Day- and nighttime injection of a nitric oxide synthase inhibitor elicits opposite sleep responses in rats

Previous studies suggest that nitric oxide (NO) may play a role in sleep regulation, particularly in the homeostatic process. The present studies were undertaken to compare the sleep effects of injecting a NO synthase (NOS) inhibitor when homeostatic sleep pressure is naturally highest (light onset) or when it is at its nadir (dark onset) in rats. Sleep, electroencephalogram delta-wave activity during nonrapid eye movement sleep (NREMS), also known as slow-wave activity (SWA), and brain temperature responses to three doses of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME ; 5, 50, and 100 mg/kg) injected intraperitoneally at light or dark onset were examined in rats (n = 6 to 8). The effects of 5 mg/kg L-NAME were determined in both normal and vagotomized (VX) rats. Light onset administration of 50 mg/kg L-NAME decreased NREMS amounts and suppressed SWA and increased rapid eye movement sleep (REMS) amounts. At dark onset, L-NAME injection also dose dependently suppressed SWA; however, unlike light onset injections, both NREMS and REMS amounts were increased after all three doses. Sleep responses to 5 mg/kg L-NAME were not different in control and VX rats, suggesting that the sleep effects of L-NAME are not mediated through the activation of sensory vagal mechanisms. The present findings suggest that timing of the injection is a major determinant of the sleep responses observed after systemic L-NAME injection in rats.     

Phosphatidylinositol 3-kinase inhibitor suppresses inducible nitric oxide synthase expression in bronchiole epithelial cells in asthmatic rats

Inducible nitric oxide synthase (iNOS) is known to produce nitric oxide (NO), which is a main contributor to asthmatic airway inflammation. Recent studies have shown that phosphatidylinositol 3-kinase (PI3K) is ubiquitously expressed in airway epithelial cells and its inhibition could relieve airway inflammation and hyperresponsiveness. This study aimed to explore the interaction of PI3K and NO signaling in allergic asthma. We investigated the effects of PI3K inhibitor wortmannin on iNOS expression in bronchiole epithelial cells and NO, IL-4 and IFN-γ levels in lung tissues of asthmatic rat model, which was prepared by 10% OVA solution sensitization and 1% OVA aerosol challenge. Our results showed that the ratio of eosinophils to total cells in BALF, PI3K activity, NO and IL-4 levels in lung tissues was increased after OVA sensitization and challenge, but then was attenuated by the administration of wortmannin. In contrast, IFN-γ level in lung tissues was decreased after OVA sensitization and challenge and increased after the administration of wortmannin. The expression of iNOS protein in bronchiole epithelial cells, iNOS mRNA level and iNOS activity in lung tissues was markedly upregulated after OVA sensitization and challenge, but the upregulation was significantly antagonized by wortmannin. Taken together, these data provide evidence that PI3K functions upstream to modulate iNOS/NO signaling, which then promotes the development of airway inflammation in asthmatic animal model. PI3K inhibitor wortmannin could lead to reduced iNOS expression and NO production, therefore inhibiting airway inflammatory responses.     

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 zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase

In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Marta?sek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.     

中枢和外周给一氧化氮合酶抑制剂对AVP降温作用的影响

目的 :探讨一氧化氮 (NO)在精氨酸加压素 (AVP)降温中的作用。方法 :用数字体温计测量大鼠的结肠温度 ,每次间隔 30min ,观察了中枢和外周给一氧化氮合酶抑制剂L 硝基精氨酸甲酯 (L NAME)对AVP引起降温作用的影响。结果 :①分别静脉注射AVP(4μg·kg- 1 )和L NAME(30mg·kg- 1 )后均可引起明显的降温效应 ,而静脉注射AVP后立即给L NAME对AVP的降温效应无明显影响。②侧脑室注射L NAME(1mg·kg- 1 )可引起体温明显升高 ,但当联合给AVP和L NAME时 ,侧脑室注射L NAME可明显阻断静脉注射AVP引起的降温效应。结论 :中枢内源性NO在AVP引起的降温过程中起重要的作用。另外 ,侧脑室注射一氧化氮合酶抑制剂L NAME有明显的升温效应 ,提示中枢性NO对正常体温的下调有紧张性调节作用     

Bacterial lipopolysaccharide signaling through Toll-like receptor 4 suppresses asthma-like responses via nitric oxide synthase 2 activity

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-gamma. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12(-/-) or IFN-gamma(-/-) mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.     

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.     

The role of inducible nitric oxide synthase inhibitor on the arteriolar hyporesponsiveness in hemorrhagic-shocked rats

Hemorrhagic shock (HS) has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, NO has been implicated to cause hyporesponsiveness of blood vessel in vitro towards vasoconstrictors in refractory (decompensated) HS. In our in vivo model, we examined the effects of aminoguanidine (AG), a known iNOS inhibitor, with angiotensin II (ANG II), a vasoconstrictor, following hemorrhagic shock decompensatory phase (HSDP) on percentage survival, vascular responsiveness, mean arterial blood pressure (MABP), heart rate and mean nitrate/nitrite levels in anaesthetized rats. HSDP (3 h) was achieved via constant pressure method (40-45 mmHg). MABP and heart rate was measured via the left carotid artery. Plasma collected from HSDP rats was used to measure nitrate/nitrite levels. Vascular hyporeactivity to ANG II was carried out using HSDP aortic strips, precontracted with KCl and noradrenaline. Sham-operated rats served as controls. HSDP rats decreased percentage survival, vascular contractility to ANG II and noradrenaline, MABP, heart rate while showing increased levels of nitrate/nitrite. Infusion of AG with ANG II, increased percentage survival and had reversed these cardiovascular effects of HSDP rats. This study indicates that excessive NO formation from iNOS activity induces vascular hyporeactivity and decompensation in HSDP. This might suggest that selective NOS inhibitor, AG, when coupled with ANG II, show reduction in NO's effect in HSDP.     

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.     

Intracerebroventricular injection of neuronal and inducible nitric oxide synthase inhibitors attenuates fever due to LPS in rats.

Nitric oxide (NO) has been shown to be an important mediator of febrile response to lipopolisaccharide (LPS). To clarify the role of different isoforms of NO synthase (NOS) in febrile response to immune challenge, effects of selective iNOS and nNOS inhibitors on fever to LPS were examined in freely moving biotelemetered rats. Vinyl-L-NIO (N(5) - (1-Imino-3-butenyl) - ornithine (vL-NIO), a neuronal nitric oxide synthase (nNOS) inhibitor, and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor, were injected intracerebroventricularly at a dose of 10 microg/rat just before intraperitoneal injection of LPS at a dose of 50 microg/kg. Both inhibitors injected at a selected doses had no effect on normal day-time body temperature (T(b)) and normal night-time T(b). vinyl-L-NIO and aminoguanidine injected intracerebroventricularly at a dose of 10 microg/animal suppressed the LPS-induced fever in rats. The fever index calculated for rats pretreated with v-LNIO or with aminoguanidine and injected with LPS was reduced by 43% and 72%, respectively, compared to that calculated for water-pretreated and LPS-injected rats. Whereas vL-NIO partly attenuated both phases of febrile rise in T(b), administration of aminoguanidine into the brain completely prevented fever induced by LPS. These data indicate that activation of iNOS inside the brain is not only responsible for triggering but also for maintaining of LPS-induced fever in rats. It is, therefore, reasonable to hypothesize that, activation of iNOS inside the brain is more important in fever development than activation of nNOS.     

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。     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

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.     

脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用

在乌拉坦麻醉的成年ＳＤ大鼠上,用玻璃微电极细胞外记录的方法,观察了脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用。结果发现：脑室内注射一氧化氮供体硝普钠对下丘脑室旁核中的加压素神经元产生剂量依赖性抑制作用;脑室内注射一氧化氮合酶抑制剂对加压素神经元也产生抑制作用。上述两种药物对催产素神经元均无作用。这些结果提示：一氧化氮可能在调节加压素和催产素神经元活动中起着不同的作用。     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.     

Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats

We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. l-Nitroarginine methyl ester (l-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.     

Systemic nitric oxide synthase inhibition improves coronary flow reserve to adenosine in patients with significant stenoses

We studied the impact of systemic infusion of the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on coronary flow reserve (CFR) in patients with coronary artery disease (CAD). We have previously demonstrated that CFR to adenosine was significantly increased after systemic infusion of L-NMMA in normal volunteers but not in recently transplanted denervated hearts. At baseline, myocardial blood flow (MBF; ml x min(-1) x g(-1)) was measured at rest and during intravenous administration of adenosine (140 microg x kg(-1) x min(-1)) in 10 controls (47 +/- 5 yr) and 10 CAD patients (58 +/- 8 yr; P < 0.01 vs. controls) using positron emission tomography and (15)O-labeled water. Both MBF measurements were repeated during intravenous infusion of 10 mg/kg L-NMMA. CFR was calculated as the ratio of MBF during adenosine to MBF at rest. CFR was significantly higher in healthy volunteers than in CAD patients and increased significantly after L-NMMA in controls (4.00 +/- 1.10 to 6.15 +/- 1.35; P < 0.0001) and in patients, both in territories subtended by stenotic coronary arteries (>70% luminal diameter; 2.06 +/- 1.13 to 3.21 +/- 1.07; P < 0.01) and in remote segments (3.20 +/- 1.23 to 3.92 +/- 1.62; P < 0.05). In conclusion, CFR can be significantly increased in CAD by a systemic infusion of L-NMMA. Similarly to our previous findings in normal volunteers, this suggests that adenosine-induced hyperemia in CAD patients is constrained by a mechanism that can be relieved by systemic NOS inhibition with L-NMMA.     

Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.     

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.     

Ventricular hypertrophy and arterial hemodynamics following deprivation of nitric oxide in rats

In the present study, we elucidated the possible role of hemodynamic parameters and chemical factors in the development of ventricular hypertrophy (VH) following chronic nitric oxide (NO) deprivation with Nomega-nitro-L-arginine methyl ester (L-NAME). Impedance spectral analysis was used to obtain the arterial hemodynamics including the steady and pulsatile components. Body weight (BW), left ventricular (LV) weight (LVW), LVW/BW ratio, LV collagen volume fraction (LVCVF), cyclic GMP, and nitrite/nitrate were measured. The extent of VH was evaluated by the LW/BW, total number, numerical density, and size of cardiomyocytes. Sprague-Dawley rats were given L-NAME 10, 20, and 40 mg/kg/day from the age of 10 to 18 weeks. Control and age-matched rats were given vehicle for the same period. Treatment of L-NAME for 8 weeks caused a dose-dependent increase in tail cuff pressure and a reduction in BW with increases in LVW, LVW/BW, number, numerical density, and size of myocytes. There was elevation of aortic pressure with decreases in cardiac output, and arterial compliance. The total peripheral resistance, characteristic impedance and pulse wave reflection were increased. Histological finding revealed severe myocardial hypertrophy and fibrosis with fibroblast infiltration. The LVCVF was increased, while LV cGMP and nitrite/nitrate were reduced in a dose-dependent manner. The results suggest that chronic NOS blockade causes hypertension, impairment of large vessel properties, and VH. The development of VH may result partly from the decreases in cGMP and nitrite/nitrate in the ventricle. Correlation analysis indicates that the extent of VH is equally related to the steady and pulsatile hemodynamics.