The effects of the lithium salt of GABA on the subcellular metabolic profile of L-arginine in the prefrontal cortex and striatum of rats during chronic stress

Our previous data suggest that depression-like behavior of rats, which is induced by the 14-daylong unpredictable influence of stress factors, is accompanied by a sustained increase in the contents of L-arginine and nitric oxide (NO) and its stable metabolites, as well as stimulation of the inducible isoform of NO synthase (iNOS) and suppression of the activity of its constitutive NOS isoforms (cNOS) in the cytosol and mitochondria of the prefrontal cortex (PFC) and striatum. A single intraperitoneal injection of GABA lithium salt (at a dose of 0.9 mg/kg) on the second day of the post-stress period normalized the levels of L-arginine and active forms of nitrogen in cellular compartments from the PFC and striatum. Although this treatment did not influence the stress-induced shifts in the activity of cNOS, it prevented stimulation of the iNOS/NO system of mitochondria due to blockage of NO hyperproduction and nitrosative stress, whose development results in the disturbance of energetic processes in these brain areas.     

Methylene blue inhibits the increase of inducible nitric oxide synthase activity induced by stress and lipopolysaccharide in the medial basal hypothalamus of rats

In infection bacterial products such as lipopolysaccharides (LPS) induce inducible nitric oxide synthase (iNOS) that produces large quantities of NO toxic to the invading organisms, but also often has toxic effects on host cells. Therefore, inhibition of iNOS activity might be beneficial in combatting these adverse effects. To determine if methylene blue (MB), an oxidizing agent that inactivates iNOS, would reduce the iNOS levels in the medial basal hypothalami (MBH) of conscious male rats, LPS (5 mg/kg) was injected intravenously (i.v.), and after 3 h they were injected i.v. with either MB (3 mg/kg) or saline and the effects on iNOS in the MBH determined. iNOS was measured by conversion of labeled arginine into citrulline by incubating MBH in the absence of calcium (Ca(2+)) since iNOS does not require Ca(2+) for activation. The results indicate that iNOS was induced by the injection of saline, but the induction by LPS was much greater, an increase of 10-fold above that of control sham-operated animals. Both the induction of iNOS from the stress of saline injections and LPS were completely eliminated by MB indicating that MB might be beneficial in preventing injury to brain tissue following LPS injection. There was no effect of either LPS or MB on the Ca(2+)-dependent constitutive NOS activity.     

Appearance of inducible nitric oxide synthase in the rat central nervous system after rabies virus infection and during experimental allergic encephalomyelitis but not after peripheral administration of endotoxin

The cellular localization of inducible (iNOS) and constitutive (cNOS) nitric oxide synthase was studied in rats by immunocytochemical techniques involving specific iNOS and cNOS directed antibodies and by NADPH-diaphorase histochemistry. Paraformaldehyde-fixed vibratome sections of brains and cryostat sections of peripheral lymph nodes were studied of rats treated with endotoxin (2.5 μg/kg or 2.5 mg/kg i.v.), rats infected with rabies virus, and rats exposed to experimental allergic encephalomyelitis (EAE). Endotoxin-treated animals showed no appearance of immunoreactive iNOS (ir-iNOS) cells in the brain with the exception of a few microglial cells near the median eminence and some meningeal macrophages. In the same animals however, iNOS-immunoreactive cells were found in peripheral lymph nodes. Neurons that stain positive for cNOS and for NADPH-diaphorase could be observed in brains of control as well as of endotoxin-treated animals with a similar distribution and staining intensity. In contrast, animals that had been infected with rabies virus or subjected to EAE, showed the appearance of ir-iNOS-positive cells in several brain areas. These cells are located near blood vessels and lesion sites. The majority of these cells are GSA-I-B4 isolectin-positive and therefore are likely to represent macrophages. Our data suggest that increased production of nitric oxide may play a role in the altered brain functions in rabies-infected and EAE rats. On the contrary, increased nitric oxide production is probably not involved in the non-specific symptoms of sickness induced by endotoxin. © 1995 Wiley-Liss, Inc.     

一氧化氮合酶活性与抑郁症的相关性

目的通过对抑郁症患者一氧化氮合酶（NOS）活性进行检测，从而研究和探讨一氧化氮合酶、一氧化氮（NO）与抑郁症之间的关系。方法采用分光光度法检测抑郁症患者治疗前后的一氧化氮合酶NOS及其亚型（结构型cNOS、诱导型iNOS）的活性，并与正常对照组比较。结果抑郁症组的NOS、cNOS活性显著低于正常对照组；治疗组的NOS、cNOS活性高于抑郁症（无显著性），但治疗后缓解组的NOS、cNOS活性均显著高于治疗前。各组iNOS的活性无显著差异。结论抑郁症病人的NOS活性下降，而且主要是结构型cNOS活性下降，经治疗缓解后有所提高。因此，NOS和NO很有可能在抑郁症的发病过程中起着重要作用。     

Nitric oxide synthase in cerebral ischemia

The results of our continuing studies on the role of nitric oxide (NO) in cellular mechanisms of ischemic brain damage as well as related reports from other laboratories are summarized in this paper. Repetitive ip administration ofN ^G-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor, protected against neuronal necrosis in the gerbil hippocampal CA1 field after transient forebrain ischemia with a bell-shaped response curve, the optimal dose being 3 mg/kg. Repeated ip administration of L-NNA also mitigated rat brain edema or infarction following permanent and transient middle cerebral artery (MCA) occlusion with a U-shaped response. The significantly ameliorative dose-range and optimal dose were 0.01–1 mg/kg and 0.03 mg/kg, respectively. Studies using a NO-sensitive microelectrode revealed that NO concentration in the affected hemisphere was remarkably increased by 15–45 min and subsequently by 1.5–4 h after MCA occlusion. Restoration of blood flow after 2 h-MCA occlusion resulted in enhanced NO production by 1–2 h after reperfusion. Administration of L-NNA (1 mg/kg, ip) diminished the increments in NO production during ischemia and reperfusion, leading to a remarkable reduction in infarct volume. In brain microvessels obtained from the affected hemisphere, Ca²⁺-dependent constitutive NOS (cNOS) was activated significantly at 15 min, and Ca²⁺-independent inducible NOS (iNOS) was activated invariably at 4 h and 24 h after MCA occlusion. Two hour reperfusion following 2 h-MCA occlusion caused more than fivefold increases in cNOS activity with no apparent alterations in iNOS activity. Thus, we report here based on available evidence that there is good reason to think that NOS activation in brain microvessels may play a role in the cellular mechanisms underlying ischemic brain injury.     

Effect of bacterial lipopolysaccharide on the reproductive axis of prepubertal and peripubertal female rats. Ontogenic changes in the immune-neuroendocrine interactions

The immune, endocrine and nervous systems are closely interrelated, which allows the organism to respond to different types of stress such as infection. Chronic infectious and inflammatory conditions are often accompanied by an impaired reproductive function. Leptin, a hormone produced by adipose tissue, exerts a regulatory function on the reproductive axis. It has homology with other proinflammatory cytokines and could be modified by lipopolysaccharide (LPS). Therefore, these studies were designed to investigate the effect of LPS administration on the neuroendocrine mechanisms involved in the regulation of the reproductive axis during sexual maturation. Fifteen- and 30-day-old female rats were injected with a single dose of LPS 250 microg/kg (i.p.) and then nitric oxide synthase (NOS) activity, hypothalamic excitatory/inhibitory amino acids and Gn-RH content, serum LH and leptin concentration were studied. In 15-day-old female rats LPS treatment did not modify hypothalamic inducible (iNOS) and constitutive (cNOS) NOS activity, Gn-RH, glutamate (GLU) and GABA content. Also serum LH and leptin levels were not modified. In 30-day-old rats LPS increased iNOS and cNOS activity (p < 0.001) and hypothalamic Gn-RH content (p < 0.001). At this age hypothalamic GABA content was significantly decreased (p < 0.001) without changes in GLU content, and serum LH (p < 0.001) and leptin (p < 0.0001) decreased significantly. In summary, current studies have demonstrated that LPS administration to 15- and 30-day-old female rats results in a different response of the hypothalamus-pituitary-gonadal axis and of the adipose tissue, demonstrating an ontogenic response of the immune-neuroendocrine system to LPS administration.     

Nitric Oxide Synthase Inhibition Delays Axonal Degeneration and Promotes the Survival of Axotomized Retinal Ganglion Cells

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.     

急性脑梗死患者血浆神经肽Y与血清一氧化氮合酶水平的变化及其临床意义

目的探讨急性脑梗死(ACI)患者血浆神经肽Y(NPY)、血清一氧化氮合酶(NOS)水平的变化及其临床意义。方法分别用放射免疫法、化学比色法、酶法检测30例ACI患者(ACI组)(发病<48 h和第12 d)及27名正常对照者(NC组)血浆NPY、血清结构型NOS(cNOS)、诱导型NOS(iNOS)及三酰甘油(TG)水平,头颅MRI检查测量脑梗死灶体积,应用美国国立卫生研究院卒中量表(NIHSS)评定神经功能缺损程度。结果与NC组比较,ACI组NPY水平差异无统计学意义;血清cNOS水平发病<48 h显著降低(P<0.01),并与梗死体积、同期NIHSS评分呈负相关(r=-0.368、-0.457,均P<0.05),与NIHSS评分差值呈正相关(r=0.466,P<0.05);血清iNOS水平发病<48 h显著升高(P<0.01),与梗死体积、同期NIHSS评分及两次检测的血TG水平呈正相关(r=0.390、0.362、0.411、0.395,均P<0.05),与NIHSS评分差值呈负相关(r=-0.461,P<0.05)。NPY差值与cNOS差值呈负相关(r=-0.369,P<0.05)。结论 ACI患者发...     

Expression of endothelial and inducible NOS-isoforms is increased in Alzheimer’s disease, in APP23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology

The nitric oxide-synthesizing enzyme nitric oxide synthase (NOS) is present in the mammalian brain in three different isoforms, two constitutive enzymes (i.e., neuronal, nNOS, and endothelial eNOS) and one inducible enzyme (iNOS). All three isoforms are aberrantly expressed in Alzheimer’s disease giving rise to elevated levels of nitric oxide apparently involved in the pathogenesis of this disease by various different mechanisms including oxidative stress and activation of intracellular signalling mechanisms. It still is a matter of debate, however, whether the abnormal expression of NOS isoforms has some primary importance in the pathogenetic chain and might thus be a potential therapeutic target or only reflects a secondary effect that occurs at more advanced stages of the disease process. To tackle this question, we analysed the expression of both eNOS and iNOS in patients with sporadic AD, in transgenic mice expressing human amyloid precursor protein (APP) with the Swedish double mutation under control of the Thy1 promotor (APP23 mice), and after electrolytic cortical lesion in rat, an experimental paradigm associated with elevated expression of APP. In all three conditions, an astrocytosis was induced accompanied by a strong increase of both iNOS and eNOS. Both NOS isoforms were frequently though not always colocalized. Thus, based on the expression pattern of NOS isoforms three types of astrocytes, expressing only one of the two isoforms or both together could be distinguished. In both AD and transgenic mice eNOS-expressing astrocytes exceeded iNOS-expressing astrocytes in number. Astrocytes with elevated levels of iNOS or eNOS were constantly seen in direct association with Aβ-deposits in AD and transgenic mice and were found in the vicinity of the lesion site in the rat cortex. The results of the present study show that expression of both iNOS and eNOS is increased in activated astrocytes under experimental conditions associated with elevated expression of APP (electrolytic brain lesion) or Aβ-deposition (APP23 transgenic mice). Therefore, it is suggested that altered expression of these NOS isoforms being part of AD pathology is secondary to the amyloid pathology and might not be primarily involved in the pathogenetic chain though it might contribute to the maintenance, self-perpetuation and progression of the neurodegenerative process.     

Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS). which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some infiltrating inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.