Dithiotreitol specifically inhibits metabotropic responses of glutamate and depolarizing agents in rat brain synaptoneurosomes (original) (raw)
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Journal of Neurochemistry
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
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-
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.
Characterization of the cholinergic stimulation of phosphoinositide hydrolysis in rat brain slices
The Journal of Neuroscience, 1984
The stimulation of the formation of inositol phosphates by various cholinergic agonists and antagonists was studied in rat brain cortical slices. Incubation of the slices with [3H]inositol led to the incorporation of radioactivity into inositol lipids. The accumulation of inositol phosphates was then followed in the presence of 8 mM lithium which blocks the hydrolysis of inositol phosphate. The release of inositol phosphate was linear up to 15 min when stimulated by 1 mM carbachol. Acetylcholine, muscarine, and methacholine also stimulated the release of inositol phosphates with about the same efficacy as carbachol. Oxotremorine, arecoline, pilocarpine, and bethanechol were not as effective as carbachol at stimulating the accumulation of inositol phosphates. Indicative of partial agonist activity, oxotremorine and pilocarpine inhibited the maximal response induced by carbachol. Muscarinic antagonists atropine, scopolamine, and pirenzepine blocked the stimulation by acetylcholine in contrast to nicotinic antagonists, which had no effect. The brain regional response to carbachol-stimulated inositol phosphate release varied widely with large responses observed in the striatum, cerebral cortex, and hippocampus. Smaller responses were seen in the brainstem, hypothalamus, and cerebellum. Although carbachol stimulated inositol phosphate release in cortical slices in the absence of added calcium, EGTA completely blocked the response. These results suggest that the previously characterized stimulation of the incorporation of "'Pi into phosphatidylinositol by cholinergic agonists in synaptosomes (Fisher, S. K., P. D. Klinger, and B. W. Agranoff (1983) J. Biol. Chem. 258: 7358-7363) is due to the initial hydrolysis of inositol lipids. This response may be coupled to the Ml subtype of muscarinic receptor and may represent the initial step in an intraneuronal cascade involving the activation of phospholipid-calcium-dependent protein kinase by the liberated diacylglycerol.
Neurochemical Research, 1991
The relatively recent discovery that excitatory amino acids (EAAs), besides their known action as fast synaptic transmitters via the opening of receptor-associated ion channels, also exert their effects by stimulating the phosphoinositide breakdown (1,2) has required a redefinition of the number and the nature of EAAs receptor subtypes and has opened the way for new roles in brain physiology and pathophysiology (for a review, see 3). In addition to the ionotropic receptor types, namely the N-methyl-D-aspartate (NMDA), the RS,x-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), also called quisqualate/kainate and the kainate (KA) subtypes (4,5), the existence of two metabotropic receptors has been proposed (for a review, see 6). The first one is preferentially activated by quisqualate (QA), not blocked by any of the EAA antagonists of the ionotropic receptors (7,8) and is tentatively named for the moment the quisqualate metabotropic receptor (QAm). The second one preferentially binds ibotenate (IBO), is inhibited by 2-amino-4-phosphonobutyrate (L-AP4) (2,9) and is referred to in this paper as the IBO metabotropic receptor (IBOm). It must be emphasized that the activation of the ionotropic NMDA receptor may also lead to an enhanced phosphoinositide hydrolysis . The transduction machinery, which starts up with the receptor activation to yield the second messengers, inositol phosphates (IPs) and diacylglycerol (DAG), is a multistep mechanism (for reviews, see 10-12). One of these steps is thought to
The Journal of Physiology, 1992
The cellular mechanism by which acetylcholine (ACh) potentiates neuronal responses to N-methyl-D-aspartate (NMDA) was investigated in CAI neurones of hippocampal slices using current-and voltage-clamp techniques. 2. Loading cells with 5'-guanylylimidodiphosphate (GppNHp) caused a gradual increase in response to NMDA. Pulses of ACh accelerated this increase. Guanosine 5'-0-(2-thiodiphosphate) (GDP,/S) blocked the potentiating effect of ACh on responses to NMDA. 3. Acute LiCl caused a gradual decrease in the potentiating effect of ACh, while the potentiation was completely prevented by 3 day chronic 6 mequiv/kg (i.P.) LiCl treatment and restored by acute treatment with 10 mM-inositol. 4. Loading cells with a general protein kinase inhibitor, H-7, enhanced the potentiating effect of ACh on responses to NMDA and blocked the effect of ACh on the after-hyperpolarization (AHP). 5. Ultraviolet irradiation of cells loaded with a photolabile inositol 1,4,5trisphosphate (InsP3) caused a transient increase in responses to NMDA, while penetrating cells with active InsP3-containing pipettes caused a gradual BAPTAsensitive increase in responses to NMDA. 6. Reducing the rate of InsP3 metabolism, with 2,3-diphosphoglyceric acid (DPG), caused an increase and prolongation of the potentiating effect of ACh, while blocking the InsP3 receptor with heparin prevented the cholinergic potentiation. 7. NMDA, by itself, potentiated subsequent responses to NMDA, an effect that was blocked when [Ca2+]i was chelated with BAPTA. NMDA and ACh were also found to compete in potentiating responses to NMDA. Finally, the cholinergic potentiation was blocked when cells were loaded with BAPTA. 8. We propose that activation of the InsP3 branch of the phosphoinositide pathway potentiated responses to NMDA and that InsP3 exerted this effect by elevating [Ca2+]j.
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.
Inositol hexakisphosphate-mediated regulation of glutamate receptors in rat brain sections
Hippocampus, 2001
D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), one of the most abundant inositol phosphates within cells, has been proposed to play a key role in vesicle trafficking and receptor compartmentalization. In the present study, we used in vitro receptor autoradiography, subcellular fractionation, and immunoblotting to investigate its effects on ␣-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and Nmethyl-D-aspartate (NMDA) receptors. Qualitative and quantitative analysis of 3 H-AMPA binding indicated that incubation of frozen-thawed brain sections with InsP6 at 35°C enhanced AMPA receptor binding in several brain regions, with maximal increases in the hippocampus and cerebellum. Moreover, saturation kinetics demonstrated that InsP6-induced augmentation of AMPA binding was due to an increment in the maximal number of AMPA binding sites. At the immunological level, Western blots performed on crude mitochondrial/synaptic (P2) fractions revealed that InsP6 (but not InsP5 and InsP3) treatment increased glutamate receptor (GluR)1 and GluR2 subunits of AMPA receptors, an effect that was associated with concomitant reductions in microsomal (P3) fractions. Interestingly, the InsP6-induced modulation of AMPA receptor binding was blocked at room temperature, and pretreatment with heparin also dampered its action on both AMPA receptor binding and GluR subunits. These effects of InsP6 appear to be specific to AMPA receptors, as neither 3 H-glutamate binding to NMDA receptors nor levels of NR1 and NR2A subunits in P2 and P3 fractions were affected. Taken together, our data strongly suggest that InsP6 specifically regulates AMPA receptor distribution, possibly through a clathrin-dependent process. Hippocampus 2001;11:673-682.
Effect of Thiol Reagents on Phosphoinositide Hydrolysis in Rat Brain Synaptoneurosomes
European Journal of Neuroscience, 1993
Some divalent ions, such as Cd2+ and Zn2+, are able to stimulate phosphoinositide (PI) breakdown and to inhibit receptor-mediated PI metabolism. These ions are also known to react with the free -SH groups of proteins. This prompted us to investigate the effects of more potent sulphhydryl reagents, Hg2+ and p-chloromercuric benzosulphonic acid (PCMBS), on the inositol phosphate (IP) accumulation triggered by the neuroactive substances: glutamate, carbachol and K + , using synaptoneurosomes from 8-day-old rat forebrains. Hg2+ and PCMBS, depending on their concentration, had two distinct effects on IP accumulation: at low doses, Hg2+ (from 1 to 10 pM) and PCMBS (0.1 mM) by themselves stimulated PI breakdown, inhibited glutamate-elicited IP accumulation and had additive effects with respect to carbachol-induced IP stimulation. At higher doses, Hg2+ (from 0.01 to 1 mM) inhibited both basal and neuroactive substance-stimulated IP accumulation. PCMBS (1 mM), provoked only an inhibition of the agonist-stimulated IP formation. Monitoring membrane potential and intracellular Ca2+ with the fluorescent dyes diSC2(5) and fura2, respectively, indicated that these mercurials could strongly depolarize the synaptoneurosomal membrane and produce a Ca2+ influx dependent on extracellular Ca2+. The stimulatory effects of low concentrations of mercurials on PI turnover could be linked to the depolarization they provoke and the subsequent Ca2+ rise, which in turn is known to stimulate some phospholipase C enzymes. The inhibitory effects observed at high concentrations might be due to a loss of activity of proteins involved in PI breakdown, as all receptor-mediated IP accumulations were inhibited.