Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The neurotoxic effect of glutamate in cultured mouse mesencephalic dopaminergic neurons was investigated. Neuron-rich cell cultures were prepared from 13-14-day-old fetal mouse ventral mesencephalic tissue. Cultures were exposed to glutamate for 10 min and evaluated for glutamate neurotoxicity (GNT) 18-24 hr later by tyrosine hydroxylase (TH) immunostaining, microtubule associated protein-2 (MAP2) immunostaining, and radiolabeled dopamine uptake assay. In glutamate-exposed cultures, the number of TH-positive neurons and the level of dopamine uptake were reduced to 40% (35-45%) and 50% (47-52%), respectively, of control cultures. The number of MAP2-positive neurons was also reduced to 47%, indicating that the GNT was not restricted or selective to dopaminergic neurons. It is concluded that GNT was mediated by the N-methyl-D-aspartic acid (NMDA) receptor from the following observations: 1) GNT was completely blocked by MK-801, an NMDA receptor antagonist; 2) NMDA itself was as toxic as glutamate; 3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor, did not block GNT; 4) kainate did not show neurotoxicity at a low concentration; and 5) two modulators of the NMDA receptor, 7-chlorokynurenic acid and magnesium, were effective in blocking GNT. Protective effects of phorbol myristate acetate, a tumor promoter, and gangliosides (GM1 and GT1b) on GNT were also demonstrated. Possible interactions between GNT and several protein kinase cascades were also investigated. Forskolin, an activator of adenyl cyclase and protein kinase A, showed some protective effect on GNT. But okadaic acid, an inhibitor of phosphatases, and genistein, a tyrosine kinase inhibitor, did not show any protective effect. These results suggest that 1) glutamate is capable of causing neuronal death in the substantia nigra; 2) GNT on dopaminergic neurons is mainly mediated by the NMDA receptor under the conditions of our study; 3) protein kinase C translocation is a key mechanism of GNT; and 4) there is an interplay of a signal transduction system in the pathomechanism of GNT.
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PMID:Glutamate neurotoxicity in mesencephalic dopaminergic neurons in culture. 790 39

Purified striatal synaptosomes were superfused continuously with L-[3,5-3H]tyrosine to measure simultaneously the synthesis ([3H]water formed during the conversion of [3H]tyrosine into [3H]DOPA) and the release of [3H]dopamine ([3H]DA). Glutamate (10(-3) M) and NMDA (10(-3) M, in the absence of Mg2+) stimulated the release of [3H]DA, but they reduced the efflux of [3H]water. This reduction of [3H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [3H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [3H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [3H]DA synthesis and release were mimicked by ionomycin (10(-6) M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.
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PMID:Opposite presynaptic regulations by glutamate through NMDA receptors of dopamine synthesis and release in rat striatal synaptosomes. 791 26

Experiments were performed to clarify the excitatory mechanism of adenosine in superior colliculus slices. Postsynaptic field potential was recorded in the superficial gray layer after stimulation to the optic layer. Application of adenosine to the perfusion medium at a concentration of 0.2 microM or 100 microM enhanced the amplitude of the postsynaptic field potential and the excitatory effect of adenosine was counteracted by application of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) (100 microM), a non-selective protein kinase inhibitor or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) (100 microM), a selective protein kinase A inhibitor. The presence of adenosine at a concentration of 0.2 microM or 100 microM during electrical stimulation to slices of the superficial gray layer increased the release of glutamate 2.5 and 2.3 times, respectively, and this increase was suppressed in Ca(2+)-free medium or by applying tetrodotoxin (1 microM) to the medium. Application of H-7 or HA1004 to the incubation medium inhibited the enhancement of the glutamate release. These results suggest that the excitatory effect of adenosine in the superior colliculus is due to an increase in glutamate release and involves the protein kinase system.
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PMID:Adenosine facilitates glutamate release in a protein kinase-dependent manner in superior colliculus slices. 791 48

Repetitive activation of hippocampal mossy fibers evokes a long-term potentiation (LTP) of synaptic responses in pyramidal cells in the CA3 region that is independent of N-methyl-D-aspartate receptor activation. Previous results suggest that the site for both the induction and expression of this form of LTP is presynaptic. Experimental elevation of cyclic adenosine 3',5'-monophosphate (cAMP) both mimics and interferes with tetanus-induced mossy fiber LTP, and blockers of the cAMP cascade block mossy fiber LTP. It is proposed that calcium entry into the presynaptic terminal may activate Ca(2+)-calmodulin-sensitive adenylyl cyclase I which, through protein kinase A, causes a persistent enhancement of evoked glutamate release.
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PMID:Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP. 791 82

Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its downstream target, mitogen-activated protein kinase (MAPK). In this study, we used stable overexpression of MAPKK and its mutants in NIH 3T3 cells to study effects on downstream components of the MAPK signaling cascade and to correlate them to physiological responses. We have mutated the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine residues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effect on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90rsk activation in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellular proliferation was examined and the different effects were accompanied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control of cellular proliferation and morphology and also establish that phosphorylation of serine 222 is essential for MAPKK activation together with the phosphorylation of an additional serine(s) (probably serine 218).
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PMID:Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII. 792 75

The transmitter glutamate is thought to be used by all vertebrate photoreceptors to drive the second-order neurons of the retina, horizontal and bipolar neurons. Dopamine, an endogenous retinal neurotransmitter localized to amacrine and interplexiform cells, has previously been shown to enhance glutamate-gated currents in retinal horizontal cells. In the present study we demonstrate that bipolar cells, like horizontal cells, possess glutamate receptors that are modulated by dopamine. We then identify some components of the pathway through which dopamine acts. We used whole-cell patch recording to measure how bath-applied dopamine modulated the currents elicited by puffs of transmitter solutions at bipolar cell dendrites. Excitatory amino acid-gated currents were evoked by pressure ejecting 1 mM glutamate or 10 microM kainate for 40 msec through a micropipette positioned at the dendrites of bipolar cells. Bath-applied dopamine (20 microM) enhanced the response to glutamate in OFF bipolar cells in the retinal slice by 40% and in isolated OFF bipolar cells by 65%. We also explored the components of the intracellular pathway mediating this modulation. Response enhancement was blocked by the D1 receptor antagonist SCH23390, but not by the D2 receptor antagonist spiperone, suggesting that the enhancement by dopamine is mediated by a D1 receptor. GDP-beta-S, a G-protein inactivator, blocked the enhancing action of dopamine, suggesting that the D1 receptor activated a G-protein to enhance the glutamate-gated current. Both 8-(4-chlorophenylthio)adenosine, a cAMP analog, and the addition of the catalytic subunit of protein kinase A (PKA) to the recording pipette enhanced glutamate-gated currents, while H-7, a PK inactivator, and PKI20amide, a PKA-specific inhibitor, blocked the enhancing action of dopamine. These data suggest that dopamine acts at D1 receptors in the dendrites of bipolar cells to activate adenyl cyclase, which through cAMP enhances a glutamate-gated current in bipolar cell dendrites. Thus, dopamine may modulate synaptic transmission from photoreceptors to OFF bipolar cells.
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PMID:Dopamine enhances a glutamate-gated ionic current in OFF bipolar cells of the tiger salamander retina. 793 65

Consequent to agonist exposure, many G protein-coupled receptors undergo sequestration or internalization. Results with receptors linked to adenylate cyclase, such as the beta 2-adrenergic receptor, or receptors linked to phospholipase C (PLC) have provided conflicting results regarding the role of second messenger-dependent (i.e., protein kinase A or C) and -independent (i.e., beta-adrenergic receptor kinase) kinases in mediating this process. Recent results for truncated and mutated gastrin-releasing peptide (GRP) receptors (GRP-R), as well as muscarinic cholinergic receptors, suggest that activation of protein kinase C may be needed for full receptor internalization. Nearly all G protein-coupled receptors studied to date, including the GRP-R, possess two highly conserved amino acids that are important in mediating receptor-G protein coupling to second messengers, i.e., arginine in the proximal second intracellular loop and alanine in the distal third intracellular loop. We selectively mutated each of these residues in the GRP-R to determine their importance for activation of PLC. Site-directed mutagenesis was performed to change arginine at position 139 to glycine (R139G mutant) and alanine at position 263 to glutamate (A263E mutant), with stable cell lines being created by transfection of the wild-type or mutated receptor cDNA into BALB/3T3 fibroblasts. Both R139G (Kd = 12.0 +/- 1.6 nM) and A263E (Kd = 12.2 +/- 1.7 nM) had a lower affinity for bombesin than did wild-type GRP-R (Kd = 1.4 +/- 0.4 nM); however, characteristic stoichiometries for the binding of agonists to this receptor were maintained equally in all three cell lines (bombesin > GRP >> neuromedin B). The wild-type GRP-R exposed to bombesin increased [3H]inositol phosphates (a measure of PLC activation) approximately 4-fold, with an EC50 of 5.1 +/- 2.2 nM. In contrast, [3H]inositol phosphates were not significantly increased in cells expressing R139G or A263E receptors, demonstrating that Arg139 and Ala263 are required for GRP-R activation of PLC. However, when receptor internalization at 37 degrees was assessed by ligand acid-stripping studies, 53 +/- 2% of A263E receptors were internalized at 90 min, compared with 85 +/- 5% of wild-type GRP-R, whereas only 10 +/- 3% of R139G receptors were internalized. Preincubation of either mutant cell line with 100 nM 12-O-tetradecanoylphorbol-13-acetate markedly increased internalization rates, such that at 90 min 62 +/- 2% of R139G receptors and 82 +/- 1% of A263E receptors were internalized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes. 793 30

The inhibitory action of a glutamate agonist, quisqualate, in association with the intracellular signal transduction, was electrophysiologically examined in identified Euhadra neurons. Quisqualate dose-dependently induced a slow outward current (Quis current) which was blocked by tetraethylammonium. This current was suppressed by intracellular injection of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and was enhanced by a CaMKII inhibitor, KN-62. However, no significant changes in the Quis current were observed when the catalytic subunit of protein kinase A (PKA) or the protein kinase C (PKC) fragment (530-558) was intracellularly applied; or using a PKA inhibitor, H-8, or a PKC inhibitor, staurosporine. These results suggest a novel mechanism linked to CaMKII, by which quisqualate induces an outward potassium current.
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PMID:Involvement of a Ca2+/calmodulin-dependent protein kinase II-associated mechanism in the induction of an outward potassium current by quisqualate. 795 2

The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16-51 mol of Pi/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, Pi determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of Pi/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.
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PMID:Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein. 796 54

The adenylate cyclase system has been implicated in taste transduction. The purpose of this study was to determine whether application of modulators of the adenylate cyclase system to the tongue alter taste responses. Integrated chorda tympani (CT) recordings were made in gerbils to bitter, sweet, salty, sour, and glutamate tastants before and after a 4-min application of four types of modulators of the adenylate cyclase system. The four types of modulators tested were: a) NaF, a compound that promotes dissociation of GTP binding protein; b) forskolin, a powerful stimulant of adenylate cyclase; c) 8-bromoadenosine 3' :5'-cyclic monophosphate sodium salt (8BrcAMP) and N6,2'-O-dibutyryl-adenosine 3' :5'-cyclic monophosphate sodium salt (DBcAMP), two membrane permeable forms of cAMP; and d) 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride) (H-8), which are protein kinase inhibitors. The tast compounds tested were: NaCl (30 mM), monosodium glutamate-MSG (50 mM), sucrose (30 mM), HCl (5 mM and 10 mM), KCl (300 mM), quinine HCl (30 mM), MgCl2 (30 mM), erythromycin (0.7 mM and 1 mM), HCl (5 mM and 10 mM), and urea (2 M). The main findings were as follows. NaF (20 mM) significantly inhibited responses to bitter compounds up to 35% and enhanced the response to sucrose by 30%. NaCl (20 mM), used as a control for NaF, inhibited most responses up to 78% with no enhancement of sucrose as seen with NaF. 8BrcAMP (1.16 mM) reduced the responses to bitter-tasting quinine HCl, MgCl2, and erythromycin but not to urea.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulators of the adenylate cyclase system can alter electrophysiological taste responses in gerbil. 797 5


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