Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 μg/ml) and tumor necrosis factor-α (TNF-α, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-α exerted a transient effect on the GPX activity. The treatment with SNAP (200 μM) or sodium nitroprusside (200 μM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-α, and aminoguanidine blocked the effect. In cells metabolically labeled with ⁷⁵Se, LPS + TNF-α accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Nitric oxide is not involved in the control of vasopressin release during acute forced swimming in rats

Summary. Neurons of the hypothalamo-neurohypophyseal system (HNS) are known to contain high amounts of neuronal nitric oxide (NO) synthase (nNOS). NO produced by those neurons is commonly supposed to be involved as modulator in the release of the two nonapeptides vasopressin (AVP) and oxytocin into the blood stream. Previous studies showed that forced swimming fails to increase the release of AVP into the blood stream while its secretion into the hypothalamus is triggered. We investigated here whether hypothalamically acting NO contributes to the control of the AVP release into blood under forced swimming conditions. Intracerebral microdialysis and in situ hybridization were employed to analyze the activity of the nitrergic system within the supraoptic nucleus (SON), the hypothalamic origin of the HNS. A 10-min forced swimming session failed to significantly alter the local NO release as indicated both by nitrite and, the main by-product of NO synthesis, citrulline levels in microdialysis samples collected from the SON. Microdialysis administration of NO directly into the SON increased the concentration of AVP in plasma samples collected during simultaneous forced swimming. In an additional experiment the effect of the defined stressor exposure on the concentration of mRNA coding for nNOS within the SON was investigated by in situ hybridization. Forced swimming increased the expression of nNOS mRNA at two and four hours after onset of the stressor compared to untreated controls. Taken together, our results imply that NO within the SON does not contribute to the regulation of the secretory activity of HNS neurons during acute forced swimming. Increased nNOS mRNA in the SON after forced swimming and the increase in AVP release in the presence of exogenous NO under forced swimming points to a possible role of NO in the regulation of the HNS under repeated stressor exposure.Current address: Departments of Behavioral Neuroscience and Neurology, Oregon Health & Science University, Portland, OR 97239, U.S.A.     

Psychosocial stress affects the involvement of prostaglandins and nitric oxide in the lipopolysaccharide-induced hypothalamic-pituitary-adrenal response.

The role of prostaglandins and nitric oxide (NO), generated after peripheral lipopolysaccharide (LPS) administration, in the adaptation of hypothalamic-pituitary-adrenal (HPA) axis under stressful circumstances remains to be elucidated. The aim of the present study was to assess the effect of chronic repetitive restraint or social crowding stress on the involvement of nitric oxide and prostaglandins in the LPS-induced pituitary-adrenocortical response. Male Wistar rats were restrained in metal tubes 2 x 10 min/day or crowded in cages for 7 days prior to treatment. All compounds were injected i.p., cyclooxygenase (COX) and nitric oxide synthase (NOS) inhibitors 15 min before LPS. Two hrs after injection LPS induced a significant increase in ACTH and corticosterone secretion. Repeated restraint impaired more potently than crowding stress the LPS-induced HPA-response. Indomethacin, a non-selective COX inhibitor, considerably reduced the LPS-induced HPA response in non-stressed rats and to a lesser extent diminished this response in repeatedly restrained or crowded rats. Neuronal NOS inhibitor, Nomega-nitro-L-arginine decreased the LPS-induced HPA response, more potently in control than crowded rats. Aminoguanidine, an iNOS inhibitor, diminished the LPS-elicited ACTH response in crowded rats. These results indicate that prostaglandins and NO generated by neuronal and inducible NOS are involved in the LPS-induced HPA axis response under basal conditions and during its adaptation to chronic social stress circumstances.     

L-Arginine uptake affects nitric oxide production and blood flow in the renal medulla

Experiments were performed to determine whether L-arginine transport regulates nitric oxide (NO) production and hemodynamics in the renal medulla. The effects of renal medullary interstitial infusion of cationic amino acids, which compete with L-arginine for cellular uptake, on NO levels and blood flow in the medulla were examined in anesthetized rats. NO concentration in the renal inner medulla, measured with a microdialysis-oxyhemoglobin trapping technique, was significantly decreased by 26-44% and renal medullary blood flow, measured by laser Doppler flowmetry, was significantly reduced by 20-24% during the acute renal medullary interstitial infusion of L-ornithine, L-lysine, and L-homoarginine (1 micromol.kg(-1).min(-1) each; n = 6-8/group). In contrast, intramedullary infusion of L-arginine increased NO concentration and medullary blood flow. Flow cytometry experiments with 4-amino-5-methylamino-2',7'-difluorescein diacetate, a fluorophore reactive to intracellular NO, demonstrated that L-ornithine, L-lysine, and L-homoarginine decreased NO by 54-57% of control, whereas L-arginine increased NO by 21% in freshly isolated inner medullary cells (1 mmol/l each, n > 1,000 cells/experiment). The mRNA for the cationic amino acid transporter-1 was predominantly expressed in the inner medulla, and cationic amino acid transporter-1 protein was localized by immunohistochemistry to the collecting ducts and vasa recta in the inner medulla. These results suggest that L-arginine transport by cationic amino acid transport mechanisms is important in the production of NO and maintenance of blood flow in the renal medulla.     

眶额叶区5-HT与Glu、NO在急性强迫游泳应激抑郁症模型中的关系

目的：探讨眶额叶区5-羟色胺（5-HT）与谷氨酸（Glu）、一氧化氮（N0）在急性强迫游泳应激抑郁症模型中的相互作用。方法：雄性SD大鼠随机分为对照组及各种药物注射组,强迫游泳制造大鼠应激性抑郁模型,眶额叶区微量注射各组药物,敞箱实验及游泳测试观察大鼠的抑郁样行为表现。结果：①与对照组比,注射Glu使大鼠强迫游泳不动时间显著增加;注射NMDA受体拮抗剂（MK-801）使大鼠强迫游泳不动时间减少;与Glu组比,MK-801预注射后Glu注射使大鼠强迫游泳不动时间减少;②与5-HT组比,MK-801预注射后5-HT注射使大鼠强迫游泳不动时间增加;③与对照组比,注射L-精氨酸（L-Ars）使大鼠强迫游泳不动时间显著增加;注射NOS抑制剂（L-NAME）（10μg/μl）使大鼠强迫游泳不动时间减少;L-NAME（20μg／μl）注射使大鼠强迫游泳不动时间增加;L-NAME（40μg/μl）注射使大鼠强迫游泳不动时间增加;④与L-NAME（10μg/μl）组比较,5-HT1A受体拮抗剂spipemne预注射后LNAME（10μg／μl）注射使大鼠强迫游泳不动时间增加。结论：眶额叶（OFC）区Glu含量的增加能够诱发抑郁,其作用可能主要是通过NMDA受体实现的,Glu经NMDA受体引发抑郁的同时还可能通过调节突触后膜上5-HT1A受体减弱5-HT的抗抑郁作用;OFC区NO可通过调节5-HT神经元进而参与抑郁的发生。     

Galanin affects vasopressin and oxytocin release from the hypothalamo-neurohypophysial system in haemorrahaged rats.

The effect of centrally administered galanin (Gal; 100 pM i.c.v.) on the hypothalamo-neurohypophysial storage as well as blood plasma level of vasopressin and oxytocin was estimated in haemorrhaged (1 ml per 100 g b.w.) male Wistar rats. Gal i.c.v. treatment did not alter vasopressin and oxytocin content both in the hypothalamus and neurohypophysis as well as their concentration in blood plasma of not haemorrhaged rats. Haemorrhage decreased the hypothalamic and neurohypophysial vasopressin and oxytocin storage but increased the neurohormones plasma level in animals injected with vehicle solution. During the haemorrhage, the increase in plasma vasopressin and oxytocin was inhibited in rats previously treated i.c.v. with galanin. The hypothalamic and neurohypophysial vasopressin as well as oxytocin content significantly increased in animals treated with galanin and subsequently haemorrhaged. These results suggest that galanin may have a regulatory role in the hypothalamo-neurohypophysial function especially under condition of hypovolemia.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Involvement of neuromedin S in the oxytocin release response to suckling stimulus

We recently identified neuromedin S (NMS) from the rat hypothalamus as an endogenous ligand for the FM-4/TGR-1 receptor distinct from neuromedin U. In the present study, we examined the role of NMS in the oxytocin release response to suckling stimulation by rat pups. Intracerebroventricular (icv) injection of NMS induced cFos expression in the paraventricular nucleus and supraoptic nucleus. Double immunohistochemical analysis revealed induction of cFos expression in a proportion of oxytocinergic neurons in both nuclei. In addition, icv injection of NMS stimulated oxytocin release dose-dependently in intact rats, and increased milk secretion in lactating rats. On the other hand, icv injection of anti-NMS antiserum into lactating rats significantly suppressed suckling-induced milk ejection. These results suggest that, in the rat, endogenous NMS plays an important role in the oxytocin release response to the suckling stimulus.     

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.     

Role of nitric oxide in the nicotine-induced pituitary-adrenocortical response.

Nitric oxide (NO) is a major signaling molecule and biological mediator of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the role of NO formed by endothelial (e), neuronal (n) and inducible (i) nitric oxide synthase (NOS) in the stimulatory effect of nicotine on the HPA axis in rats under basal conditions. Also possible interaction of NOS systems with endogenous prostaglandins (PG) in that stimulation was assessed. NOS and cyclooxygenase inhibitors were administered i.p. 15 min prior to nicotine (2, 5 mg/kg i.p.). Plasma ACTH and serum corticosterone levels were measured 1 h after nicotine injection. NOS blockers given alone did not markedly affect the resting ACTH and corticosterone levels. L-NAME (2-10 mg/kg), a broad spectrum NOS inhibitor considerably and dose dependently enhanced the nicotine-induced ACTH and corticosterone secretion. L-NNA (2 mg/kg) and 7-nitroindazole (7-NI 20 mg/kg), neuronal NOS inhibitors in vivo also significantly augmented the nicotine-induced ACTH and corticosterone levels. L-arginine greatly impaired the nicotine-induced hormone responses and reversed the L-NNA elicited enhancement of the nicotine-evoked ACTH and corticosterone response. In contrast to the constitutive eNOS and nNOS antagonists, an inducible NOS antagonist guanethidine (50-100 mg/kg i.p.) did not substantially affect the nicotine-elicited pituitary-adrenocortical responses. Indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase blocker abolished the L-NAME and L-NNA-induced enhancement of the nicotine-evoked ACTH and corticosterone response. These results indicate that NO is an inhibitory mediator in the HPA axis activity. Inhibition of its generation by eNOS and nNOS significantly enhances the nicotine-induced HPA response. Under basal conditions iNOS is not involved in the nicotine-induced ACTH and corticosterone secretion. Prostaglandins play an obligatory role in the response of HPA axis to systemic nicotine administration.     

The role of nitric oxide in the hypothalamic control of LHRH and oxytocin release, sexual behavior and aging of the LHRH and oxytocin neurons

Nitric oxide (NO) affects reproductive processes both at the level of the brain and reproductive tract and this review is focused on its role as an essential regulator of the hypothalamic control of reproduction. The data gathered indicate that glutamate stimulates noradrenergic neurons which subsequently activate NO-ergic cells via alpha1-adrenergic receptors. The released NO diffuses into luteinizing hormone-releasing hormone (LHRH) terminals where it triggers LHRH secretion by activation of guanylyl cyclase and cyclooxygenase. The NO released by estrogen-stimulated NO-ergic ventromedial neurons plays a crucial role in the regulation of sexual behavior. Furthermore, an increased expression of inducible nitric oxide synthase in the LHRH and oxytocin neurons underlies the destructive action of NO on the aging of the hypothalamic neuroendocrine pathways. Within the hypothalamo-hypophyseal system, NO exerts an inhibitory effect in the control of oxytocin secretion. This action seems to employ an indirect mechanism by which NO may modulate the release of GABA. This review provides an overview of the role of NO in hypothalamic control of LHRH and oxytocin release, aging of the LHRH and oxytocin neurons and sexual behavior.     

Gender-dependent response in blood pressure changes following the inhibition of nitric oxide synthase

Many studies employed L-NAME (N(G)-nitro-L-arginine methyl ester), an L-arginine antagonist and nitric oxide (NO) synthase (NOS) inhibitor, to produce hypertension experimentally in male animals. It is not known whether females respond similarly. We thus examined the effect of long-term oral administration of L-NAME on body weight (BW), blood pressure (BP), and heart rate (HR) of both female and male rats. We found that L-NAME induced significant increase in mean BP (MAP) in both genders, however, L-NAME-treated females (F*) exhibited a significantly higher elevation than males (M*) did. This difference persisted for 5 wks and then diminished. L-NAME was thus withdrawn and a rapid decrease of MAP was observed. MAP of F* decreased less and thus remained higher than M* for 5 wks. MAP of control rats (F and M) remained unchanged during the period. Systolic BP (SBP) altered in a similar pattern. We also found that HR decreased immediately after L-NAME administration and that HR of F* was significantly less reduced. These findings indicate that L-NAME induced a more pronounced response in females than males, consistent with the view that females are more dependent on NOS activity for their regulation of BP.     

Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.     

An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP

A novel microflow technique is used to demonstrate that a weakened oxidant defense system found in diabetic erythrocytes leads to decreased levels of deformation-induced release of adenosine triphosphate (ATP) from erythrocytes. Addition of an oxidant to rabbit erythrocytes resulted in a 63% decrease in deformation-induced ATP release before eventually recovering to a value that was statistically equivalent to the initial value. Inhibition of glucose-6-phosphate dehydrogenase prevents recovery from the oxidant attack. Finally, results indicated that the ATP release from the erythrocytes of type II diabetics (91 nM +/- 10 nM) was less than half of that measured from the erythrocytes of healthy controls (190 +/- 10 nM). These data suggest that the antioxidant status of erythrocytes is a critical determinant in the ability of these cells to release ATP, a known nitric oxide stimulus.     

Hemoglobin and red blood cells alter the response of expired nitric oxide to mechanical forces

Expired nitric oxide (NO(e)) varies with hemodynamic or ventilatory perturbations, possibly due to shear stress- or stretch-stimulated NO production. Since hemoglobin (Hb) binds NO, NO(e) changes may reflect changes in blood volume and flow. To determine the role of blood and mechanical forces, we measured NO(e) in anesthetized rabbits, as well as rabbit lungs perfused with buffer, red blood cells (RBCs) or Hb following changes in flow, venous pressure (P(v)), and positive end-expiratory pressure (PEEP). In buffer-perfused lungs decreases in flow and P(v) reduced NO(e), but NO(e) rose when RBCs and Hb were present. These findings are consistent with changes in vascular NO production, whose detection is obscured in blood-perfused lungs by the more dominant effect of Hb NO scavenging. PEEP decreased NO(e) in all perfused lungs but increased NO(e) in live rabbits. The NO(e) fall with PEEP in isolated lungs is consistent with flow redistribution from alveolar septal capillaries to extra-alveolar vessels and decreased surface area or a direct, stretch-mediated depression of lung epithelial NO production. In live rabbits, increased NO(e) may reflect blood flow reduction and decreased Hb NO scavenging and/or autonomic responses that increase NO production. We conclude that blood and systemic responses render it difficult to use NO(e) changes as an accurate measure of lung tissue NO production.     

Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide.

Rhodobacter sphaeroides 2.4.1, which is incapable of denitrification, has been found to carry nnrR, the nor operon, and nnrS, which are utilized for denitrification in R. sphaeroides 2.4.3. The gene encoding nitrite reductase was not found in 2.4.1. Expression of beta-galactosidase activity from a norB-lacZ fusion was activated when cells of 2.4.1 were incubated with NO-producing bacteria. This result indicates that the products of nnrR and the genes flanking it are utilized when 2.4.1 is growing in an environment where denitrification occurs.     

Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes.

Androgens are known to influence penile erection and nitric oxide synthase (NOS) activity in cavernosal tissue homogenates. The present study was an assessment of the effects of castration and androgen replacement on the in vivo release of nitric oxide (NO), and of the simultaneously recorded intracavernosal pressure (ICP) changes elicited by electrostimulation of the cavernosal nerves (SCN) in the anesthetized rat. The extracellular levels of NO in the corpora were monitored electrochemically using porphyrin microsensors. The content of NOS isoenzymes in corporal homogenates was determined by immunoblotting. The responses of castrated rats with or without testosterone (T) implants were compared to those of intact animals. Castration virtually abolished both the NO and the ICP responses to SCN. There was a concomitant significant decrease in the content of both the neuronal (nNOS) and the endothelial (eNOS) isoenzymes in the cavernosal tissue. All these effects of castration were prevented by T replacement. The NO response to SCN was positively correlated with the levels of nNOS and eNOS, especially when the values of the two isoforms were added (r = 0.71, P < 0.001). These data suggest that the facilitatory action of androgens on penile erection involves the up-regulation of both constitutive NOS isoenzymes in the corpora cavernosa.