Targeting and translocation of endothelial nitric oxide synthase

This review explores advances in our understanding of the intracellular regulation of the endothelial isoform of nitric oxide synthase (eNOS) in the context of its dynamically regulated subcellular targeting. Nitric oxide (NO) is a labile molecule, and may play important biological roles both within the cell in which it is synthesized and in its interactions with nearby cells and molecules. The localization of eNOS within the cell importantly influences the biological role and chemical fate of the NO produced by the enzyme. eNOS, a Ca2+/calmodulin-dependent enzyme, is subject to a complex pattern of intracellular regulation, including co- and post-translational modifications and interactions with other proteins and ligands. In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targeting of the protein to caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation. In resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. However, following agonist activation, eNOS dissociates from caveolin, and nearly all the eNOS translocates to structures within the cell cytosol; following more protracted incubations with agonists, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The agonist-induced internalization of eNOS is completely abrogated by chelation of intracellular Ca2+. These rapid receptor-mediated effects are seen not only for "classic" eNOS agonists such as bradykinin, but also for estradiol, indicating a novel non-genomic role for estrogen in eNOS activation. eNOS targeting to the membrane is labile, and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Effects of a neuronal nitric oxide synthase inhibitor on lipopolysaccharide-induced fever

It has been demonstrated that nitric oxide (NO) has a thermoregulatory action, but very little is known about the mechanisms involved. In the present study we determined the effect of neuronal nitric oxide synthase (nNOS) inhibition on thermoregulation. We used 7-nitroindazole (7-NI, 1, 10 and 30 mg/kg body weight), a selective nNOS inhibitor, injected intraperitoneally into normothermic Wistar rats (200-250 g) and rats with fever induced by lipopolysaccharide (LPS) (100 µg/kg body weight) administration. It has been demonstrated that the effects of 30 mg/kg of 7-NI given intraperitoneally may inhibit 60% of nNOS activity in rats. In all experiments the colonic temperature of awake unrestrained rats was measured over a period of 5 h at 15-min intervals after intraperitoneal injection of 7-NI. We observed that the injection of 30 mg/kg of 7-NI induced a 1.5oC drop in body temperature, which was statistically significant 1 h after injection (P<0.02). The coinjection of LPS and 7-NI was followed by a significant (P<0.02) hypothermia about 0.5oC below baseline. These findings show that an nNOS isoform is required for thermoregulation and participates in the production of fever in rats.

Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

Induction of Fos protein immunoreactivity by spinal cord contusion

The objective of the present study was to identify neurons in the central nervous system that respond to spinal contusion injury in the rat by monitoring the expression of the nuclear protein encoded by the c-fos gene, an activity-dependent gene, in spinal cord and brainstem regions. Rats were anesthetized with urethane and the injury was produced by dropping a 5-g weight from 20.0 cm onto the exposed dura at the T10-L1 vertebral level (contusion group). The spinal cord was exposed but not lesioned in anesthetized control animals (laminectomy group); intact animals were also subjected to anesthesia (intact control). Behavioral alterations were analyzed by Tarlov/Bohlman scores, 2 h after the procedures and the animals were then perfused for immunocytochemistry. The patterns of Fos-like immunoreactivity (FLI) which were site-specific, reproducible and correlated with spinal laminae that respond predominantly to noxious stimulation or injury: laminae I-II (outer substantia gelatinosa) and X and the nucleus of the intermediolateral cell column. At the brain stem level FLI was detected in the reticular formation, area postrema and solitary tract nucleus of lesioned animals. No Fos staining was detected by immunocytochemistry in the intact control group. However, detection of FLI in the group submitted to anesthesia and surgical procedures, although less intense than in the lesion group, indicated that microtraumas may occur which are not detected by the Tarlov/Bohlman scores. There is both a local and remote effect of a distal contusion on the spinal cord of rats, implicating sensory neurons and centers related to autonomic control in the reaction to this kind of injury.

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-µm sections at 120-µm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 ± 206, N = 6; 1020 ± 150, N = 6; nonhandled and handled, respectively), paraventricular (584 ± 85, N = 8; 682 ± 62, N = 9) or periventricular (743 ± 118, N = 7; 990 ± 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

Nitric oxide synthase blockade and body fluid volumes

The influence of chronic nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on body fluid distribution was studied in male Wistar rats weighing 260-340 g. Extracellular, interstitial and intracellular spaces, as well as plasma volume were measured after a three-week treatment with L-NAME (~70 mg/kg per 24 h in drinking water). An increase in extracellular space (16.1 ± 1.1 vs 13.7 ± 0.6 ml/100 g in control group, N = 12, P<0.01), interstitial space (14.0 ± 0.9 vs 9.7 ± 0.6 ml/100 g in control group, P<0.001) and total water (68.7 ± 3.9 vs 59.0 ± 2.9 ml/100 g, P<0.001) was observed in the L-NAME group (N = 8). Plasma volume was lower in L-NAME-treated rats (2.8 ± 0.2 ml/100 g) than in the control group (3.6 ± 0.1 ml/100 g, P<0.001). Blood volume was also lower in L-NAME-treated rats (5.2 ± 0.3 ml/100 g) than in the control group (7.2 ± 0.3 ml/100 g, P<0.001). The increase in total ratio of kidney wet weight to body weight in the L-NAME group (903 ± 31 vs 773 ± 45 mg/100 g in control group, P<0.01) but not in total kidney water suggests that this experimental hypertension occurs with an increase in renal mass. The fact that the heart weight to body weight ratio and the total heart water remained constant indicates that, despite the presence of high blood pressure, no modification in cardiac mass occurred. These data show that L-NAME-induced hypertension causes alterations in body fluid distribution and in renal mass.

Substance P immunoreactivity in the lumbar spinal cord of the turtle Trachemys dorbigni following peripheral nerve injury

Immunoreactive substance P was investigated in turtle lumbar spinal cord after sciatic nerve transection. In control animals immunoreactive fibers were densest in synaptic field Ia, where the longest axons invaded synaptic field III. Positive neuronal bodies were identified in the lateral column of the dorsal horn and substance P immunoreactive varicosities were observed in the ventral horn, in close relationship with presumed motoneurons. Other varicosities appeared in the lateral and anterior funiculi. After axotomy, substance P immunoreactive fibers were reduced slightly on the side of the lesion, which was located in long fibers that invaded synaptic field III and in the varicosities of the lateral and anterior funiculus. The changes were observed at 7 days after axonal injury and persisted at 15, 30, 60 and 90 days after the lesion. These findings show that turtles should be considered as a model to study the role of substance P in peripheral axonal injury, since the distribution and temporal changes of substance P were similar to those found in mammals.

Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg) elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade) and complete autonomic blockade (38 ± 3, 55 ± 6, 54 ± 5, 45 ± 5 mmHg, respectively; N = 9, P = NS). L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 ± 8, 38 ± 5, 46 ± 6 bpm, respectively; N = 9, P = NS). Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 ± 3 bpm, P<0.05 vs intact animals, N = 9). Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9) caused a similar increase in blood pressure (45 ± 5, 38 ± 3, 44 ± 9 mmHg, respectively; P = NS) and heart rate (31 ± 4, 34 ± 3, 35 ± 4 bpm, respectively; P = NS). Addition of L-NAME (500 µM) to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9). In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.

Expression of neuronal nitric oxide synthase in the developing superficial layers of the rat superior colliculus

We investigated the level of expression of neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of the rat superior colliculus during early postnatal development. Male and female Lister rats ranging in age between the day of birth (P0) and the fourth postnatal week were used in the present study. Two biochemical methods were used, i.e., in vitro measurement of NOS specific activity by the conversion of [³H]-arginine to [³H]-citrulline, and analysis of Western blotting immunoreactive bands from superior colliculus homogenates. As revealed by Western blotting, very weak immunoreactive bands were observed as early as P0-2, and their intensity increased progressively at least until P21. The analysis of specific activity of NOS showed similar results. There was a progressive increase in enzymatic activity until near the end of the second postnatal week, and a nonsignificant tendency to an increase until the end of the third week was also observed. Thus, these results indicated an increase in the amount of nNOS during the first weeks after birth. Our results confirm and extend previous reports using histochemistry for NADPH-diaphorase and immunocytochemistry for nNOS, which showed a progressive increase in the number of stained cells in the superficial layers during the first two postnatal weeks, reaching an adult pattern at the end of the third week. Furthermore, our results suggested that nNOS is present in an active form in the rat superior colliculus during the period of refinement of the retinocollicular pathway.

Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

The objective of the present study was to determine the relationship between nitric oxide synthases (NOS) and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) <35% (N = 10), and from patients with CAD and LVEF >60% (N = 10) during cardiac surgery. NOS activity was measured by the conversion of L-[H³]-arginine to L-[H³]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS) activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 ± 0.18 vs 1.51 ± 0.31 pmol mg-1 min-1 (P < 0.0001) and 0.37 ± 0.08 vs 0.78 ± 0.09 relative cDNA absorbance at 320 nm (P < 0.0001), respectively. In contrast, inducible NOS (iNOS) activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 ± 0.90 vs 1.54 ± 0.65 pmol mg-1 min-1 (P < 0.0001) and 2.19 ± 0.27 vs 1.43 ± 0.13 cDNA absorbance at 320 nm (P < 0.0001), respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 µM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 µM L-NOARG from 73 ± 5.1 to 19 ± 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Inducible nitric oxide synthase and tumor necrosis factor-alpha in delayed gastric emptying and gastrointestinal transit induced by lipopolysaccharide in mice

Gastrointestinal motility disturbances during endotoxemia are probably caused by lipopolysaccharide (LPS)-induced factors: candidates include nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß, and interleukin-6. Flow cytometry was used to determine the effects of LPS and these factors on gastric emptying (evaluated indirectly by determining percent gastric retention; %GR) and gastrointestinal transit (GIT) in male BALB/c mice (23-28 g). NO (300 µg/mouse, N = 8) and TNF-alpha (2 µg/mouse, N = 7) increased (P < 0.01) GR and delayed GIT, mimicking the effect of LPS (50 µg/mouse). During early endotoxemia (1.5 h after LPS), inhibition of inducible NO synthase (iNOS) by a selective inhibitor, 1400 W (150 µg/mouse, N = 11), but not antibody neutralization of TNF-alpha (200 µg/mouse, N = 11), reversed the increase of GR (%GR 78.8 ± 3.3 vs 47.2 ± 7.5%) and the delay of GIT (geometric center 3.7 ± 0.4 vs 5.6 ± 0.2). During late endotoxemia (8 h after LPS), both iNOS inhibition (N = 9) and TNF-alpha neutralization (N = 9) reversed the increase of GR (%GR 33.7 ± 2.0 vs 19.1 ± 2.6% (1400 W) and 20.1 ± 2.0% (anti-TNF-alpha)), but only TNF-alpha neutralization reversed the delay of GIT (geometric center 3.9 ± 0.4 vs 5.9 ± 0.2). These findings suggest that iNOS, but not TNF-alpha, is associated with delayed gastric emptying and GIT during early endotoxemia and that during late endotoxemia, both factors are associated with delayed gastric emptying, but only TNF-alpha is associated with delayed GIT.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8%; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2%; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95% CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Hippocampal output is increased in affective disorders and is mediated by increased glutamatergic input via N-methyl-D-aspartate (NMDA) receptor and moderated by antidepressant treatment. Activation of NMDA receptors by glutamate evokes the release of nitric oxide (NO) by the activation of neuronal nitric oxide synthase (nNOS). The human hippocampus contains a high density of NMDA receptors and nNOS-expressing neurons suggesting the existence of an NMDA-NO transduction pathway which can be involved in the pathogenesis of affective disorders. We tested the hypothesis that nNOS expression is increased in the human hippocampus from affectively ill patients. Immunocytochemistry was used to demonstrate nNOS-expressing neurons in sections obtained from the Stanley Consortium postmortem brain collection from patients with major depression (MD, N = 15), bipolar disorder (BD, N = 15), and schizophrenia (N = 15) and from controls (N = 15). nNOS-immunoreactive (nNOS-IR) and Nissl-stained neurons were counted in entorhinal cortex, hippocampal CA1, CA2, CA3, and CA4 subfields, and subiculum. The numbers of Nissl-stained neurons were very similar in different diagnostic groups and correlated significantly with the number of nNOS-IR neurons. Both the MD and the BD groups had greater number of nNOS-IR neurons/400 µm² in CA1 (mean ± SEM: MD = 9.2 ± 0.6 and BD = 8.4 ± 0.6) and subiculum (BD = 6.7 ± 0.4) when compared to control group (6.6 ± 0.5) and this was significantly more marked in samples from the right hemisphere. These changes were specific to affective disorders since no changes were seen in the schizophrenic group (6.7 ± 0.8). The results support the current view of the NMDA-NO pathway as a target for the pathophysiology of affective disorders and antidepressant drug development.