Nitric oxide inhibition unmasks ischemic myocardium-derived vasoconstrictor signals activating endothelin type A receptor of coronary microvessels

NO plays an important role in the compensatory increase in coronary flow conductance against myocardial ischemia, and NO bioavailability is impaired in various diseases. We tested the hypothesis that, when NO production is inhibited, vasoconstrictor signals from the ischemic myocardium are unmasked. We investigated the involvement of endothelin type A (ETA) receptors in the transduction of the constrictor signal. To detect coronary vasoactive signals derived from ischemic myocardium, we used a bioassay system in which an isolated rabbit coronary microvessel (detector vessel, DV) was placed on beating myocardium perfused by the left anterior descending coronary artery (LAD) of an anesthetized open-chest dog (n = 38). The DV was pressurized to 60 cmH2O throughout the experiment and observed with an intravital microscope equipped with a floating objective. After the intrinsic tone of the DV was established, vehicle (n = 7), Nomega-nitro-L-arginine (L-NNA, 100 micromol/l; n = 13), L-NNA + BQ-123 (a selective ETA receptor blocker, 1 micromol/l; n = 7), or BQ-123 alone (1 micromol/l; n = 7) was superfused onto the DV. Thereafter, the LAD of the beating heart was occluded. Coronary occlusion produced significant dilation of the DV by 10 +/- 4%. When L-NNA was applied, the DV significantly constricted by 12 +/- 5% in response to LAD occlusion, and BQ-123 abolished the vasoconstriction. Pretreatment with BQ-123 alone produced an enhancement of the ischemia-induced dilation. We conclude that ischemic myocardium releases transferable vasomotor signals that produce coronary microvascular constriction during the blockade of NO production and the constrictor signal is mediated by ETA receptors.     

Nitric oxide synthase in guinea pig sympathetic ganglia: Correlation with tyrosine hydroxylase and neuropeptides

Nitric oxide synthase (NOS) has previously been reported in a small population of postganglionic sympathetic neurons in the guinea pig. The present study of paravertebral ganglia and the inferior mesenteric ganglion aimed to classify these neurons according to their content of neuropeptides (calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide) and the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, by means of immunohistochemical and histochemical double-labelling techniques. NOS-containing neurons belonged to the non-catecholaminergic population of postganglionic neurons, and partial coexistence was found with neuropeptide Y and vasoactive intestinal peptide immunoreactivities but not with calcitonin gene-related peptide. However, most of the NOS-containing neurons contained none of the neuropeptides, thus representing a hitherto unrecognized population of postganglionic neurons. The findings show that NOS is localized to small but neurochemically highly specific populations of postganglionic neurons, which most likely reflects an association with target- and function-specific pathways.     

Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo.

Inhibition of nitric oxide (NO) synthase activity by L-NG-Nitroarginine (NO2Arg) in brain preparations is not reversed by dialysis and is enhanced by prolonged preincubation of NO2Arg with the enzyme. By contrast, the weaker inhibition by NO2Arg of macrophage NO synthase is fully reversible. NO2Arg inhibits NO synthase activity in the brain after i.p. administration of 5 or 50 mg/kg. This in vivo inhibition also appears to be irreversible. The potent in vivo inhibition of central NO synthase by NO2Arg may facilitate studies of the physiologic function of NO as a neuronal messenger.     

Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.

The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and gamma-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock.     

Nitric oxide and cerebral blood flow responses to hyperbaric oxygen.

We have tested the hypothesis that cerebral nitric oxide (NO) production is involved in hyperbaric O(2) (HBO(2)) neurotoxicity. Regional cerebral blood flow (rCBF) and electroencephalogram (EEG) were measured in anesthetized rats during O(2) exposure to 1, 3, 4, and 5 ATA with or without administration of the NO synthase inhibitor (N(omega)-nitro-L-arginine methyl ester), L-arginine, NO donors, or the N-methyl-D-aspartate receptor inhibitor MK-801. After 30 min of O(2) exposure at 3 and 4 ATA, rCBF decreased by 26-39% and by 37-43%, respectively, and was sustained for 75 min. At 5 ATA, rCBF decreased over 30 min in the substantia nigra by one-third but, thereafter, gradually returned to preexposure levels, preceding the onset of EEG spiking activity. Rats pretreated with N(omega)-nitro-L-arginine methyl ester and exposed to HBO(2) at 5 ATA maintained a low rCBF. MK-801 did not alter the cerebrovascular responses to HBO(2) at 5 ATA but prevented the EEG spikes. NO donors increased rCBF in control rats but were ineffective during HBO(2) exposures. The data provide evidence that relative lack of NO activity contributes to decreased rCBF under HBO(2), but, as exposure time is prolonged, NO production increases and augments rCBF in anticipation of neuronal excitation.     

Role of renal sympathetic nerve activity in hypertension induced by chronic nitric oxide inhibition

Nitric oxide levels are diminished in hypertensive patients, suggesting nitric oxide might have an important role to play in the development of hypertension. Chronic blockade of nitric oxide leads to hypertension that is sustained throughout the period of the blockade in baroreceptor-intact animals. It has been suggested that the sympathetic nervous system is involved in the chronic increase in blood pressure; however, the evidence is inconclusive. We measured renal sympathetic nerve activity and blood pressure via telemetry in rabbits over 7 days of nitric oxide blockade. Nitric oxide blockade via N(omega)-nitro-L-arginine methyl ester (L-NAME) in the drinking water (50 mg x kg(-1) x day(-1)) for 7 days caused a significant increase in arterial pressure (7 +/- 1 mmHg above control levels; P < 0.05). While the increase in blood pressure was associated with a decrease in heart rate (from 233 +/- 6 beats/min before the L-NAME to 202 +/- 6 beats/min on day 7), there was no change in renal sympathetic nerve activity (94 +/- 4 %baseline levels on day 2 and 96 +/- 5 %baseline levels on day 7 of L-NAME; baseline nerve activity levels were normalized to the maximum 2 s of nerve activity evoked by nasopharyngeal stimulation). The lack of change in renal sympathetic nerve activity during the L-NAME-induced hypertension indicates that the renal nerves do not mediate the increase in blood pressure in conscious rabbits.     

Cyclooxygenase inhibition attenuates sympathetic responses to muscle stretch in humans

Passive muscle stretch performed during a period of post-exercise muscle ischemia (PEMI) increases muscle sympathetic nerve activity (MSNA), and this suggests that the muscle metabolites may sensitize mechanoreceptors in healthy humans. However, the responsible substance(s) has not been studied thoroughly in humans. Human and animal studies suggest that cyclooxygenase products sensitize muscle mechanoreceptors. Thus we hypothesized that local cyclooxygenase inhibition in exercising muscles could attenuate MSNA responses to passive muscle stretch during PEMI. Blood pressure (Finapres), heart rate, and MSNA (microneurography) responses to passive muscle stretch were assessed in 13 young healthy subjects during PEMI before and after cyclooxygenase inhibition, which was accomplished by a local infusion of 6 mg ketorolac tromethamine in saline via Bier block. In the second experiment, the same amount of saline was infused via the Bier block. Ketorolac Bier block decreased prostaglandin synthesis to approximately 34% of the baseline. Before ketorolac Bier block, passive muscle stretch evoked significant increases in MSNA (P < 0.005) and mean arterial blood pressure (P < 0.02). After ketorolac Bier block, passive muscle stretch did not evoke significant responses in MSNA (P = 0.11) or mean arterial blood pressure (P = 0.83). Saline Bier block had no effect on the MSNA or blood pressure response to ischemic stretch. These observations indicate that cyclooxygenase inhibition attenuates MSNA responses seen during PEMI and suggest that cyclooxygenase products sensitize the muscle mechanoreceptors.     

Nitric oxide synthase inhibition does not affect regulation of muscle sympathetic nerve activity during head-up tilt

To test the hypothesis that systemic inhibition of nitric oxide (NO) synthase does not alter the regulation of sympathetic outflow during head-up tilt in humans, in eight healthy subjects NO synthase was blocked by intravenous infusion of NG-monomethyl-L-arginine (L-NMMA). Blood pressure, heart rate, cardiac output, total peripheral resistance (TPR), and muscle sympathetic nerve activity (MSNA) were recorded in the supine position and during 60 degrees head-up tilt. In the supine position, infusion of L-NMMA increased blood pressure, via increased TPR, and inhibited MSNA. However, the increase in MSNA evoked by head-up tilt during L-NMMA infusion (change in burst rate: 24 +/- 4 bursts/min; change in total activity: 209 +/- 36 U/min) was similar to that during head-up tilt without L-NMMA (change in burst rate: 23 +/- 4 bursts/min; change in total activity: 251 +/- 52 U/min, n = 6, all P > 0.05). Moreover, changes in TPR and heart rate during head-up tilt were virtually identical between the two conditions. These results suggest that systemic inhibition of NO synthase with L-NMMA does not affect the regulation of sympathetic outflow and vascular resistance during head-up tilt in humans.     

Nitric oxide synthase inhibition decreases tolerance to hyperoxia in newborn rats

We evaluated the effects of sustained perinatal inhibition of NO synthase (NOS) on hyperoxia induced lung injury in newborn rats. N(G)-nitro-Larginine-methyl-ester (L-NAME) or untreated water was administered to pregnant rats for the final 7 days of gestation and during lactation; followed by postnatal exposure to hyperoxia (>95% O(2)) or room air. The survival rate of L-NAME treated pups when placed in > 95% O(2) at birth was significantly lower than controls from day 4 (L-NAME, 87%; control pups, 100%, p < 0.05) to 14 (L-NAME, 0%; control pups, 53%, p < 0.05). Foetal pulmonary artery vasoconstriction was induced by L-NAME with a decrease in internal diameter from 0.88 +/- 0.03 mm to 0.64 +/- 0.01 mm in control vs. L-NAME groups (p < 0.05), respectively. We conclude that perinatal NOS inhibition results in pulmonary artery vasoconstriction and a decreased tolerance to hyperoxia induced lung injury in newborn rats.     

Nitric oxide regulatory role in sensitized guinea pig trachea

Nitric oxide (NO) has been cited to play an important regulatory role in airway function. Moreover, the NO synthase expression in models of inflammation is documented. The aim of this study was to investigate, in vitro, the NO modulation of cholinergic responses in sham-sensitized and ovalbumin-sensitized guinea pig trachea by using L-arginine (L-ARG), a precursor of NO synthesis, and L-Ng-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase.Our results showed that NO's ability to modulate cholinergic responses in oval-buminsensitized guinea pig trachea is lost. Indeed L-ARG and L-NAME modify acetylcholine sensitivity in sham-sensitized guinea pig but not in ovalbumin-sensitized guinea pig.     

Nitric oxide modulates the responses of osteoclast formation to static magnetic fields

Nitric oxide (NO) is involved in osteoclast differentiation. Our previous studies showed that static magnetic fields (SMFs) could affect osteoclast differentiation. The inhibitory effects of 16 T of high SMF (HiMF) on osteoclast differentiation was correlated with increased production of NO. We raised the hypothesis that NO mediated the regulatory role of SMFs on osteoclast formation. In this study, 500 nT of hypomagnetic field (HyMF), 0.2 T of moderate SMF (MMF) and 16 T of high SMF (HiMF) were utilized as SMF treatment. Under 16 T, osteoclast formation was markedly decreased with enhanced NO synthase (NOS) activity, thus producing a high level of NO. When treated with NOS inhibitor N-Nitro-L-Arginine Methyl Ester (L-NAME), NO production could be inhibited, and osteoclast formation was restored to control group level in a concentration-dependent manner. However, 500 nT and 0.2 T increased osteoclast formation with decreased NOS activity and NO production. When treated with NOS substrate L-Arginine (L-Arg) or NO donor sodium nitroprusside (SNP), the NO level in the culture medium was obviously elevated, thus inhibiting osteoclast differentiation in a concentration-dependent manner under 500 nT or 0.2 T. Therefore, these findings indicate that NO mediates the regulatory role of SMF on osteoclast formation.     

Nitric oxide affects plant mitochondrial functionality in vivo

In this report, we show that nitric oxide affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway. The residual respiration depends on the alternative pathway and novel synthesis of alternative oxidase occurs. These modifications are associated with depolarisation of the mitochondrial membrane potential and release of cytochrome c from mitochondria, suggesting a conserved signalling pathway in plants and animals. This signal cascade is triggered at the mitochondrial level and induces about 20% of cell death. In order to achieve a higher level of cell death, the addition of H(2)O(2) is necessary.     

Nitric oxide derived from sympathetic nerves regulates airway responsiveness to histamine in guinea pigs

Matsumoto, Koichiro, Hisamichi Aizawa, Shohei Takata,Hiromasa Inoue, Naotsugu Takahashi, and Nobuyuki Hara.Nitric oxide derived from sympathetic nerves regulates airwayresponsiveness to histamine in guinea pigs. J. Appl.Physiol. 83(5): 1432-1437, 1997.Nitric oxide(NO), which can be derived from the nervous system or the epithelium ofthe airway, may modulate airway responsiveness. We investigated how NOderived from the airway nervous system would affect the airwayresponsiveness to histamine and acetylcholine in mechanicallyventilated guinea pigs. An NO synthase inhibitor N^G-nitro-L-argininemethyl ester (L-NAME) (1 mmol/kgip) significantly enhanced airway responsiveness to histamine but notto acetylcholine. Its enantiomerD-NAME (1 mmol/kg ip), incontrast, had no effect. TheL-NAME-induced airwayhyperresponsiveness was still observed in animals pretreated withpropranolol (1 mg/kg iv) and atropine (1 mg/kg iv). Pretreatment withthe ganglionic blocker hexamethonium (2 mg/kg iv) completely abolishedenhancing effect of L-NAME on airway responsiveness. Bilateral cervical vagotomy did not alter theL-NAME-induced airwayhyperresponsiveness, whereas sympathetic stellatectomy completelyabolished it. Results suggest that NO that was presumably derived fromthe sympathetic nervous system regulates airway responsiveness tohistamine in guinea pigs.

     

Adenosine modulation of vasoconstrictor responses to stimulation of sympathetic nerves and norepinephrine infusion in the superior mesenteric artery of the cat

Vasoconstriction induced by sympathetic nerve stimulation and by norepinephrine infusion in the superior mesenteric artery of cats anesthetized with pentobarbital was inhibited by adenosine infusions in a dose-related way. The responses to nerve stimulation were not inhibited to a greater extent than the responses to norepinephrine, thus suggesting no presynaptic modulation of sympathetic nerves supplying the resistance vessels of the feline intestinal vascular bed. Blockade of adenosine receptors using 8-phenyltheophylline did not alter the degree of constriction induced by nerve stimulation or norepinephrine infusion, indicating that in the fasted cat, endogenous adenosine co-released or released subsequent to constriction does not affect the peak vasoconstriction reached. Isoproterenol caused similar degrees of vasodilation as adenosine but did not show significant antagonism of the pooled responses to nerve stimulation or norepinephrine infusion; there was no tendency for the degree of dilation induced by isoproterenol to correlate with the inhibition of constrictor responses. Thus, the effect of adenosine on nerve- and norepinephrine-induced constriction is not secondary to nonspecific vasodilation.     

Nitric oxide inhibition abolishes sleep-wake differences in cerebral circulation

Nitric oxide (NO), being produced by active neurones and also being a cerebral vasodilator, may couple brain activity and blood flow in sleep, particularly during active sleep (AS), which is characterized by widespread neural activation and markedly elevated cerebral blood flow (CBF) compared with quiet wakefulness (QW) and quiet sleep (QS). This study examined CBF and cerebral vascular resistance (CVR) in lambs (n = 6) during spontaneous sleep-wake cycles before and after infusion of N(omega)-nitro-L-arginine (L-NNA), an inhibitor of NO synthase. L-NNA infusion produced increases in CVR and decreases in CBF during all sleep-wake stages, with the greatest changes occurring in AS (DeltaCVR, 88 +/- 19%; DeltaCBF -24 +/- 8%). The characteristic CVR and CBF differences among AS, QS, and QW disappeared within 1-3 h of L-NNA infusion, but had reappeared by 24 h despite persisting cerebral vasoconstriction. These experiments show that NO promotes cerebral vasodilatation during sleep as well as wakefulness, particularly during AS. Additionally, NO is the major, although not sole, determinant of the CBF differences that exist between sleep-wake states.     

Nitric oxide in skeletal muscle: inhibition of nitric oxide synthase inhibits walking speed in rats.

Nitric oxide (NO*) is a multifunctional messenger molecule generated by a family of enzymes called the nitric oxide synthases (NOSs). Although NOSs have been identified in skeletal muscle, specifically brain NOS (bNOS) and endothelial NOS (eNOS), their role has not been well clarified. The goals of this investigation were to (1) characterize the immunoreactivity, Ca(2+) dependence, and activity of NOS in human and rat skeletal muscle and (2) using a rat model, investigate the effect of chronic blockade of NOS on skeletal muscle structure and function. Our results showed that both human and rodent skeletal muscle had NOS activity. This NOS activity was similar to that of the endothelial and brain NOS isoforms in that it was calcium-dependent. However, Western blot analysis consistently showed that a polyclonal antibody raised against a peptide sequence of human inducible NOS (iNOS) reacted with a protein with a molecular weight (95 kDa) that was different from that of other NOS isoforms. RT-PCR analysis identified the mRNA expression of not only eNOS and bNOS but also iNOS in human and rat muscle. Inhibition of nitric oxide synthase in rats with N(omega)-nitro-L-arginine methyl ester (L-NAME) resulted in a progressive, severe reduction in walking speed (30-fold reduction in walking velocity at day 22, P < 0.001), muscle fiber cross-sectional area (40% reduction at day 22, P < 0.001), and muscle mass (40% reduction in dry weight at day 22, P < 0.01). Rats fed the same regimen of the enantiomer of L-NAME (d-NAME) had normal motor function, muscle fiber morphology, and muscle mass. Taken together, these results imply that there may be a novel nitric oxide synthase in muscle and that NO. generated from muscle may be important in muscle function.