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In rat cerebellar granule cells, glutamate induced rapid activation of c-Jun N-terminal kinase (JNK) and p38 kinase to phosphorylate c-Jun (at Ser63) and p53 (at Ser15), respectively, and a subsequent marked increase in activator protein-1 (AP-1) binding that preceded apoptotic death. These glutamate-induced effects and apoptosis could largely be prevented by long-term (7 days) pretreatment with 0.5-2 mm lithium, an antibipolar drug. Glutamate's actions could also be prevented by known blockers of this pathway, MK-801 (an NMDA receptor blocker), SB 203580 (a p38 kinase inhibitor) and curcumin (an AP-1 binding inhibitor). The concentration- and time-dependent suppression of glutamate's effects by lithium and curcumin correlated well with their neuroprotective effects. These results suggest a prominent role of JNK and p38, as well as their downstream AP-1 binding activation and p53 phosphorylation in mediating glutamate excitotoxicity. Moreover, the neuroprotective effects of lithium are mediated, at least in part, by suppressing NMDA receptor-mediated activation of the mitogen-activated protein kinase pathway. more...

Antibodies directed against the C-terminal and the N-terminal regions of the mu-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the mu receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the mu receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating approximately 70% of solubilized rat brain mu receptors as determined by [3H][D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to mu receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein a subunits, it was determined that G(alpha)o, G(alpha)i1, G(alpha)i3, and to a lesser extent G(alpha)i2, but not G(alpha)s or G(alpha)q11, were coimmunoprecipitated with the mu receptor. Inclusion of GTPgammaS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins. more...

High-affinity mu-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the mu-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][D-Ala2,N-Me-Phe4,Gly-Dl5]-enkephalin([3H]DAMGO), a mu-selective opioid agonist, with high affinity (KD = 1.90 +/- 0.93 nM; Bmax = 629 +/- 162 fmol/mg of protein). Of the membrane-associated [3H]-DAMGO binding sites, 29 +/- 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 microM guanosine 5'-O-(3-thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 +/- 0.39 nM, Bmax = 920 +/- 154 fmol/mg of protein). Guanosine 5'-O-(3-thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 microM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [D-Pen2, D-Pen5]-enkephalin, a delta-selective opioid agonist, and U50488H, a kappa-selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of < 1 microM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two-site than a one-site model with KDhigh = 2.17 +/- 1.5 nM, Bmax = 648 +/- 110 fmol/mg of protein and KDlow = 468 +/- 63 nM, Bmax = 253 +/- 84 fmol/mg of protein. In the presence of 10 microM guanosine 5'-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with KD = 815 +/- 33 nM, Bmax = 965 +/- 124 fmol/mg of protein. more...