Impairment of neuronal nitric oxide synthase-dependent dilation of cerebral arterioles during chronic alcohol consumption

BACKGROUND: Although chronic alcohol consumption impairs endothelial nitric oxide synthase-dependent reactivity of cerebral arterioles, the effect of alcohol consumption on vasodilation in response to activation of neuronal nitric oxide synthase (nNOS) has not been examined. Thus, our first goal was to determine whether chronic alcohol consumption impairs nNOS-dependent reactivity of pial arterioles. Our second goal was to examine potential mechanisms for impaired responses of pial arterioles during chronic alcohol consumption. METHODS: Sprague Dawley rats were fed liquid diets with or without alcohol for 8 to 12 weeks. By using intravital microscopy, we measured the diameter of pial arterioles in response to nNOS-dependent agonists--NMDA and kainate (KA)--in the absence and presence of N(G)-monomethyl-L-arginine (L-NMMA) or 7-nitroindazole (7-NI). We also measured responses of pial arterioles to nitroglycerin. Next, using Western blot analysis, we measured protein levels of the NMDA receptor subunit, KA receptor subunit, and nNOS protein in cerebral microvessels, parietal cortex, cerebellum, and brainstem of non-alcohol-fed and alcohol-fed rats. RESULTS: Topical application of NMDA (100 and 300 microM) and KA (100 and 300 microM) produced dose-related dilation of pial arterioles in non-alcohol-fed and alcohol-fed rats. However, the magnitude of vasodilation in response to NMDA and KA, but not nitroglycerin, was significantly less in alcohol-fed compared with non-alcohol-fed rats. Topical application of L-NMMA (10 microM) or 7-NI (10 microM) significantly inhibited dilation of pial arterioles in response to NMDA and KA in non-alcohol-fed rats. In alcohol-fed rats, only NMDA-induced vasodilation was inhibited by L-NMMA. In addition, we found that NMDA receptor subunit and KA receptor subunit protein levels increased in the parietal cortex and cerebellum of alcohol-fed compared with non-alcohol-fed rats. However, no significant difference in protein level of nNOS was observed between non-alcohol-fed and alcohol-fed rats. CONCLUSIONS: Our findings suggest that chronic alcohol consumption impairs nNOS-dependent dilation of pial arterioles via a mechanism that appears to be unrelated to quantitative changes in NMDA receptors, KA receptors, or nNOS. Because the regulation of cerebral blood flow is influenced by neuronal activation, impaired reactivity of cerebral blood vessels to neuronal activation may contribute to the pathogenesis of cerebrovascular disorders observed during chronic alcohol consumption.     

Modulation of spinal nociception by GluR5 kainate receptor ligands in acute and hyperalgesic states and the role of gabaergic mechanisms

GluR5 receptors modulate spinal nociception, however, their role in nociceptive hypersensitivity remains unclear. Using behavioural and electrophysiological approaches, we have investigated several GluR5 ligands in acute and hyperalgesic states. Furthermore, as the GABAergic system plays a role in GluR5 mediated effects in the brain, we also analysed the interaction between GluR5 agonists and GABA(A) antagonists in the spinal cord. In young rats in vivo, the GluR5 selective agonist ATPA was antinociceptive and antihyperalgesic in a model of inflammatory hyperalgesia (ED(50) approximately 4.6 and approximately 5.2 mg/kg, respectively), whereas the GluR5/GluR6 agonist SYM2081 was only antihyperalgesic. ATPA, but not SYM2081, was also able to inhibit nociceptive motoneurone responses in anaesthetised adult rats after intrathecal administration. In hemisected spinal cords in vitro, SYM2081 was inactive, whereas ATPA and another GluR5 agonist, (S)-5-iodowillardiine, inhibited nociceptive reflexes (EC(50) 1.1+/-0.4 micro M and 0.36+/-0.05 micro M, respectively). Both GluR5 agonists also inhibited motoneurone responses to repetitive dorsal root stimulation and their cumulative depolarisation, a correlate of wind-up. The GABA(A) antagonists bicuculline (10 micro M) and SR95531 (1 micro M) enhanced polysynaptic responses to single stimuli but abolished the cumulative depolarisation. Both bicuculline and SR95531 significantly attenuated the inhibition of nociceptive responses by 1 micro M ATPA (by approximately 50%). We conclude that selective GluR5 kainate receptor activation inhibits spinal nociception and its sensitisation caused by ongoing peripheral nociceptive drive. GABA(A) receptors are involved in tonic inhibition of segmental responses, but contribute to their sensitisation by repetitive primary afferent stimulation. Furthermore, there is a cross-talk between the two systems, presumably due to GluR5-mediated activation of GABAergic inhibitory interneurones in the spinal cord.     

Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA

(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring (S)-glutamic acid ((S)-Glu) receptors. We report here the separation of (+)- and (−)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and >99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of (S)-(+)-ATPA and (R)-(−)-ATPA. (S)- and (R)-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6+ KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, (S)-ATPA showed AMPA receptor agonist effects (EC₅₀=23 μM) approximately twice as potent as those of ATPA. (R)-ATPA antagonized depolarizations induced by AMPA (K_i=253 μM) and by (S)-ATPA (K_i=376 μM), and (R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid (K_i=301 μM and 1115 μM). At cloned AMPA receptors, (S)-ATPA showed agonist effects at GluR3 and GluR4 with EC₅₀ values of approximately 8 μM and at GluR1 (EC₅₀=22 μM), producing maximal steady state currents only 5.4–33% of those evoked by kainic acid. (R)-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with K_i values of 33–75 μM. (S)-ATPA produced potent agonist effects at GluR5 (EC₅₀=0.48 μM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, (S)-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by (S)-ATPA and kainic acid were not significantly different. (R)-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither (S)- nor (R)-ATPA showed significant effects at GluR6. (S)-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6+KA2 receptors, whereas (R)-ATPA was inactive. Thus, (S)- and (R)-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA (S)-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.     

Developmental variation of the permeability to Ca2+ of AMPA receptors in presumed hilar glial precursor cells

Glial cells in the hilus of the dentate gyrus of the rat were investigated using the patch-clamp technique in acute slices of the hippocampal formation. According to their voltage-gated current patterns, two classes of glial cells — putative astrocytes and presumed glial precursor cells — were apparent. The glutamate receptor agonists kainate, glutamate, and -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) evoked inward currents at a holding potential of –70 mV in astrocytes and presumed glial precursor cells. Inward currents could also be induced in nucleated patches, indicating a direct action on glial receptors. In presumed hilar glial precursor cells, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 M) blocked the kainate-induced current, while it was partially inhibited by Zn²⁺ (2 mM) and Evans Blue (10 M). Cyclothiazide (100 M), in contrast, potentiated this current, indicating the presence of AMPA receptors. In 90% of the presumed glial precursor cells the excitatory amino-acid-evoked current voltage (I/V) relations were linear or outwardly rectifying and reversed close to 0 mV, which is characteristic for non-specific cation channels. To determine the permeability to Ca²⁺, I/V relations were determined in a Na⁺-free solution containing 40 mM Ca²⁺ and showed reversal potentials of a wide variation ranging from –63 mV to + 1 mV with corresponding P _Ca/ P _Cs permeability ratios of between 0.09 and 2.10. Statistical analysis revealed that the permeability to Ca²⁺ significantly decreased with an advance in age (r=–0.596; n=21; P<0.01). These data suggest that the Ca²⁺ influx mediated by the activation of AMPA receptors expressed in presumed hilar glial precursor cells is dependent on the developmental stage.     

An immunocytochemical study of glutamate receptors and glutamine synthetase in the hippocampus of rats injected with kainate

Immunocytochemistry was used to study the distribution of the kainate receptors GluR1, GluR2/3 and GluR4 and of the N-methyl-d-aspartate (NMDA) receptor NMDAR1 as well as the astrocyte markers glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) in the hippocampus of normal and kainate-lesioned rats. Hippocampal pyramidal neurons and dentate granule neurons were labelled heavily for GluR1 and GluR2/3, but only lightly for GluR4. Dense GluR4 immunopositivity was, however, observed in oligodendrocyte-like glial cells. Hippocampal pyramidal neurons and dentate granule neurons were moderately labelled for NMDAR1. Intravenous kainate injections resulted in a decrease in GluR1 and GluR2/3 immunoreactivity on the apical dendrites of pyramidal neurons as early as 7 h postinjection. At 18 h, there was a marked reduction in GluR1 and GluR2/3 receptors in the terminal tuft of dendrites of most hippocampal pyramidal neurons in the affected area, although some cells showed labelling in other portions of the apical dendrites and in basal dendrites. Immunostaining for GluR4 and NMDAR1 was also reduced at this time. At postinjection day 3, only the cell bodies and the basal dendrites of a few scattered pyramidal cells were labelled. Taken together, these results indicate a progressive loss of glutamate receptors, which affects the apical dendritic tree before the basal dendritic tree. The decrease in receptor immunoreactivity could be due to a downregulation of the receptors, since it occurred as early as 7 h postlesion, before cell death was evident in Nissl-stained sections. At long intervals after kainate injection, all pyramidal cells at the centre of the lesion showed a lack of glutamate receptor staining, and no partially labelled pyramidal cells were observed. The periphery of the lesion, however, contained many partially labelled pyramidal neurons among the unlabelled cells and had features of early lesions. The present study also showed an early decrease in GS immunoreactivity in the affected CA fields of the hippocampus (18 h to 3 days postinjection), followed by a medium-term increase (5–68 days) and a late decrease in GS immunoreactivity (81 days). The decrease in GS immunoreactivity at 81 days is not due to an absence of astrocytes, since GFAP staining showed many densely labelled astrocytes in the affected CA field.     

Inhibition by the chromaffin cell-derived peptide serine-histogranin in the rat's dorsal horn

The heptadecapeptide histogranin, synthesized by adrenal chromaffin cells, is implicated in the analgesia produced by transplanting chromaffin cells into the spinal cord, including block of hyperalgesia mediated by NMDA-subtype glutamate receptors. To examine the neurophysiological basis for this analgesia, we applied the stable analog [Ser¹]-histogranin (SHG) by iontophoresis near extracellularly recorded wide-dynamic range (WDR) neurons in anesthetized rats. When SHG was applied during peripheral electrical stimulation of A and C fibers at 0.1 Hz, the C-fiber response was significantly inhibited but the A-fiber response was unaffected. SHG also opposed the NMDA-receptor-dependent post-tetanic facilitation (wind-up) of C-fiber responses produced by increasing the rate of peripheral afferent stimulation to 1 Hz for 20 s. To test whether block of NMDA-subtype receptors could be wholly or partially responsible for this suppression, SHG was applied during sequential pulsed iontophoresis of three agonists targeting distinct excitatory synaptic receptors: NMDA, kainate and substance P. All three excitatory effects were reversed by SHG; this reversal outlasted the 10–30 min observation period when higher SHG doses were applied (>60 nA). Histogranin therefore probably produces prolonged spinal analgesia by opposing the basal and potentiating synaptic effects of C-fibers on dorsal horn neurons. Actions besides or in addition to NMDA-receptor antagonism (e.g., agonism at inhibitory postsynaptic receptors or block of voltage-gated cation channels on C-fibers) are implied by the diversity of excitatory transmitters opposed by SHG.     

Antioxidant N-acetylcysteine inhibits the activation of JNK3 mediated by the GluR6–PSD95–MLK3 signaling module during cerebral ischemia in rat hippocampus

Cerebral ischemia induces kainate receptor glutamate receptor 6 (GluR6) binding to the postsynaptic density protein 95 (PSD95), which in turn anchors mixed lineage kinase 3 (MLK3) via SH3 domain in rat brain. MLK3 subsequently activates c-Jun NH₂-terminal kinase (JNK) via MAP kinase kinases (MKKs). In this study, we investigated the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3, and the phosphorylation of JNK3 during cerebral ischemia in rat hippocampus CA1. Our results indicate that the GluR6–PSD95–MLK3 complex quickly enhanced at 5 min of ischemia and peaked at 10 min of ischemia, and then gradually reduced with the prolonged time of ischemia. Interestingly, the combination of MLK3 and JNK3 gradually increased from 5 min to 30 min of ischemia. JNK3 phosphorylation first increased and then attenuated in cytosol, suggesting the translocation of activated JNK3 to nucleus during ischemia. To further investigate the possible mechanism of JNK3 activation, antioxidant N-acetylcysteine (NAC) was given to the rats 20 min prior to ischemia. Results indicate that NAC distinctly inhibited the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3 and JNK3 activation. Taken together, these finding indicate that ischemic stimulation results in JNK3 activation through the GluR6–PSD95–MLK3 signaling module, and that the activation of JNK3 is closely related to oxidative stress.     

Ca(2+)-permeable AMPA receptors and selective vulnerability of motor neurons

To evaluate the role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis (ALS), we compared the sensitivity of motor neurons and that of dorsal horn neurons to kainic acid (KA). Short exposure to KA resulted in the death of motor neurons, while dorsal horn neurons were unaffected. This selective motor neuron death was completely dependent on extracellular Ca²⁺ and insensitive to inhibitors of voltage-operated Ca²⁺ or Na⁺ channels. It was also completely inhibited by the specific AMPA antagonist LY300164 and by Joro spider toxin (JSTx), a selective blocker of AMPA receptors that lack the edited GluR2 subunit. KA selectively killed those motor neurons that stained positive for the Co²⁺ histochemical staining, a measure for the presence of Ca²⁺-permeable AMPA receptors. These results suggest that Ca²⁺ entry via Ca²⁺-permeable AMPA receptors is responsible for the selective motor neuron death. As the Ca²⁺ permeability of the AMPA receptor is regulated by its GluR2 subunit, we stained motor neurons for GluR2. Immunoreactivity was present in all motor neurons, albeit to a variable degree. However, double-staining experiments demonstrated that motor neurons clearly expressing GluR2, also expressed Ca²⁺-permeable AMPA receptors. This indicates that despite the abundant expression of GluR2, this subunit is excluded from a subset of AMPA receptors and that the activation of these receptors is responsible for the selective motor neuron death.     

Alteration of Bcl-2 expression in the nigrostriatal system after kainate injection with or without melatonin co-treatment

In order to understand further the role of the anti-apoptotic Bcl-2 proto-oncogene protein in excitotoxin-induced brain injury and possible interaction between Bcl-2 and the antioxidant melatonin, the expression of Bcl-2 in various brain parts was studied after intrastriatal injection of kainate (KA, 2.5 nmol) with or without co-treatment of melatonin (10 mg/kg, intraperitoneally (i.p.)). Three days after unilateral injection of KA to the striatum in the rat, a dramatic direct cytotoxic effect was observed, as indicated an expression of Bcl-2 immunoreactivity in TUNEL- and OX-42-positive cells in the KA-injected striatum and traumatized cortical region. A less severe detrimental effect was also observed in the ipsilateral substantia nigra and peritraumatic cortex, as reflected by an upregulation of Bcl-2-immunostained neurons. Surprisingly, a reduction in Bcl-2-immunoreactive neurons that was accompanied by a less severe loss of tyrosine hydroxylase-immunoreactive neurons in the nigrostriatal pathway was observed after co-treatment with melatonin. Western blot analysis confirmed that Bcl-2 expression is elevated in striatum and cortex on the lesioned side, and that its expression was attenuated substantially after systemic administration of melatonin. The results showing an upregulation of Bcl-2 in nigral neurons and reactive microglia after KA lesion are consistent with the view that Bcl-2 is protective in function in the central nervous system.