A specific transduction mechanism for the glutamate action on phosphoinositide metabolism via the quisqualate metabotropic receptor in rat brain synaptoneurosomes: I. External Na+ requirement (original) (raw)
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Journal of Neurochemistry, 1991
In this article, we demonstrate that an increase in intracellular Ca2+ concentration may represent a specific common step(s) in the mechanism@) of action of glutamate (Glu) and depolarizing agents on formation of inositol phosphates (IPS) in 8-day-old rat forebrain synaptoneurosomes. In fact, A23 187, a Caz+ ionophore, induces a dose-dependent accumulation of IPS, which is not additive with that evoked by Glu and K+ but is slightly synergistic with that induced by carbachol. In addition, Glu and K+ augment the intracellular Ca2+ concentration in synaptoneurosome preparations as measured by the fura-2 assay. The absence of external Ca2+ decreases basal and Glu-, and K+-stimulated formation of Ips. Cd2+ (100 p M ) fully inhibits both Glu-and K+-evoked formation of IPS without affecting the carbachol-elicited response of IPS. Zn2+ inhibits Glu-and K+-stimulated accumulation of IPS (IC50 -0.4 mM) but with a lower affinity than Cd2+ (IC50 -0.035 mM). The organic Ca" channel
Journal of Neurochemistry, 1991
The characteristics of the transduction mecha-nism@) activated by glutamate (Glu) via the quisqualate metabotropic receptor, as well as by depolarizing agents, to trigger formation of inositol phosphates (IPS) were investigated in S-day-old rat forebrain synaptoneurosomes. The replacement of external Na+ by various compounds (Li+, Tris+, N-methyl-D-glucamine+, and sucrose) induces an increase in basal accumulation of IPS and depolarizes synaptoneurosome membranes. Under these conditions, Glu-and K+-induced accumulations of IPS are inhibited, whereas the carbachol (Carb)-elicited response of IPS parallels the basal one. Agents increasing Na+ influx, such as veratridine and monensin, de-
European Journal of Neuroscience, 1995
The regulation of intracellular Ca2+ concentration ([Ca2+Ii) by glutamate rnetabotropic receptors (mGluR) was studied in 8-day-old rat forebrain synaptoneurosomes using spectrofluorirnetric methods. Here we demonstrate that metabotropic glutamate agonists induce in rat brain synaptoneurosornes a Ca2+ influx largely dependent upon the presence of Ca2+ in the external medium. The pharmacological profile of this influx is strongly correlated with the pharmacological profile of the activation of phosphoinositide hydrolysis, i.e. quisqualic acid > > 1 S,BR-amino-l -dicarboxylate-l,3 cyclopentane = glutamate. This rnetabotropic glutamate receptor-induced Ca2+ influx is insensitive to voltage-dependent Ca2+ channel antagonists and occurs through a Mn2+ impermeant pathway. The study of the rapid kinetics shows that this influx is triggered after a 300 ms delay compared with that elicited by depolarizing agents and Ca2+ ionophore A23187. In order to assess further if mGluR stimulate this influx through the recruitment of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores, we have tested the effect of thapsigargin on membrane potential and intracellular Ca2+ simultaneously. Thapsigargin induces a depolarization of the synaptoneurosomal membrane followed by a massive Ca2+ influx, occurring via a Mn2+ nonpermeant route. This depolarizing effect is sensitive to the presence of the intracellular Ca2+ chelator [I ,2-bis(2-aminophenoxy)ethane-N,N,N',~-tetraacetoxymethyl ester], and partially sensitive to extracellular Na+, but insensitive to the presence of extracellular Ca2+. Taken together, our data suggest that mGluR stimulate self-maintained increases of [Ca2+Ii in rat forebrain synaptoneurosornes via the activation of a multistep mechanism, sequenced in the following steps: (i) mGluR-induced IP3 synthesis; (ii) IP3-stimulated intracellular Ca2+ release; (iii) Ca2+-activated non-specific cation channel, leading to local depolarization and a Ca2+ influx; and (iv) activation of Ca2+-sensitive phospholipase C.
Journal of Neurochemistry, 1990
The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 m M sodium and various concentrations of added calcium (0-1 .O mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 'H-inositol phosphates decreased with decreasing extracellular calcium. Potassium-and norepinephrine-stimulated formation of 'H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or Nmethyl-D-glucamine to disrupt Na'/Ca2+ exchange resulted in a large increase in the formation of 'H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 'H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3Hinositol phosphates from that in the presence of 1 mM carbachol or I 0 0 jd4 norepinephrine revealed that the carbacholstimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-0-tetradecanoylphorbol 13-acetate inhibited norepinephrine-and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 'H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis. These results also support previous evidence of two separate and distinct pathways for stimulating phosphoinositide-linked phospholipase C (PLC) activity in brain: One pathway appears to involve a phosphoinositideassociated guanine nucleotide binding protein-PLC coupling process, and the other a direct activation of PLC by an increase in intracellular calcium.
Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown
Neurochemistry International, 1996
The dependence on Ca 2+ of basal, glutamate-and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca 2+ concentrations were buffered by bis- (~-aminophenoxy)-ethane-N,N,N',N'-tetraacefic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca 2+ buffering on intracellular and extracellular Ca 2+ concentration i and [CaZ+]e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca 2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca2+]~. Basal inositol phosphate (IP) formation was slightly affected by this [CaZ+]~ buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca 2+ ions with BAPTA produced a reduction of both [Ca2+]e and [Ca2+]i. Basal IP accumulation was maximally inhibited by 50%. The carbachoMnduced PI hydrolysis was completely inhibited in the presence of 200 #M BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca:+]~ of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca2+]e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca2÷-insensitive way.
A modulation of glutamate-induced phosphoinositide breakdown by intracellular pH changes
Neuropharmacology, 1997
The influence of intracellular pH (pHi) changes on the formation of inositol phosphate metabolites (IPs) produced by glutamatergic stimulation was studied in g-day-old rat brain synaptoneurosomes. For this purpose pHi was .measured using 2',7'-bis-(2-carboxyl)-5,6-carboxyfluorescein (BCECF) fluorimetric assay in parallel with the basal and receptor-mediated formations of inositol monophosphate (IPl) and inositol bisphosphate (IP2:). We found that glutamate (1 mM), which induces a transient acidification (ApH = -0.05), produces an identical accumulation of IPl and IP2. K+ (30 mM), which provokes an alkalinization of the internal medium (ApH = +0.22), mainly leads to the formation of IPl metabolites. Paired combinations of glutamate with 1,5 and 10 mM NH4+ finally result in an alkalinization of the intrasynaptoneurosomal medium. These combinations produce a strong decrease of the IP2 level concomitant with an increase of the IPl formation, compared to the levels of IPl and IP2 evoked by glutamate alone. The total amount of IPs (IPl+IP2) produced by these combinations is not different from that obtained with glutamate alone. Paired combinations of carbachol with NH4+ produce an identical alkalinization to that produced by NH4+ alone. These combinations produce an increased IPl accumulation, while the IP2 formation is slightly decreased. When the internal medium lis acidified by dimishing the external concentration of Na+, the ratio IPl/IP2 produced after metabotropic glutamate receptor (mGluR) activation is shifted to lower values, while it is not affected for the muscarinic stimulation. These data suggest that the mGluR-associated pathway in synaptoneurosomes is sensitive to pHi shifts, while the muscarinic receptor-associated pathway is less altered when pHi is manipulated. It may be proposed that pH-sensitive inositol phosphate dephosphorylating systems, i.e. phosphatases, are associated with mGluRs in this preparation.
Journal of Neurochemistry, 1993
L-Glutamate(3-1,000pM)and(IS,3R)-l-aminocyclopentane-I ,3-dicarboxylic acid (I S.3R-ACPD: 10-1.000 p M) , a selective agonist for the metabotropic glutamate receptor, stimulated the formation of inositol 1,4.5trisphosphate in a concentration-dependent manner. L-Glutamate was half as efficacious as IS,3R-ACPD. Nmethyl-D-aspartate (NMDA: I n M to 1 mM) did not significantly influence the response to a maximally effective concentration of IS.3R-ACPD (100 p M). On the other hand, coapplication of (R S)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA: 1-300 nbf) produced a concentration-and time-dependent inhibition of the 1S.3R-ACPD effect. with a maximal inhibition (97%) at 100 nM. Ten micromolar 6-cyano-7-nitroquinoxaline-2.3-dione. an antagonist of the AMPA receptor, blocked the inhibitory effect of AMPA. Reduced extracellular calcium concentration. as well as 10 p M nimodipine, an L-type calcium channel antagonist, inhibited the AMPA influence on the IS.3R-ACPD response. W-7, a calcium/calmodulin antagonist, prevented the inhibition by AMPA. whereas H-7. an inhibitor of protein kinase C, had no effect. These data suggest that activation of AMPA receptors has an inhibitory influence on inositol 1.4.5-trisphosphate formation mediated by stimulation of the metabotropic glutamate receptor. The mechanism of action involves calcium influx through Ltype calcium channels and possible activation of calcium/calmodulin-dependent enzymes. Key Words: 1-Aminocyclopentane-1.3-dicarboxylic acid-Desensitization-H-7-Hippocampus-Ar-Methyl-~aspartate-W-7. Lonart G. et al. (R.S)-ru-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors mediate a calciumdependent inhibition of the metabotropic glutamate receptor-stimulated formation of inositol 1,4,5-trisphosphate.
European Journal of Pharmacology, 1997
w 3 x The effect of glutamate on the accumulation of H inositol phosphates was examined in oligodendrocyte progenitor cultures prepared Ž . from rat brains. Glutamate, and the analogues a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid AMPA and kainate, caused a w 3 x Ž . concentration-and time-dependent increase in H inositol trisphosphate IP formation and the effect was blocked by 6-cyano-7-nitro-45 2q channel blockers, diltiazem, nifedipine and CdCl , partially prevented the glutamate-induced H IP accumulation as well as Ca 2 3
Experimental Brain Research, 1989
The Ca 2+ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca 2+, ([Ca2+]i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca 2 +, suggesting that L-glutamate promotes mobilization of Ca 2 + from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca2+]i was, in part, mediated by an increase in the plasma membrane permeability to Ca 2+. This Ca 2+ influx was not affected by the CaZ+-channel antagonist 1-Verapamil. However, L-Verapamil did block the increase in [Ca2+]i seen after depolarization of the cells with potassium. The Ca 2 + response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris-and tetrakisphosphates (IPI, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca 2 +]i.