and (c) subcutaneously (SC) insulin-injected rats (n = 5). We showed that centrally administered insulin produced increase in the urinary output of sodium (from 0.15 M NaCl: 855.6 +/- 85.1 Delta%.min(-1) to 126 ng insulin: 2055 +/- 310.6 Delta%.min(-1)) and potassium (126 ng: from 0.15 M NaCl: 460.4 +/- 100 Delta%.min(-1) to 126 ng insulin: 669 +/- 60.8 Delta%.min(-1)). The urinary sodium excretion response to i.c.v. 126 ng insulin microinjection was significantly abolished by previous systemic treatment of animals with 15 mg/kg/day L-NAME (from vehicle + 126 ng insulin: 1935 +/- 258.3 Delta%. min(-1) to L-NAME + 126 ng insulin: 582.3 +/- 69.6 Delta%. min(-1)). In addition, we showed that insulin-induced natriuresis occurred by increasing post-proximal tubule sodium rejection (FEPP(Na)), despite an unchanged glomerular filtration rate (C(Cr)). The current data suggests the novel concept that CNS NO-dependent neural pathways may play an instrumental role on efferent insulin-sensitive nerve activity from periventricular region. Speculatively, it seems interesting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature, and that defects in this efferent signal could result in insulin central resistance, inability of renal tubules to handle the hydro electrolyte balance and hypertension.     

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro‐inflammatory cytokines that up‐regulate iNOS contribute to pre‐eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre‐eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham‐operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N‐[3‐(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8‐isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ~30 mmHg in RUPP rats, and 1400 W attenuated this increase by ~50% (P < 0.05). While RUPP increased plasma 8‐isoprostane levels, aortic ROS levels, and NADPH‐dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre‐eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.     

Platelet activation and modulation of the induction of nitric oxide synthase in the conscious rat.

Injection of lipopolysaccharide (LPS) (Salmonella W. Typhosa i.v. bolus) into conscious rats, induced a rapid drop of circulating platelets analogous to that induced by ADP. The animals showed a small fall in mean arterial blood pressure (MABP), an increase in heart rate and a significant increase in plasma nitrite and nitrate level. This result is consistent with the stimulation of an inducible NO synthase (i-NOS). The administration of the stable prostacyclin analogue, iloprost plus ADP or LPS, significantly protected against the decrease in free platelet number induced by ADP or LPS. The plasma nitrite and nitrate level stimulated by LPS was significantly reduced by iloprost and also by prostacyclin. These results are consistent with an inhibition of i-NOS by agents that increase the intracellular level of cAMP. The administration of the NO donor S-Nitroso-N-acyl-D-penicillamine (SNAP) plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP and by LPS. SNAP did not decrease the plasma nitrite and nitrate level stimulated by LPS; furthermore it induced a significant increase of heart rate, without affecting MABP, suggesting a direct accelerating effect of NO on the sino-atrial node. The administration of S-nitroso-glutathione (GSNO), a stable nitrosothiol, plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP but not by LPS. GSNO significantly reduced the plasma nitrite and nitrate level stimulated by LPS. These data demonstrate that the L-Arginine: NO pathway in vivo may be modulated by prostanoids and that compounds which increase cAMP, such as iloprost, are able to protect against LPS-induced early thrombocytopenia.     

Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle.

The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no l-NAME ingestion and acute exercise, rest plus l-NAME, and rest without l-NAME. The exercised rats ran on a treadmill for 53 +/- 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-gamma coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.     

Nitric oxide protects nitric oxide synthase function from hydroxyl radical-induced inhibition

The interdependent relationships among nitric oxide synthase (NOS), its coenzyme, cofactors and nitric oxide (NO(free radical) were studied using electron paramagnetic resonance spectroscopy. It was found that superoxide-dependent hydroxyl free radical (OH(free radical), derived from NOS coenzyme and cofactors, inhibits NOS activity, and that endogenous NO(free radical) generated by NOS scavenges OH(free radical) and protects NOS function. These results reveal a new role for NO(free radical) that may be important in NOS function and cellular free radical homeostasis.