nNOS mRNA Research Articles

Losartan reduces central and peripheral sympathetic nerve activity in a rat model of neurogenic hypertension.

We have developed a new model of neurogenic hypertension in the rat, in which hypertension is caused by injecting 50 microL of 10% phenol in the lower pole of one kidney. Administration of phenol in the kidney causes an immediate and persistent rise in blood pressure (BP), norepinephrine (NE) secretion from the posterior hypothalamic nuclei (PH), and renal sympathetic nerve activity (RSNA). Because angiotensin II (Ang II) is known to stimulate central and peripheral sympathetic nervous system (SNS) activity, we have tested the hypothesis that losartan, a specific Ang II AT1 receptor antagonist, may lower BP, at least in part, by SNS inhibition. To this end, we studied the effects of losartan on BP and SNS activity following intrarenal phenol injection. Central SNS activity was measured by NE secretion from the PH using a microdialysis technique, and peripheral SNS activity was measured by direct recording of renal nerve activity. At the end of the experiments, brains were isolated and interleukin (IL)-1beta and nitric oxide synthase (NOS) mRNA gene expression was measured by RT-PCR in the PH, paraventricular nuclei (PVN), and locus ceruleus (LC). The intrarenal injection of phenol raised BP, as well as central and renal SNS activity, but reduced the abundance of IL-1beta and neuronal NOS (nNOS) mRNA in the PH, PVN, and LC. Whether injected intravenously or in the lateral ventricle, losartan caused a significant (P<0.01) and dose-dependent inhibition of the effects of phenol on BP, NE secretion from the PH, and RSNA. Losartan also caused a significant (P<0.01) and dose-dependent rise in IL-1beta and nNOS-mRNA gene expression in the PH, PVN, and LC of phenol-injected rats. In conclusion, these studies have shown that the intrarenal injection of phenol causes a rise in central and renal SNS activity and a decrease in IL-1beta and nNOS-mRNA in the PH, PVN, and LC. Losartan prevented the rise in BP and SNS activity, as well as the decrease in IL-1beta and nNOS mRNA abundance caused by phenol. These studies have demonstrated that the antihypertensive action of losartan in the phenol renal injury model is largely mediated by inhibition of central and peripheral SNS activity and suggest that activation of IL-1beta and nNOS, 2 important modulators of central SNS activity, mediates the inhibitory action of losartan on SNS activity.

Suppression of Nitric Oxide (NO)-Dependent Behavior by Double-Stranded RNA-Mediated Silencing of a Neuronal NO Synthase Gene

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies.

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene.

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies.

Nitric oxide and nitric oxide synthases in the fetal cerebral cortex of rats following transient uteroplacental ischemia

The effect of transient uteroplacental ischemia on nitric oxide (NO) levels, enzymatic activity, and expression of NO synthase (NOS) isoforms was studied in fetal rat brains. Fetuses were subjected to ischemia by clamping the uterine arteries for 5 min on gestational day 17 (GD17). At different times after ischemia, fetuses were delivered by Cesarean section under anesthesia to obtain the brains. Transient uteroplacental ischemia produced a time dependent increase in nitrite levels in the brain, reaching a maximum value (300±25% of baseline) 24 h after uterine artery occlusion and remaining elevated as long as 48 h. Significantly increased nitrite levels were found in the cerebral cortex but not in the mesencephalon and cerebellum. The ischemia-induced increment in nitrite levels was totally blocked by either l-NAME (10 mg/kg) or AMT (0.65 mg/kg) administered i.p. 1 h before uterine artery occlusion. Both Ca 2+-dependent and Ca 2+-independent NOS activities in the cerebral cortex remained significantly increased with respect to controls after 24 h following the ischemia. Reverse transcriptase-polymerase chain reaction showed augmented levels of mRNAs for both nNOS and iNOS when compared with controls at 8 h after ischemia. At 36 h, nNOS mRNA returned to basal levels whereas eNOS mRNA levels increased and iNOS mRNA remained elevated. Our results show that the three NOS isoforms participate in increasing NO levels after transient ischemia and suggest a biphasic and differential regulation of the expression of constitutive NOS isoforms in the rat cerebral cortex.

비용조직에서 산화질소합성효소들의 발현

Background and Objective: The pathogenesis of nasal polyp is poorly understood. Nitric oxide (NO) plays a major role in a number of physiologic function and may be cytotoxic in high concentrations. NO is formed by the oxidative deamination of L-arginine by nitric oxide synthases (NOS). Three NOS isoforms have been identified in human. Constitutive forms of NOS are present in vascular endothelial cells (Type III NOS, eNOS) and neurons in the brain and the peripheral nervous system (Type I NOS, nNOS) whereas the inducible form (Type II NOS, iNOS) is transcriptionally induced by cytokines in macrophages, neutrophils, mast cells, smooth-muscle cells, and fibroblasts. The aim of this study was to detect and localize three NOS isoforms expression in nasal polyp tissues, and compare these findings with inferior nasal turbinate tissues. Materials and Methods: The authors examined the expression and localization of three NOS isoforms in nasal mucosal specimens from patients undergone elective nasal turbinectomy (n=10) and nasal polypectomy (n=23). The mRNA expressions of three NOS isoforms were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern hybridization. The protein expression of three NOS isoforms were examined by immunohistochemistry. Statistics were analysed using Wilcoxon rank sum test. Results: Semi-quantitative RT-PCR Southern analysis of RNA obtained from 23 surgical specimens of nasal polyps demonstrated that the mRNA expressions of iNOS and eNOS were significantly increased in nasal polyps compared with inferior turbinates. The nNOS mRNA was similarly expressed in nasal polyps and inferior turbinates. The immunohistochemical studies revealed that the immunoreactivity to iNOS protein was mainly localized to epithelium, whereas eNOS protein to vascular endothelium, and nNOS protein to inflammatory cell, epithelium and vascular endothelium in all specimens reviewed. The high level of expression of three NOS isoforms in the nasal polyps were demonstrated in this study. Conclusion: The authors suggest that iNOS mRNA, eNOS mRNA and their product, nitric oxide may play an important role in the formation and growth of nasal polyps. (J Clinical Otolaryngol 2002;13:85-92)

Streptozotocin-induced diabetes and the neurochemistry of vagal afferent neurons

To assess whether diabetes alters the content and/or expression of neuroactive agents and protooncogenes in afferent neurons of the vagus nerve, the nodose ganglia of streptozotocin (STZ)-induced diabetic rats were studied at 8, 16, and 24 weeks after induction of diabetes. Neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), the immediate early gene c-Jun, vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP) content and expression were measured in nodose ganglia of control, diabetic, and diabetic+insulin-treated rats using immunocytochemistry and reverse trancription-polymerase chain reaction (RT-PCR). The numbers of nNOS-immunoreactive (ir) neurons were increased in the nodose ganglion of diabetic compared to control rats at the 8- and 16-week time points. However, no change was noted in the nNOS mRNA content of the diabetic nodose ganglion at either time point. Moreover, no alterations in the numbers of vagal efferent NOS-containing neurons (labeled with NADPH-diaphorase histochemistry) were noted in the dorsal motor nucleus of the vagus (DMV) or the nucleus ambiguus (NA) of control, diabetic, and diabetic+insulin-treated rats at any time point. Neither the numbers of TH-ir neurons nor the content of TH mRNA was altered in the diabetic rats at the 8- and 16-week time points. However, 24 weeks of diabetes resulted in a reduction in the numbers of TH-ir neurons in the diabetic nodose ganglia when compared to control, an effect not seen in diabetic rats receiving insulin. The number of nodose ganglion neurons labeled for the protooncogene, c-Jun, was small yet slightly increased in the diabetic nodose ganglia at the 8-week time point and was reversed with insulin treatment. The increase in c-Jun-ir neurons was not found at 16 or 24 weeks of diabetes. VIP-ir and CGRP-ir were unchanged at any of the time points. These data show that diabetes affects the content of some, but not all, neuroactive agents in the nodose ganglion and may reflect a modest level of diabetes-induced damage and/or alterations in axonal transport in the vagus nerve.

Nitric oxide synthase and nitric oxide production in in vivo-derived mast cells

Nitric oxide (NO) is a potent mediator synthesized by a variety of cells involved in inflammatory reactions. We investigated the expression of NO synthase (NOS) in rat peritoneal mast cells (PMC). Small amounts of eNOS mRNA were detected basally, whereas neither mRNA for iNOS nor nNOS was detected in unstimulated PMC. Following stimulation by antigen, interferon-gamma (IFN-gamma), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-gamma, or anti-CD8, nitrite production was increased significantly (8.4+/-0.6, 7.6+/-0.9, and 6.6+/-0.9 microM/2x10(5) cells/48 h NO2-, respectively; P<0.01), whereas unstimulated PMC released 2.1 +/- 0.3 microM/2 x 10(5) cells/48 h NO2-. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.

Sex difference in the expression and regulation of nitric oxide synthase gene in the rat preoptic area.

Neuronal nitric oxide synthase (nNOS) mRNA-positive cells were visualized by non-isotopic in situ hybridization histochemistry in the organum vasculosum of the lamina terminalis (OVLT) and the preoptic area (POA) in gonadectomized juvenile female and male rats. In the rostral POA (rPOA) at the level of the anteroventral periventricular nucleus, nNOS mRNA-positive cells were distributed in an inverted V-shaped area over the third ventricle and were in close proximity to cell bodies of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons. In the caudal POA (cPOA) at the level of the medial preoptic nucleus, no topological association existed between GnRH and nNOS. Throughout the rPOA, both the number and the area of nNOS mRNA positive cells were significantly larger in the gonadectomized females than in the gonadectomized males. Treatment with estradiol for 2 days, followed by progesterone in the next morning, which caused an increase in serum luteinizing hormone 6 h later, induced a significant reduction of the nNOS mRNA expression in the rPOA in the female but not in the male rat at the time of sacrifice. In the OVLT and the cPOA, ovarian steroids had no effect on nNOS mRNA expression of both sexes. The results indicate that nNOS mRNA expression in the rPOA is sexually dimorphic and regulated by ovarian steroids in a sex specific manner.

Nitric oxide synthase isoform expression in acute versus chronic anti-Thy 1 nephritis

Two inbred Lewis rat substrains (LEW/Moe, LEW/Maa) were identified responding differently to induction of anti-Thy 1 glomerulonephritis (aThy 1-GN). LEW/Moe rats show an acute mesangioproliferative glomerulonephritis with rapid healing of glomerular lesions within four weeks, while LEW/Maa rats develop severe glomerular injury followed by chronic glomerular sclerosis and persistent albuminuria. We investigated whether the glomerular expression pattern of nitric oxide synthase (NOS) isoforms could explain these substrain-related differences. Rats (N = 5 to 7 per group) were investigated in a time course experiment. Severity of aThy 1-GN was determined by albuminuria measurements, glomerular matrix score and microaneurysm formation. Glomerular gene expression of NOS isoforms was determined by semiquantitative RT-PCR. Inducible NOS (iNOS) activity was determined in cultured glomeruli and peritoneal macrophages. Neuronal NOS (nNOS) protein expression was detected by Western blotting and enzyme histochemistry. Plasma renin activity (PRA) was measured by RIA. Induction of iNOS expression and activity was found significantly increased and sustained in LEW/Maa vs. LEW/Moe rats associated with an increased number of infiltrating macrophages and with an increased capacity of iNOS-expression and iNOS-activation by isolated macrophages in LEW/Maa rats. Glomerular nNOS mRNA and nNOS protein expression were constitutively increased in LEW/Maa rats. Renal nNOS localization was restricted to the macula densa region in both substrains and associated with increased PRA in LEW/Maa rats. No difference in glomerular endothelial NOS-mRNA expression between the substrains was observed. Increased glomerular iNOS and nNOS expression were associated with chronic anti-Thy 1 glomerulonephritis in LEW/Maa rats and may contribute to glomerular damage by separate mechanisms.

Downregulation of neuronal nitric oxide synthase and interleukin-1beta mediates angiotensin II-dependent stimulation of sympathetic nerve activity.

There is substantial evidence that angiotensin II (Ang II) enhances sympathetic nervous system (SNS) activity. We recently observed that nitric oxide and interleukin-1beta (IL-1beta) exert a tonic inhibitory action on central SNS activity. Moreover, in 2 rat models of neurogenic hypertension, one caused by intrarenal injection of phenol and the other by 5/6 nephrectomy, we observed that losartan, an Ang II type 1 receptor blocker, inhibits SNS activity and increases the abundance of IL-1beta and the neuronal isoform of nitric oxide synthase (nNOS) in the posterior hypothalamic nuclei (PH), paraventricular nuclei (PVN), and locus ceruleus (LC). This raises the possibility that the stimulatory effects of Ang II on central SNS activity may be mediated by inhibition of nNOS and IL-1beta. To test this hypothesis, we studied the effect of an intracerebroventricular (ICV) infusion of Ang II on blood pressure (BP), norepinephrine (NE) secretion from the PH, renal SNS activity (RSNA), and abundance of IL-1beta and nNOS mRNA in the PH, PVN, and LC of normal Sprague-Dawley rats. Finally, we measured the concentration of nitrite/nitrate in the dialysate collected from the PH after Ang II or vehicle. ICV infusion of Ang II (100 ng/kg body wt dissolved in 10 microL of artificial cerebrospinal fluid) raised BP, RSNA, and NE secretion from the PH compared with control rats. Ang II reduced the abundance of IL-1beta and nNOS mRNA in the PH, PVN, and LC. Pretreatment with losartan (10 microg/kg body wt dissolved in 10 microL of aCSF) given ICV 20 minutes before Ang II abolished the effects of Ang II on BP, RSNA, and NE secretion from the PH and IL-1beta and nNOS mRNA. Ang II also decreased the secretion of NO from the PH. In conclusion, these studies suggest that Ang II inhibits the expression of IL-1beta and nNOS in the brain. Because locally produced NO exerts a tonic inhibitory action on SNS activity, the decrease in NO expression caused by Ang II results in greater SNS activity.

Site-specific gene expression of nNOS variants in distinct functional regions of rat gastrointestinal tract.

5' mRNA variants of neuronal nitric oxide synthase (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOSalpha) or alternative splicing encoding NH(2)-terminally truncated proteins (nNOSbeta/gamma) that lack the PDZ/GLGF domain for protein-protein interaction of nNOSalpha. We studied the expression of 5' nNOS mRNA forms and nNOS-interacting proteins (postsynaptic density protein-95; PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with COOH- and NH(2)-terminal nNOS antibodies. 5' nNOS mRNA variants showed a site-specific expression along the gastrointestinal tract with presence of all forms (nNOSalpha-a, -b, -c; nNOSbeta) in the muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of nNOSalpha-a and nNOSbeta mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOSalpha and nNOSbeta cDNAs revealed proteins of ~155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and NH(2)-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell-specific expression and distinct compartmentalization of nNOS proteins. Observed distribution of 5' nNOS mRNA variants and proteins argue for a complex control of nNOS expression by usage of separate promoters, cell- and site-specific splicing mechanisms, and translational initiation. These mechanisms could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOSalpha knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.

Role of nNOS in regulation of renal function in hypertensive Ren-2 transgenic rats

The present study was performed to evaluate the role of neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) during the developmental phase of hypertension in transgenic rats harboring the mouse Ren-2 renin gene (TGR). The first aim of the present study was to examine nNOS mRNA expression in the renal cortex and to assess the renal functional responses to intrarenal nNOS inhibition by S-methyl-L-thiocitrulline (L-SMTC) in heterozygous TGR and in age-matched transgene-negative Hannover Sprague-Dawley rats (HanSD). The second aim was to evaluate the role of the renal sympathetic nerves in mediating the renal functional responses to intrarenal nNOS inhibition. Thus, we also evaluated the effects of intrarenal L-SMTC administration in acutely denervated TGR and HanSD. Expression of nNOS mRNA in the renal cortex was significantly increased in TGR compared with HanSD. Intrarenal administration of L-SMTC decreased the glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion and increased renal vascular resistance (RVR) in HanSD. In contrast, intrarenal inhibition of nNOS by L-SMTC did not alter GFR, RPF or RVR and elicited a marked increase in sodium excretion in TGR. This effect of intrarenal L-SMTC was not observed in acutely denervated TGR. These results suggest that during the developmental phase of hypertension TGR exhibit an impaired renal vascular responsiveness to nNOS derived NO or an impaired ability to release NO by nNOS despite enhanced expression of nNOS mRNA in the renal cortex. In addition, the data indicate that nNOS-derived NO increases tubular sodium reabsorption in TGR and that the renal nerves play an important modulatory role in this process.

Lack of a Role of Neuronal Nitric Oxide Synthase in the Regulation of the Renal Function in Rats Fed a Low-Sodium Diet

Background/Aims: It has been shown that nitric oxide (NO) generated from neuronal NO synthase (nNOS) counteracts angiotensin II mediated vasoconstriction in the pre- and in the postglomerular microcirculation. Previous studies have demonstrated that the nNOS expression in the macula densa of the renal cortex is enhanced by dietary salt restriction. In view of the well-known fact that dietary salt restriction also leads to an activation of the renin-angiotensin system, the present study was performed to assess the role of nNOS-derived NO in the regulation of the renal function in rats maintained on control (C) and low-salt (LS) diets. Methods: Groups of rats were fed either the C or the LS diet. On day 13 after adaptation to the appropriate diet, renal clearance studies were performed to determine the effects of acute nNOS inhibition either by S-methyl-L-thiocitrulline (L-SMTC) or by N^ω-propyl-L-arginine (L-NPA) on renal hemodynamics and sodium excretory function. In separate groups of rats maintained on either the C or the LS diet, the mRNA levels of nNOS and of renin in the renal cortex were examined using the semiquantitative reverse-transcriptase polymerase chain reaction. Results: Intrarenal infusion of vehicle (0.9% saline; 4 µl/min) did not change glomerular filtration rate (GFR), renal plasma flow (RPF), or sodium excretion in either C diet or LS diet rats. Acute intrarenal infusion of L-SMTC (0.3 mg/h) and L-NPA (0.01 mg/h) decreased GFR (–14 ± 5 vs. –13 ± 3%), RPF (–19 ± 6 vs. –17 ± 5%), and sodium excretion (–17 ± 5 vs. –16 ± 4%) in C diet rats as compared with control values (p < 0.05). In contrast, in LS rats, intrarenal administration of either L-SMTC or L-NPA did not cause significant changes in GFR, RPF, and sodium excretion. Furthermore, the mRNA expression for nNOS in the renal cortex was moderately increased in LS rats as compared with C rats (densitometric ratios of nNOS mRNA/GAPDH mRNA 0.31 ± 0.01 vs. 0.22 ± 0.04, p < 0.05), in parallel with the renin expression (renin mRNA/GAPDH mRNA ratios 1.4 ± 0.2 vs. 1.0 ± 0.1, p < 0.05). Conclusions: These results indicate that in normotensive rats kept on a normal salt intake nNOS-derived NO modulates both afferent and efferent arteriolar tones. In contrast, rats on an LS diet exhibit an impaired renal vascular responsiveness to nNOS-derived NO or an impaired ability to release NO by nNOS despite enhanced expression of nNOS mRNA in the renal cortex. In addition, the lack of effect of acute nNOS inhibition on renal function suggests that NO derived by nNOS does not participate in counteracting the vasoconstrictor influences of elevated circulating and/or intrarenal angiotensin II levels on pre- and postglomerular microcirculation in rats on an LS diet.

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.

Reduced functional expression and molecular synthesis of inducible nitric oxide synthase in rostral ventrolateral medulla of spontaneously hypertensive rats.

We demonstrated recently that the prevalence of neuronal (nNOS) over inducible (iNOS) nitric oxide synthase activity at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, and the associated dominance of sympathoexcitation over sympathoinhibition underlie the maintenance of sympathetic vasomotor outflow by the endogenous NO. Here, we evaluated the hypothesis that a significant downregulation of iNOS at the RVLM may play a crucial role in the genesis of augmented sympathetic vasomotor tone during hypertension. Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats anesthetized with propofol were used. Compared with SHR, the hypotension, bradycardia, or depression in sympathetic vasomotor tone induced by bilateral microinjection of lipopolysaccharide (5 or 10 ng) into the RVLM of WKY rats exhibited significantly shorter-onset latency, appreciably steeper slope, and a greater incidence of mortality. All these effects of lipopolysaccharide (10 ng) were significantly blunted by coadministration of the selective iNOS inhibitor S-methylisothiourea (250 pmol). Reverse transcription-polymerase chain reaction and Western blot analyses further revealed significantly lower iNOS mRNA and protein levels at the ventrolateral medulla in SHR under basal conditions or on activation by lipopolysaccharide (10 ng). Conversely, nNOS mRNA and protein levels remained constant in the RVLM and were comparable in both strains of rats. We conclude that a significant downregulation in both functional expression and molecular synthesis of iNOS at the RVLM may underlie the augmented sympathetic vasomotor tone during hypertension.

Major role for neuronal NO synthase in curtailing choroidal blood flow autoregulation in newborn pig.

We examined whether nitric oxide (NO) generated from neuronal NO synthase (nNOS) contributes to the reduced ability of the newborn to autoregulate retinal blood flow (RBF) and choroidal blood flow (ChBF) during acute rises in perfusion pressure. In newborn pigs (1-2 days old), RBF (measured by microsphere) is autoregulated over a narrow range of perfusion pressure, whereas ChBF is not autoregulated. N(G)-nitro-L-arginine methyl ester (L-NAME) or specific nNOS inhibitors 7-nitroindazole, 3-bromo-7-nitroindazole, and 1-(2-trifluoromethyl-phenyl)imidazole as well as ganglionic blocker hexamethonium, unveiled a ChBF autoregulation as observed in juvenile (4- to 6-wk old) animals, whereas autoregulation of RBF in the newborn was only enhanced by L-NAME. All NOS inhibitors and hexamethonium prevented the hypertension-induced increase in NO mediator cGMP in the choroid. nNOS mRNA expression and activity were three- to fourfold higher in the choroid of newborn pigs than in tissues of juvenile pigs. It is concluded that increased production of NO from nNOS curtails ChBF autoregulation in the newborn and suggests a role for the autonomic nervous system in this important hemodynamic function, whereas, for RBF autoregulation, endothelial NOS seems to exert a more important contribution in limiting autoregulation.

Dose-related alteration in nitric oxide synthase mRNA expression induced by amphetamine and the full D1 dopamine receptor agonist SKF-82958 in mouse striatum

Neuronal nitric oxide synthase (nNOS) is normally expressed in one population of intrinsic interneurons of the striatum. Production of nitric oxide in the nNOS-containing neurons is sensitive to dopamine stimulation. Using quantitative in situ hybridization, the present study investigated the alteration in basal nNOS mRNA expression in striatal nNOS-containing neurons of mice treated with the psychostimulant amphetamine or a full D1 dopamine receptor agonist, SKF-82958. A single systemic injection of amphetamine induced a dose-related change in striatal nNOS mRNA expression. Whereas amphetamine at 4 mg/kg decreased basal levels of nNOS mRNA in both the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum, the drug at a higher dose (12 mg/kg) increased nNOS expression in the two regions. Similarly, an acute systemic injection of SKF-82958 decreased and increased nNOS mRNA levels in the dorsal and ventral striatum at 2 and 4 mg/kg, respectively. These data indicate that constitutive nNOS expression in nitric oxide-producing neurons of the mouse striatum is regulated by dopaminergic transmission. Altered nNOS expression may result in changes in nitric oxide synthesis and thus contribute to biological actions of dopamine stimulants.

Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metabolite.

Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 micromol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways.

The Ras-ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells.

We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)-treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we conclude that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction.

Differential nNOS gene expression in salt-sensitive and salt-resistant Dahl rats.

Several indications exist to suggest that an impaired production of nitric oxide might have a role in the development of salt-sensitive hypertension. To examine whether the gene expression of the nitric oxide synthases (NOS) is altered in the salt-sensitive Dahl rat compared with that in the salt-resistant Dahl rat. The abundance of NOS mRNA was measured by RNase protection assay in different organs of salt-resistant and salt-sensitive Dahl rats. In addition, the zonal expression of NOS genes in the kidney under salt load and salt restriction was determined. The abundance of endothelial NOS mRNA was similar between the salt-resistant and salt-sensitive Dahl rat strains in all organs. Inducible NOS mRNA was not detectable by RNase protection assay in any organ. Neuronal NOS (nNOS) mRNA expression, however, was about 50% lower in brain and kidney of salt-sensitive Dahl rats than in salt-resistant Dahl rats. Within the kidney, nNOS mRNA levels were significantly decreased in salt-sensitive Dahl rats compared with those in salt-resistant Dahl rats, in cortex, outer and inner medulla (50, 40 and 30%, respectively) under all dietary conditions. A comparison of renal nNOS gene expression in Dahl rats with that in salt-insensitive Sprague- Dawley rats revealed that the abundance of renal nNOS was similar in salt-sensitive Dahl and Sprague-Dawley rats, but was increased in salt-resistant Dahl rats relative to that in Sprague-Dawley rats. These data suggest that nNOS gene expression is increased in salt-resistant Dahl rats compared with that in salt-sensitive Dahl rats. This increased nNOS expression of the salt-resistant Dahl strain might play a part in compensating for a defect of renal salt excretion in the Dahl strains.