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Notes: Abstract: The joint, but not independent, activation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic glutamate receptors induces liberation of arachidonic acid from cultured mouse striatal neurones. We examined whether blocking AMPA receptor desensitisation with cyclothiazide would modify this response. Cyclothiazide strongly potentiated the combined AMPA/(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-evoked release of arachidonic acid (EC50 of ∼7 µM) but did not modulate the basal, ACPD, or NMDA response. The enhanced liberation of arachidonic acid, observed in the presence of cyclothiazide, was due to the appearance of a genuine AMPA response that was independent of an associative activation of metabotropic receptors. The potentiated and nonpotentiated AMPA responses were inhibited by both competitive [2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(f)quinoxaline] and 2,3-benzodiazepine noncompetitive (GYKI 53655 and GYKI 52466) receptor antagonists. Cyclothiazide was equally effective at potentiating the AMPA response in either the presence or absence of glucose, suggesting that the increased glutamate-evoked arachidonic acid release observed in these cells under conditions of glucose deprivation is not due to reduced AMPA receptor desensitisation. The enhanced liberation of arachidonic acid measured in the presence of cyclothiazide appeared to result from a large (fourfold) elevation of the AMPA-induced increase in intracellular calcium level. Therefore, an AMPA-evoked mobilisation of arachidonic acid could potentially contribute to non-NMDA receptor-mediated neurotoxicity, which has been observed in neuronal cells in the presence of cyclothiazide.

Notes: As shown by autoradiography, peripheral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a dose-dependent decrease of [3H]SCH 23390 and [3H]prazosin high-affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin-sensitive α1-adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA-sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA-sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA-sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (-20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%. Demonstrating further a role of noradrenergic transmission of the α1 type in the regulation of the cortical D1 receptor sensitivity, the blockade of α1-adrenergic receptors by prazosin prevented the appearance of the EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase in groups of rats exhibiting similar decreases in the density of D1 binding sites (-20%). The DA-sensitive adenylate cyclase activity was significantly lower in cortices of animals pretreated with prazosin than in those not treated with the α1 antagonist. These results are discussed in relation to previous studies indicating that prazosin pretreatment also abolishes the locomotor hyperactivity induced by bilateral electrolytic lesions of the ventral teg-mental area.

Notes: : Striatal neurons from the mouse brain embryo grown in primary culture express high levels of cyclic AMP (cAMP)-dependent protein kinase (PK A) activity. To study the modulation of PKA in intact neurons, a rapid method based on Zn2+-protein precipitation was developed. This strategy allowed analysis of the stimulation of PKA under conditions of intracellular cAMP concentration increases. Whereas increases up to 1 μM lead to an activation, large and sustained accumulations of cAMP result in a loss of the enzyme activity. With 8-bromo-cAMP (8-Br-cAMP) at 100 μM, the PKA refractoriness occurs within 2 min. It is rapidly reversible because incubation of treated neurons in fresh medium leads to a complete recovery of PKA activity within 30 min. The decrease in assayable PKA does not involve an activation of phosphatases because the histone dephosphorylation rate is not affected by 8-Br-cAMP treatment Finally, not only 8-Br-cAMP-but also forskolin-and vasoactive intestinal peptide-induced increases in intracellular cAMP concentration can lead to the PKA desensitization. Altogether, these data unravel a new mechanism of PKA regulation.

Notes: By means of the push-pull cannula method, the outflow of endogenous amino acids was studied in the striatum of halothane-anesthetized rats. Addition of K + ions (30 mM for 4 min) to the superfusion fluid increased the release of aspartate (+116%), glutamate (+ 217%), taurine (+109%), and γ-aminobutyric acid (GABA) (−429%) whereas a prolonged decrease in the outflow of glutamine (−28%) and a delayed reduction in the efflux of tyrosine (−25%) were observed. In the absence of Ca2-, the K+-induced release of aspartate, glutamate, and GABA was blocked whereas the K + -induced release of taurine was still present. Under these conditions, the decrease in glutamine efflux was reduced and that of tyrosine was abolished. Local application of tetrodotoxin (5 μM) decreased only the outflow of glutamate (-25%). One week following lesion of the ipsilateral sensorimotor cortex the spontaneous outflow of glutamine and of tyrosine was enhanced. Despite the lack of change in their spontaneous outflow, the K +-evoked release of aspartate and glutamate was less pronounced in lesioned than in control animals, whereas the K + -evoked changes in GABA and glutamine efflux were not modified. Our data indicate that the push-pull cannula method is a reliable approach for the study of the in vivo release of endogenous amino acids. In addition, they provide further evidence for a role for glutamate and aspartate as neuro-transmitters of corticostriatal neurons.

Notes: Push-pull cannulae were implanted in both substantiae nigrae and caudate nuclei of the halothane-anesthetized cat. The release of total protein, acetylcho-linesterase, and nonspecific cholinesterases was examined. Following direct application of potassium to one substantia nigra, changes occurred in the local release of total protein and acetylcholinesterase, but not nonspecific cholinesterases; changes also were observed in both caudate nuclei and the contralatera/ substantia nigra. The local evoked release of acetylcholinesterase and of total protein differed in the extent to which they were calcium-dependent. Control studies suggest that release of these compounds, both spontaneous and evoked, is related, at least in part, to neuronal activity. The significance of the neuronal release of proteins is discussed.

Notes: Abstract: l-Glutamate stimulates the liberation of arachidonic acid from mouse striatal neurons via the activation of N-methyl-d-aspartic acid (NMDA) receptors and by the joint stimulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic receptors. In this study, we investigated whether starving cultured mouse striatal neurons of glucose would modify glutamatergic receptor-mediated arachidonic acid release. Glucose deprivation for 30 min led to enhancement of the NMDA-evoked release of arachidonic acid, compared with that observed in the presence of glucose. This enhanced response depended on both the concentration of glucose and the length of time of glucose deprivation. The enhanced NMDA response appeared to result from both a release of glutamate and the subsequent additional release of arachidonic acid due to the activation of AMPA and metabotropic receptors. Indeed, the increased NMDA response was completely reversed when extracellular glutamate was enzymatically removed. Moreover, glucose deprivation potentiated the combined AMPA/metabotropic receptor-evoked release of arachidonic acid, even in the absence of extracellular glutamate. However, removing glucose did not improve the calcium rise induced by AMPA or NMDA. The ATP-evoked release of arachidonic acid from striatal astrocytes was not altered by glucose starvation. In summary, glucose deprivation affected two properties of striatal neurons: (a) it induced an NMDA-evoked release of glutamate from striatal neurons and (b) it selectively potentiated the AMPA/(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-evoked release of [3H]arachidonic acid without altering the authentic NMDA-mediated response.

Notes: Abstract: PEA-15 has recently been identified as a major phosphoprotein in astrocytes and an endogenous substrate for protein kinase C. This 15-kDa protein exists under three molecular forms, an unphosphorylated form, N, and two phosphorylated forms, Pa and Pb. Ȧntisera were raised against synthetic peptides corresponding to the internal sequences of the mouse protein containing the two specific phosphorylation sites and affinity-purified antibodies were used for immunoblotting. PEA-15 was found mainly in the cytosol, but its protein kinase C-phosphorylated form, Pb, was also detectable in association with the membrane and remained with the fraction that contains stabilized microtubules. Abundant in astrocytes, particularly in the hippocampus, PEA-15 was also detected in all cultured brain cell types examined, indicating a more ubiquitous distribution of the protein, further demonstrated by its detection in the eye and in the lung. Parallel to the increase in expression levels, phosphorylation of PEA-15 also increased during development. This paralleled results obtained in primary cultures, where PEA-15 levels increase with cell maturation. Finally, physiological importance of PEA-15 phosphorylation was illustrated by immunoreactivity observed in brain homogenates of different mammals, birds, amphibians, and fish.

Notes: The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]- substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihy-dropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyper-polarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KC1 canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.