[Ca2+]i regulation by glutamate receptor agonists in cultured chick retina cells

AbstractThe effect of glutamate receptor agonists on the intracellular free calcium concentration ([Ca2+]i), measured with Indo-1, was studied in populations of cultured chick embryonic retina cells. The agonists of the ionotropic glutamate receptors,N-methyl-d-aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) increased the [Ca2+]i through a composite effect, comprising Ca2+ permeating the receptor-associated channels, and Ca2+ entering through voltage-gated Ca2+ channels. Furthermore, the [Ca2+i responses to NMDA and AMPA also involved Ca2+ release from intracellular stores, which could not be mobilized by stimulation of the metabotropic receptor

On-line Detection of Glutamate Release from Culture Chick Retinospheroids

AbstractA continuous fluorometric assay was adapted to measure the release of endogenous glutamate from cultured chick retinospheroids. The results obtained with this technique are compared with the release of [3H]d-aspartate from monolayer cultures of chick retina cells. It is shown that although excitatory amino acids may be released in a Ca2+-dependent manner, most of the neurotransmitter release from cultured retina cells occurs by reversal of the glutamate transporter. The presence of extracellular Ca2+ may actually inhibit glutamate release by the cells present in the retinospheroids, or the [3H]d-aspartate release by cells in monolayers, when veratridine is the depolarizing agent. Copyright © 1996 Elsevier Science Ltd

Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 μM AMPA (α-amino-3-hydroxy-5-methyl-isoxazole-4- propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 μM [Leu31,Pro34]NPY, 300 nM NPY13–36 or 1 μM 500 nM NPY(19–23)- 1 3 4 6 31 32 34 (Gly ,Ser ,Gln ,Thr ,Ala ,Aib ,Gln )-PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 μM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid-induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor-mediated neurodegeneration in the hippocampu

Regulation of AMPA Receptors by Phosphorylation

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+ - and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity

Differential Expression of Syntaxin 1A and 1B by Noradrenergic and Adrenergic Chromaffin Cells

The expression and localization of syntaxin isoforms 1A and 1B in adrenergic and noradrenergic chromaffin cells were examined by both immunoblot analysis and confocal immunofluorescence microscopy. Syntaxin 1A was found in higher levels in noradrenergic cells, whereas syntaxin 1B was similarly expressed in most noradrenergic and adrenergic cells. However, some heterogeneity was observed within each catecholaminergic phenotype. Although the majority of adrenergic cells appeared to express low levels of syntaxin 1A, about 7% was strongly stained for syntaxin 1A. A subpopulation of noradrenergic cells, about 17%, expressed greater levels of syntaxin 1B. Syntaxin 1B labeling showed a punctate appearance in the cytoplasm, whereas syntaxin 1A appeared predominantly localized to the plasma membrane. These data show differences in the exocytotic machinery of the two subtypes of chromaffin cells that may underlie some of the distinct characteristics of adrenaline and noradrenaline secretion

Functional interaction between neuropeptide Y receptors and modulation of calcium channels in the rat hippocampus

We investigated the functional interaction between neuropeptide Y (NPY) receptors using nerve terminals and cultured rat hippocampal neurons, and we evaluated the involvement of voltage-gated Ca2+ channels (VGCCs) in NPY receptors-induced inhibition of Ca2+ influx and glutamate release. The KCl-evoked release of glutamate from hippocampal synaptosomes was inhibited by 1 [mu]M NPY and this effect was insensitive to either BIBP3226 (Y1 receptor antagonist) or L-152,804 (Y5 receptor antagonist), but was sensitive to BIIE0246 (Y2 receptor antagonist). We could also pharmacologically dissect the NPY receptors activity by using Y1, Y2 and Y5 receptor agonists ([Leu31,Pro34]NPY, NPY13-36, NPY (19-23)-(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)-pancreatic polypeptide (PP), respectively), and in all the cases we observed that these agonists could inhibited the KCl-induced release of glutamate. However, the selective and specific co-activation of both Y1 and Y2 or Y2 and Y5 receptors resulted in non-additive inhibition, and this effect was prevented in the presence of the Y2 antagonist, but was insensitive to the Y1 or Y5 receptor antagonist. Moreover, as we previously showed for Y1 receptors, we also observed that the activation of Y5 receptors inhibited the glutamate release in the dentate gyrus and CA3 subregion, without significant effect in the CA1 subregion of the hippocampus. The same qualitative results were obtained when we investigated the role of NPY Y1 and Y2 receptors in modulating the changes in [Ca2+]i due to KCl depolarisation in cultured hippocampal neurons. The inhibitory effect of nitrendipine (L-type VGCC blocker) or [omega]-conotoxin GVIA ([omega]-CgTx; N-type VGCC blocker) was not potentiated by the simultaneous activation of Y1 or Y2 receptors. Moreover, the exocytotic release of glutamate was inhibited by [omega]-agatoxin IVA ([omega]-Aga; P-/Q-type VGCC blocker), and this VGCC blocker did not potentiate Y1, Y2 or Y5 receptor-mediated inhibition of glutamate release. Also, the effect of ionomycin in inducing the exocytotic release of glutamate from hippocampal synaptosomes was insensitive to the activation of NPY receptors. In the present paper, we identified a role for NPY Y1, Y2 and Y5 receptors in modulating the exocytotic release of glutamate and the [Ca2+]i changes in the rat hippocampus. In conditions of co-activation, there appears to exist a physiological cross-talk between Y1 and Y2 and also between Y2 and Y5 receptors, in which Y2 receptors play a predominant role. Moreover, we also show that Y1 and Y2 receptors exert their inhibitory action by directly modulating L-, N-, and P-/Q-type VGCCs, whereas the inhibition of glutamate release mediated by the Y5 receptors seems to involve P-/Q-type VGCCs.http://www.sciencedirect.com/science/article/B6T0C-47RRRP0-C/1/70e1c3a1dbd6c37bea36062e48bf4a0

Ca2(+)-dependent binding of tamoxifen to calmodulin isolated from bovine brain

The interaction of the antiestrogen tamoxifen (Tx) with calmodulin (CaM) was investigated by cross-linking between the protein and [3H] tamoxifen aziridine. We observed that CaM binds Tx in a Ca2(+)-dependent manner and that two components are involved in the binding, with apparent dissociation constants (Kd) of about 6 nM and 9 microM. The high affinity binding site has a maximal capacity of 25 pmol/mg protein, whereas the low affinity binding site has a Bmax value of 120 nmol/mg protein. The stimulatory effect of Ca2+ is maximal at the pCa value of 5, and it is noncompetitively inhibited by Mg2+. In the micromolar range, the cation-dependent interaction of Tx with CaM exhibits positive cooperativity (nH = 1.4) and it is specific in the sense that it is inhibited by unlabeled Tx and by the CaM antagonist trifluoperazine. In contrast, no specificity was observed for the Tx binding, which is cation independent. Tx in the nanomolar range forms complexes with CaM which can be visualized by fluorography after electrophoretic separation in a polyacrylamide gel. Furthermore, CaM antagonism of Tx was observed with respect to inhibition of the CaM effect on the RBC membrane (Ca2(+) + Mg2+)-ATPase. The results indicate that Tx may alter Ca2(+)-dependent processes by interacting directly with Ca

Mechanisms of Action of Carbamazepine and Its Derivatives, Oxcarbazepine, BIA 2-093, and BIA 2-024

Carbamazepine (CBZ) has been extensively used in the treatment of epilepsy, as well as in the treatment of neuropathic pain and affective disorders. However, the mechanisms of action of this drug are not completely elucidated and are still a matter of debate. Since CBZ is not very effective in some epileptic patients and may cause several adverse effects, several antiepileptic drugs have been developed by structural variation of CBZ, such as oxcarbazepine (OXC), which is used in the treatment of epilepsy since 1990. (S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), which were recently developed by BIAL, are new putative antiepileptic drugs, with some improved properties. In this review, we will focus on the mechanisms of action of CBZ and its derivatives, OXC, BIA 2-093 and BIA 2-024. The available data indicate that the anticonvulsant efficacy of these AEDs is mainly due to the inhibition of sodium channel activity