Impact of the estrus cycle and reduction in estrogen levels with aromatase inhibition, on renal function and nitric oxide activity in female rats

Estradiol increases mRNA and/or protein expression of the nitric oxide synthase (NOS) isoforms in a variety of tissues including kidney. In this study we determined the relationship between cyclical variations in estradiol levels and renal function and total NO production in the virgin female rat. In addition, we used an aromatase inhibitor (Anastrozole), to inhibit synthesis of estradiol from testosterone. Estradiol levels were higher in proestrus vs. diestrus, and were markedly suppressed by 7 days treatment with aromatase inhibitor. There was no difference in total NO production (from urinary and plasma nitrate + nitrite = NO_X) between proestrus and diestrus but aromatase inhibition resulted in increases in total NO production. The renal cortical NOS activity and protein abundance also increased in aromatase-inhibited female rats. There were no differences in blood pressure (BP) in any group but the renal vascular resistance (RVR) was low in proestrus, increased in diestrus and did not change further after aromatase inhibition. In summary, the cyclical changes in renal function correlate with estradiol but not NO levels. Pharmacologic castration with aromatase inhibition leads to a marked increase in total and renal NOS. This contrasts to earlier work where surgical castration causes decreased NOS.     

Physical conditioning modulates rat cardiac vascular endothelial growth factor gene expression in nitric oxide-deficient hypertension

Many individuals with cardiac diseases undergo periodic physical conditioning with or without medication to improve cardiovascular health. Therefore, this study investigated the interaction of physical training and chronic nitric oxide synthase (NOS) inhibitor (nitro-L-arginine methyl ester, L-NAME) treatment on blood pressure (BP), cardiac vascular endothelial factor (VEGF) gene expression, and nitric oxide (NO) systems in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10mg/kg, s.c. for 8 weeks), and (4) ET+L-NAME. BP was monitored with tail-cuff method. The animals were sacrificed 24h after last treatments and hearts were isolated and analyzed. Physical conditioning significantly increased respiratory exchange ratio, cardiac NO levels, NOS activity, endothelial eNOS, and inducible iNOS protein expression as well as VEGF gene expression. Training also caused depletion of cardiac malondialdehyde (MDA) levels indicating the beneficial effects of the training. Chronic L-NAME administration resulted in a depletion of cardiac NO level, NOS activity, and eNOS, nNOS, and iNOS protein expressions, as well as VEGF gene expression (2-fold increase in VEGF mRNA). Chronic L-NAME administration also enhanced cardiac MDA levels indicating cardiac oxidative injury. These biochemical changes were accompanied by increases in BP after L-NAME administration. Interaction of training and NOS inhibitor treatment resulted in normalization of BP and up-regulation of cardiac VEGF gene expression. The data suggest that physical conditioning attenuated the oxidative injury caused by chronic NOS inhibition by up-regulating the cardiac VEGF and NO levels and lowering the BP in rats.     

Chronic nitric oxide exposure alters the balance between endothelium-derived relaxing factors released from rat renal arteries: prevention by treatment with NOX-100, a NO scavenger

We investigated whether nitric oxide (NO) exposure alters the balance between NO and endothelium-derived hyperpolarizing factor (EDHF) released from rat renal arteries. To produce states of acutely or chronically excessive NO, lipopolysaccharide (LPS) was administered intraperitoneally to rats in a single dose of 4 mg/kg (LPS-single group) or in stepwise doses of 0.5, 1.0 and 2.0 mg/kg every other day (LPS-repeated group). On the day after LPS treatment, the protein levels of inducible NO synthase (iNOS) and endothelial NOS (eNOS) were measured, and the relaxation responses were determined in the renal arteries. The protein levels of iNOS markedly increased in both LPS-treated groups, while those of eNOS significantly increased in the LPS-repeated group compared with those in the respective control groups. In both LPS-treated groups, the relaxations in response to acetylcholine (ACh) and sodium nitroprusside remained unchanged. The ACh-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester, a NOS inhibitor, or by 1H-[1, 2, 4-] oxadiazole [4, 3-a] quinoxalin-1-one, a soluble guanylyl cyclase inhibitor, i.e. EDHF-mediated relaxations were significantly impaired in the LPS-repeated group but not in the LPS-single group, indicating increase in NO-mediated relaxation in the LPS-repeated group. These changes in the protein levels and EDHF-mediated relaxations induced by ACh observed in the LPS-repeated group were restored by treatment with NOX-100, a NO scavenger. These results suggest that persistent but not acute excessive NO exposure in rats impairs EDHF-mediated relaxation in renal arteries, leading to a compensatory upregulation of the eNOS/NO pathway.     

早期经验对大鼠脑区一氧化氮合酶活性的影响

目的　探讨NO与早期饲养环境所引起脑效应的关系。方法　将断乳大鼠在丰富环境 (EC)和单调环境 (IC)中饲养 30d。环境暴露后通过NADPH -黄递酶组化方法对海马齿状回 (DEN)和大脑皮层NOS活性进行定量测定以及对大鼠进行Morris水迷宫作业训练。结果　EC大鼠与IC大鼠相比 ,海马齿状回 (DEN)和大脑皮层NOS活性明显下降 ,迷宫测试表明EC大鼠的空间认知显著优于IC大鼠。在环境暴露期间隔日注射一氧化氮合酶 (NOS)抑制物L -NAME(50mg/kg) ,未引起EC或IC大鼠认知行为的明显改变 ,但导致DEN和大脑皮层NOS活性的不同改变。结论　NO可能与早期经验脑效应有关。     

Human eNOS gene delivery attenuates cold-induced elevation of blood pressure in rats

We previously showed that chronic cold exposure inhibits endothelial nitric oxide synthase (eNOS) expression and decreases nitric oxide (NO) production. The aim of the present study was to evaluate the possible role of the NO system in the development of cold-induced hypertension (CIH) by testing the hypothesis that adenoviral delivery of human eNOS gene increases NO production and attenuates CIH in rats. The effect of in vivo delivery of adenovirus carrying human eNOS full-length cDNA (rAdv.heNOS) on CIH was tested using four groups of Sprague-Dawley rats (6 rats/group). Blood pressure (BP) did not differ among the four groups during the control period at room temperature (24 degrees C). Two groups of rats received intravenous injection of rAdv.heNOS (1 x 10(9) plaque-forming units/rat), and the other two groups received the same dose of rAdv.LacZ to serve as controls. After gene delivery, one rAdv.heNOS-treated group and one rAdv.LacZ-treated group were exposed to cold (6 degrees C) while the remaining groups were kept at 24 degrees C. We found that the BP of the rAdv.LacZ group increased significantly within 1 wk of exposure to cold and reached a peak level at week 5 (152.2 +/- 6.4 mmHg). In contrast, BP (118.7 +/- 8.4 mmHg) of the cold-exposed rAdv.heNOS group did not increase until 5 wk after exposure to cold. The rAdv.heNOS increased plasma and urine levels of NO significantly in cold-exposed rats, which indicates that eNOS gene transfer increased NO production. Notably, rAdv.heNOS decreased plasma levels of norepinephrine and plasma renin activity in cold-exposed rats, which suggests that eNOS gene transfer may decrease the activities of the sympathetic nervous system and the renin-angiotensin system. Immunohistochemical analysis showed that the transferred human eNOS was expressed in both endothelium and adventitia of mesenteric arteries. We conclude that 1) eNOS gene transfer attenuates CIH by increasing NO production and inhibiting the sympathetic nervous system and the renin-angiotensin system; and 2) the NO system appears to mediate this nongenetic, nonpharmacological, nonsurgical model of hypertension.     

Decreased Nitric Oxide Production in the Rat Brain after Chronic Arsenic Exposure

Chronic arsenic exposure is associated with nervous system damage, vascular disease, hepatic and renal damage as well as different types of cancer. Alterations of nitric oxide (NO) in the periphery have been detected after arsenic exposure, and we explored here NO production in the brain. Female Wistar rats were exposed to arsenite in drinking water (4–5 mg/kg/day) from gestation, lactation and until 4 months of age. NOS activity, NO metabolites content, reactive oxygen species production (ROS) and lipid peroxidation (LPx) were determined in vitro in the striatum, and NO production was estimated in vivo measuring citrulline by microdialysis. Exposed animals showed a significantly lower response to NMDA receptor stimulation, reduction of NOS activity and decreased levels of nitrites and nitrates in striatum. These markers of NO function were accompanied by significantly higher levels of LPx and ROS production. These results provide evidence of NO dysfunction in the rat brain associated with arsenic exposure.     

Alterations of NO synthase isoforms in brain and kidney of rats with genetic and salt hypertension

Both brain and peripheral nitric oxide (NO) play a role in the control of blood pressure and circulatory homeostasis. Central NO production seems to counteract angiotensin II-induced enhancement of sympathetic tone. The aim of our study was to evaluate NO synthase (NOS) activity and protein expression of its three isoforms--neuronal (nNOS), endothelial NOS (eNOS) and inducible (iNOS)--in two brain regions involved in blood pressure control (diencephalon and brainstem) as well as in the kidney of young adult rats with either genetic (12-week-old SHR) or salt-induced hypertension (8-week-old Dahl rats). We have demonstrated reduced nNOS and iNOS expression in brainstem of both hypertensive models. In SHR this abnormality was accompanied by attenuated NOS activity and was corrected by chronic captopril treatment which prevented the development of genetic hypertension. In salt hypertensive Dahl rats nNOS and iNOS expression was also decreased in the diencephalon where neural structures important for salt hypertension development are located. As far as peripheral NOS activity and expression is concerned, renal eNOS expression was considerably reduced in both genetic and salt-induced hypertension. In conclusions, we disclosed similar changes of NO system in the brainstem (but not in the diencephalon) of rats with genetic and salt-induced hypertension. Decreased nNOS expression was associated with increased blood pressure due to enhanced sympathetic tone.     

Vascular and renal effects of dopamine during extracellular volume expansion: Role of nitric oxide pathway

OBJECTIVE: The aim of the study was to determine the possible role of NO-system activation in vascular and renal effects of the dopaminergic system and the probable interaction between both systems during acute volume expansion in rats. DESIGN AND METHODS: Expanded (10% bw) and non-expanded anaesthetized male Wistar rats were treated with haloperidol, a DA receptor antagonist (3 mg/kg bw, ip). Mean arterial pressure, diuresis, natriuresis, renal plasma flow, glomerular filtration rate, nitrites and nitrates excretion (NOx) were determined. NADPH diaphorase activity was measured using a histochemistry technique in kidney, aorta and renal arteries. NOS activity in kidney and aorta from expanded and non-expanded animals was determined with L-[U14C]-arginine substrate, in basal conditions and after DA (1 microM) administration. RESULTS: The hypotensive effect of L-arg and hypertension induced by L-NAME were not modified by haloperidol. This blocker reverted the increase in diuresis, natriuresis and RPF induced by L-arg in both groups. Dopaminergic blockade induced a decrease in NOx excretion and in NADPH-diaphorase activity in glomeruli, proximal tubule and medullar collecting duct and in endothelium and vascular smooth muscle of renal arteries. DA induced an increase in NOS activity in renal medulla and cortex in both groups, but no changes in the aorta were observed. CONCLUSIONS: Our results suggest that renal DA would be associated with the renal response induced by NO during extracellular volume expansion. NO-system activation would be one of the mechanisms involved in renal DA activity during saline load, but NO appears not to be involved in DA vascular effects.     

Prenatal stress induces alterations in cerebellar nitric oxide that are correlated with deficits in spatial memory in rat’s offspring

Prenatal stress (PS) has been linked to abnormal cognitive, behavioral and psychosocial outcomes in both animals and humans. Since PS has been shown to induce a cerebellar cytoarchitectural disarrangement and cerebellar abnormalities that have been linked to an impairment of behavioral functions, the aim of the present work was to investigate whether the exposure to PS in a period in which the cerebellum is still immature can induce behavioral deficits in the adult and whether this alterations are correlated with changes in nitric oxide (NO) and cellular oxidative mechanisms in offspring’s cerebellum. Our results show impairments in spatial memory and territory discrimination in PS adult rats. PS offspring also displayed alterations in cerebellar nitric oxide synthase (NOS) expression and activity. Moreover, a correlation between spatial memory deficits and the increase in NOS activity was found. The results found here may point to a role of cerebellar NO in the behavioral alterations induced by stress during early development stages.     

血立停胶囊对早孕大鼠RU486药流后NO、NOS表达的影响

目的:研究血立停胶囊对早孕大鼠RU486药物流产后的子宫平滑肌一氧化氮(NO)及一氧化氮合酶(NOS)水平变化的影响。方法:选择妊娠Wistar大鼠,随机分为5组,即对照组,米非司酮组,大剂量血立停组,小剂量血立停组,催产素组。于妊娠第7天,开始相应处理,妊娠第14天分别监测早孕大鼠RU486药物流产后的子宫平滑肌一氧化氮(NO)及一氧化氮合酶(NOS)水平后处死。结果:大剂量血立停可明显降低大鼠子宫肌组织匀浆中NO、NOS含量,与对照组比较差异有显著性(P<0.05)。结论:血立停胶囊可降低子宫肌组织匀浆中NO、NOS水平,从而起到对药物流产后阴道出血的治疗作用。     

The Role of Nitric Oxide in the Neuroprotection of Limb Ischemic Preconditioning in Rats

Brief limb ischemia was reported to protect neurons against injury induced by subsequent cerebral ischemia-reperfusion, and this phenomenon is known as limb ischemic preconditioning (LIP). To explore the role of nitric oxide (NO) in neuroprotection of LIP in rats, we observed changes in the content of nitric oxide (NO) and activity of NO synthase (NOS) in the serum and CA1 hippocampus of rats after transient limb ischemic preconditioning (LIP), and the influence of N^G-nitro-l-arginine methylester (l-NAME), a NOS inhibitor, on the neuroprotection of LIP against cerebral ischemia-reperfusion injury. Results showed that NO content and NOS activity in serum increased significantly after LIP compared with the sham group. The increase showed a double peak pattern, in which the first one appeared at time 0 (immediate time point) and the second one appeared at 48 h after the LIP (P < 0.01). The NO content and NOS activity in the CA1 hippocampus in LIP group showed similar change pattern with the changes in the serum, except for the first peak of up-regulation of NO content and NOS activity appeared at 6 h after LIP. Pretreatment with l-NAME before LIP blocked the neuroprotection of LIP against subsequent cerebral ischemic insult. The blocking effect of l-NAME was abolished with pretreatment of l-Arg. These findings indicated that NO may be associated with the tolerance of pyramidal cells in the CA1 hippocampus to ischemia induced by LIP in rats.     

Increased Nitric Oxide Synthase Activities and l-[3H]Arginine Uptake in Brain Following Portacaval Anastomosis

Abstract: Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal-systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval-shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS), in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham-operated controls. As l -arginine transport is a prerequisite for nitric oxide production, we also studied l -[³H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval-shunted and sham-operated rats. l -[³H]Arginine uptake was significantly increased (by ∼50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting.     

Mechanisms underlying sex differences in progressive renal disease

Men with nondiabetic renal disease exhibit a faster rate of decline in renal function compared with women. To investigate this sex difference in renal disease progression, our research group has been studying the renal wrap (RW) model of hypertension in rats. Compared with RW female rats, the glomerulosclerosis index, mean glomerular volume, and proteinuria were greater (3.1-, 1.7-, and 1.8-fold, respectively) in RW males under conditions in which no differences in the degree of hypertension were detected, suggesting that sex differences may exist in the mechanisms underlying renal injury, independent of blood pressure. Gonadal steroids contribute to these sex differences, because orchidectomy attenuated and ovariectomy exacerbated the severity of renal injury, whereas dihydrotestosterone and 17β-estradiol (E₂) replacement prevented these respective effects. Chronic renal disease is associated with impairment in nitric oxide (NO) signaling and elevated levels of superoxide. Sex differences were observed in RW-induced changes in renal nitric oxide synthesis (NOS) protein abundance. Whereas RW had no effect on NOS in the female kidney, endothelial NOS was elevated and neuronal NOS was decreased in the male kidney, suggesting that renal injury may cause dysfunction in NO metabolism in the male. Sex differences in superoxide signaling were also observed. Renal cortical nicotinamide adenine dinucleotide phosphate oxidase activity was 1.3-fold higher in RW males than in RW females, and ovariectomy increased enzyme activity 1.4-fold, whereas E₂ replacement prevented this effect. These changes in enzyme activity were mirrored by changes in protein abundance of the p22^phox regulatory subunit. Our findings suggest that E₂ may protect the female kidney from hypertension-associated renal disease by attenuating injury-induced superoxide production.     

Role of cardiovascular nitric oxide system in C-type natriuretic peptide effects

The aims were to evaluate the role of cardiovascular nitric oxide (NO)-system in C-type natriuretic peptide (CNP) actions and to investigate receptor types and signaling pathways involved in this interaction. Wistar rats were infused with saline or CNP. Mean arterial pressure (MAP) and nitrites and nitrates (NOx) excretion were determined. NO synthase (NOS) activity and NOS expression (Western blot) were analyzed in atria, ventricle and aorta. CNP decreased MAP and increased NOx excretion. CNP estimulated NOS activity, inducing no changes on cardiac and vascular endothelial NOS expression. NOS activity induced by CNP was abolished by suramin and calmidazoliumand but it is not modified by anantin. CNP would interact with NPR-C receptor coupled via G proteins leading to the activation Ca(2+)-calmodulin dependent endothelial NOS, increasing NO production which would induce the reduction in cardiac myocyte contractility and ANP synthesis and secretion in right atria and the relaxation of vascular smooth muscle.     

Mitochondrial function and nitric oxide metabolism are modified by enalapril treatment in rat kidney

The renal and cardiac benefits of renin-angiotensin system (RAS) inhibition in hypertension exceed those attributable to blood pressure reduction, and seem to involve mitochondrial function changes. To investigate whether mitochondrial changes associated with RAS inhibition are related to changes in nitric oxide (NO) metabolism, four groups of male Wistar rats were treated during 2 wk with a RAS inhibitor, enalapril (10 mg x kg(-1) x day(-1); Enal), or a NO synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME) (1 mg x kg(-1) x day(-1)), or both (Enal+L-NAME), or were untreated (control). Blood pressure and body weight were lower in Enal than in control. Electron transfer through complexes I to III and cytochrome oxidase activity were significantly lower, and uncoupling protein-2 content was significantly higher in kidney mitochondria isolated from Enal than in those from control. All of these changes were prevented by L-NAME cotreatment and were accompanied by a higher production/bioavailability of kidney NO. L-NAME abolished mitochondrial NOS activity but failed to inhibit extra-mitochondrial kidney NOS, underscoring the relevance of mitochondrial NO in those effects of enalapril that were suppressed by L-NAME cotreatment. In Enal, kidney mitochondria H(2)O(2) production rate and MnSOD activity were significantly lower than in control, and these effects were not prevented by L-NAME cotreatment. These findings may clarify the role of NO in the interactions between RAS and mitochondrial metabolism and can help to unravel the mechanisms involved in renal protection by RAS inhibitors.     

口服L-瓜氨酸对大鼠勃起功能的影响

给予雄性SD大鼠口服不同剂量组的L-瓜氨酸8周后,检测电刺激大鼠阴茎海绵体神经海绵体内压(ICP)的变化;比色法检测血清和组织中的一氧化氮(NO)含量、一氧化氮合酶(NOS)活性及血清中超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)含量等。结果高剂量(4.5 g.kg-1)L-瓜氨酸组ICP、NO含量、NOS活性显著增高;低、高剂量组SOD活性、GSH含量变化差异均无显著性。     

一氧化氮的功能及其作用机制(Ⅰ)——性质与功能

一氧化氮(nitric oxide,NO)是第一个被发现的参与细胞信号转导的气体信号分子。NO参与的生命活动非常广泛,在神经、免疫、呼吸等系统中发挥着重要作用。很久以来,一氧化氮合酶(nitric oxide synthase,NOS)被认为是人体内合成NO的主要途径,其活性受到严格的调控。直到最近,人们才发现亚硝酸盐(nitrite,NO2-)也可以参与体内NO的合成。本综述总结NO的相关性质与功能,并简介亚硝酸盐的研究进展。     

Interaction between endogenous nitric oxide and carbon monoxide in the pathogenesis of recurrent febrile seizures

The aim of the study was to investigate the interaction between nitric oxygenase (NOS)/nitric oxide (NO) and heme oxygenase (HO)/carbon monoxide (CO) system in the pathogenesis of recurrent febrile seizures (FS). On a rat model of recurrent FS, the ultrastructure of hippocampal neurons was observed under electron microscopy, and expression of neuronal NOS (nNOS) in hippocampus and NO formation in plasma were examined after treatment with ZnPP-IX, an HO-1 inhibitor. In the ultrastructure of hippocampal neurons, the expression of HO-1 in hippocampus and CO formation in plasma were examined after treatment with L-NAME, a NOS inhibitor. We found that hippocampal neurons were injured after recurrent FS. The gene and protein expression of nNOS and HO-1 increased markedly in hippocampus in FS rats, while CO formation in plasma increased markedly and the concentration of NO in plasma increased slightly. ZnPP-IX could worsen the neuronal damage of recurrent FS rats. However, it further increased the expression of nNOS and endogenous production of NO obviously. L-NAME alleviated the neuronal damage of recurrent FS rats, but decreased the expression of HO-1 and CO formation. The results of this study suggested that endogenous NOS/NO and HO/CO systems might interact with each other and therefore play an important regulating role in recurrent FS brain damage.     

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.