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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.

Lithium, the major drug used to treat manic depressive illness, robustly protects cultured rat brain neurons from glutamate excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors. The lithium neuroprotection against glutamate excitotoxiciy is long-lasting, requires long-term pretreatment and occurs at therapeutic concentrations of this drug. The neuroprotective mcchanisms involve inactivation of NMDA receptors, decreased expression of pro-apoptotic proteins, p53 and Bax, enhanced expression of the cytoprotective protein, Bcl-2, and activation of the cell survival kinase, Akt. In addition, lithium pretreatment suppresses glutamate-induced loss of the activities of Akt, cyclic AMP-response element binding protein (CREB), c-Jun - N-terminal kinase (JNK) and p38 kinase. Lithium also reduces brain damage in animal models of neurodegenerative diseases in which excitotoxicity has been implicated. In the rat model of stroke using middle cerebral artery occlusion, lithium markedly reduces neurologic deficits and decreases brain infarct volume even when administered after the onset of ischemia. In a rat Huntington's disease model, lithium significantly reduces brain lesions resulting from intrastriatal infusion of quinolinic acid, an excitotoxin. Our results suggest that lithium might have utility in the treatment of neurodegenerative disorders in addition to its common use for the treatment of bipolar depressive patients.

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.

This report describes a modulatory action of lithium and glutamate on the activity of serine/threonine kinase Akt-1. Lithium is most commonly used to treat bipolar disorder, but the mechanism of its therapeutic action remains unknown. We have recently demonstrated that lithium protects against glutamate-induced excitotoxicity in cultured brain neurons and in an animal model of cerebral ischemia. This study was undertaken to investigate the role of Akt-1, activated by the phosphatidylinositol 3-kinase (PI 3-K) signaling pathway, in mediating glutamate excitotoxicity and lithium protection in cerebellar granule cells. High levels of phosphorylation and activity of Akt-1 were detected in cerebellar neurons cultured in the presence of serum. Protracted treatment with selective PI 3-K inhibitors, wortmannin and LY294002, abolished Akt-1 activity and induced neuronal death that could be reduced by long-term lithium pretreatment. Exposure of cells to glutamate induced a rapid and reversible loss of Akt-1 phosphorylation and kinase activity. These effects were closely correlated with excitotoxicity and caspase 3 activation and were prevented by phosphatase inhibitors, okadaic acid and caliculin A. Long-term lithium pretreatment suppressed glutamate-induced loss of Akt-1 activity and accelerated its recovery toward the control levels. Lithium treatment alone induced rapid increase in PI 3-K activity, and Akt-1 phosphorylation with accompanying kinase activation, which was blocked by PI 3-K inhibitors. Lithium also increased the phosphorylation of glycogen synthase kinase-3 (GSK-3), a downstream physiological target of Akt. Thus, modulation of Akt-1 activity appears to play a key role in the mechanism of glutamate excitotoxicity and lithium neuroprotection.

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.

We tested how chronic antidepressant treatments (chronic imipramine, chronic electroconvulsive shock (ECS) and chronic ECS given after chronic imipramine) affect the feedback inhibition of alpha 1-adrenoceptor activity (measured with inositol phosphate (IP) accumulation after stimulation with noradrenaline). The inhibitory effect of a 12-O-tetradecanoylphorbol 13-acetate (TPA) on IP response was found to be due to protein kinase C (PKC) activation, as it was abolished by specific protein kinase inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Chronic ECS completely abolished the inhibition by TPA of inositol phosphate response to noradrenaline, while chronic imipramine reversed the feedback and led to potentiation of responses by TPA to intermediate concentrations of noradrenaline. Subsequent chronic ECS abolished the imipramine-induced reversal of TPA inhibition and lead to the changes similar as observed after ECS alone. The present results may suggest why in some cases, in which imipramine therapy is ineffective, subsequent ECS may be clinically beneficial.

The effects of glutamate, NMDA and quisqualate on carbachol- and norepinephrine-elicited formation of inositol phosphate (IP) were evaluated in slices prepared from the cerebral cortex of 3- and 24-month Sprague-Dawley rats. Glutamate, NMDA, and quisqualate antagonized the IP response to carbachol in a concentration-dependent fashion. This antagonism was more pronounced in aged than in young rats, both for glutamate (IC5O 0.114 and 0.210 mM) and NMDA (IC5O 0.0029 and 0.127 mM), but not for quisqualate. Glutamate (but not NMDA) also antagonized in a concentration-dependent fashion the IP response to norepinephrine, IC50s were 0.061 and 0.126 mM for aged and young rats, respectively; quisqualate had an inhibitory effect only at 1 mM concentration in the two age-groups, while in aged rats some stimulatory effect was present at 0.1 mM concentration. Glutamate, NMDA and quisqualate (1 mM) did not affect basal IP accumulation in either young or aged rats; quisqualate, however, at 0.1 mM concentration had some stimulatory effect, more pronounced in aged rats. This effect was probably responsible for the biphasic effect of quisqualate in this age-group. The most important finding consists of the demonstration of an age-related increase in the inhibitory effects of NMDA on carbachol-induced IP accumulation. This implies an altered modulation of cholinergic post-receptor mechanisms by glutamatergic mechanisms.

We have studied the in-vitro effect of centpropazine on cerebral cortical noradrenergic receptors measured as the accumulation of second messengers, cyclic AMP and inositol phosphate, stimulated by noradrenaline, and the binding to alpha 1- and beta-adrenoceptors. Centpropazine inhibited inositol phosphate, but not the cyclic AMP accumulation in the cerebral cortical slices of the rat. It moderately antagonized the specific binding of [3H]prazosin, but did not affect the specific binding of the beta-adrenoceptor ligand, [3H]CGP 12177, to cerebral cortical membranes.

Stimulation of phosphatidylinositol hydrolysis by noradrenaline requires the presence of calcium in the medium during the whole incubation and preincubation procedure, while carbachol-induced inositol phosphate formation is equally efficient in calcium-free and calcium-containing medium. This finding confirms the notion that phosphatidylinositols mobilized after stimulation of various receptors are present in different metabolic pools: the pool mobilized by alpha 1 adrenoceptors is apparently formed from myo-inositol incorporated into membrane phospholipids by a calcium-dependent process.

In both the rat and Acomys cahirinus the adrenergic cyclic AMP generating system in the brain is dependent not only on beta-, but also on alpha-adrenoceptors. The relative role of alpha-adrenoceptors is much greater in the Acomys cahirinus. This feature makes the Acomys an interesting animal model for investigating the role of alpha-beta-adrenoceptor coupling in generation of cyclic AMP and the mechanism of action of antidepressant treatment.

The present study was undertaken to examine the effect of electroshock on adrenoceptor-mediated cAMP and inositol phosphate accumulation in rat brain cerebral cortical slices. Under the conditions of these experiments, isoproterenol-induced cAMP accumulation was unaltered by electroshock, although there was a significant reduction in the norepinephrine- and isoproterenol + 6-fluoronorepinephrine-stimulated responses. No change in alpha-adrenoceptor-mediated inositol phosphate accumulation was noted. The results indicate that electroshock selectively modifies an alpha-adrenoceptor system in brain that differs from that associated with inositol phosphate accumulation.

Chronic administration of imipramine with drinking water to Wistar rats for two months did not affect the [3H]dihydroalprenolol binding to lymphocytic membranes, though the treatment depressed the radioligand binding to cerebral cortical membranes. The result suggests that the beta-adrenoceptor downregulation caused by the antidepressant in the central nervous system is not reflected in the lymphocytes.