Biomedical subjects

J Bockaert

Publications and source records attributed to J Bockaert.

At least 163 records · Page 9Linked to original sources

PubMed1992

The metabotropic glutamate receptor (MGR): pharmacology and subcellular location.

A pharmacological characterization of the metabotropic glutamate receptor (MGR) was performed in striatal neurons. Among the excitatory amino acid receptor antagonists tested, only D, L-2-amino-3-phosphonopropionate (D, L-AP3) inhibited QA-induced inositol phosphate (InsP) formation in a competitive manner (mean pKi = 4.45 +/- 0.43, n = 4). However, this drug was a partial agonist of MGR since it stimulated the inositol-phosphate formation. We found that D, L-AP3 also inhibited NMDA-induced calcium increase, in a competitive manner (mean pIC50 = 4.34 +/- 0.22, n = 8, and mean pKi = 3.7 +/- 0.11 n = 5). 1 mM of the ionotropic agonists alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA) or domoate (DO) (100 microM or higher) induced a significant InsP formation in striatal neurons. The InsP responses induced by all these agonists were totally blocked by the phorbol ester phorbol-12,13-dibutyrate (PdBu), but not by atropine or prazosin. Agonist-induced increases of intracellular calcium concentrations ([Ca2+]i) were insensitive to PdBu, suggesting that all these substances were able to stimulate the MGR in striatal neurons. Trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD) evoked dose-dependent inositol phosphate formations with an EC50 of 29 microM but had no significant effect on NMDA or AMPA receptors, as measured by the patch clamp technique. In the presence of 30 microM of AMPA, trans-ACPD induced a significant release of arachidonic acid (AA) in striatal neurons. No important AA release was observed by any of these agonists alone. 56 mM K+ did not mimic AMPA in this associative ionotropic/metabotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.

3T3 Cells↗

PubMed1992

Sodium nitroprusside blocks NMDA receptors via formation of ferrocyanide ions.

The effects of a nitric oxide (NO) donor, sodium nitroprusside (SNP), on N-methyl-D-aspartate (NMDA) receptors were assessed by optical measurements of intracellular calcium concentration ([Ca2+]i) and patch-clamp techniques in cultured central neurons. SNP selectively blocked NMDA-mediated currents and increases in [Ca2+]i. SNP inhibited the binding of [3H]-CGS 19755. The blockade of NMDA responses by SNP was prevented by CPP or APV which are selective competitive NMDA receptor antagonists. These effects were not necessarily mediated by NO, since they were mimicked by ferrocyanide ions, the NO companion photolysis product of SNP.

Animals↗

PubMed1992

A nitric oxide (NO) synthase inhibitor accelerates amygdala kindling.

In response to NMDA receptor activation, hippocampal, striatal and cerebellar neurons synthesize nitric oxide (NO), which in turn elevates cGMP levels via guanylate cyclase. NO is increasingly being considered as a transsynaptic retrograde messenger, involved in neuronal plasticity. The effect of an inhibitor of NO synthase, L-NG-nitroarginine (NOArg), was studied on amygdala kindling and on kindled seizures in rats. NOArg increased kindling rate, particularly in its initial period, but did not modify seizure severity in previously kindled rats, although we have no definitive explanation for this effect. However, an enhanced post-synaptic excitability could be attributed to the blockade of the negative feed-back exerted by NO on the NMDA receptor.

Amino Acid Oxidoreductases↗

PubMed1992

Chronic NO synthase inhibition fails to protect hippocampal neurones against NMDA toxicity.

Since nitric oxide (NO) is supposed to mediate excitotoxicity in various brain structures, the effects of two NO synthase inhibitors were studied on rat hippocampal lesions induced by the focal injection of N-methyl-D-aspartate (NMDA). Although both drugs (NG-nitro-L-arginine methyl ester: L-NAME and L-NG-nitroarginine: L-NOARG) were given twice daily for 4 days before NMDA injection, at doses which are known to profoundly inhibit NO synthase activity, no significant decrease of NMDA-induced damage could be observed. These results do not confirm the current hypothesis of a NO involvement in NMDA toxicity at least on hippocampal neurons, in vivo.

Amino Acid Oxidoreductases↗

PubMed1992

The 5-HT4 receptor subtype inhibits K+ current in colliculi neurones via activation of a cyclic AMP-dependent protein kinase.

1. The aim of the present study was to examine the effect of 5-hydroxytryptamine (5-HT) on K+ current in primary culture of mouse colliculi neurones and to identify the 5-HT receptor subtype that could be involved in this effect. 2. The voltage-activated K+ current of the neurones was partially blocked by 8-bromo adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP). This effect was mimicked by 5-HT and the action of 5-HT could be antagonized by H7, a non specific protein kinase inhibitor, and by PKI, the specific cyclic AMP-dependent protein kinase blocker. 3. A similar cyclic AMP-dependent blockade of the K+ current was found with renzapride (BRL 24,924) and other 5-HT4 receptor agonists such as cisapride, BIMU 8, zacopride and 5-methoxytryptamine (5-MeOT). ICS 205,930, the classical 5-HT4 receptor blocker, could not be used in this study because it inhibited the studied K+ current by itself. However, the novel 5-HT4 receptor antagonist, DAU 6285 blocked the effects of 5-HT and renzapride on the K+ current. 4. The current was insensitive to the 5-HT1 and 5-HT3 receptor agonists (8-hydroxy-2-(di-n-propylamino) tetralin, RU 24,969, carboxamidotryptamine, 2-CH3-5-HT) as well as to 5-HT1, 5-HT2 and 5-HT3 antagonists (methiothepin, ketanserin, ondansetron [GR 38,032]). Moreover, these antagonists did not affect the actions of the tested 5-HT4 receptor agonists. 5. The present results show that part of the voltage-activated K+ current in mouse colliculi neurones is cyclic AMP-sensitive and the blockade of the current by 5-HT involves the 5-HT4 receptor subtype.The putative implication of 5-HT4 receptors in neuronal plasticity, via a blockade of K+ channels, is discussed.

Animals↗

PubMed1992

Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas.

1. The effect of L-glutamate has been studied on insulin secretion by the isolated perfused pancreas of the rat. The glutamate receptor subtype involved has been characterized. 2. In the presence of a slightly stimulating glucose concentration (8.3 mM), L-glutamate (5 x 10(-5)-4 x 10(-3) M) induced an immediate, transient and concentration-dependent insulin response. On the other hand, in the presence of a non stimulating glucose concentration (2.8 mM), L-glutamate (10(-3) M) did not modify the basal insulin secretion. 3. The three non-NMDA receptor agonists, kainate (10(-4)-10(-3) M), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 5 x 10(-5)-10(-4) M) and quisqualate (5 x 10(-6)-5 x 10(-5) M) all provoked a transient and concentration-dependent insulin response from pancreas perfused with 8.3 mM glucose. Compared with glutamate, kainate exhibited a similar efficacy, whereas AMPA and quisqualate elicited only a 3 fold lower maximal insulin response. In contrast, NMDA (10(-4)-10(-3) M) was ineffective. 4. An antagonist of non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 x 10(-5) M) totally prevented the stimulatory effect of L-glutamate (4 x 10(-4) M) and kainate (2 x 10(-4) M). In contrast, the NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ((+) MK801) was without effect. 5. The insulin secretory effect of glutamate (4 x 10(-4) M) was not affected by atropine (3 x 10(-7) M) or tetrodotoxin (3 x 10(-6) M). 6. Quisqualate at a high maximally effective concentration (4 x 10(-4) M) inhibited glutamate (10(-3) M) or kainate (4 x 10(-4) M)-induced insulin release. 7. This study shows that L-glutamate stimulates insulin secretion in rat pancreas, by acting on an excitatory amino acid receptor of the AMPA subtype.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1992

Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors.

This article describes a new approach for determining the role of endogenous guanine nucleotide binding (G) protein subunits in signal transduction. Sequential patch-clamping was applied to BSA gradient-enriched cultured lactotropes from lactating rats, first to dialyze antisense oligodeoxyribonucleotides (AS) directed against G alpha protein mRNAs and 48 h later to record ion-current responses to the PRL release inhibitor, dopamine. The effectiveness and specificity of action of six types of AS were determined by their effects on the in vitro translation of alpha o, alpha i1, alpha i2, alpha i3, and alpha s. The specificity of AS could be enhanced by replacing guanine by cytosine bases within the center core of AS and by maximizing the number of mismatches against nontargeted mRNAs within the extremities of AS. A total of 59 out of 240 cells could be investigated using the sequential patch clamp procedure in the absence of antibiotics. The typical decrease of the voltage-activated calcium current in response to 10 nM dopamine was diminished or abolished by AS, in correlation with the inhibition of in vitro translation of the alpha o subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against alpha i3 but not alpha o mRNA. Control experiments showed that culture conditions or loss of receptor affinity for dopamine were not responsible for the loss of response. The results suggest that dopamine D2 receptors are linked via alpha o to calcium channels and via alpha i3 to potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine4 receptors.

In various species, including humans, 5-hydroxytryptamine (5-HT) has been shown to exert positive chronotropic and inotropic cardiac effects through different types of receptors. The goal of the present study was to investigate the regulation by 5-HT of voltage-gated Ca2+ channels in human atrial myocytes and to characterize the receptor involved. Cardiomyocytes isolated enzymatically and mechanically were voltage-clamped using the whole-cell configuration of the patch-clamp technique. Extracellular perfusion of 5-HT increased Ca2+ current (ICa) amplitude with a EC50 (0.1 microM) similar to that observed with isoprenaline. The effects of 5-HT were blocked by the addition of protein kinase A inhibitor in the pipette. In addition, the effects of 5-HT, isoprenaline, and intracellular cAMP on ICa were not additive. These results support the hypothesis that the inotropic effect of 5-HT in human atrial myocytes is related to an increase of ICa via an elevation of intracellular cAMP levels and stimulation of cAMP-dependent protein kinase. The effects of 5-HT were not blocked by antagonists of 5-HT1 (methiothepin), 5-HT2 (ketanserin), or 5-HT3 (ICS 205-930 at a low concentration) receptors. The benzamide derivatives renzapride and zacopride and the azabicyclobenzimidazolone derivative BIMU 8 increased ICa, but less efficiently than did 5-HT or 5-methoxytryptamine. Moreover, ICS 205-930 at high concentrations (greater than 1 microM) completely antagonized the effects of 5-HT. Thus, the pharmacology of the 5-HT receptor involved in an increase of ICa in human atrial myocytes resembles that recently described for the 5-HT4 receptor. In atrial myocytes dissociated from rat, rabbit, guinea pig, or frog, 5-HT at high concentrations had no effect on Ca2+ currents, suggesting that the distribution of 5-HT4 receptors in cardiac tissues is species dependent.

Animals↗

PubMed1992

Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons.

Exposure of mouse colliculi neurons to selective 5-hydroxytryptamine (5-HT)4 agonists was accompanied by a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Half-maximal desensitization occurred after 2 min. Only exposure of neurons to selective 5-HT4 agonists led to a potent desensitization of the 5-HT4-mediated response. Neurons exposed to other agents, like isoproterenol, vasoactive intestinal peptide, or forskolin, that increase cAMP levels did not undergo any desensitization of 5-HT4 receptors. Activation of protein kinase A with either 8-bromo-cAMP or dibutyryl-cAMP or application of inhibitors of protein kinase A-dependent phosphorylation did not change the rate of 5-HT4-induced desensitization. No shift to lower potency of 5-HT4 agonists in the concentration-response curve was observed. These results suggest that 5-HT4 receptor agonists induced homologous but not cAMP-mediated heterologous desensitization. A good correlation was found between the affinities of nine 5-HT4 agonists and their abilities to desensitize the adenylyl cyclase response. This may indicate that homologous desensitization is a function of the mean occupancy time of the receptors by agonists. When permeabilized neurons were loaded with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), 5-HT4 receptor desensitization was reduced by 30-40%. Interestingly, Zn2+, an other inhibitor of beta ARK, totally prevented 5-HT4-induced desensitization. Pretreatment of neurons with concanavalin A, reported to inhibit sequestration of beta-adrenergic receptors from the cell surface, reduced the desensitization process by 70%. These data suggest that both sequestration and phosphorylation by beta ARK, or another specific agonist-dependent receptor kinase, are involved in homologous desensitization of 5-HT4 receptors coupled to adenylyl cyclase.

Animals↗

PubMed1992

Both enantiomers of 1-aminocyclopentyl-1,3-dicarboxylate are full agonists of metabotropic glutamate receptors coupled to phospholipase C.

We tested the effects of two enantiomers of a glutamate analogue, (trans)-1-aminocyclopentyl-1,3-dicarboxylate (t-ACPD), in striatal and cerebellar neurons in primary culture, as well as in Xenopus oocytes injected with cerebellar rat RNA. In the presence of MK-801, to avoid N-methyl-D-aspartate receptor activation, and 3 microM tetrodotoxin, both enantiomers [(1R,3S)- and (1S,3R)-t-ACPD] stimulated inositol phosphate (InsP) formation both in striatal neurons after 9-11 days in vitro [EC50, 3.7 +/- 1.1 microM, three experiments, and 33 +/- 7.5 microM, three experiments; maximal stimulatory effects, 252 +/- 15%, 13 experiments, and 269 +/- 15% of basal InsP formation, 14 experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively] and in cerebellar granule cells after 9-11 days in vitro [EC50, 50 +/- 18 microM, four experiments, and 307 +/- 92 microM, four experiments; maximal stimulatory effects, 401 +/- 71%, eight experiments, and 423 +/- 75% of basal InsP formation, eight experiments, for (1R,3S)- and (1S,3R)-t-ACPD, respectively]. These effects were not additive, indicating that both enantiomers acted at the same receptor molecule. When we monitored t-ACPD-induced increases in intracellular Ca2+ concentration ([Ca2+]i) with fura-2 ratio-imaging, we found that both enantiomers could elicit similar increase in [Ca2+]i, in the presence of 1 microM MK-801 and 3 microM tetrodotoxin; these effects were also observed in the absence of external Ca2+. Moreover, in Xenopus oocytes injected with adult rat cerebellar RNA, both drugs elicited oscillatory increases of a Ca(2+)-dependent chloride conductance, with similar efficacy, with (1R,3S)-t-ACPD being the more potent isomer. These data are in contradiction to previous reports showing that, in "immature" cerebellar neurons and adult hippocampal slices, (1S,3R)-t-ACPD was either the only active enantiomer or a full agonist of metabotropic receptors, with (1R,3S)-t-ACPD being ineffective or a partial agonist. However, performing these experiments in immature (2-3 days in vitro) striatal or cerebellar neurons, we found that only (1S,3R)-t-ACPD was active in stimulating [Ca2+]i.

Animals↗

PubMed1992

Specific antibodies against Go isoforms reveal the early expression of the Go2 alpha subunit and appearance of Go1 alpha during neuronal differentiation.

We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on neuroblastoma cells, the Go2 alpha subunit was almost exclusively present in undifferentiated cells, and differentiation induced the appearance of the Go1 alpha subunit, with a reduction in the amount of Go2 alpha polypeptide. Thus, the evolution of the two Go alpha subunits during cell differentiation, unambiguously identified with specific antibodies, suggests that neuronal differentiation is responsible for the on/off switch of the expression of the Go alpha isoforms and indicates that Go1 alpha, rather than Go2 alpha, is involved in neurotransmission.

Amino Acid Sequence↗

PubMed1992

Transfection of human 5-hydroxytryptamine1A receptors in NIH-3T3 fibroblasts: effects of increasing receptor density on the coupling of 5-hydroxytryptamine1A receptors to adenylyl cyclase.

Human serotonin [5-hydroxytryptamine (5-HT)1A] receptors have been transfected in NIH-3T3 cells, and their pharmacology and coupling to adenylyl cyclase have been analyzed. Three cellular preparations were used, 1) monoclonal cell lines (clones 6, 2B, and 4B), expressing 45, 280, and 500 fmol of 5-HT1A receptors/mg of protein, respectively; 2) clones 6, 2B, and 4B in which the concentration of 5-HT1A receptors was increased after stimulation of the glucocorticoid-inducible promoter with dexamethasone; and 3) polyclonal cell lines that expressed an increasing amount of 5-HT1A receptor as a function of cell passage. The transfected 5-HT1A receptors inhibited basal, forskolin-stimulated, and isoproterenol-stimulated adenylyl cyclase. The inhibition was dependent on the receptor density expressed, increasing from 60% at low density (45 fmol/mg) to 90% at a density higher than 280 fmol/mg. The pharmacology of the 5-HT1A receptor was studied, with particular attention being paid to the behavior of some agonists. These pharmacological characteristics are similar to those of 5-HT1A receptors in hippocampus but different from those of 5-HT1A in cerebral cortex. Analysis of the potencies and efficacies of the full agonist 5-HT and the partial agonist ipsapirone, as a function of receptor density in the three cellular populations used, revealed that 1) the efficacies of the full and partial agonists increased with the receptor density; 2) the EC50 values of the full and partial agonists were not shifted to the left when the receptor density was increased (based on the increase in efficacy and considering the classical pharmacological models of receptor-drug action, a 9-10-fold shift was expected); and 3) the ratio between the efficacies of the full agonist 5-HT and the partial agonist ipsapirone was not modified when the receptor concentration was increased or when the GTP-binding protein availability was decreased. The results indicate that neither the classical nor the operational model of drug-receptor action can be used to describe the coupling of 5-HT1A receptors to adenylyl cyclase in transfected NIH-3T3 cells. One of the explanations could be that 5-HT1A receptors and GTP-binding proteins are coupled in functional domains (almost precoupled), rather than distributed in homogeneous compartments in which they are free to diffuse.

3T3 Cells↗

PubMed1991

Amino acids 367-376 of the Gs alpha subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 alpha subunit.

Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the alpha subunit could be indirect by means of the other beta gamma subunits or polydisperse multimers. In the activated state, although the alpha subunit is dissociated from other subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins alpha subunits, the C-terminal domain of Gs alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs alpha C-terminal domain. We first deleted codons 2-6 of Gi1 alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 alpha by different domains of the Gs alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs alpha). Our results showed that addition of amino acids 367-376 of Gs alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 alpha.

Amino Acid Sequence↗

PubMed1991

Metabolism of two Go alpha isoforms in neuronal cells during differentiation.

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Pharmacological characterization of the quisqualate receptor coupled to phospholipase C (Qp) in striatal neurons.

A detailed pharmacological characterization of the quisqualate (QA) receptor coupled to phospholipase C (Qp) was performed in striatal neurons. The experiments were carried out in the presence of the ionotropic antagonists MK-801 (1 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM), concentrations that block N-methyl-D-aspartate (NMDA) or alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in these cells. QA, ibotenate and trans-1-aminocyclopentyl-1,3-dicarboxylate (ACPD) evoked dose-dependent inositol phosphate formations with EC50 values of 0.3, 6.7 and 29 microM, respectively. QA and ibotenate had the same maximal effect (295.7 +/- 17.9% of basal, n = 6) whereas the efficacy of ACPD was somewhat lower (70.2 +/- 8.9% of the maximal quisqualate effect, n = 4). The QA-, ibotenate- and ACPD-induced maximal effects were not additive, and the inositol phosphate formations induced by high concentrations of L-aspartate (L-ASP), AMPA, kainate (KA) and domoate (DO) (100 microM or higher) were also not additive. The inositol phosphate responses induced by all these agonists were totally blocked by the phorbol ester phorbol 12,13-dibutyrate (PdBu), but not by atropine or prazosin suggesting that all these substances were able to stimulate the Qp excitatory amino acid receptor in striatal neurons. Of the excitatory amino acid receptor antagonists tested, only D,L-2-amino-3-phosphonopropionate (D,L-AP3) inhibited QA-induced InsP formation in a competitive manner (mean pKi = 4.45 +/- 0.43, n = 4). However, this drug was also a partial agonist of the Qp receptor since it stimulated the inositol phosphate formation. We found that D,L-AP3 also inhibited NMDA-induced calcium increase, in a competitive manner (mean pIC50 = 4.34 +/- 0.22, n = 8, and mean pKi = 3.7 +/- 0.11, n = 5). The Qp excitatory amino acid receptor in striatal neurons therefore closely resembles Qp receptors with high potency for agonists as described in striatal and retinal slices and synaptoneurosomes, and has several pharmacological differences compared to the Qp receptors which have low potency for agonists described in hippocampal and cortical slices, cerebellar granule cells, astrocytes and rat brain mRNA-injected oocytes.

2-Aminoadipic Acid↗

PubMed1991

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.

Adenosine Diphosphate Ribose↗

PubMed1991

Treatment of intact striatal neurones with cholera toxin or 8-bromoadenosine 3',5'-(cyclic)phosphate decreases the ability of pertussis toxin to ADP-ribosylate the alpha-subunits of inhibitory and other guanine-nucleotide-binding regulatory proteins, Gi and Go. Evidence for two distinct mechanisms.

Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 micrograms/ml, 22 h) decreases the subsequent ADP-ribosylation of the alpha subunit of the guanine-nucleotide-binding regulatory protein Go (Go alpha) and the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory protein (Gi alpha) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin. The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones. Treatment of cells with 0.1 microM 8-bromoadenosine 3',5'-(cyclic)phosphate (BrcAMP) for 16 h, or with 0.1 mM BrcAMP for 5 min, mimicked the effect of cholera toxin on the ADP-ribosylation of Go alpha and Gi alpha in vitro. However, the two agents seem to act through distinct mechanisms. The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin. In addition, measurements of the pI of the Go alpha deduced from immunoblots of two-dimensional gels performed using a specific antibody directed against Go alpha suggest that treatment of neurones with cholera toxin induces ADP-ribosylation of Go alpha in intact cells, while BrcAMP does not.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗