ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09792.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—The alkylating agentN‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) is a peptide‐coupling agent that is being used to inactivate irreversibly α2‐adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l2‐imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10−8‐10−5M) markedly decreased (20–90%) the specific binding of the selective antagonist [3H]R821002 to α2‐adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to l2‐imidazoline sites. In EEDQ‐pretreated membranes (10−5M, 30 min at 25°c), the density of l2‐imidazoline sites (Bmax= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10−6M(‐)‐adrenaline (Bmax= 83 ± 4 fmol/mg of protein), and both densities were lower (24%,p<0.05) than the total native density of [3H]idazoxan binding sites (Bmax= 107 ± 6 fmol/mg of protein) (l2‐imidazoline sites plus a2‐adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [3H]‐R821002 to α2‐adrenoceptors, but also the binding of [3H]idazoxan to l2‐imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ‐induced α2‐adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ‐induced inactivation of l2‐imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase‐A sites labeled by [3H]Ro 41–1049 or that of monoamine oxidase‐B sites labeled by [3H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [3H]idazoxan to l2‐imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ‐pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l2‐imidazoline sites when using [3H]‐imidazoline ligands that also recognize α2‐adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l2‐imidazoline sites probably by an indirect mechanism. Key Words: Brain l2‐imidazoline sites—[3H]‐Idazoxan—α2‐Adrenoceptors—[3H] R821002—N‐Ethoxyc

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09793.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—The present study was designed to examine the effects of chronic cocaine administration on the extracellular response of serotonin (5‐HT) and dopamine (DA) to a peripheral cocaine injection using in vivo brain microdialysis in awake rats. Two different dual probe preparations were used: One group of animals had guide cannulae aimed at the ventral tegmental area (VTA) and nucleus accumbens (N ACC) and a second group of animals had guide cannulae aimed at the dorsal raphe nucleus (DRN) and N ACC. Rats from both groups were given daily injections of either cocaine (20 mg/kg i.p.) or saline (0.9%; 0.05 ml/kg i.p.) for 10 consecutive days. On day 11, baseline dialysate levels of DA, 5‐HT, dihydroxyphenylacetic acid, and 5‐hydroxyindoleacetic acid were obtained from either the N ACC and VTA or the N ACC and DRN, followed by a 10 mg/kg i.p. cocaine injection and an additional 150 min of dialysate sampling. The percent baseline increases of both 5‐HT and DA were significantly higher in the N ACC, VTA, and DRN of animals that received daily injections of cocaine compared with saline controls (p<0.05, in each region). Maximum dialysate 5‐HT concentrations after cocaine challenge were significantly higher in the N ACC and VTA (p<0.05) and DRN (p<0.01) of chronically treated animals compared with saline controls. Maximum dialysate DA concentrations were significantly higher in the N ACC and DRN (p<0.05) of chronically treated animals compared with saline controls. There was no significant difference between acute and chronic animals in the maximum dialysate DA concentration from the VTA after cocaine challenge. 5‐HT was significantly more sensitized in the 5‐HT cell body region (DRN) than the N ACC terminal field (p<0.05), whereas DA was significantly more sensitized in the N ACC terminal field than the DA cell bodies of t

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09794.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—Chronic administration of ethanol results in the development of tolerance and dependence. The molecular mechanism underlying these behavioral actions of ethanol is poorly understood. Several lines of evidence have suggested that some of the pharmacological actions of ethanol are mediated via a potentiation of GABAergic transmission. Chronic ethanol administration results in a reduction in the GABAAreceptor‐mediated36Cl−uptake in cortical synaptoneurosomes and primary cultured neurons. We and others have shown that it also results in a 40‐50% reduction in GABAAreceptor α‐subunit mRNA levels in the rat cerebral cortex. In the present study, we investigated the expression of α1, α2, and α3subunits of the GABAAreceptor in the cerebral cortex and the α1subunit in the cerebellum by immunoblotting using polyclonal antibodies raised against α1‐, α2‐, and α3‐subunit polypeptides following chronic ethanol treatment. These results reveal that chronic ethanol administration to rats results in a 61 ± 4% reduction in level of the GABAAreceptor α1subunit (51 kDa), 47 ± 8% reduction in level of the α2subunit (53 kDa), and 30 ± 7% reduction in level of the α3subunit (59 kDa) in the cerebral cortex and a 56 ± 5% reduction in content of the α1subunit in the cerebellum. In summary, this ethanol‐induced reduction in content of the GABAAreceptor α subunits may underlie alterations in the GABAAreceptor function and could be related to cellular adaptation to the functi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09795.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—Recent studies have identified at least two homologous mitogen‐activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e.,trans‐(±)‐1‐amino‐1,3‐cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)‐α‐(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not byN‐methyl‐d‐aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABAAreceptor‐mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by e

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09796.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—The presynaptic regulation of stimulated dopa‐mine release from superfused rat striatal synaptosomes by opioids and γ‐aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D2autoreceptors, calcium‐dependent K+‐stimulated [3H]dopamine release was inhibited through activation of a homogeneous population ofk‐opioid receptors in view of the potent inhibitory effect of thek‐selective agonist U69.593 (EC500.2 nM) and its antagonism by norbinaltorphimine. Neither μ‐nor δ‐selective receptor agonists affected release of [3H]‐dopamine. In addition, GABA potently inhibited the evoked [3H]dopamine release (EC500.4 nM) through activation of GABAAreceptors in view of the GABA‐mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro‐toxin and bicuculline, and the absence of an effect of the GABABreceptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release‐inhibitory effect, indicating that these receptors and the presynaptic D2receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonistsN‐methyl‐d‐aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [3H]dopamine release, which was subject tok‐opioid receptor‐mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally cont

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09797.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—The levels of hydroxyl radicals and oxidized GSH have been examined as indices of oxidative stress in young (3 months), middle‐aged (15 months), and old (20–24 months) gerbil brain hippocampus, cortex, and striaturn. The hydroxyl radical stress was estimated by measuring the salicylate hydroxyl radical trapping products 2,5‐and 2,3‐dihydroxybenzoic acid. The stress was significantly higher in all three brain regions in middle‐aged and old gerbils versus young animals (66.0%). Regional comparisons showed that the stress was significantly higher in cortex than in either the hippocampus or striatum of the middle‐aged and old gerbils (32.0%). The ratio of oxidized to total GSH also increased progressively in middle‐aged and old animals in all three brain regions (p<0.05, 41.1%), further indicating a general age‐related increase in oxidative stress. Parallel to this age‐related increase in oxidative stress, a significant, albeit slight (8%), decrease in neuronal number in hippocampal CA1 region was observed in both the middle‐aged and old animals. Possible differences in antioxidant levels were also examined. Total GSH levels were similar across age groups (variance<12%). However, the regional comparison showed that it was highest in striatum in all age groups. The levels of a‐tocopherol (vitamin E) were significantly higher in the middle‐aged and old animals in all three regions (70.4%). Vitamin E was highest in the hippocampus and the differences between the hippocampus and the cortex and striatum increased with age. Although of a lesser magnitude, significant increases in hippocampal total ascorbic acid level were also noted with age (pstriatum for all age groups. The difference in ascorbic acid level between hippocampus and cortex also increased with age (64.4%). The results suggest that the general age‐related, regionally specific increases in oxidative stress stimulate the accumulation of antioxidants. It is interesting that the hippocampus, which is selectively vulnerable to various insults such as ischemia, epilepsy, and insulin‐induced hypoglycemia, exhibits the greatest age‐related increase in vitamin E and ascorbic acid, perhaps reflective of a greater impact of the progressive increa

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09798.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—The mRNA levels of secretogranin II, chromo‐granin B, and VGF were compared in brains of control and AF64A‐treated rats. This toxin induces specific lesions of the septohippocampal cholinergic pathway. As a consequence of this treatment, the Chromogranin B message was elevated in the dentate gyrus granule cells of the hippocampus. In the paraventricular nucleus of the hypothalamus, a concomitant elevation of the messages of secretogranin II and corticotropin‐releasing factor occurred in the parvocellular neurons, and an increase of those of secretogranin II and VGF occurred in a subgroup of magnocellular neurons. Further increases for secretogranin II were seen in the amygdaloid nuclei and the reticular thalamic nuclei and increases for Chromogranin B in the temporal cortex, substantia nigra compacta, and ventral tegmental area. These results indicate that the toxin‐induced lesion of the cholinergic pathway innervating the hippocampus apparently leads to the stimulation of several defined groups of neurons that react with an increase in the mRNA levels of their secretory peptides. We suggest that changes in mRNA expression of these peptides are useful parameters for defining neurons under chronic stimulation. Key Words: Secretory peptides—Large dense core vesicles—Corticotropin releasing factor—Septohippocampal cholinergic system—

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09799.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2‐type pur‐inergic receptor. ATP‐induced norepinephrine release was inhibited 80% when extracellular Ca2+was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2‐methylthio‐ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP‐induced secretion was inhibited by Mg2+, and this inhibition was overcome by the addition of excess ATP suggesting that ATP4‐was the active ligand. ATP‐induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12‐O‐tetradecanoylphorbol 13‐acetate. The stimulatory effects of 12‐O‐tetradecanoyl‐phorbol 13‐acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α‐32P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP‐γS and BzATP but not GTP blocked labeling of the proteins by [32P]BzATP. Labeling of the 50‐kDa protein was more sensitive to competition by 2‐methylthio‐ATP than the other labeled proteins, suggesting that the 50‐kDa protein represents the P2receptor responsib

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09800.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY

摘要:

Abstract—The glutathione level and the factors affecting this level were investigated in fetal rat brain cells in a primary culture. Early in the culture, the glutathione level of the brain cells decreased, but after 5 h it began to increase. This increase was not observed in a cystine‐free medium and was prevented by excess glutamate. Cystine was taken up in freshly isolated brain cell suspensions, and its rate increased during the culture. The cystine uptake was mediated by a Na+‐independent, glutamate‐sensitive route previously found in various types of cells and designated as system x−c. The uptake of cystine is a crucial factor in maintaining the glutathione level of the cells under culture, because it provides cysteine for the cells for glutathione synthesis. Cysteine was undetectable in the medium before the culture, but it appeared, though at a very low level, when the brain cells were cultured there. The source of this cysteine was the cystine in the medium. Presumably the decrease in the glutathione level of the cells in the early stage of the culture resulted from the fact that the medium did not contain cysteine. The enhancement of the cystine uptake during culture may constitute a protective mechanism against the oxidative stress to which the cultured cells are exposed. Regulation of the glutathione level in fetal brain cells in vivo by the transport of cystine and cysteine is

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1993.tb09801.x

出版商:Blackwell Publishing Ltd    

年代:1993

数据来源: WILEY