Infrequent cellular coexistence of NADPH-diaphorase and calretinin in the neurosecretory nuclei and adjacent areas of the rat hypothalamus

Colocalization of the calcium-binding protein calretinin and NADPH-diaphorase activity at the cellular level was studied in the magnocellular secretory nuclei of the rat hypothalamus using sequential immunocytochemical and histochemical staining of the same sections. A low degree of colocalization of these markers was observed in certain cellular subpopulations within all the areas considered (supraoptic, paraventricular, circular and both fornicals nuclei and in the hypothalamic area located between the supraoptic and paraventricular nuclei). However, since in the paraventricular nucleus both markers were expressed by different neuronal populations, the coexistence was almost non-existent in some subdivisions of this nucleus. This rare coexistence strongly suggests that NADPH-diaphorase and calretinin are related to different functions shared by restricted hypothalamic neuronal populations.     

NADPH-diaphorase and cytosolic urea cycle enzymes in the rat accessory olfactory bulb

The nitric oxide cycle consists of nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase to form nitric oxide. We have examined the colocalization of nitric oxide synthase and the cytosolic urea cycle enzymes (argininosuccinate synthetase, argininosuccinate lyase and arginase) in the accessory olfactory bulb of the rat by using a double labeling procedure combining reduced-nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) reaction with fluorescent immunocytochemistry. Each glomerulus showed a different NADPH-d activity, and those with the strongest NADPH-d activities were assembled in the caudomedial part of the accessory olfactory bulb. Argininosuccinate synthetase-like immunoreactive glomeruli were distributed in the caudomedial part of the accessory olfactory bulb, and most of them were also strongly NADPH-d positive. The mitral or tufted cells were argininosuccinate synthetase-, argininosuccinate lyase- and arginase-like immunoreactive, but were not NADPH-d positive. The granule cells were NADPH-d positive or argininosuccinate lyase-like immunoreactive, but were not argininosuccinate synthetase- or arginase-like immunoreactive. Some granule cells were both NADPH-d positive and argininosuccinate lyase-like immunoreactive. The results indicate the heterogeneity of glomeruli of the accessory olfactory bulb with respect to the distribution of these enzymes. The granule cells have nitric oxide synthase and argininosuccinate lyase, and thus may efficiently produce nitric oxide.     

Melatonin and estradiol effects on food intake, body weight, and leptin in ovariectomized rats

OBJECTIVE: The study in ovariectomized (Ovx) rats, as a model of menopausal status, of the effects of melatonin (M) and/or estradiol (E), associated or not with food restriction, on body weight (BW) and serum leptin levels. METHODS: Female SD rats (200-250 g) were Ovx and treated with E, M, E+M or its diluents. Control sham-Ovx rats were treated with E-M diluents. After 7 weeks being fed ad libitum, the animals were exposed for 7 more weeks to a 30% food restriction. We measured: food intake, BW, nocturnal and diurnal urinary excretion of sulphatoxymelatonin (aMT6s), leptin in midday and midnight blood samples, glucose, total cholesterol, LDL, HDL and triglycerides. RESULTS: Day/night rhythm of aMT6s excretion was preserved in all cases. The increase of aMT6s excretion in M-treated animals basically affected the nocturnal period. In animals fed ad libitum, E fully prevented Ovx-induced increase of BW, leptin and cholesterol. Melatonin reduced food intake and partially prevented the increase of BW and cholesterol, without changing leptin levels. Under food restriction, M was the most effective treatment in reducing BW and cholesterol. Leptin levels were similar in M, E or E+M treated rats, and lower than in untreated Ovx rats. CONCLUSIONS: Our result gives a preliminary experimental basis for a post-menopausal co-treatment with estradiol and melatonin. It could combine the effectiveness of estradiol (not modified by melatonin) with the positive effects of melatonin (improvement of sleep quality, prevention of breast cancer, etc.). The possible beneficial effects of melatonin which could justify its use, need to be demonstrated in clinical trials.     

Melatonin inhibits amphetamine-induced nitric oxide synthase mRNA overexpression in microglial cell lines

Amphetamine (AMPH) derivatives are the most commonly abused drugs. Chronic or intermittent AMPH abuse may create temporary or permanent disturbances in the dopaminergic system of the brain that may predispose individuals to Parkinsonism. AMPH induces a massive release of dopamine from synaptic vesicles and then generates reactive oxygen species (ROS). Furthermore, nitric oxide (NO), produced in the central nervous system (CNS) mediated by the activation of microglia, appears to play a critical role in stress-induced brain damage. In the present study, we examined the involvement of NO in the neurotoxic effects of AMPH, to investigate the hypothesis that altered nitric oxide synthase (NOS) function was involved. AMPH at a concentration of 0.4-3.2mM has a cytotoxic effect on highly aggressively proliferating immortalized (HAPI) cells, a rat microglial cell line. The effect of AMPH on increasing inducible NOS (iNOS) mRNA in HAPI microglial cells is concentration-dependent. Pretreatment with either S-methylisothiourea (S-MT), a selective iNOS inhibitor, or melatonin, a major secretory product of pineal gland, counteracted the over expression of iNOS induced by AMPH in a concentration-dependent manner. The induction of iNOS by AMPH in microglial cells could be an important source of NO in CNS inflammatory disorders associated with the death of neurons and oligodendrocytes. Administration of exogenous melatonin will be beneficial, as it reduces iNOS mRNA expression, and may, therefore, be able to be used as a neuroprotective agent in toxicity induced by AMPH or other immunogens.     

Effect of exercise on blood pressure in rats with chronic NOS inhibition

Regular training lowers blood pressure in hypertensive humans and other animals. We investigated the response to 4 weeks of treadmill exercise training in hypertensive male Wistar rats receiving the nitric oxide synthase inhibitor N ^ω-nitro-L-arginine methyl ester (L-NAME). The rats were on either a short- (4 weeks) or long-term (10 weeks) L-NAME treatment protocol and were subjected to running exercise that started concomitantly in the short-term group and in the 6th week in the long-term group. Four weeks of exercise training induced a fall in mean arterial pressure in both the short- [mean (SEM) 137.6 (4.0) mmHg] and long-term hypertensive groups [161.4 (2.3) mmHg] compared to their sedentary hypertensive controls [160.4 (3.3) mmHg and 176.8 (8.9) mmHg, respectively]. Exercise also increased muscle nitric oxide synthase activity in both of the trained hypertensive groups. Muscle nitrite levels were higher in the exercising short-term hypertensive group compared to both the sedentary control and the sedentary hypertensive groups, and were not different between the sedentary and exercising long-term hypertensive groups. Increased wall thickness of the aortic and mesenteric vessels was observed in the hypertensive groups, but was prevented in the exercising long-term hypertensive group. In rat, exercise reduces the elevated blood pressure in L-NAME-induced hypertension via increasing nitric oxide synthase activity. Changes in vessel structure with exercise training may also be involved in the blood-pressure-lowering effects. Electronic Publication     

哌唑嗪对自发性高血压大鼠下丘脑nNOS表达的影响

目的研究哌唑嗪延缓自发性高血压大鼠(SHR)病理生理进程是否与调控下丘脑内神经元型一氧化氮合酶(nNOS)的表达有关。方法 8周龄雄性SHR,持续给予哌唑嗪1个月后,检测平均动脉压(MAP)和心率(HR)以及进行肾脏组织化学染色,并应用免疫荧光染色结合显微图像分析及Western blot测定下丘脑nNOS的表达水平。结果与未给药同龄SHR相比,给药SHR组MAP和HR明显降低,肾脏纤维化损害程度减轻,下丘脑nNOS蛋白水平升高,室旁核和视上核内nNOS阳性神经元含量增加,但均未达到正常同龄Wistar大鼠水平。结论哌唑嗪可能通过调节下丘脑nNOS发挥中枢降压作用,进而部分逆转自发性高血压发病进程。     

实验性精索静脉曲张大鼠睾丸组织和血清一氧化氮和一氧化氮合酶的改变

目的：检测实验性精索静脉曲张大鼠睾丸组织和血清NO含量和NOS活性的改变，以进一步阐明精索静脉曲张的发生机制。方法：通过手术方法建立大鼠精索静脉曲张模型，检测NO和NOS的变化。结果：实验组左侧睾丸组织NO含量显著高于对照组左侧睾丸含量；实验组血清NOS活力明显低于对照组。结论：实验性精索静脉曲张大鼠睾丸组织和血清NO和NOS有改变。     

S-甲基异硫脲对阿霉素致大鼠心肌组织超氧化物歧化酶、谷胱甘肽过氧化物酶活性改变的影响

目的:探讨S-甲基异硫脲(SMT)对阿霉素(ADM)所致大鼠心肌组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)活性改变的影响。 方法:给大鼠腹腔注射ADM(10.0 mg·kg^-1)1次，给ADM处理的大鼠静脉注射不同剂量的SMT(每天1次，共3次)进行干预。用分光光度法测定心肌组织丙二醛(MDA)含量、NO2-/NO3-含量、锰超氧化物歧化酶(MnSOD)活性、铜-锌超氧化物歧化酶（Cu-ZnSOD）活性、谷胱甘肽过氧化物酶(GPx)活性、一氧化氮合酶活性；用酶的速率法测定血清肌酸激酶(CK)的同功酶CK-MB活性；用逆转录-聚合酶链反应方法检测心肌组织MnSOD mRNA、Cu-ZnSOD mRNA、GPx mRNA、诱导型一氧化氮合酶(iNOS)mRNA表达。 结果:SMT(5.0，10.0，20.0 mg· kg^-1)3个干预组心肌组织MDA含量、NO2-/NO3-含量、iNOS活性及血清CK-MB活性明显低于ADM组(P<0.01)，而其心肌组织MnSOD mRNA、Cu-ZnSOD mRNA、GPx mRNA表达水平及其酶活性明显高于ADM组(P<0.01)。 结论:SMT拮抗ADM抑制心肌组织MnSOD mRNA、Cu-ZnSOD mRNA、GPx mRNA表达而拮抗ADM抑制这些酶的活性。     

诱生型一氧化氮合酶的变化在高胆固醇血症大鼠肾损害中的作用

目的:探讨高胆固醇血症大鼠肾组织诱生型一氧化氮合酶(iNOS)的变化与脂质肾损害的关系。方法:采用生化法检测血脂、尿蛋白、尿及肾皮质中一氧化氮含量,免疫组化法及逆转录—多聚酶链法检测肾组织中iNOS的表达强度及水平。TUNEL法检测肾组织中凋亡细胞。结果:8周时高脂组大鼠血清总胆固醇、低密度脂蛋白胆固醇、24h尿蛋白定量及尿一氧化氮量、肾皮质一氧化氮含量明显高于对照(P＜0.05)。肾皮质iNOSmRNA表达上调(P＜0.01)。肾皮质一氧化氮量与24h尿蛋白定量呈正相关(P＜0.05)。肾皮质iNOS积分光度与肾组织细胞凋亡指数呈正相关(P＜0.05)。肾组织细胞凋亡指数与24h尿蛋白含量呈正相关(P＜0.01)。结论:iNOS表达增强与脂质肾损害有关,可能通过引起细胞凋亡导致脂质肾损害。     

左旋精氨酸对被动吸烟孕鼠及胎仔发育的作用及其与NO、NOS的关系

目的了解左旋精氨酸对被动吸烟所致大鼠宫内发育迟缓及胎仔生长发育的作用,通过其与NO、NOS的关系探讨其作用机理.方法利用被动吸烟法建立大鼠IUGR模型,孕鼠随机分为5组:对照组、模型组、低剂量左旋精氨酸组、中剂量左旋精氨酸组、高剂量左旋精氨酸组.孕21天剖宫取胎,观察胎盘重、仔重、仔脑重、仔肝重;检测孕鼠血清及胎盘组织中NO含量及iNOS、cNOS活性.结果与对照组比较,模型组孕鼠胎盘重及仔重、仔脑重、仔肝重均明显降低(P<0.01),孕鼠血清及胎盘组织NO含量下降,iNOS活性升高,cNOS活性下降(P<0.01).低剂量及中剂量左旋精氨酸组与模型组比较各指标均有改善,但高剂量左旋精氨酸组与模型组比较无明显差异.结论孕中期应用一定剂量左旋精氨酸可有效防治被动吸烟孕鼠IUGR的发生并对胎仔发育有显著促进与保护作用,其作用与NO/NOS系统有关系.     

Nicotinamide induces male-specific body weight loss in the postnatal period through molecular regulation of the hypothalamus and liver

Molecular mechanisms of body weight control have been discovered recently and much research focuses on the hypothalamic regulation of food intake and the hepatic regulation of glucose utility. We previously reported that postnatal nicotinamide treatment reduced brain dopamine and body weight. To further investigate the differential effects of nicotinamide-mediated body weight loss, nicotinamide (i.p. 100 mg/kg) was injected into postnatal and adult mice twice a week for 4 weeks. Interestingly, following nicotinamide treatment, male postnatal mice displayed reduced body weight and spontaneous motor activity. No significant changes were observed in adult and postnatal female mice or adult male mice following nicotinamide treatment. In male postnatal mice, hypothalamic agouti-related peptide (AGRP) and proopiomelanocortin (POMC) levels were increased in the arcuate nucleus following nicotinamide treatment. Neuropeptide Y (NPY) levels were unchanged in both male and female mice. Additionally, nicotinamide-injected male postnatal mice had increased glucose 6-phosphatase (G6Pase) and decreased phosphoenolpyruvate carboxykinase (PEPCK) expression in liver. These results indicate that hypothalamic POMC and hepatic PEPCK are important molecules that mediate nicotinamide-induced weight loss in postnatal male mice.     

Low and infrequent expression of nitric oxide synthase/NADPH-diaphorase in neurons of the human supraoptic nucleus: a histochemical study

The gas nitric oxide is a messenger in brain signaling. In the hypothalamo-hypophyseal system nitric oxide is involved in the control of the expression and/or release of peptide hormones (corticotropin-releasing hormone, gonadotropin-releasing hormone, vasopressin and oxytocin). Nitric oxide synthase (NOS), the enzyme generating nitric oxide, is abundantly present in the magnocellular nuclei of the rat hypothalamus. Its localization in the human hypothalamus is less well studied. Hence, we investigated the anatomical distribution of neuronal nitric oxide synthase in the human supraoptic nucleus by use of immunohistochemical and enzyme histochemical techniques. The immunohistochemical localization of NOS was studied in 31 matched human hypothalami (13 control cases, eight depressed patients and ten schizophrenics). NADPH-diaphorase studies were carried out on seven additional hypothalami (three normal brains, four schizophrenics). Apparent inter-individual differences exist with regard to the occurrence of the enzyme in supraoptic neurons. In a majority of cases no immunostaining or histochemical reaction for the enzyme was observed. In seven cases (three controls, two schizophrenics, two depressives) a population of nitrergic nerve cells was seen in the dorsomedial part of the nucleus. This group of cells also stained for NADPH-diaphorase. Also, there were a few NOS-immunopositive neurons scattered throughout the nucleus. Additionally, thin NADPH-diaphorase positive fibers were observed to cross the nucleus. Our data show that, unlike the rat, the human supraoptic nucleus contains only a small number of nitrergic neurons. No correlation was found between the expression of the enzyme in supraoptic neurons and the psychiatric status of the patients.     

Developmental pattern of NADPH-diaphorase activity and nitric oxide-stimulated cGMP immunoreactivity in the frontal rat cortex and its role in functional recovery from aspiration lesions

The present study was undertaken to (1) explore the cortical nitric oxide (NO)-system during postnatal development and (2) to see whether or not the NO-system reacts differentially after neonatal and adult lesions of the medial prefrontal cortex. Three aspects of the NO-system were studied, i.e., NADPH-diaphorase (NADPH-d) activity, sodium nitroprusside (SNP)- and N-methyl- -aspartate (NMDA)-stimulated cGMP-immunoreactivity (cGMP-IR). It was shown that: (1) the development of NADPH-d activity containing cells is continued in the period from P6 until P21; (2) during the same period, large developmental changes take place in basal, and SNP- or NMDA-stimulated cGMP-IR in the cortex. These changes are regionally specific and follow the general cortical developmental pattern; and (3) aspiration lesions do not induce major changes in the distribution of NADPH-d activity or cGMP-IR, either basal, SNP- or NMDA-stimulated.     

Role of nitric oxide in thermoregulation during septic shock: involvement of vasopressin

We tested the hypothesis that the nitric oxide (NO) pathway in the central nervous system (CNS) plays a role in hypothermia, as well as in the febrile response during experimental septic shock, by regulating vasopressin (AVP) release. Experiments were performed on male Wistar rats treated with N ^G-nitro-l-arginine methyl ester (l-NAME), a non-selective NO synthase (NOS) inhibitor, injected intracerebroventricularly (250 µg/1 l) 30 min before lipopolysaccharide (LPS) 1.5 mg/kg i.v. injection. One hour after LPS administration we observed a significant drop in body temperature (hypothermic response), followed by a temperature increase after the second hour (febrile response), which remained until the end of the experiment. Increased plasmatic AVP levels were concomitantly observed during hypothermia, nearly returning to basal levels during the febrile phase. When l-NAME was administered with LPS, plasmatic AVP concentrations remained high throughout the experiment, hypothermia was accentuated and the febrile response was abolished. Additionally, pre-treatment with -mercapto-,-cyclopentamethylenepropionyl¹, O-Et-Tyr², Val⁴, Arg⁸-vasopressin, an AVP V1 receptor blocker (10 µg/kg) administered i.v., reduced hypothermia and exacerbated the febrile response to endotoxin. In conclusion, our data indicate that the central NO pathway plays an inhibitory role in AVP release during experimental septic shock, which seems to be critical for the thermoregulation during this pathophysiological state.     

大鼠下丘脑大细胞分泌系统内一氧化氮合酶与催产素的共存

目的　为进一步阐明一氧化氮 (NO)与催产素 (OT)在下丘脑神经内分泌调节中的作用提供形态学资料。方法　用NADPH d组织化学方法 ,并结合免疫组织化学相结合技术 ,观察大鼠下丘脑大细胞分泌系统内一氧化氮合酶 (NOS)与OT免疫阳性神经元的分布与共存关系。结果　NOS与OT在下丘脑室旁核、视上核等下丘脑各大神经分泌核区内的分布基本类似 ,并呈部分共存关系。并且初次在第三脑室室周区内观察到NOS/OT双重反应阳性触液神经元。结论　研究结果提示NO与OT在有关下丘脑生殖与性行为的神经内分泌调节活动中起着重要的介质作用。     

Effect of focal cerebral ischemia on nitric oxide synthase expression in rats

Time- and cell-type-dependent immunohistochemical activity of nitric oxide synthase (NOS) was investigated in rat cerebral cortex following focal ischemia and the local concentration of nitric oxide (NO) was measured. NO concentration increased 2 min after the ischemia. Brain NOS-immunoreactive neurons increased in number 5 min after the ischemia. Endothelial cell NOS immunoreactivity was first detected in vascular endothelial cells and astrocytes 5 min after the ischemia, and it increased again during 60 min to 4 days after the ischemia in reactive astrocytes. Inducible NOS immunoreactivity was detected in astrocytes, vascular endothelium, and microglia/macrophages at the periphery of the ischemic core during 2–4 days after the ischemia.This study was presented at the 28th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Osaka, October 17–19, 1996.     

The effects of estradiol on body weight and food intake in normal weight VMH-lesioned rats.

The effects of estradiol on food intake and body weight were examined in ovariectomized and VMH-lesioned, ovariectomized rats that were prevented from supranormal weight gain by food restriction. Estradiol injections that were effective in reducing weight in supranormal weight, ovariectomized rats had no effect on weight in normal weight, ovariectomized or VMH-lesioned, ovariectomized rats. Estradiol did not prevent hyperphagia and weight gain in VMH-lesioned, ovariectomized rats when they were provided with ad lib food.     

脑干及下丘脑内一氧化氮合酶（NOS）阳性神经元向海马的投射

采用还原型尼克酰胺嘌呤二核苷酸脱氢酶（NADPH－d）与辣根过氧化物酶（HRP）相结合方法，追踪脑干及下丘脑内NOS阳性神经元向海马的投射。结果表明：中缝背核有较多的NOS阳性神经元投射到背海马；中缝背核、乳头体上核及蓝斑有NOS阳性神经元投射到腹海马。这些核团向海马发出投射纤维的NOS阳性神经元占其总投射量的1／3左右。     

Coexistence of NADPH-diaphorase and tyrosine hydroxylase in the mesencephalic catecholaminergic system of the Japanese quail

Previous studies have shown the presence of a large number of tyrosine hydroxylase immunoreactive neurons and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase positive elements within the mesencephalic and pontine regions of the Japanese quail. In the present study histochemical and immunohistochemical procedures reveal that cells expressing at least one of these two neurochemical markers coexist throughout a large part of the substantia nigra and of the area ventralis of Tsai. Also about 40% of the neurons in these two regions that contain immunoreactive tyrosine hydroxylase also exhibit NADPH-diaphorase activity. This is not a general property of the quail catecholaminergic system: in the locus coeruleus (the main noradrenergic group) there is a complete separation between these two neuronal populations. The number of neurons expressing either neurochemical marker is not different between males and females in any of the regions that have been investigated. NADPH-diaphorase is known to be an indicator of the enzyme nitric oxide synthase; these results therefore suggest that nitric oxide may play an important role in the regulation of the activity of a significant part of the avian mesencephalic dopaminergic system.