Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after local cerebral ischemia in rat

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2–48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24–48 h after ischemia, neurons in the ischemia lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.     

Involvement of nitric oxide in re-innervation of rat molar tooth pulp following transection of the inferior alveolar nerve

The aim of this study was to elucidate whether nitric oxide (NO) is involved in re-innervation of rat molar tooth pulp following transection of the inferior alveolar nerve. The inferior alveolar nerves (IAN) of rats were transected unilaterally under anesthesia with chloral hydrate. The animals received horseradish peroxidase (HRP) application to mandibular molar tooth pulps on both sides and were fixed by transvascular perfusion. The average number of labeled cells on each side of the trigeminal ganglion was not significantly different [101±11 (mean±S.E.M.; n=6, left) and 89±11 (n=6, right)]. With HRP application on postoperative day 3, the ratio of the number of labeled neurons in the transected vs. non-transected (contralateral) sides was 31.5±5.8% (n=11). The i.p. administration of N^ω-nitro-

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-arginine methyl ester (

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-NAME; 100 mg/kg, once a day for a period of 4 days), but not

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-NAME, significantly decreased the ratio of the number of labeled neurons (10.1±7.0%, n=10).

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-Arginine (300 mg/kg, i.p., once a day for a period of 4 days) slightly increased the number of labeled neurons on the transected side. Clonidine (25 μg/kg, i.p., once a day for a period of 4 days) failed to exhibit any significant effect on nerve regeneration. In the trigeminal ganglion ipsilateral to the transected IAN on postoperative day 4, NADPH-diaphorase (NADPH-d)-positive neurons had significantly increased. On the other hand, no changes in NADPH-d were observed in the superficial layers of the subnucleus caudalis of the spinal trigeminal nucleus from where primary neurons innervating the mammalian tooth pulp project. These results suggest that NO is involved in several mechanisms related to neuronal regeneration.     

3,4-diaminopyridine-evoked noradrenaline release in rat hippocampal slices: facilitation by endogenous or exogenous nitric oxide

The involvement of nitric oxide (NO) in the evoked release of noradrenaline (NA) was studied in rat hippocampal slices preincubated with [³H]NA and stimulated with 3,4-diaminopyridine (3,4-DAP; 200 μM) for 2 min. The 3,4-DAP-evoked [³H]overflow was enhanced by the NO synthase substratel-arginine, but not byd-arginine; it was reduced by the NO synthase inhibitorN^G-mitro-l-arginine, whichl-arginine. Also drugs known to produce NO in-vitro, like sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) andS-nitroso-N-acetylpenicillamine (SNAP) enhanced the 3,4-DAP-evoked NA release. The NO scavenger hemoglobin showed no significant effects when given alone, but reduced or abolished, respectively, the facilitatory effects of SNP, or SNAP andl-arginine. The cyclic GMP derivatives 8-Br-cGMP and Sp-8-p-chlorophenylthioguanosine-3′,5′-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) also acted facilitatory, whereas the corresponding Rp-enantiomer of the latter compound was inactive, but antagonized the effect of Sp-8-pCPT-cGMPS. NA release evoked by 3,4-DAP (10 μM) from rat hippocampus synaptosomes was not affected byl-arginine orN^G-nitro-l-arginine but slightly increased by SNAP and Sp-8-pCPT-cGMPS. Antagonists at NMDA, non-NMDA and metabotropic glutamate receptors neither affected the 3,4-DAP-evoked NA release nor the facilitatory effect ofl-arginine. From these findings we conclude that endogenously formed NO facilitates 3,4-DAP-evoked NA release in rat hippocampus, possibly by a cyclic GMP dependent mechanism; NMDA receptor stimulation and glutamate release seem not to be involved in this phenomenon.     

Evidence for the involvement of nitric oxide and nitric oxide synthase in the modulation of opioid-induced antinociception and the inhibitory effects of exposure to 60-Hz magnetic fields in the land snail

The attenuation of opioid peptide-mediated antinociception is a well-established effect of extremely low frequency (ELF) electromagnetic fields with alterations in calcium channel function and/or calcium ion flux and protein kinase C activity being implicated in the mediation of these effects. The present study was designed to examine the effects of nitric oxide (NO) and calcium ion/calmodulin-dependent nitric oxide synthase (NOS) on opioid-induced antinociception and their involvement in mediating the inhibitory effects of exposure to ELF magnetic fields. We observed that enkephalinase (SCH 34826)-induced, and likely enkephalin-mediated, antinociception in the land snail, Cepaea nemoralis, as measured by the enhanced latency of a foot withdrawal response to a thermal (40°C) stimulus, was reduced by the NO releasing agent, S-nitro-N-acetylpenicillamide (SNP), and enhanced by the NO synthase inhibitor, N^G-nitro- -arginine methyl ester ( -NAME). Exposure of snails to an ELF magnetic field (15 min, 60 Hz, 141 μT peak) also reduced the enkephalinase-induced antinociception. The inhibitory effects of the 60-Hz magnetic field were significantly reduced by the NO synthase inhibitor, -NAME, and significantly enhanced by the NO releasing agent, SNP, at dosages which by themselves had no evident effects on nociceptive sensitivity. These results suggest that: (1) NO and NO synthase have antagonistic effects on opioid-induced analgesia in the snail, Cepaea and (2) the inhibitory effects of ELF magnetic fields on opioid analgesia involve alteration in NO and NO synthase activity.     

Vasodilatory actions of calcitonin gene-related peptide and nitric oxide in parenchymal microvessels of the rat hippocampus

Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent dilators in a variety of vascular beds. Recent evidence suggests that NO may serve as an intermediary messenger for CGRP and/or CGRP may serve as an intermediary messenger for NO in the expression of vasodilation. The present study was designed to provide an initial characterization of the responses to NO and CGRP in parenchymal microvessels and to determine whether NO and/or CGRP act as intermediaries for one another. Microvessels in the parenchyma of in vitro hippocampal slices from rat brain were examined using computer-assisted videomicroscopy. The resting diameter of the microvessels ranged from 9 to 26 μm. Treatment with the nitric oxide synthase inhibitor, N^G-nitro-l-arginine ( -NNA; 100 μM) constricted vessels to 64.2% ± 3.0% of resting luminal diameter. Sodium nitroprusside (SNP; 1 μM), a donor of NO, reversed the -NNA-induced vasoconstriction by 77.0% ± 15.0%. CGRP alone (10 nM) elicited a small but significant vasodilatory effect on resting vascular tone (2.3% ± 0.6%). In the presence of -NNA, CGRP elicited a significant dose-dependent vasodilatory response, and 10 nM CGRP elicited a sizeable response, reversing the -NNA-induced constriction by 84.3% ± 15.5%. This CGRP-induced dilation was inhibited by pretreatment with the CGRP receptor antagonist, CGRP fragment (8–37) (1 μM). In contrast, pretreatment with 1 μM CGRP fragment (8–37) did not attenuate the SNP-induced dilation in the presence of -NNA. Taken together, these findings demonstrate that CGRP and NO are potent dilators of parenchymal microvessels, and that NO provides a substantial relaxant effect on resting tone. In addition, the results indicate that CGRP is not a necessary intermediary in NO-induced dilation, and that NO is not a necessary intermediary in CGRP-induced dilation in parenchymal microvessels.     

Excitatory action of N-methyl- -aspartate on the rat locus coeruleus is mediated by nitric oxide: an in vivo voltammetric study

Biochemical, electrophysiological and behavioural studies have provided evidence that activation of N-methyl- -aspartate (NMDA) receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) in precipitated opioid withdrawal. Recently, it was demonstrated that central administration of nitric oxide (NO) synthase inhibitors suppresses this hyperactivity suggesting that NO mediates the NMDA receptor activation of LC in opioid withdrawal. Using a combination of microdialysis and in vivo voltammetry, this study examined whether local application of NMDA to the LC in opioid naive animals mimics the NO-dependent LC response seen in opioid withdrawal. In the urethane anaesthetized rat, perfusion of the LC (2 μl min⁻¹) with a solution of NMDA (5 mmol) via a microdialysis probe for 9 min resulted in a rapid and robust increase (290.1±32.2% above baseline) in the catechol oxidation current (CA·OC) recorded from the LC using differential normal pulse voltammetry (DNPV). The NMDA microdialysis also produced a large increase in the blood pressure (150.4±6.9% above baseline). An injection of the non-competitive NMDA receptor antagonist (+)MK-801 (0.5 mg kg⁻¹ i.v.), given 45 min after the start of NMDA application, rapidly returned both the CA·OC signal and the blood pressure response to baseline levels. Pretreatment of animals with intraventricular nitric oxide synthase (NOS) inhibitor, N^ω-nitro- -arginine methyl ester ( -NAME) (100 μg) significantly inhibited NOS activity in the LC, PAG-PVG and cerebellum. This dose of -NAME, administered prior to application of NMDA by microdialysis abolished the NMDA-induced rise in the CAOC recorded in the LC and the increase in systolic blood pressure. The results show that in voltammetry experiments, NMDA produces hyperactivity of LC and hypertension, responses that are dependent upon the synthesis of NO. Thus, in opioid naive rats, regional NMDA application via microdialysis mimics characteristics of the LC response that occur during the antagonist-precipitated opioid withdrawal.     

Inhibitory effects of hypoxia and adenosine on N-methyl-d-aspartate-induced pial arteriolar dilation in piglets

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-

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-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were 100 μm. Topical application of NMDA at concentrations of 10⁻⁵, 5×10⁻⁵ and 10⁻⁴ M resulted in dose-dependent vasodilation (9±2, 18±2 and 29±2% above baseline, respectively, n=21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O₂). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28±2 vs. 2±1%, respectively to 10⁻⁴ M, n=7) while sodium nitroprusside (SNP, 10⁻⁴ M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10⁻⁴ M; n=5) or SNP (10⁻⁵ M; n=4), or when SNP (10⁻⁷ M) was coapplied with NMDA (n=6). Conversely, coapplication of adenosine (10⁻⁶ M) attenuated NMDA responses (31±5 vs. 20±3%, n=7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.     

Warm cells revealed by microfluorimetry of Ca in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca²⁺ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca²⁺]_i from 52.5±9.5 nM (mean±S.D., n=18) to 171.0±15.6 nM in cells between 15 and 25 μm in diameter. The depletion of extracellular Ca²⁺ diminished the Ca²⁺ elevation. The Na⁺-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Nuclear and cytoplasmic Ca signals in developing rat dorsal root ganglion neurons studied in excised tissue

Confocal microscopy and the Ca²⁺-sensitive fluorescent dye fluo-3 were used to study subcellular Ca²⁺ signals in embryonic, neonatal, and adult dorsal root ganglion (DRG) neurons in excised dorsal rooot ganglia. Optical images obtained from isolated' whole embryonic and neonatal ganglia revealed a marked variability in the resting Ca²⁺ signals of different neurons as compared to signals in adult neurons which were uniformly faint. Many of the embryonic and neonatal neurons displayed nuclear Ca²⁺ signals at rest which were larger than those in the cytoplasm. Embryonic DRG neurons showed a significant increase in nuclear and cytoplasmic fluorescence in response to depolarization with elevated extracellular potassium or electrical stimulation. A single brief electrical stimulus was sufficient to elicit nuclear Ca²⁺ signals in a subset of the embryonic neurons. The depolarization-induced Ca²⁺ signals were blocked by removal of extracellular Ca²⁺, but not by treatment with 2,5-di (tert-butyl)- 1,4 benzohydroquinone (DTBHQ), a compound which depletes intracellular Ca²⁺ stores. The intensity of the depolarization-induced Ca²⁺ signals declined significantly between the late embryonic (E18–E20) and early postnatal time periods (P0–P1). The nuclear and cytoplasmic Ca²⁺ signals of the embryonic DRG neurons in the excised tissue preparation occur at a time of intense target innervation, suggesting a role for Ca²⁺ signals in the development and maturation of rat DRG neurons.     

Increased inducible nitric oxide synthase protein but limited nitric oxide formation occurs in astrocytes of the hph-1 (tetrahydrobiopterin deficient) mouse

It has been suggested that decreased tetrahydrobiopterin (BH₄) availability may be a useful tool for limiting excessive nitric oxide (NO) formation. In order to test this hypothesis we utilised cultured astrocytes derived from the brain of the hph-1 (BH₄ deficient) mouse. In response to treatment with lipopolysaccharide and interferon-γ (LPS/γIFN) levels of BH₄ doubled in both wild type and hph-1 astrocytes. However, levels of BH₄ in hph-1 astrocytes remained only 25% of the wild type astrocytes. Nitric oxide formation, measured with an NO-electrode, was 45% less from LPS/γIFN stimulated hph-1 astrocytes compared with wild type stimulated astrocytes. In contrast, iNOS specific activity and iNOS protein were enhanced in hph-1 stimulated astrocytes by 40 and 60%, respectively when compared with wild type. In conclusion it appears that whilst a decrease in BH₄ may limit NO release per se, the possibility and consequences of long term `over' induction of iNOS protein requires further consideration.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

α2-Adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a potent vasodilator, is produced in dorsal root ganglia (DRG) neurons which extend nerves peripherally to blood vessels and centrally to the spinal cord. We previously reported that neuronal CGRP expression is significantly reduced in the spontaneously hypertensive rat (SHR) which could contribute to the elevated BP. Other studies suggest that the enhanced activity of the sympathetic nervous system in the SHR may mediate, at least in part, this reduction in neuronal CGRP expression via activation of α₂-adrenoreceptors (α₂-AR) on DRG neurons. To test this hypothesis in vitro we employed primary cultures of adult rat DRG neurons. Neuronal cultures were initially exposed (24 h) to either the α₂-AR agonist UK 14,304 (10⁻⁶ M) or vehicle; however, no changes in CGRP mRNA content or immunoreactive CGRP (iCGRP) release were observed. Using the rationale that in vivo DRG neurons receive a continuous supply of target tissue derived nerve growth factor (NGF), which stimulates CGRP synthesis, the cultured neurons were treated (24 h) with either vehicle, NGF (25 ng/ml) alone, or NGF plus UK. NGF treatment increased CGRP mRNA accumulation 5.5±0.9-fold (p<0.001) and iCGRP release 2.9±0.4-fold (p<0.001) over control levels. The stimulatory effects of NGF were markedly attenuated, but not abolished, by UK (NGF+UK vs. control, CGRP mRNA, 2.9±0.4-fold, p<0.05; iCGRP, 1.7±0.2-fold, p<0.05). These values were also significant (p<0.05) when compared to NGF treatment alone. Experiments performed using the α₂-antagonist yohimbine confirmed that the effects of UK were mediated by the α₂-AR. These results, therefore, demonstrate that α₂-AR activation attenuates the stimulatory effects of NGF on CGRP expression in DRG neurons.     

Ultrastructural localization and comparative distribution of nitric oxide synthase and N-methyl-d-aspartate receptors in the shell of the rat nucleus accumbens

Nitric oxide (NO), the diffusible gas formed by nitric oxide synthase (NOS) has been implicated in the enhanced locomotor activity attributed mainly to increased dopamine release in the shell of the nucleus accumbens (Acb). Furthermore, the release of both NO and dopamine are known to be altered by agonists of N-methyl-d-aspartate (NMDA) type glutamate receptors in this region. We examined the cellular sites of NO synthesis and the sites of potential relevancy for functional associations between neurons containing NOS and the NMDA receptor in the shell of the Acb. This was achieved by dual ultrastructural immunogold and immunoperoxidase labeling of antisera raised against the brain form of NOS and the NMDARI subunit of the NMDA receptor in this region of rat brain. NOS-like immunoreactivity (NOS-LI) was seen throughout the cytoplasm of isolated medium-large somata, aspiny dendrites and axon terminals. In 217 NOS-labeled profiles, NMDARI-like immunoreactivity (NMDARI-LI) was colocalized in 17% of somata and dendrites. Additionally, 35% of NOS-labeled dendrites apposed glial processes containing NMDARI-LI, and 29% apposed axon terminals containing NMDARI-LI. NOS-labeled terminals more rarely colocalized NMDARI or apposed NMDARI-labeled glial processes or dendrites. These results provide anatomical evidence that, in the shell of the Acb, NMDA receptors are localized so as to directly modulate the output of neurons producing NO as well as to influence other neurons and glia having the greatest access to the released gas.     

NADPH-diaphorase/nitric oxide synthase containing neurons in normal and Parkinson's disease putamen

Summary Nitric oxide (NO) is thought to be involved in neurodegenerative processes. Concerning Parkinson's disease (PD) it remains to be elucidated, if NO contributes to pathological alterations in the striatum. The present study evaluates the post-mortem putamen of PD patients and control subjects for distribution patterns of NO-synthase containing neurons, using the NADPH-diaphorase technique. The ratio of positively stained neurons and the total number of cells (control: 1,120±69 per mm², n=5; PD: 575±164mm², n=5) shows striking differences between controls and PD patients. Our findings give reason to conclude that NADPH-diaphorase positive structures may have pathogenetic importance in degenerative processes in PD putamen.     

SnCl(2) reduces voltage-activated calcium channel currents of dorsal root ganglion neurons of rats

Stannous dichloride (SnCl₂) occurs in the environment where it has been especially enriched in aquatic ecosystems. Furthermore, it is used in food manufacturing (e.g. for stabilizing soft drinks or as an anti-corrosive substance) and in nuclear medicine where it is employed as a reducing agent for technecium-99m (99mTc) and therefore is applied intravenously to human beings.SnCl₂ is known to have toxic effects on the nervous system which can be related to alterations of intracellular calcium homeostasis ([Ca²⁺]_i). In this study the whole cell patch clamp technique is used on dorsal root ganglion neurons of 3-week-old “Wistar” rats to evaluate the effects of SnCl₂ on voltage-activated calcium channel currents (I_Ca(V)).I_Ca(V) were reduced concentration-dependently by SnCl₂ (1–50 μM). 1 μM SnCl₂ reduced I_Ca(V) by 8.1 ± 4.5% (peak current) and 19.2 ± 8.9% (sustained current), whereas 50 μM inhibited I_Ca(V) by 50.6 ± 4.3% (peak current) and 55.6 ± 11.3% (sustained current). Sustained currents were slightly but not significantly more reduced than peak currents. The effect appeared not to be reversible. The threshold concentration was below 1 μM.The current–voltage relation did not shift which is an indication that different calcium channel subtypes were equally affected. There was a slight but not significant shift of the activation/inactivation curves towards the depolarizing direction.We conclude that voltage-gated calcium channels are affected by Sn²⁺ similarly to other divalent metal cations (e.g. Pb²⁺ or Zn²⁺).The reduction of I_Ca(V) could be related to the neurotoxic effects of SnCl₂.     

Modulation by protein kinase C of nitric oxide and cyclic GMP formation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells withl-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 ± 0.03 nmol/h/dish (2.5 × 10⁶ cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 ± 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with staurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg²⁺, N-methyl-d-aspartate and, to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C, an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.     

Involvement of nitric oxide in the deregulation of cytosolic calcium in cerebellar neurons during combined glucose-oxygen deprivation

Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca²⁺]_i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca²⁺]_i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca²⁺]_i, which returned to near basal levels on restoration of O₂-containing medium. Prolonged hypoxic conditions (>60 min) caused irreversible elevation of [Ca²⁺]_i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake (“point of no return”). Pretreatment withN ^G-nitro-l-arginine methyl ester (l-NAME, 100 μM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca²⁺]_i. The hypoxia-induced elevation of [Ca²⁺]_i was also greatly attenuated ifl-NAME (100 μM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca²⁺]_i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the “point of no return”, as characterized above, greatly potentiated [Ca²⁺]_i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca²⁺]_i and subsequent cell death, was not blocked by NOS inhibitor,l-NAME.     

Abdominal vagotomy attenuates interleukin-1β-induced nitric oxide release in the paraventricular nucleus region in conscious rats

Nitric oxide (NO) has recently been shown to modulate the hypothalamic–pituitary–adrenal axis response to interleukin-1β (IL-1β). We measured levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the hypothalamic paraventricular nucleus (PVN) region using an in vivo brain microdialysis technique in conscious rats. Intraperitoneally administered IL-1β produced a significant increase in both NO₂⁻ and NO₃⁻ levels in the PVN region. We also examined the possible involvement of the abdominal vagal afferent nerves in this effect. In abdominal-vagotomized rats, the increase was significantly attenuated compared to that in sham-operated rats. Our results suggest that the abdominal vagal afferent nerves are involved in intraperitoneally administered IL-1β-induced NO release in the PVN region.     

A new type of Ca channel blocker, NC-1100, inhibits the low- and high-threshold Ca currents in the rat CNS neurons

The effect of a new type of organic Ca²⁺ channel blocker, NC-1100 [(±)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)ethanol dihydrochloride], on both low- and high-threshold Ca²⁺ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca²⁺ current (HVAI_Ca) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC₅₀) were 1.3 × 10⁻⁵ and 9.1 × 10⁻⁶M in external solution containing 10 and 2.5 mM Ca²⁺, respectively. The NC-1100 also decreased the low-threshold Ca²⁺ current (LVAI_Ca) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and / or decreasing the extracellular Ca²⁺ concentration to a physiological range (2.5 mM). The IC₅₀ value decreased to 7.7 × 10⁻⁷M in external solution containing 2.5 mM Ca²⁺ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca²⁺ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca²⁺ channel but also the inactivated one.     

NADPH-diaphorase reactivity in articular afferents of a normal and inflamed knee joint in the cat

The distribution of NADPH-diaphorase was studied in retrogradely labelled dorsal root ganglion cells innervating the knee joint of the cat. A strong staining reaction was found in7.5 ± 1.9% (mean ± S.D. of 9 normal joints and 6393 labelled perikarya) of the articular afferents. An acute inflammation (32 h) significantly increased this proportion to10.9 ± 2.2% (mean ± S.D. of 5 inflamed joints and 3933 labelled perikarya). The diameter distribution of the somata with a positive NADPH-diaphorase reaction ranged from 18 to 46 μm with a maximum at 24–28 μm. These data indicate that a small proportion of knee joint primary afferents may be able to release nitric oxide playing a role in synaptic transmission and in regulatory functions within the peripheral tissue under normal and pathophysiological conditions.