Blockade of nitric oxide formation impairs adrenergic-induced ACTH and corticosterone secretion.

It has been suggested that adrenergic agents might modulate the L-arginine-NO pathway. Sympathomimetic agonists enhance the basal release of NO, and noradrenaline increases the synthesis of nitric oxide synthase (NOS) in the medial basal hypothalamus in vitro. In the present study possible involvement of NO in central stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by adrenergic agents was investigated in conscious rats. The nitric oxide synthase blocker N(omega)-nitro-L-arginine methyl ester (L-NAME 2 and 10 microg) was administered intracerebroventricularly (i.c.v.) 15 min before the adrenergic agonist given by the same route; 1 h later the rats were decapitated. Plasma levels of ACTH and corticosterone were measured. L-NAME significantly diminished the ACTH and corticosterone response to phenylephrine (30 microg), an alpha1-adrenergic receptor agonist. These hormone responses to clonidine (10 microg), an alpha2-receptor agonist, were dose-dependently suppressed or totally abolished by L-NAME. A significant rise in the ACTH and corticosterone secretion induced by isoprenaline (10 microg), a beta-adrenergic receptor agonist, was only moderately diminished by pretreatment with L-NAME. These results indicate that NOS is considerably involved in central stimulation of the HPA axis by alpha1- and alpha2-adrenergic receptor agonists, and that NO mediates the stimulatory action of these agonists on ACTH and corticosterone secretion. The stimulation induced by beta-adrenergic receptors is only moderately affected by endogenous NO.     

一氧化氮合酶抑制剂L-NAME对大鼠脑缺血耐受诱导的影响

采用大鼠四血管闭塞全脑缺血耐受模型和脑组织切片形态学方法,观察应用一氧化氮合酶(NOS)抑制剂L—NAME对大鼠海马CAl区脑缺血耐受(BIT)诱导的影响,在整体水平探讨一氧化氮(NO)在BIT诱导中的作用。54只Wistar大鼠凝闭双侧推动脉后分为6组：(1)假手术组(n＝6);分离双侧颈总动脉,但不阻断脑血流;(2)损伤性缺血组(n＝6)：全脑缺血10min;(3)预缺血 损伤性缺血组(n＝6)：脑缺血预处理(CIP)3min,再灌注72h后行全脑缺血10min;(4)L—NAME组;分别于CIP前1h和后1、12及36h腹腔注射L—NAME(5mg／kg),每个时间点6只动物,其余步骤同预缺血 损伤性缺血组;(5)L—NAME L—精氨酸组(n＝6)：于CIP前1h腹腔注射L—NAME(5mg／kg)和L—精氨酸(300mg／kg),其它步骤同L—NAME组;(6)L—NAME 损伤性缺血组(n＝6)：于腹腔注射L—NAME(5mg／kg)72h后行全脑缺血10min。实验结果表明,(1)单纯10min全脑缺血可使海马CAl区组织学分级增加(表明损伤加重),神经元密度降低(P＜0．01);(2)预缺血 损伤性缺血组的海马CAl区组织学分级、神经元密度与假手术组相比,无显著性差别(P＞0．05);(3)L—NAME组中,应用L—NAME后海马CAl区组织学分级增加,神经元密度降低,与预缺血 损伤性缺血组相比有显著性差异(P＜0．05),表明L—NAME可阻断CIP对神经元的保护作用;(4)L—NAME L—精氨酸组与L—NAME组相比,海马CAl区组织损伤明显减轻(P＜0．05),但与预缺血 损伤性缺血组相比仍有显著性差别(P＜0．05),提示L-精氨酸可部分逆转L—NAME的作用;(5)L—NAME 损伤性缺血组的组织学表现与损伤性缺血组相同(P＞0．05)。这些结果表明,在整体情况下N0参与BIT的诱导。与CIP前1h及后1、12h给予L—NAME组相比,CIP后36h给予L—NAME对CIP保护作用的阻断效应明显减弱,提示N0在CIP后较早阶段即开始参与BIT的诱导。     

Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.     

The influence of nitric oxide synthase inhibitor L-NAME on bones of male rats with streptozotocin-induced diabetes

The pathophysiological processes underlying the development of diabetic osteopenia has not hitherto been elucidated. Induction of streptozotocin diabetes leads in our experiments to decrease of bone density, ash, mineral content and to thinner cortical width compared to control male rats. In order to investigate the pathogenetic role of bone resorption by osteoclasts in streptozotocin-induced diabetes, we determined the circulating levels of tartrate-resistant acid phosphatase (TRAP), a biochemical marker for bone resorption. Plasma TRAP values in diabetic rats did not differ from their corresponding controls. Streptozotocin diabetes by itself did not have any effect on the weight of seminal vesicles which are highly testosterone-dependent. Low doses of nitric oxide cause bone resorption, but higher doses of NO inhibit bone resorbing activity. We examined the effect of L-NAME (inhibitor of nitric oxide production) after six weeks of administration to diabetic rats. There was no further significant loss of bone mineral density, ash and mineral content or tibia weight in diabetic rats treated with L-NAME. L-NAME itself did not decrease bone metabolism. In our study no evidence of an increased bone resorption was found. Our results have indicated that a predominance of bone resorption over bone formation is not involved in the pathogenesis of diabetes-associated osteopenia. Inhibition of NO neither increased osteoclastic activity (TRAP) nor induced osteopenia in L-NAME-treated rats. This suggests a possibility that NO is not involved in the pathogenesis of diabetic osteopenia.     

Influence of nitric oxide synthase inhibition on vasopressin and corticosterone secretion during water deprivation in rats

Nitric oxide (NO) is a short-lived radical that functions as a neurotransmitter in the central nervous system and plays a physiological role in the regulation of hypothalamic–pituitary–adrenal axis and vasopressinergic axis. In the present study, we aimed to investigate the interaction between the generation of NO and vasopressin (AVP) and corticosterone release after 3 days of water deprivation in rats. Animals were previously treated with intraperitoneal (i.p.) saline or l-nitro-arginine methyl ester (L-NAME) injection. l-NAME is a nonspecific inhibitor of nitric oxide synthases. In control rats given i.p. saline or l-NAME, hypothalamic, pituitary, and plasma AVP levels and plasma corticosterone did not change from baseline levels (p > 0.05). Three days of water deprivation increased significantly the corticosterone levels in plasma (p < 0.01) and AVP levels in hypothalamus and plasma (p < 0.01), but not in pituitary, which showed a significant decrease. These variations were concomitant with the elevation of nitrates/nitrates in plasma. l-NAME injection abolished significantly (p < 0.01) the elevation of plasma corticosterone and hypothalamic AVP levels induced by water deprivation. These findings showed that in water-deprived rats, nitric oxide synthase inhibition by l-NAME inhibits corticosterone and vasopressin release, suggesting a potent stimulatory role of NO.     

Role of prostaglandins and nitric oxide in the lipopolysaccharide-induced ACTH and corticosterone response.

This study was designed to determine the role of endogenous prostaglandins (PG) and nitric oxide (NO) in the lipopolysaccharide (LPS)-induced ACTH and corticosterone secretion in conscious rats. LPS (0.5 and 1 mg/kg) given i.p. stimulated the hypothalamic-pituitary-adrenocortical (HPA) activity measured 2 h later. A non-selective cyclooxygenase inhibitor indomethacin (10 mg/kg i.p.), piroxicam (2 mg/kg i.p.), a more potent antagonist of constitutive cyclooxygenase (COX-1) and compound NS-398 (2 mg/kg i.p.), a selective inhibitor of inducible cyclooxygenase (COX-2) given 30 min before LPS (1 mg/kg i.p.) significantly diminished both the LPS-induced ACTH and corticosterone secretion. COX-2 blocker was the most potent inhibitor of ACTH secretion (72.3%). Nomega-nitro-L-arginine methyl ester (L-NAME 2 and 10 mg/kg i.p.), a non-selective nitric oxide synthase (NOS) blocker given 15 min before LPS did not substantially alter plasma ACTH and corticosterone levels 2 h later. Aminoguanidine (AG 100 mg/kg i.p.), a selective inducible nitric oxide synthase (iNOS) inhibitor, considerably enhanced ACTH and corticosterone secretion induced by a lower dose (0.5 mg/kg) of LPS and did not significantly alter this secretion after a larger dose (1 mg/kg) of LPS. L-NAME did not markedly affect the indomethacin-induced inhibition of ACTH and corticosterone response. By contrast, aminoguanidine abolished the indomethacin-induced reduction of ACTH and corticosterone secretion after LPS. These results indicate an opposite action of PG generated by cyclooxygenase and NO synthesized by iNOS in the LPS-induced HPA-response.     

Impaired duodenal bicarbonate secretion in diabetic rats. Salutary effect of nitric oxide synthase inhibitor

We previously reported the impaired HCO₃⁻ secretion and the increased mucosal susceptibility to acid in the duodenum of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the salutary effect of the NO synthase inhibitor L-NAME (N^G-nitro-L-arginine methyl ester) on these changes and compared it with those of insulin. Animals were injected streptozotocin (STZ: 70 mg/kg, ip) and used after 1, 3–4, and 5–6 weeks of diabetes with blood glucose levels of > 300 mg/dL. Under urethane anesthesia the HCO₃⁻ secretion was measured in the proximal duodenal loop using a pH-stat method and by adding 10 mM HCl. L-NAME (20 mg/kg × 2) or insulin (4 units/rat) was administered sc for 4–5 weeks, starting 1 week after STZ treatment. The duodenal HCO₃⁻ secretory responses to various stimuli such as mucosal acidification (10 mM HCl for 10 min), 16,16-dimethyl prostaglandin E₂ (dmPGE₂: 10 μg/kg, iv), and vagal stimulation (0.5 mA, 2 ms, 3 Hz) were significantly decreased in STZ-treated rats, depending on the duration of diabetes. Repeated administration of L-NAME, starting from 1 week after STZ treatment, significantly reduced blood glucose levels toward normal values and restored the HCO₃⁻ responses to various stimuli in STZ rats, the effects being similar to those observed after supplementation of insulin. Diabetic rats developed duodenal lesions after perfusion of the duodenum with 150 mM HCl for 4 h, but this ulcerogenic response was significantly inhibited by the repeated treatment with L-NAME as well as insulin. We conclude that L-NAME is effective in ameliorating hyperglycemic conditions in STZ-diabetic rats, similar to insulin, and restores the impaired HCO₃⁻ secretion and the increased mucosal susceptibility to acid in diabetic rat duodenums.     

Effect of nitric oxide synthase inhibitor on diaphragmatic function after resistive loading

We studied the effect of a nitric oxide synthase inhibitor, Nomega-Nitro-L-arginine-methyl-ester (L-NAME), on in vitro diphragmatic function both at rest (control) or after inspiratory resistive loading (IRL). Sprague-Dawley rats were anesthetized, instrumented, and then the following experimental groups: (1) controls; (2) L-NAME (100 mg/kg/body weight intravenously alone); (3) IRL alone; and (4) L-NAME + IRL. The IRL protocol consisted of applying a variable resistor to the inspiratory limb of a two-way valve at 70% of maximal airway pressure until apnea. After the experiment, the animals were sacrificed and diaphragmatic strips were obtained for activity of constitutive nitric oxide synthase (cNOS) and measurements of in vitro contractile properties: tetanic (Po) and twitch tensions (Pt). cNOS activity was significantly decreased in the L-NAME and L-NAME + IRL groups (P < or = 0.05) as compared with control and IRL groups. L-NAME alone did not affect Po or Pt. However, in both IRL groups, with and without was a significant decrease in Po and Pt. This reduction was comparable in both groups. In summary, our data showed that L-NAME resulted in a significant decrease cNOS activity, but in vitro contractility was impaired.     

The non specificity of specific nitric oxide synthase inhibitors.

L-NAME (Nw-Nitro-L-arginine methylester) and L-NMMA (NG- Monomethyl-L-arginine, monoacetate) are used widely as nitric oxide (NO) synthase inhibitors. Because of their functional groups (alcohols, amines and carboxylates), it appeared that they could interact with iron in a variety of systems. Using three in vitro models we observed these two compounds had inhibitory effects on cytochrome C reduction by ferrous iron, by ferrous iron accelerated by an unsaturated fatty acid or by epinephrine. This suggests that L-NAME and L-NMMA could have effects in iron containing systems found intracellularly apart from their inhibition of (NO) synthesis.     

Cocaine induced secretion of ACTH, beta-endorphin, and corticosterone

The effect of intraperitoneal administration of cocaine on the concentrations of hypothalamic corticotropin releasing factor like-immunoreactivity (CRF-LI), plasma ACTH, beta-endorphin, and corticosterone was investigated. Groups of rats were injected with 20 mg/kg cocaine HCI or 0.9% NaCl and then killed 0, 10, 20, 30 or 60 minutes later. Hypothalamic CRF-LI, plasma ACTH, beta-endorphin, and corticosterone concentrations were determined by radioimmunoassay. A significant increase in plasma ACTH, beta-endorphin, and corticosterone concentrations was observed after cocaine administration. In contrast, cocaine had no significant effect on hypothalamic CRF-LI concentrations. Intravenous administration of 0.5 and 2.0 mg/kg cocaine to rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine, and pentobarbital elicited a significant increase in plasma corticosterone concentrations. These results demonstrate that cocaine induces the release of ACTH, beta-endorphin, and corticosterone and suggest that this response is mediated at the pituitary level.     

Acute pancreatitis,expression of inducible nitric oxide synthase and defective insulin secretion

A high level of nitric oxide (NO) produced by inducible NO synthase (iNOS) is involved in pancreatic beta-cell dysfunction and apoptosis. In the present study, we examined whether iNOS is also expressed in beta cells after induction of acute pancreatitis (AP) in the rat. Pancreatic islets taken from AP animals and incubated for 60 min in the presence of 20.0 mmol/l glucose showed a decreased insulin secretory response to glucose. The basal insulin release at 1.0 mmol/l glucose was also moderately reduced. Experiments on the dynamics of insulin secretion from perfused pancreas revealed an impairment of both first and second phase of glucose-stimulated insulin release after the induction of AP. Confocal microscopy demonstrated that most of the beta cells in pancreas of rat with AP expressed strong immunoreactivity for iNOS. This was further confirmed by biochemical and Western blot analysis that showed a marked increase in iNOS protein expression and enzyme activity concomitant with a modest reduction in the cNOS protein and activity. Although the mechanisms underlying the defective insulin secretory response of beta cells seen during the early stage of AP are complex, the present finding suggests that the expression of iNOS and a marked iNOS-derived NO production in the beta cells may play at least a contributory role in the impairment of beta-cell function.This study was supported by the Swedish Medical Research Council (14X-4286), the Swedish Diabetes Association, and the Crafoord, Albert Påhlsson and Magnus Bergvall Foundations     

Hyperactivity caused by a nitric oxide synthase inhibitor is countered by ultra-wideband pulses.

Potential action of ultra-wideband (UWB) electromagnetic field pulses on effects of N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on nociception and locomotor activity was investigated in CF-1 mice. Animals were injected IP with saline or 50 mg/kg L-NAME and exposed for 30 min to no pulses (sham exposure) or UWB pulses with electric field parameters of 102+/-1 kV/m peak amplitude, 0.90+/-0.05 ns duration, and 160+/-5 ps rise time (mean+/-S.D.) at 600/s. Animals were tested for thermal nociceptive responses on a 50 degrees C surface and for spontaneous locomotor activity for 5 min. L-NAME by itself increased mean first-response (paw lift, shake, or lick; jump) and back-paw-lick response latencies and mean locomotor activity. Exposure to UWB pulses reduced the L-NAME-induced increase in back-paw-lick latency by 22%, but this change was not statistically significant. The L-NAME-induced hyperactivity was not present after UWB exposure. Reduction and cancellation of effects of L-NAME suggest activation of opposing mechanism(s) by the UWB pulses, possibly including increase of nitric oxide production by NOS. The action, or actions, of UWB pulses appears to be more effective on locomotor activity than on thermal nociception in CF-1 mice.     

Abrogation of bleomycin-induced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice.

The effects of aminoguanidine (AG), a specific inhibitor of inducible nitric oxide synthase, on the bleomycin (BL)-induced lung fibrosis was evaluated in mice. The animals were placed into five groups: saline (SA)-instilled drinking water (SA+H(2)O), saline-instilled drinking water containing 0.5%AG (SA+0.5%AG), BL-instilled drinking water (BL+H(2)O), BL-instilled drinking water containing 0.2%AG (BL+0.2%AG), and BL-instilled drinking water containing 0.5%AG (BL+0.5%AG). The mice had free access to H(2)O or H(2)O containing AG and lab chow ad lib 2 days prior to intratracheal (IT) instillation of BL (0.07U/mouse/100 microL) or an equivalent volume of sterile isotonic saline. The mice in the SA+0.5%AG group consumed the greatest amount of AG without any ill effects than the mice in any other group. There were no differences in any of the measured biochemical determinants between the SA+H(2)O and SA+0.5%AG control groups. The IT instillation of BL in the BL+H(2)O group caused significant increases in the lipid peroxidation, hydroxyproline content, and prolyl hydroxylase activity of lungs and influx of inflammatory cells in the broncheoalveolar lavage fluid (BALF) as compared to both control groups. The intake of aminoguanidine by mice in the BL+0.5%AG group caused significant reductions in the BL-induced increases in all measured biochemical indices of lung fibrosis without any effects on the influx of inflammatory cells in the BALF. In fact, AG in both BL-treated groups additionally increased the total cell counts in the BALF from mice in the BL+0.2%AG and BL+0.5%AG groups as compared to the BL+H(2)O group. Histopathological evaluation of the lungs revealed that the mice in the BL+0.5%AG group had markedly fewer fibrotic lesions than mice in the BL+H(2)O group. These results demonstrate that aminoguanidine minimizes the BL-induced lung fibrosis at both the biochemical and the morphological level and support our earlier hypothesis that the production of nitric oxide plays a significant role in the pathogenesis lung fibrosis caused by BL.     

A potent inhibitor of inducible nitric oxide synthase, ONO-1714, a cyclic amidine derivative

(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K(i) of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID(50) value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent.     

Circadian influences on feeding-induced changes in ACTH and corticosterone secretion in rats.

Food ingestion has a variable influence on pituitary-adrenal hormone secretion depending on the species studied and/or the experimental design. In this study, changes in plasma concentrations of ACTH and corticosterone following 30 min of food ingestion were compared in both 24 h fasted and ad libitum fed rats tested during either the early light cycle or the early dark cycle. In the dark, food ingestion caused significant decreases in both ACTH (to 80% of control) and corticosterone (to 32% of control) in both fasted and ad libitum fed rats. In contrast, in the light, food ingestion by the fasted animal resulted in a doubling of corticosterone concentrations. Such a response was not seen in ad libitum fed animals; however, these animals ate very little during the test. There were no significant changes in ACTH during the light phase. These data indicate that time of day has a significant impact on the responses of the pituitary-adrenal system to food ingestion. This circadian effect may be due to the influence of the endogenous levels of ACTH/corticosterone existing at the time of food ingestion.     

Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor,NG-nitro-L-arginine methyl ester

The hypothesis that an arginine-nitric oxide (NO) synthase-NO system mediates the morphine abstinence syndrome was tested in adult male rats implanted subcutaneosly for 3 days with one morphine (75 mg) pellet followed by naloxone-precipitated withdrawal (0.5 mg/kg). Injection with a NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (NAME, 100 mg/kg subcutaneous), shortly before naloxone-induced withdrawal significantly inhibited abstinence signs by 25–80%. Continuous infusion of NAME via subcutaneous osmotic pumps during the development of morphine physical dependence and during naloxone-precipitated withdrawal also inhibited morphine abstinence signs. In addition, treatment with isosorbide dinitrate, a NO donor, induced a quasi morphine-abstinence syndrome (QMAS) that was significantly suppressed by implantation of a morphine pellet 3 days before isosorbide dinitrate treatment. These results indicate that NO mediates part of the expression of the morphine abstinence syndrome.     

NOSIP, a novel modulator of endothelial nitric oxide synthase activity.

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。