Different regulation of phospholipase D activity in glioma C6 cells by sphingosine, propranolol, imipramine and phorbol ester.

In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.

Involvement of protein kinase C in the mechanism of in vitro effects of imipramine on generation of second messengers by noradrenaline in cerebral cortical slices of the rat.

Imipramine did not significantly inhibit the noradrenaline or isoproterenol-induced cyclic AMP accumulation in rat cerebral cortical slices, but inhibited the potentiation of this response by protein kinase C activator, a phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate. In low concentrations (0.1-1 microM) it prevented the inhibitory effect of the phorbol ester on accumulation of inositol phosphate induced by noradrenaline, while in higher concentrations it inhibited the response by itself. Imipramine did not bind to beta-adrenoceptors but was an effective blocking agent of alpha 1-adrenoceptors (Ki = 38.1 nM). The data suggest that imipramine acts within the noradrenergic cyclic AMP generating system on two targets: inhibiting protein kinase C and blocking the alpha 1-adrenoceptor; both actions may reduce the alpha-adrenoceptor potentiation of beta-adrenoceptor-mediated cyclic AMP generation.

Norepinephrine-independent regulation of GRII mRNA in vivo by a tricyclic antidepressant.

Desipramine (DMI), a tricyclic antidepressant drug used in the treatment of depression, has been shown to increase steady-state levels of glucocorticoid receptor type II (GRII) mRNA in vitro and in vivo. To determine whether this effect is secondary to norepinephrine (NE) reuptake inhibition i.e., increases in synaptic NE induced by DMI, GRII mRNA levels were assayed in rat hippocampus following neurotoxic lesioning of NE neurons with DSP4. Chronic DMI treatment significantly increased GRII mRNA levels to the same degree in lesioned and non-lesioned animals. In contrast to DMI, the non-tricyclic antidepressant fluoxetine had no effect on GRII mRNA. These results provide evidence which demonstrates that a tricyclic antidepressant can regulate steady-state mRNA levels in vivo by a mechanism which is independent of its effects on synaptic monoamine levels.

Antidepressants: past, present and future.

Since the discovery of first antidepressants in mid-1950's, the field has been intensively studied. Several new classes of compounds emerged and several hypotheses on the mechanism of their action were proposed. The novel antidepressants are either selective and reversible monoamine oxidase inhibitors, (e.g., moclobemide), or selective serotonin reuptake inhibitors (e.g., citalopram or paroxetine), or serotonin and noradrenaline reuptake inhibitors (e.g. , venlafaxine). Recently neuropeptides (e.g., thyrotropin-releasing hormone,TRH) or antagonists of neuropeptide receptors (e.g., tachykinin NK(1) receptor) undergo clinical tests. Several hypotheses proposed the predominant involvement of one or few neurotransmitter receptors in the mechanism of antidepressant action, but it is now assumed that several distinct receptor mechanisms' trigger different but converging intracellular signal cascades that activate transcription factors, which, in turn, promote the expression of genes encoding for proteins, that play a crucial role in restoring of neuronal functions involved in mood regulation.

Reserpinization enhances electroconvulsive treatment effects on cortical alpha 1-adrenoceptors.

Repeated electroconvulsion shock (ECS) for 7 days given to mildly reserpinized rats strongly elevated the density of [3H]prazosin-labelled alpha 1-adrenoceptors and depressed the EC50 for norepinephrine-stimulated inositol phosphate accumulation, while the effects of either treatment given alone were small or negligible. Alterations in alpha 1-adrenoceptor activity caused by ECS could possibly be a function of receptor sensitivity during drug treatment.

Reversal by imipramine of beta-adrenoceptor up-regulation induced in a chronic mild stress model of depression.

Male Wistar rats were subjected to a chronic mild stress procedure involving different stress stimuli applied for 8 weeks. During this time the consumption of 1% sucrose solution was monitored at weekly intervals. After the first 3 weeks, when stressed animals displayed a reduction of sucrose consumption, the control and stressed groups were divided into subgroups receiving daily placebo or imipramine (10 mg/kg/day) treatment. After 5 weeks of treatment, 24 h after the last injection, the rats were killed and beta-adrenoceptor density and affinity in cortical membrane preparations and the accumulation of cyclic AMP in cortical slices stimulated with noradrenaline were assessed. While in stressed placebo-treated rats the sucrose consumption remained reduced, in the imipramine-treated group the level of consumption gradually returned to control values. The stressed placebo-treated rats also displayed an increase in cortical beta-adrenoceptor density (by 34%) with no changes in affinity, and an increase (22%) in the cyclic AMP response to noradrenaline in cortical slices. Imipramine, which in non-stressed rats did not affect sucrose intake but depressed the beta-adrenoceptor density and the cyclic AMP response, reversed the stress-induced decrease in sucrose consumption and the increase in the beta-adrenoceptor density; at physiological noradrenaline concentrations it also reduced the enhanced cyclic AMP response. The results suggest that the chronic mild stress procedure produces behavioral and biochemical changes consistent with a realistic model of depression in animals.

The influence of electroshock on adrenoceptor function in rat brain cerebral cortex: selectivity for the alpha-adrenoceptor site.

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.

Increased responsiveness of the cerebral cortical phosphatidylinositol system to noradrenaline and carbachol in senescent rats.

The responsiveness of cerebral cortical alpha 1-adrenoceptors and cholinergic muscarinic M1 receptors was assessed in young (3 months) and aged (24 months) male Sprague-Dawley rats. The measure of responsiveness was the accumulation of inositol phosphate (IP) formed in [3H]myo-inositol-preloaded cerebral cortical slices in the presence of lithium, following stimulation with various concentrations of noradrenaline (1-300 microM) and carbachol (5-1000 microM). In old rats the maximum response to noradrenaline was higher by 80%, and that to carbachol by 33%, indicating an increased responsiveness of the investigated receptors in senescence.

Lack of beta adrenoceptor desensitization in brain following the dual noradrenaline and serotonin reuptake inhibitor venlafaxine.

Venlafaxine, a dual amine reuptake inhibitor, was utilized to delineate the role of the individual aminergic components of the 'serotonin/noradrenaline link' in modifying receptor-linked second messenger cascades. Venlafaxine (20 mg/kg i.p. bid for 10 days) failed to alter in normal animals either the density of beta adrenoceptors or the response of the beta adrenoceptor-coupled adenylate cyclase system to noradrenaline but significantly decreased the cyclic AMP response to noradrenaline in the brain of rats with selective depletion of brain serotonin by p-chlorophenylalanine. The studies provide evidence for a cross-talk between noradrenergic and serotonergic receptor cascades at the level of mechanisms involved in the desensitization of the beta adrenoceptor-coupled adenylate cyclase system.

Assessment of a comparison of colorimetric methods used for oxytocinase determination.

The correlation of three colorimetric oxytocinase determination methods used in clinics was compared and assessed. It was found that the new methods, the method of Kleiner and Brounet-Yager, and that of Usategui-Gomez et al., show a high correlation with respect to the Tuppy and Nesvadba method (modified by Klimek) the usefulness of which has already been proved. The method of Usategui-Gomez et al., using L-cystine-bis-p-nitroanilide as substrate, is more sensitive and has more advantages.

Altered modulation by excitatory amino acids of cortical phosphatidylinositol system stimulated by carbachol in rats poisoned by an anti-cholinesterase compound, diisopropyl fluorophosphate.

The effects of glutamate and N-methyl aspartate (NMDA) on carbachol-induced inositol phosphate (IP) accumulation were evaluated in slices of the cerebral cortex of rats treated with diisopropyl fluorophosphate (DFP) for 2 weeks. This induced an about 75% inhibition of cholinesterases. The IP accumulation induced by carbachol (expressed as ratio stimulated/basal IP content) was lower in DFP rats than in controls when incorporation of [3H]-myoinositol into membrane phospholipids and their hydrolysis were measured (no washing step between labeling and hydrolytic incubation). There were no differences in carbachol induced IP accumulation between control and DFP rats when only phosphoinositide hydrolysis was determined (hydrolytic incubation of prelabeled washed slices). When both incorporation of [3H]-myoinositol and the hydrolysis were measured, 0.5 mM glutamate and 0.1 mM NMDA caused a significant, about 40%, decrease of carbachol-induced IP accumulation in control rats; the inhibitory effects of glutamate and NMDA were not significant in DFP rats. When only hydrolytic IP accumulation by carbachol was studied, the inhibitory effects of glutamate and NMDA were very similar in control and DFP rats. Additional experiments on inositol phospholipid synthesis showed a significantly lesser [3H]-myoinositol incorporation (by about 30%) in DFP rats. This may explain the differences between the results obtained by the two methods. The overall data suggest that the attenuation of glutamate and NMDA effects in DFP-rats depends on a decrease of carbachol-induced IP accumulation or phosphoinositide synthesis rather than on the EAA specific action.

Effects of diisopropyl fluorophosphate on muscarinic-M1-receptors and on receptor-mediated responsiveness of the phosphatidyl-inositol system in the cerebral cortex of rats.

The effects of acute and repeated treatments with an anticholinesterase (anti-ChE) compound, diisopropylfluorophosphate (DFP) on M1-acetylcholine receptor (M1-AChRs) density and on M1-mediated breakdown of inositol phospholipids were studied in the cerebral cortex of rats. The DFP doses induced an about 75% inhibition of cortical ChE 48 hr after the last treatment. The acute treatment did not change Bmax of M1-AChRs (measured as 3H-pirenzepine binding), while 1-week and 2-weeks treatments induced their significant down-regulation, by 14 and 29%, respectively. The responsiveness of M1-AChRs was measured in cortical prisms as accumulation of inositol phosphate (IP) following stimulation with a cholinergic agonist, carbachol (from 10 to 1000 microM). The IP accumulation (expressed as ratio stimulated/basal IP content) was lower in acute DFP rats than in controls at few carbachol concentrations, and after 2 weeks at most carbachol concentrations. This resulted in a significant increase of EC50. The data indicate the involvement of cortical phosphatidyl inositol system during intoxication by anti-ChE agents, namely a decreased efficiency of post-receptor mechanisms.

Retrieval associated cholinergic activity and its inhibition by memory updating.

Both hippocampal cholinergic and glutamatergic systems are believed to be engaged in learning and memory. By measuring behavior and ex vivo second messenger inositol phosphate (IP) accumulation, we investigated biochemical responses of cholinergic receptors to retrieval and acquisition processes in rats trained in a spatial task. We report that in rats retrieving spatial information, carbachol--induced IP accumulation strongly and transiently increased above values observed in handled controls and rats acquiring new information, and that this increase was profoundly inhibited by N-methyl-D-aspartate (NMDA). These results suggest that memory retrieval, rather than formation of a memory trace, is related to increased responsiveness of the hippocampal cholinergic system, and that formation of a new memory trace, which updates long-term memory, inhibits this cholinergic activation, possibly by a learning-associated increase in NMDA receptor activation. Moreover, the present study shows that the distinction between acquisition and retrieval processes can be demonstrated on both a behavioral and biochemical level.

Centpropazine affinity to cortical noradrenergic receptors and effect on their responsiveness in the rat.

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.

The effect of (-)-4-(2-hydroxy-3(N-isopropylamino)-propoxyimino)-cis-carane on basal and forskolin-stimulated accumulation of cyclic AMP in the cerebral cortical slices of the rat.

(-)-4-(2-Hydroxy-3(N-isopropylamino)-propoxyimino)-cis-carane++ +, a local anaesthetic and platelet aggregation inhibitor which is much more potent than lignocaine, facilitated forskolin-induced cyclic (c) AMP accumulation in cerebral cortical slices of the rat. Lignocaine was ineffective in this respect. It is hypothesized that a cAMP-related mechanism may be involved in increased efficacy of the compound.

Avoidance learning during antidepressant withdrawal in mice.

Shuttle-box avoidance acquisition, locomotor activity and density of adrenoreceptors in the cerebral cortex have been evaluated, in CD-1 mice, during withdrawal from repeated treatment with desipramine or mianserin (5 or 14 daily injections of antidepressant drug, 10 mg kg-1). Withdrawal from mianserin did not produce any behavioural or neurochemical change. Mice withdrawn from desipramine exhibited avoidance facilitation, when training started 24 h (but not 72 or 120 h) after the last injection. Locomotor activity was not affected and no change was found in the density of beta-adrenoreceptors. An up-regulation of alpha 2- and, to a lesser extent, of alpha 1-adrenoreceptors, occurred 72 h following desipramine withdrawal. However, the assessment of the role played by these neurochemical changes in the avoidance facilitation observed during withdrawal from the antidepressant treatment requires further study.

Second messengers: rate of formation as an index of receptor reactivity.

Generation of a second messenger upon stimulation of a metabotropic membrane receptor is the first biochemical reaction in the process of translation of a signal approaching the cell into the cellular response. Thus, the measurement of the rate of generation of these intracellular chemical signals may be a convenient way to assess the functional state of the receptor. The basic methodologies for assessment of the formation of cyclic AMP and inositol phosphates are described. Examples from our laboratory show how the measurement of these messengers may be employed in studies on receptor adaptation to permanently changed neurotransmitter availability, the discrepancy between the changes of receptor densities and functional up- or down-regulation, cooperativity of receptors, mechanisms of the action of drugs, neurotransmission changes in senescence and comparative neurochemistry.

Isomer-nonspecific action of dichlorodiphenyltrichloroethane on aryl hydrocarbon receptor and G-protein-coupled receptor 30 intracellular signaling in apoptotic neuronal cells.

Extended residual persistence of the pesticide dichlorodiphenyltrichloroethane (DDT) raises concerns about its long-term neurotoxic effects. Little is known, however, about DDT toxicity during the early stages of neural development. This study demonstrated that DDT-induced apoptosis of mouse embryonic neuronal cells is a caspase-9-, caspase-3-, and GSK-3ß-dependent process, which involves p,p'-DDT-specific impairment of classical ERs. It also provided evidence for DDT-isomer-nonspecific alterations of AhR- and GPR30-mediated intracellular signaling, including changes in the levels of the receptor and receptor-regulated mRNAs, and also changes in the protein levels of the receptors. DDT-induced stimulation of AhR-signaling and reduction of GPR30-signaling were verified using selective ligands and specific siRNAs. Co-localization of the receptors was demonstrated with confocal microscopy, and the presence of functional GPR30 was detected by electrophysiology. This study demonstrates that stimulation of AhR-signaling and impairment of GPR30-signaling play important roles in the propagation of DDT-induced apoptosis during the early stages of neural development.