Neuronal Nitric Oxide Synthase Activation Is Involved in Insulin-Mediated Cardiovascular Effects in the Nucleus Tractus Solitarii of Rats

Neuronal nitric oxide synthases (nNOS) is distributed throughout the central nervous system (CNS) and has been proposed to modulate neuronal activity in the nucleus tractus solitarii (NTS). Here, we investigated whether the activation of nNOS is involved in insulin-induced cardiovascular responses in the NTS. Insulin (100 IU/ml) was unilaterally microinjected into the NTS, and the cardiovascular effects were evaluated before and after microinjection of the nNOS inhibitors 7-nitroindazole (7-NI) (5 pmol) and N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-L-NIO) (600 pmol). Western blot and immunohistochemical analyses were performed to determine nNOS phosphorylation levels after insulin or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 microinjection into the NTS. Unilateral microinjection of insulin into the NTS produced prominent depressor and bradycardic effects in WKY rats. Pretreatment with the nNOS inhibitors 7-NI and Vinyl-L-NIO attenuated the cardiovascular response evoked by insulin in Wistar-Kyoto (WKY) rats. Moreover, Western blot analysis showed a significant increase in nNOS (16.5±0.4-fold; P<0.05; n=4) phosphorylation after insulin injection, whereas the PI3K inhibitor LY294002 abolished the insulin-induced effects. In situ nNOS phosphorylation was found to be increased in the NTS after insulin injection. Furthermore, co-immunoprecipitation assay showed Akt and nNOS can bind to each other as detected by phospho-Akt^S473 and phospho-nNOS^S1416 antibodies. In vitro kinase assay showed insulin activated Akt can directly phosphorylate nNOS^S1416. These results demonstrated that nNOS may couple with the activation of the insulin receptor, via the liberation of NO, in order to participate in central cardiovascular regulation of WKY rats.     

Increased and decreased muscle tone with orexin (hypocretin) microinjections in the locus coeruleus and pontine inhibitory area

Orexin-A (OX-A) and orexin-B (OX-B) (hypocretin 1 and hypocretin 2) are synthesized in neurons of the perifornical, dorsomedial, lateral, and posterior hypothalamus. The locus coeruleus (LC) receives the densest extrahypothalamic projections of the orexin (OX) system. Recent evidence suggests that descending projections of the LC have a facilitatory role in the regulation of muscle tone. The pontine inhibitory area (PIA), located ventral to LC, receives a moderate OX projection and participates in the suppression of muscle tone in rapid-eye-movement sleep. We have examined the role of OX-A and -B in muscle-tone control using microinjections (0.1 microM to 1 mM, 0.2 microl) into the LC and PIA in decerebrate rats. OX-A and -B microinjections into the LC produced ipsi- or bilateral hindlimb muscle-tone facilitation. The activity of LC units was correlated with the extent of hindlimb muscle-tone facilitation after OX microinjections (100 microM, 1 microl) into fourth ventricle. Microinjections of OX-A and -B into the PIA produced muscle-tone inhibition. We did not observe any significant difference in the effect of OX-A and -B on muscle tone at either site. Our data suggest that OX release activates LC units and increases noradrenergic tonus in the CNS. Moreover, OX-A and -B may also regulate the activity of pontine cholinoceptive and cholinergic neurons participating in muscle-tone suppression. Loss of OX function may therefore disturb both facilitatory and inhibitory motor processes.     

Involvement of nitric oxide production via kynurenic acid-sensitive glutamate receptors in DOPA-induced depressor responses in the nucleus tractus solitarii of anesthetized rats

We have proposed the hypothesis that L-3,4-dihydroxyphenylalanine (DOPA) plays a role of neurotransmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii (NTS). In the present study, we tried to clarify whether glutamate receptors and/or nitric oxide (NO), important modulators for central cardiovascular regulation, are involved in the DOPA-induced cardiovascular responses in the nucleus. Male Wistar rats were anesthetized with urethane and artificially ventilated. Compounds or antisense oligos (17-mer) for neuronal NO synthase were microinjected into depressor sites of the unilateral nucleus. DOPA 30-300 pmol microinjected into the nucleus dose-dependently induced depressor and bradycardic responses. Prior injection of kynurenic acid (600 pmol) suppressed DOPA (300 pmol)-induced responses by approximately 80%. Prior injection of N(G)-monomethyl-L-arginine 100 nmol, a potent NO synthase inhibitor, reversibly attenuated by approximately 90% DOPA-induced responses, while the D-isomer 100 nmol produced no effect. Furthermore, prior injection of neuronal NO synthase antisense oligos (20 pmol) reversibly reduced by approximately 70% responses to DOPA. Sense or scrambled oligos produced no effect. A NO precursor L-arginine (30 nmol) induced depressor and bradycardic responses, but these responses were not affected by kynurenic acid. These results suggest important roles for glutamate receptors and NO in DOPA induced-depressor and bradycardic responses in the NTS.     

Is estradiol cardioprotection a nitric oxide-mediated effect?

BACKGROUND: Estradiol exerts a number of biological effects that support extensive observational data suggesting a protective role for estrogen in cardiovascular disease prevention. These include effects on lipid and carbohydrate metabolism, coagulation/fibrinolysis as well as a possible effect on vascular reactivity. It has been proposed that this might be mediated by vascular endothelial nitric oxide (NO) production. Accordingly, we designed complementary in-vivo and in-vitro studies to investigate this hypothesis further. METHODS: Firstly, in a group of 10 healthy post-menopausal women, bilateral venous occlusion plethysmography was used to examine forearm vasoconstrictor responses to intrabrachial N(G)-monomethyl-l-arginine (l-NMMA; a substrate inhibitor of nitric oxide synthase) both before and after 4 weeks of treatment with transdermal 17beta-estradiol (E(2)) (80 microg/day). Secondly, we examined the direct effects of acute (24 h) and chronic (7 days) treatment with E(2) (10 pmol/l and 10 nmol/l) on endothelial nitric oxide synthase (eNOS) gene expression in cultured human aortic endothelial cells. RESULTS: No significant differences were observed between the vasoconstrictor responses to l-NMMA (2, 4, 8 micromol/min) before and after E(2) treatment. Comparison of E(2)-treated endothelial cells with control cells showed no significant increase in eNOS mRNA expression following either acute or chronic estradiol treatment. CONCLUSIONS: The present studies do not provide evidence for an eNOS-mediated cardioprotective response to estrogen and therefore suggest that additional mechanisms other than the endothelial NO system may have an important role in the cardiovascular effects of estrogen.     

Involvement of protein kinase C and nitric oxide in the modulation by insulin-like growth factor-I of glutamate-induced GABA release in the cerebellum

Insulin-like growth factor-I elicits a long-term depression of the glutamate-induced GABA release in the adult rat cerebellum that lasts at least several hours. We studied whether protein kinase C and nitric oxide may be involved in this effect of insulin-like growth factor-I on GABA release since both signalling pathways have been implicated in other forms of neuromodulation in the cerebellum. By using microdialysis in the adult rat cerebellum, we found that either an inhibitor of protein kinase C (staurosporine) or of nitric oxide synthase (Nw-nitro-l-arginine methyl ester) counteracted the long-term, but not the acute effects of insulin-like growth factor-I on glutamate-induced GABA release. On the contrary, when either an activator of protein kinase C (phorbol ester), or an nitric oxide donor (l-arginine), were given with glutamate, they mimicked only the acute effects of insulin-like growth factor-I on glutamate-induced GABA release. Finally, when both protein kinase C and nitric oxide-synthase were simultaneously inhibited by conjoint administration of staurosporine and Nw-nitro-l-arginine methyl ester, a complete blockage of both the short and the long-term effects of insulin-like growth factor-I on GABA release was obtained. These results, indicate that: (i) activation by insulin-like growth factor-I of either the protein kinase C or nitric oxide-signalling pathways is sufficient for the short-term inhibition of glutamate-induced GABA release; and (ii) simultaneous activation of both the protein kinase C and the nitric oxide signalling pathways is necessary for insulin-like growth factor-I to induce a long-term depression of GABA responses to glutamate.

Thus, long-term depression of glutamate-induced GABA release by insulin-like growth factor-I in the cerebellum is mediated by simultaneous activation of both protein kinase C and nitric oxide-signalling pathways.     

Unravelling mechanisms of action of angiotensin II on cardiorespiratory function using in vivo gene transfer

We review recent and ongoing work from our laboratory that has shed novel insights into the effects of angiotensin II (ANGII) on the baroreflex at the level of the nucleus of the solitary tract (NTS). The NTS is the site of termination for baroreceptor afferents and is a potentially powerful region for neuronal modulation. ANGII applied to this nucleus attenuated the cardiac vagal and cardiac sympathetic components of the baroreceptor reflex. This effect was antagonized by blockade of either γ‐amino butyric acid receptors or nitric oxide synthase within the NTS. Interestingly, nitric oxide donors microinjected into the NTS mimicked the effect of ANGII. Using an adenovirus we showed that ANGII activated the endothelial isoform of nitric oxide synthase. The NTS was transfected to express a dominant negative truncated mutant form of endothelial nitric oxide synthase that prevented the depressant effect of ANGII on the baroreflex. Endothelial nitric oxide synthase was present in both neurones and endothelium in the NTS. A possibility is that ANGII activation of endothelial nitric oxide synthase is calcium dependent. However, in most NTS neurones tested, ANGII failed to elevate intracellular calcium concentration. We conclude that ANGII activates endothelial nitric oxide synthase to release nitric oxide which enhances γ‐amino butyric acid transmission destined for circuitry mediating the baroreflex. We discuss the contribution of endothelial cells within the nucleus of the solitary tract as a potential target for both circulating and/or centrally produced ANGII. These data have relevance to patients with essential hypertension and left heart failure, conditions in which ANGII activity is elevated and the baroreceptor reflex is depressed.     

Different effects of constitutive nitric oxide synthase and heme oxygenase on pulmonary or liver metastasis of colon cancer in mice

It has recently been reported that not only endogenous nitric oxide (NO) but also carbon monoxide (CO) produced by heme oxygenase (HO) have many physiological functions. The objective of the present study was to determine whether endogenous NO or CO is involved in the experimental pulmonary or liver metastasis of colon cancer in mice. Intravenous or intrasplenic injection of colon 26 cells from a mouse colon adenocarcinoma cell line resulted in multiple pulmonary or liver metastases. NG-nitro-l-arginine methyl ester (l-NAME), a competitive inhibitor of NO synthase (NOS), or zinc deuteroporphyrin 2, 4-bis glycol (ZnDPBG), a competitive inhibitor of HO, was administered to the mice only on the day of tumor inoculation. We assessed the number of tumor cells 24 h later and the outcome of metastases of the target organ. In the pulmonary metastasis model, l-NAME increased both the number of tumor cells 24 h later and outcome of metastases 18 days later, but did not have a significant effect on liver metastasis. On the other hand, metastasis to the liver, but not that to the lung, increased following administration of ZnDPBG. These results suggest that the activities of NOS and HO could influence experimental metastasis in an organ-specific manner. This revised version was published online in July 2006 with corrections to the Cover Date.     

Orexins/hypocretins control bistability of hippocampal long-term synaptic plasticity through co-activation of multiple kinases

Aim: Orexins/hypocretins (OX/Hcrt) are hypothalamic neuropeptides linking sleep–wakefulness, appetite and neuroendocrine control. Their role and mechanisms of action on higher brain functions, such as learning and memory, are not clear. Methods: We used field recordings of excitatory post-synaptic potentials (fEPSP) in acute mouse brain slice preparations to study the effects of orexins and pharmacological inhibitors of multiple kinases on long-term synaptic plasticity in the hippocampus. Results: Orexin-A (OX-A) but not orexin-B (OX-B) induces a state-dependent long-term potentiation of synaptic transmission (LTP_OX) at Schaffer collateral-CA1 synapses in hippocampal slices from adult (8- to 12-week-old) mice. In contrast, OX-A applied to slices from juvenile (3- to 4-week-old) animals causes a long-term depression (LTD_OX) in the same pathway. LTP_OX is blocked by pharmacological inhibition of orexin receptor-1 (OX1R) and plasticity-related kinases, including serine/threonine- (CaMKII, PKC, PKA, MAPK), lipid- (PI3K), and receptor tyrosine kinases (Trk). Inhibition of OX1R, CaMKII, PKC, PKA and Trk unmasks LTD_OX in adult animals. Conclusion: Orexins control not only the bistability of arousal states and threshold for appetitive behaviours but, in an age- and kinase-dependent manner, also bidirectional long-term synaptic plasticity in the hippocampus, providing a possible link between behavioural state and memory functions.     

Orexins and the regulation of the hypothalamic-pituitary-testicular axis

Orexins (OX), OX-A and OX-B, were initially identified as hypothalamic neuropeptides primarily involved in the control of food intake and states of arousal. Thereafter, orexins have been substantiated as putative pleiotropic regulators of a wide diversity of biological systems, including different neuroendocrine axes. Among the latter, compelling experimental evidence has recently been documented that orexins, mainly OX-A, may act at different levels of the hypothalamic-pituitary-gonadal (HPG) axis to modulate reproductive function. These actions are likely to include regulatory effects on the hypothalamic centres governing the HPG axis, as well as direct actions at the gonadal level. We review herein the experimental evidence, gathered in recent years, supporting a reproductive ‘facet’ of orexins, with special emphasis on our current knowledge of their patterns of expression and potential functional roles in the testis. Overall, the available data strongly suggest that, by acting at different levels of the HPG axis, orexins may operate as putative neuroendocrine and autocrine/paracrine regulators of gonadal function.     

Central cardiovascular effects of calcitonin gene-related peptide: interaction with noradrenaline in the nucleus tractus solitarius of rats

Summary The haemodynamic responses to microinjections of rat or human calcitonin gene-related peptide (CGRP) into the nucleus tractus solitarius (NTS) of rats were studied. 40 fmol rCGRP did not significantly modify cardiovascular parameters, but 0.2 pmol decreased blood pressure and heart rate (HR), whereas 2 pmol produced a pressor response with no effect on HR. hCGRP elicited a transient fall in blood pressure when administered at the highest dose (2 pmol), but had no effects when given at 0.2 pmol. A possible functional relationship with catecholamines was also investigated. The hypotensive response to 20 nmol noradrenaline (NA) was significantly modified by simultaneous administration of a low dose (40 fmol, ineffective alone) of rCGRP. When rCGRP (40 fmol) was coinjected simultaneously with an ineffective dose (10 pmol) of NA, a hypotensive response was observed. Our results provide evidence that rCGRP may play a role in the control of cardiovascular homeostasis in the NTS, and suggest a functional interaction between this peptide and NA.     

L-arginine ameliorates experimental autoimmune myocarditis by maintaining extracellular matrix and reducing cytotoxic activity of lymphocytes

It was previously shown that administration of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) aggravated murine viral myocarditis by increasing myocardial virus titres. Experimental autoimmune myocarditis in mice and rats mimics human fulminant myocarditis. The effects of L-arginine, a precursor of nitric oxide, upon heart failure in experimental autoimmune myocarditis were evaluated. Dietary L-arginine (L-arginine group) and L-arginine plus N(G)-nitro-L-arginine methyl ester (L-arginine + l-NAME group) were administered to C57BL/6 mice immunized with porcine cardiac myosin over 3 weeks. An untreated myocarditis group was prepared. Cardiac damage was less in the L-arginine group compared with the other two groups, as was incidence of heart failure. In addition, extracellular matrix change was less prominent in the L-arginine group. Plasma concentrations of nitric oxide were elevated in the L-arginine group. Cytotoxic activities of lymphocytes were lower in L-arginine group than in other two groups. L-arginine treatment may be effective in preventing the development of heart failure in experimental myocarditis by maintaining extracellular matrix and reducing the cytotoxic activity of lymphocytes.     

Nitric oxide modulates the firing rate of the rat supraoptic magnocellular neurons

In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect of l-arginine, a precursor for NO synthesis, and N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 muM) and dl-2-amino-5-phosphonovaleric acid (dl-AP5) (30 muM) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 muM) and saclofen (30 muM) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 muM l-arginine decreased the firing rate from 3.7+/-0.6 Hz to 1.3+/-0.3 Hz. Conversely, 100 muM l-NAME increased the firing rate from 3.0+/-0.3 Hz to 5.8+/-0.4 Hz. All points histogram analysis showed changes in resting potential from -58.1+/-0.8 mV to -62.2+/-1.1 mV in the presence of l-arginine and from -59.8+/-0.7 mV to -56.9+/-0.8 mV by l-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4+/-1.2 mV to 16.8+/-0.7 mV by l-arginine, but without significant changes by l-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation.     

Lipopolysaccharide-activated microglia induce death of oligodendrocyte progenitor cells and impede their development

Damage to oligodendrocyte (OL) progenitor cells (OPCs) and hypomyelination are two hallmark features of periventricular leukomalacia (PVL), the most common form of brain damage in premature infants. Clinical and animal studies have linked the incidence of PVL to maternal infection/inflammation, and activated microglia have been proposed to play a central role. However, the precise mechanism of how activated microglia adversely affects the survival and development of OPCs is still not clear. Here we demonstrate that lipopolysaccharide (LPS)-activated microglia are deleterious to OPCs, that is, impeding OL lineage progression, reducing the production of myelin basic protein (MBP), and mediating OPC death. We further demonstrate that LPS-activated microglia mediate OPC death by two distinct mechanisms in a time-dependent manner. The early phase of cell damage occurs within 24 h after LPS treatment, which is mediated by nitric oxide (NO)-dependent oxidative damage and is prevented by N^G-nitro-l-arginine methyl ester (l-NAME), a general inhibitor of nitric oxide synthase. The delayed cell death is evident at 48 h after LPS treatment, is mediated by cytokines, and is prevented by blocking the activity of tumor necrosis factor-alpha (TNF-α) and pro-nerve growth factor (proNGF), but not by l-NAME. Furthermore, microglia-derived insulin-like growth factor-1 (IGF-1) and ciliary neurotrophic factor (CNTF) were significantly suppressed by LPS, and exogenous IGF-1 and CNTF synergistically protected OLs from death induced by LPS-treated microglia conditioned medium, indicating that a deficiency in trophic support may also be involved in OL death. Our finding that LPS-activated microglia not only induce two waves of cell death but also greatly impair OL development may shed some light on the mechanisms underlying selective white matter damage and hypomyelination in PVL.     

Nitric oxide synthase inhibition in rostral ventrolateral medulla attenuates pressor response to psychological stress in rabbits

Nitric oxide (NO) has been critically implicated in the central regulation of autonomic function. We recently found, however, that acute (up to 30min) blockade of NO synthase (NOS) in the rostral ventrolateral medulla (RVLM) inhibited sympathetic baroreflex transmission, without altering the cardiovascular response to psychological (air-jet) stress in rabbits. In the present study, we examined the effect of the later phase (1-3h) of NOS inhibition in the RVLM on the pressor and sympathetic responses to air-jet stress in conscious rabbits. Air-jet evoked a sustained increase in blood pressure (+14+/-2mmHg), heart rate (+37+/-9beats/min) and renal sympathetic nerve activity (+52+/-8%). Bilateral microinjection of a NOS inhibitor l-NAME (10nmol) into RVLM did not affect resting parameters or stress responses during the first 30min after injection. Conversely, in the later phase of NOS inhibition, the pressor, tachycardic and renal sympathetic responses to air-jet stress were reversibly attenuated by 48-72%. Microinjection of l-NAME outside the RVLM did not change stress responses. Microinjection of glutamate (3nmol) into the RVLM induced similar pressor effects before and after l-NAME (+30+/-6mmHg and +26+/-6mmHg, respectively). Microinjection of d-NAME altered neither stress responses nor pressor response to glutamate. These results suggest that NOS inhibition in the RVLM has a dual effect on the autonomic response to psychological stress. In the early phase, NOS inhibition has little impact on this response. However, in the later phase, NOS inhibition attenuates the stress response, perhaps via indirect mechanisms such as altering the local redox state.     

NMDA receptor antagonism blocks the cardiovascular responses to microinjection of trans-ACPD into the NTS of awake rats

The possible interaction of glutamatergic metabotropic agonists and N-methyl- d -aspartate (NMDA) receptors was investigated in the nucleus tractus solitarii (NTS) of awake rats. The cardiovascular responses to unilateral microinjection of trans -1-amino-1,3-cyclopentanediocarboxylic acid ( trans -ACPD; 250 pmol/50 nL) into the NTS ( n = 8) produced hypotension (−64 ± 4 mmHg) and bradycardic (−206 ± 11 bpm) responses, which were blocked by previous microinjection of 2-amino-5-phosphonovaleric acid (AP-5; 10 nmol/50 nL), a selective antagonist of NMDA ionotropic receptors, into the same site. Intravenous injection of methyl-atropine blocked both the bradycardic and hypotensive responses to microinjection of trans -ACPD into the NTS, indicating that the hypotension was secondary to the intense bradycardic response. The data also showed that the bradycardic and hypotensive responses to microinjection of an NMDA agonist (10 pmol/50 nL) into the NTS were not affected by previous microinjection of α-methyl-4-carboxyphenylglycine (MCPG; 5 nmol/50 nL), a non-selective antagonist of metabotropic receptors. The results showing that the cardiovascular responses to microinjection of trans -ACPD into the NTS were blocked by AP-5 indicate that the responses to metabotropic agonists in the NTS involves NMDA receptors.     

Oxygen or low concentrations of nitric oxide reverse pulmonary vasoconstriction induced by nitric oxide synthesis inhibition in rabbits

The objective of this study was to investigate the role of nitric oxide and oxygen in the regulation of pulmonary vascular resistance, especially by means of substitution with nitric oxide after inhibition of endogenous nitric oxide formation. In artificially ventilated open-chest rabbits pulmonary vascular resistance at normoxic ventilation (F₁0₂= 21%) was 56±6cmH₂O ml^-1 mim-¹ 1000^-1 (mRU_L). N^w-nitro-L-arginine methyl ester (l-NAME, 30 mg kg^-1), an inhibitor of NO synthase, increased pulmonary vascular resistance to 122±17 mRU_L at normoxic ventilation. In response to l-NAME there was also an increase in mean arterial blood pressure. Exogenous nitric oxide (0.014-9 p.p.m. in the inhaled air) dose-dependently and reversibly counteracted the effect of l-NAME on pulmonary vascular resistance at normoxic ventilation, without affecting systemic blood pressure. In addition, the L-NAME-induced vasoconstriction was critically dependent on oxygen. Thus, during hypoxic ventilation (F₁O₂= 10%) the pulmonary vascular resistance was increased approximately four-fold by the presence of L-NAME (30 mg kg^-1), and increments in F,0₂ (21–100%) dose-dependently and reversibly counteracted the effect of L-NAME on pulmonary vascular resistance. Taken together these findings demonstrate that inhalation of low doses of NO may act as a replacement when endogenous NO synthesis is inhibited, and that pulmonary vasoconstriction induced by NO synthesis inhibition is likely to be the result of interference with oxygen-dependent regulatory mechanisms. Endogenous NO cooperates with oxygen to evoke a vasodilator component of the pulmonary hypoxic pressor response, balancing a hitherto unknown constrictor mechanism.     

一氧化氮对大鼠黑质致密部神经元自发放电及Glu能和GABA能传入活动的影响

目的:研究一氧化氮(NO)对大鼠黑质致密部(SNc)神经元自发放电活动及对谷氨酸(Glu)、γ-氨基丁酸(GABA)能神经纤维输入活动的影响,为进一步研究Parkinson病(PD)的发病机制奠定神经生理学基础。方法:采用微电泳技术观察NO供体硝普钠(SNP),Glu及GABA对SNc神经元自发放电活动的影响及神经递质间的相互作用。结果:微电泳SNP使81.25%(39/48)受试神经元自发放电频率加快,此兴奋性作用与微电泳电流强度正相关,随着电流强度的增加(20nA～80nA),SNc神经元自发放电频率进一步加快。在35个受试神经元上分别微电泳Glu或GABA后,可以观察到分别有31神经元兴奋或所有神经元表现为抑制作用。在已经被Glu兴奋的31个神经元上同时微电泳SNP可使SNc神经元自发放电频率进一步加快。相反,在微电泳GABA的同时应用SNP可使已经被抑制的神经元放电频率增加。结论:NO对SNc神经元产生兴奋性作用。NO与Glu、GABA能投射在SNc有汇聚作用,NO协同Glu对SNc神经元产生兴奋性作用,但拮抗GABA对SNc神经元抑制性作用。     

化学发光法测定脑组织中一氧化氮合酶的活性

在NADPH等辅助因子的参与下，一氧化氛合酶（NOS）能催化L-精氨酸生成一氧化氮（NO）。NO与过氧化氢反应生成过氧亚硝酸，过氧亚硝酸能与鲁米诺产生化学发光反应，当NO浓度为100fmol／L～Inmol／L时，发光强度与NO量成正比．根据这一原理、利用化学发光分析建立了一种新的NOS活性测定方法。用该方法测量10只大鼠脑组织中的NOS活性，其NOS活性为28．3±6．9pmolNO／mg蛋白／min。     

Effects of acute hypobaric hypoxia on the nitric oxide system of the rat cerebral cortex: Protective role of nitric oxide inhibitors

Exposure to hypobaric hypoxia produces neuropsychological disorders. The brain nitrergic system was investigated following hypobaric hypoxia in the presence or absence of nitric oxide synthase (NOS) inhibitors. Adult rats were exposed to a simulated altitude of 8325 m (27,000 ft) for 7 h and killed after 0, 1, 3, 5, and 10 days of recovery. In addition to normobaric controls, three experimental groups were studied: i) subjected to hypobaric hypoxia without inhibitors; ii) subjected to hypobaric hypoxia and treated with 7-nitroindazole; iii) subjected to hypobaric hypoxia and treated with N(omega)-nitro-l-arginine methyl ester (l-NAME). Cerebral cortex was assayed by immunohistochemistry, Western blotting, and enzymatic assays. In animals subjected to hypobaric hypoxia without inhibitors, there was an increase in neuronal nitric oxide synthase (nNOS) immunoreactivity and Ca(2+)-dependent NOS activity from 0 to 1 days of reoxygenation. In these animals, inducible nitric oxide synthase (iNOS) expression and Ca(2+)-independent activity were undetectable, but nitrotyrosine immunoreactivity was found in some neurons. Administration of either inhibitor prevented the increase in nNOS immunoreactivity and enzymatic activity provoked by hypobaric hypoxia. Concomitantly, nitrotyrosine immunoreactivity decreased progressively. In conclusion, activation of the nitrergic system constitutes a cortical response to hypobaric hypoxia and the administration of NOS inhibitors could provide new therapeutic avenues to prevent and/or treat the symptoms produced by hypobaric hypoxia.     

Evidence for the presence of GABAergic and glycine-like systems responsible for cardiovascular control in the nucleus tractus solitarii of the rat

Microinjections of γ-aminobutyric acid (GABA) and glycine, into the medial area of the nucleus tractus solitarii (NTS) of the rat, led to an increase in arterial pressure and heart rate. The GABA receptor antagonist bicuculline and the glycine receptor antagonist strychnine decreased both of these cardiovascular parameters whereas the GABA uptake inhibitor nipecotic acid produced hypertension. High K⁺ stimulation caused a calcium-dependent release of GABA and glycine from tissues in the area of the NTS. Our results suggest that GABA and glycine may modulate the cardiovascular control within the NTS.