Inhibitory effects of pertussis toxin on a depolarization-evoked Ca2+ influx in NG108-15 cells.

Depolarized stimulation 1.5-fold increased Ca2+ influx which was inhibited by pretreatment with verapamil or LaCl3. Treatment with pertussis toxin, islet-activating protein (IAP), induced a reduction in 50 mM K+-induced Ca2+ influx and stimulated adenylate cyclase (AC) activity in NG108-15 cells. However, addition of dibutyryl cAMP or forskolin treatment elevating cAMP level exerted no effects on a depolarization-induced Ca2+ influx. Dissociated B-oligomer of IAP after treatment with dithiothreitol and ATP increased a depolarization-evoked Ca2+ influx. It is suggested that inhibitory GTP-binding protein (G1) or other IAP substrate proteins could directly be involved in Ca2+ influx via voltage-sensitive Ca2+ channel.

Brain serotonin neurotransmission: an overview and update with an emphasis on serotonin subsystem heterogeneity, multiple receptors, interactions with other neurotransmitter systems, and consequent implications for understanding the actions of serotonergic drugs.

Knowledge about serotonergic neurotransmission has been expanding rapidly. Recent research has delineated 15 molecularly different serotonin receptors and multiple, discrete neuronal and nonneuronal (including endocrine) pathways and mechanisms that mediate the many functions of serotonin. Nonetheless, gaps remain regarding aspects of the anatomy and physiology of serotonin in its roles as a neurotransmitter, a neuromodulator, and a hormone. Few serotonin receptor-selective drugs are available for clinical use. A group of selective serotonin reuptake inhibitors (SSRIs) remain the agents with greatest therapeutic utility, although the mechanisms underlying their delayed efficacy, which clearly result from adaptive consequences following repeated administration rather than early uptake inhibition of serotonin by itself, are incompletely understood and appear to involve changes in signal transduction and gene expression in serotonergic and other neurotransmitter systems.

Effects of histamine on polyphosphoinositide metabolism in NG108-15 cells.

Effects of histamine on metabolism of phosphatidylinositol and its related phospholipids were studied in NG108-15 cells. Histamine (100 ?M) significantly caused formation of both inositol trisphosphate (IP(3)) and inositol bisphosphate (IP(2)) in NG108-15 cells whose phosphatidylinositol was prelabelled with [(3)H]inositol. Histamine at 1-100 ?M dose-dependently caused the degradation of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)) and phosphatidic acid (PA) in the cells prelabelled with [(3)H]arachidonic acid. Histamine degraded PI-4,5-P(2) in a biphasic manner which reached two peaks at 10 and 30 s. The degradation at 30 s was inhibited by pretreatment with 100 ?M quinacrine, while it was not affected at 10 s. In addition, 100 ?M quinacrine significantly abolished histamine-induced degradation of PA at 10 and 30 s. Although 100 ng/ml pertussis toxin pretreatment for 4 h resulted in ADP-ribosylation of 41 kDa membrane proteins, histamine-induced PI-4,5-P(2) degradation was not significantly affected by the toxin treatment. These results suggest that: (1) histamine causes polyphosphoinositide degradation by phospholipase C and further by phospholipase A(2); (2) histamine presumably stimulates PA-specific phospholipase A(2), resulting in the decrease of PA and release of arachidonic acid and/or the metabolites; (3) 41 kDa GTP-binding proteins ADP-ribosylated by pertussis toxin were not involved in histamine receptor-mediated polyphosphoinositide turnover in NG108-15.

Neurochemical and morphological studies on differentiation of NG108-15 cells by phorbol ester and forskolin.

12-O-tetradecanoylphorbol 13-acetate (TPA), forskolin or dibutyryl cAMP induced neurite outgrowth and inhibition of cell growth in NG108-15 cells. TPA, forskolin and dibutyryl cAMP significantly increased specific activity of choline acetyltransferase. Forskolin markedly stimulated cAMP accumulation, but not TPA, suggesting that forskolin could induce differentiation by increasing the cAMP content via adenylate cyclase activation, but TPA-induced differentiation seems not to be due to the raise of the cAMP level. Incubation of the cells with TPA, forskolin or dibutyryl cAMP for 24 h resulted in enhancement of 50 mM K(+)-evoked Ca(2+) influx and neurite elongation, although incubation with these agents for 1 h didn't affect these events. From these results, it is suggested that TPA and forskolin induce differentiation of NG108-15 cells to acetylcholine neurons via different mechanisms: protein kinase C activation by TPA and cAMP-dependent protein kinase activation by forskolin. In addition, it is likely that Ca(2+) channels in cells differentiated by TPA, forskolin or dibutyryl cAMP become sensitive to depolarization.

Inhibitory effects of antidepressants on NMDA-induced currents in Xenopus oocytes injected with rat brain RNA.

Although it has been reported that desipramine affects ion-channel activity of NMDA receptor/ion-channel complexes, the binding sites remain unclear. To identify the binding site, influences of desipramine on NMDA-induced current were examined in Xenopus oocytes injected with rat brain RNA and compared with those of blockers, MK-801, Zn2+ and Mg2+. Application of 100 microM desipramine irreversibly inhibited NMDA-induced inward current as well as 1 microM MK-801. Mg2+ and Zn2+ showed a reversible inhibition. Pretreatment with Mg2+ or Zn2+ abolished the irreversible inhibition of desipramine. In contrast, the irreversible inhibition of desipramine was still observed after application of Mg2+ and Zn2+. These results suggest that Mg2+/Zn2+ and desipramine bind on different sites from each other and affect the cation permeability via different mechanisms. Regarding inhibitory effects of other antidepressant drugs, imipramine and setiptiline were found to markedly inhibit NMDA current, while maprotiline, amitriptyline and lofepramine slightly inhibited the current. Mianserin, a potent antagonist of 5-HT1c receptors, however, had no influence.

Delivery of antisense oligonucleotides to neuroblastoma cells.

pH-Sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesterol hemisuccinate (3:2 mol/mol) were applied in delivery of antisense oligodeoxynucleotides (asODN) into NG 108-15 neuroblastoma and glioma cells. Fluorescently labelled asODN were entrapped in liposomes by a modified freeze-thawing method (20% encapsulation efficiency). The uptake of asODN (free or entrapped in liposomes) by NG 108-15 cells was monitored by fluorescence-activated cell sorting and confocal microscopy. Delivery of asODN was significantly improved when antisense were entrapped in liposomes as compared to free (nonliposomal) asODN. The uptake was dose-dependent and optimum was achieved after 2 h incubation.

Imipramine-induced increase in 5-HT2C receptor mRNA level in the rat brain.

Repeated oral administration of 20 mg/kg imipramine elevated the level of 5-HT2C mRNA in the rat brain. Hybridization signals in nearly all regions stained by digoxigenin-labeled antisense cRNA probe, such as the hippocampus, choroid plexus, habenular nucleus, and dorsomedial hypothalamic nucleus, were more intense following imipramine treatment. These results suggest that long-term treatment with imipramine stimulates 5-HT2C receptor gene expression.

A novel factor, TA20, involved in neuronal differentiation: cDNA cloning and expression.

Combined treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) and dibutyryl cyclic AMP (diBu-cAMP) induced significantly longer neurites than treatment with each alone in NG108-15 cells. We performed differential screening to identify genes expressed only by treatment with TPA plus diBu-cAMP but not by that with diBu-cAMP for 72 h alone in NG108-15 cells, and isolated a novel gene, TA20. Over-expression of the gene in NG108-15 and neuroblastoma N18TG-2 cells caused intense neurite elongation and suppressed cell growth. TA20 did not cause, however, any morphological changes in glioma C6Bu-1 cells. These results suggest that TA20 is a novel neuronal differentiation factor.

Diazepam binding inhibitor (DBI) gene expression in the brains of socially isolated and group-housed mice.

Diazepam binding inhibitor (DBI), a putative endogenous polypeptide ligand for benzodiazepine (BZD) receptors, has been shown to act as an inverse BZD receptor agonist in the brain. We previously suggested that the social isolation stress-induced decrease in pentobarbital sleeping time in mice was partly due to an increase in the activity of endogenous substances with an inverse BZD receptor agonist-like property such as DBI. In this study, we examined whether the DBI gene expression is affected by socially isolated stress. Consistent with the previous findings, the in situ hybridization result showed very strong signals of DBI mRNA around the regions of the third ventricle, especially the lining cells, the arcuate nucleus of the hypothalamus and the cerebellum, in both socially isolated and group-housed animals. Unexpectedly, however, semi-quantitative experiments with reverse transcription polymerase chain reaction technique revealed that socially isolated mice had significantly less expression of DBI mRNA in the hypothalamus than group-housed animals, and no difference in the expression in the other brain areas was observed between two animal groups. We discuss the relationship between the decrease of DBI mRNA expression in the hypothalamus and the decrease of GABA(A) receptor function following long-term social isolation in mice.

NMDA antagonists potentiate scopolamine-induced amnesic effect.

The effects of N-methyl-D-aspartate NMDA receptor antagonists on scopolamine-induced amnesia and on delay-interposed short-term memory performance were investigated using an 8-arm radial maze in rats. Scopolamine, a muscarinic antagonist, deteriorated the radial maze performance, while MK-801, an NMDA receptor channel blocker and CGS-19755, a competitive NMDA receptor antagonist, showed no obstruction to the spatial cognition in the non-delayed maze task. MK-801 (0.01-0.03 mg/kg, i.v.) and CGS-19755 (1-10 mg/kg, i.v.) significantly augmented scopolamine-induced deficit in the non-delayed maze task and impaired the short-term memory in the 5-min delay-interposed task. These results suggest that NMDA antagonists have a negative action on short-term memory and that the interaction between the NMDA and the central muscarinic system plays a role in modulating the cognitive function.

Hyperactivity of central noradrenergic and CRF systems is involved in social isolation-induced decrease in pentobarbital sleep.

The modulatory effects of the central noradrenergic and CRF systems on the pentobarbital-induced hypnotic activity were investigated in socially isolated mice. Pentobarbital-induced sleeping time decreased depending on the duration of isolation period and reached the minimum at 4 weeks after the isolation. The intermale aggressive behavior tested in isolated mice increased along with the decrease of hypnotic activity of pentobarbital. I.c.v. injection of CRF (corticotropin-releasing factor; 0.6-2.1 nmol) and i.p. injection of yohimbine (0.5-1 mg/kg), an alpha 2-adrenoceptor antagonist, significantly decreased the pentobarbital-induced sleeping time in group-housed but not in socially isolated mice while alpha-helical CRF9-41 (alpha hCRF; 3.3-6.5 nmol i.c.v.), a CRF antagonist, and clonidine (12.5-100 micrograms/kg i.p. and 7.5-15 nmol i.c.v.), an alpha 2-adrenoceptor agonist, recovered the hypnotic activity of pentobarbital decreased by social isolation to the level in group-housed mice without changing the activity observed in group-housed animals. alpha hCRF (6.5 nmol i.c.v.) significantly abolished the yohimbine (1 mg/kg i.p.)-induced decrease in the hypnotic activity of pentobarbital in group-housed mice. Propranolol (50-100 nmol i.c.v. and 5-10 mg/kg i.p.), a beta-adrenoceptor antagonist, and prazosin (5-10 nmol i.c.v. and 250-500 micrograms/kg i.p.), an alpha 1-adrenergic antagonist, significantly and dose-dependently recovered the hypnotic activity of pentobarbital in socially isolated mice to the level in group-housed mice.(ABSTRACT TRUNCATED AT 250 WORDS)

NMDA-but not AMPA-receptor antagonists augment scopolamine-induced spatial cognitive deficit of rats in a radial maze task.

The effect of NMDA and AMPA receptor antagonists on a scopolamine-induced spatial cognitive deficit was investigated in rats using an 8-arm radial maze. The NMDA antagonists, MK801 and CGS19755, robustly augmented scopolamine-induced deficits but had no effect on spatial cognition when administered alone. In contrast, augmentation of the scopolamine-induced deficits was not observed when the selective AMPA antagonist, YM90K, was administered with scopolamine. These results suggest that the NMDA but not AMPA subtypes of the ionotropic glutamate receptors play important roles in regulation of the central cholinergic function related to the spatial learning and memory processes.

Biphasic effects of mianserin and desipramine on serotonin-evoked current and Cl- efflux in Xenopus oocytes.

Serotonin (5-HT, 1 microM) elicited two phases of Cl- inward current in Xenopus oocytes injected with rat brain mRNA: a transient current (T-current), which was generated rapidly (within 1 min), and a sustained current (S-current), which persisted for 10 min. Each type of 5-HT-evoked response was time-dependent after mRNA injection. The T-current was generated at 20-30 h and the S-current at 30-40 h. Although mianserin at 0.1 microM completely inhibited the T-current, 10 microM mianserin was required to suppress the S-current. 5-HT also caused Cl- efflux from oocytes preloaded with 36Cl-. Cl- efflux during 1 min, corresponding to the T-current, was inhibited by 0.1 microM mianserin. A higher concentration of mianserin (10 microM) was required to block the efflux for 10 min, corresponding to the S-current, as well as the current response. Desipramine selectively inhibited the T-current and Cl- efflux for 1 min. The mechanisms underlying the different sensitivity to mianserin of oocytes injected with rat brain mRNA are discussed.

Effects of antidepressants on serotonin-evoked current in Xenopus oocytes injected with rat brain mRNA.

Serotonin (5-HT, 1 microM) induced an inward current in Xenopus oocytes injected with rat brain mRNA under steady voltage clamp conditions of -60 mV. The 5-HT response was blocked by 0.1 microM mianserin, but neither buspirone, 8-hydroxy-dipropylaminotetralin (8-OH-DPAT), trifluoromethylphenyl piperazine (TFMPP), pindolol, propranolol, spiperone, ketanserin nor ICS 205-930 exerted effects, suggesting that the 5-HTIC subtype of 5-HT receptors is involved in the current. The 5-HT-induced current was inhibited by imipramine or desipramine with IC50 values of 60 nM or 20 microM, respectively. The response was also inhibited by setiptilline, maprotiline or amoxapine at a dose of 10 microM. Imipramine at 10 microM had no effect on the acetylcholine (ACh, 1 mM)-induced current response. It has been reported that inositol triphosphate (IP3) formation and intracellular Ca2+ are involved in the 5-HT- as well as ACh-induced current and that intracellular injection of either 50 pmol IP3 or 50 pmol Ca2+ mimics the 5-HT-induced current. The response induced by intracellular injection of either 50 pmol IP3 or 50 pmol Ca2+ was not affected by 10 microM imipramine. It is suggested that imipramine, and perhaps other antidepressant drugs tested, blocks 5-HTIC receptors, subsequently inhibiting the 5-HT-evoked current.

Distinct induction of c-fos mRNA in NG108-15 cells transfected with muscarinic m1 and m3 receptors.

The differences of intracellular signalling mechanisms between muscarinic acetylcholine m1 and m3 receptors, which are coupled with polyphosphoinositide turnover, were examined by using m1- and m3-transfected NG108-15 cells. The c-fos mRNA was induced by 1 mM acetylcholine peak at 60 min in both m1 and m3 cells. The c-fos induction in m1 cells was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), but was not inhibited by prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), suggesting that intracellular Ca2+ and calmodulin are involved in the induction. The c-fos induction in m3 cells was inhibited by BAPTA-AM and prolonged treatment with TPA, but was not influenced by W-7, suggesting that protein kinase C is mainly involved in m3-induced c-fos expression. Acetylcholine induced an increase in inositol phosphates and a transient increase in the intracellular concentration of Ca2+ in both m1 and m3 cells. Sustained stimulation of acetylcholine strongly increased the inositol monophosphate content in m3 cells, but that of inositol trisphosphate and inositol diphosphate in m1 cells. These results suggest that the difference between m1- and m3-induced c-fos mRNA induction mechanisms is due to the difference in respective properties in polyphosphoinositide turnover.

Inhibitory effects of botulinum toxin on 5-HT1C receptor-induced Cl- current in Xenopus oocytes.

Several low molecular weight G proteins have been identified, but their functional roles remain unclear. To clarify the involvement of low molecular weight G protein in receptor-stimulated turnover of polyphosphoinositide (PI) turnover, influences of botulinum toxins on serotonin (5-HT)-stimulated Cl- current mediated by PI turnover were investigated using Xenopus oocytes injected with rat brain mRNA. Treatment with botulinum toxin C, D or purified ADP-ribosyltransferase of botulinum toxin (botulinum toxin C3 enzyme) inhibited the 5-HT-induced Cl- current in oocytes, and ADP-ribosylated 23 kDa proteins. Both botulinum toxin C3 enzyme-induced inhibition of the current and ADP-ribosylation were suppressed by pretreatment with antibotulinum toxin C3 enzyme antibody. Botulinum toxin D treatment of oocytes was ineffective in the response of Cl- current induced by injection of 50 pmol inositol 1,4,5-trisphosphate and 50 pmol Ca2+. It is suggested that low molecular weight G proteins ADP-ribosylated by botulinum toxin C3 enzyme are involved in phospholipase C activation in Xenopus oocytes.

Inhibitory effects of corymine, an alkaloidal component from the leaves of Hunteria zeylanica, on glycine receptors expressed in Xenopus oocytes.

We previously reported that corymine, an alkaloidal compound extracted from the leaves of Hunteria zeylanica native to Thailand, potentiated convulsions induced by either picrotoxin or strychnine. Therefore, to clarify the mechanism of action of corymine, the effects of corymine on gamma-aminobutyric acid (GABA) and glycine receptors were examined. We used Xenopus oocytes expressing these receptors and the two-electrode voltage-clamp method. The receptors expressed in oocytes injected with rat brain and spinal cord RNA showed the pharmacological properties of GABAA and glycine receptors, respectively. Corymine (1-100 microM) partially (20-30%) reduced the GABA responses in oocytes injected with rat brain RNA, while marked (up to 80%) dose-dependent reductions were observed in the glycine responses in oocytes injected with rat spinal cord RNA. These observations suggest that corymine was more effective against the glycine receptors than the GABA receptors. The ED50 of corymine on the glycine response was 10.8 microM. Corymine, at 30 microM, caused a shift to the right, with a lower maximal response, of the glycine concentration-response curve. This indicated that the action of corymine on glycine receptors is neither competitive nor purely non-competitive. These observations suggest that a binding site other than the glycine recognition site of the glycine receptors is the site of action of corymine.

A possible mechanism underlying corymine inhibition of glycine-induced Cl- current in Xenopus oocytes.

We previously reported that corymine, an alkaloid extracted from the leaves of Hunteria zeylanica native to Thailand, inhibited glycine-induced chloride current using a receptor expression model of Xenopus oocytes. In this study, we investigated the mechanism underlying the inhibitory action of this alkaloid on glycine current using the same model. Corymine inhibited glycine current in a noncompetitive fashion. Co-application with strychnine, a competitive glycine receptor antagonist, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, corymine decreased the ED50 value of strychnine, but did not change that of DIDS. Moreover, the inhibitory effects of corymine and either strychnine or DIDS were additive. The desensitization phase of glycine current showed two exponentials and corymine preferentially inhibited the fast component, whereas strychnine affected both of them to the same extent and DIDS preferentially inhibited the slow component. When these drugs were applied repeatedly, the inhibitory effects of corymine and strychnine were not use-dependent and reversible, while the effect of DIDS was use-dependent and irreversible. The inhibitory effect of corymine on gamma-aminobutyric acid (GABA) current was less potent than the effect on glycine current, while this alkaloid failed to affect acetylcholine and serotonin currents. These results demonstrate that corymine inhibits glycine-gated CI- channels by interacting with the site different from that of DIDS.

Inhibitory effects of KN-62, a specific inhibitor of Ca/calmodulin-dependent protein kinase II, on serotonin-evoked C1-current and 36-C1-efflux in Xenopus oocytes.

To clarify the details of the involvement of Ca(2+)-calmodulin in gating of C1-channels, effects of a novel calmodulin-dependent protein kinase II (CaMK II) inhibitor, KN-62, on C1-current and C1-efflux induced by serotonin (5-HT) were investigated in Xenopus oocyte injected with rat brain mRNA. 5-HT evoked inward current on voltage clamp condition at -60 mV in a concentration-dependent manner. The 5-HT (1 microM)-evoked current was blocked by preperfusion with 1 microM KN-62 also inhibited acetylcholine- and inositol 1,4,5-trisphosphate-evoked current. Furthermore, 5-HT enhanced Cl- efflux about 2.5-fold from the oocyte preinjected with 36Cl-, and the effects were inhibited by KN-62 as well. These results suggest that CaMK II is activated by Ca(2+)-calmodulin and opens Cl- channels to induce CL- efflux in Xenopus oocytes.

Possible involvement of botulinum ADP-ribosyltransferase sensitive low molecular G-protein on 5-hydroxytryptamine (5-HT)-induced inositol phosphates formation in 5-HT2c cDNA transfected cells.

To clarify the involvement of botulinum ADP-ribosyltransferase sensitive low molecular G-proteins in 5-hydroxytryptamine (5-HT)-induced stimulation of phosphatidylinositol turnover, we examined the effects of 5-HT on inositol phosphates formation in COS 7 cells transfected with 5-HT2c receptor cDNA, but did not in non-transfected or vector-transfected cells. A typical 5-HT2c receptor antagonist mianserin (0.3-3 microM) inhibited the 5-HT-induced inositol phosphates formation. Treatment with botulinum toxin D preparation (20 micrograms/ml, 8 h) that contained botulinum C3 ADP-ribosyltransferase, blocked the 5-HT-induced inositol phosphate formation, although botulinum toxin A preparation that did not contain the enzyme did not have an influence. These results support our previous findings suggesting that low molecular weight G-proteins ADP-ribosylated by botulinum ADP-ribosyltransferase are involved in phospholipase C activity.