Michihisa Tohda

Pteropodine and isopteropodine positively modulate the function of rat muscarinic M1 and 5-HT2 receptors expressed in Xenopus oocyte

Pteropodine and isopteropodine are heteroyohimbine-type oxindole alkaloid components of Uncaria tomentosa (Willd.) DC, a Peruvian medicinal plant known as cats claw. In this study, the effects of these alkaloids on the function of Ca(2+)-activated Cl(-) currents evoked by stimulation of G protein-coupled muscarinic M(1) acetylcholine and 5-HT(2) receptors were studied in Xenopus oocytes in which rat cortex total RNA was translated. Pteropodine and isopteropodine (1-30 microM) failed to induce membrane current by themselves. However, these alkaloids markedly enhanced the current responses evoked by both acetylcholine and 5-hydroxyhyptamine (5-HT) in a concentration-dependent and reversible manner with the maximal effects at 30 microM. Pteropodine and isopteropodine produced 2.7- and 3.3-fold increases in the acetylcholine response with EC(50) values of 9.52 and 9.92 microM, respectively, and 2.4- and 2.5-fold increases in the 5-HT response with EC(50) values of 13.5 and 14.5 microM, respectively. In contrast, in oocytes injected with total RNA from the rat cerebellum or spinal cord, neither alkaloid had an effect on the metabotropic current responses mediated by glutamate receptor(1 and 5) (mGlu(1/5)) receptors or ionotropic responses mediated by N-methyl-D-aspartate, kainic acid or glycine. Pteropodine and isopteropodine (10 microM) significantly reduced the EC(50) values of acetylcholine and 5-HT that elicited current responses, but had no effect on the maximal current responses elicited by acetylcholine and 5-HT. On the other hand, mitraphylline, a stereoisomer of pteropodine, failed to modulate acetylcholine- and 5-HT-induced responses. These results suggest that pteropodine and isopteropodine act as positive modulators of muscarinic M(1) and 5-HT(2) receptors.

Protective effect of Oren-gedoku-to (Huang-Lian-Jie-Du-Tang) against impairment of learning and memory induced by transient cerebral ischemia in mice.

The protective effect of Oren-gedoku-to (OGT; Huang-Lian-Jie-Du-Tang), a traditional Chinese medicine, against impairment of learning and memory induced by transient cerebral ischemia was investigated in mice. The cerebral ischemia caused a reduction of step-down latency and an increase of step-down errors in the passive avoidance task. Pretreatment with oral administration of OGT (2, 4 or 8 g of herbs per kg) once daily for 5 days prolonged the step-down latency significantly and decreased the step-down errors as compared with those of sham-operated controls. In the Morris water maze test, the cerebral ischemia caused an increase in the latency until finding the platform in the training trial and a decrease in the percentage of swimming in the quadrant of the former platform in the probe trial. Oren-gedoku-to (OGT; 2, 4 and 8 g/kg, p. o.) shortened the latency of escaping markedly onto the platform in the training trial and increased the percentage of crossing the former platform quadrant in the probe trial. A reference drug, tacrine (0.5 and 1.0 mg/kg, p.o.), prevented the reduction of step-down latency in the passive avoidance task and shortened the escape latency in the Morris water maze task. Furthermore, OGT significantly protected against cerebral ischemia-induced reduction in the acetylcholine (ACh) content of the cerebral cortex, hippocampus and striatum. These results indicate that the protective effects of OGT against the impairment of learning and memory induced by transient cerebral ischemia may be associated with preventing the decrease in the ACh content of the mouse brain.

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.

CNS inhibitory effects of barakol, a constituent of Cassia siamia Lamk

The present study determined the pharmacological profile of barakol, a major constituent of Cassia siamea Lamk., in rodent behavioral and neurochemical tests. Barakol reduced spontaneous locomotor activity, increased the number of sleeping animals and prolonged the thiopental-induced sleeping time, indicating a sedative effect. As for interactions between barakol and convulsants (pentylenetetrazole (PTZ), picrotoxin, bicuculline and strychnine), only a high dose (100 mg/kg, i.p.) of barakol slightly prolonged the latency of clonic convulsion induced by picrotoxin. This suggests that the sedative effect may not be induced via the GABA or glycine systems. There was no evidence of an anxiolytic effect of barakol in the plus-maze test. However, barakol (25-100 mg/kg, i.p.) could suppress methamphetamine (1 mg/kg, i.p.)-induced hyper-locomotor activity in a dose-dependent manner, indicating an effect on the dopaminergic system. In a microdialysis study, the dose of barakol (100 mg/kg) that inhibited spontaneous locomotor activity in mice did not affect the basal levels of extracellular dopamine (DA) or its metabolites in the striatum. However, pretreatment with barakol (100 mg/kg, i.p.) decreased the maximal dopamine release and dopamine turnover induced by methamphetamine (1 mg/kg, i.p.). This finding indicates that the inhibitory effect of barakol on dopamine release may account for the blocking effect of barakol on the striatum-related behavior induced by methamphetamine.

Involvement of Growth-Associated Protein-43 with Irreversible Neurite Outgrowth by Dibutyryl Cyclic AMP and Phorbol Ester in NG108–15 Cells

Simultaneous treatment with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and dibutyryl cyclic AMP (diBu‐cAMP) for 72 h induced neurites in NG108–15 cells significantly longer than treatment with each alone. Treatment for 72 h with both drugs induced irreversible neurite extension and a decline in protein kinase C activity, although neurites extended by diBu‐cAMP alone disappeared after the withdrawal of the drug. The expression of growth‐associated protein‐43 (GAP‐43) mRNA was also observed by a combined application of TPA and diBu‐cAMP. The increased level of GAP‐43 mRNA induced by treatment with both drugs for 72 h was maintained at least 24 h after withdrawal of the drugs. In cells transfected with GAP‐43 cDNA, neurites induced by treatment with diBu‐cAMP alone for 72 h were maintained at least 48 h after removal of the drugs. These results suggest that GAP‐43 could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in the accumulation of GAP‐43.

Increase in Neurite Formation and Acetylene line Release by Transfection of Growth‐Associated Protein‐43 cDNA into NG108–15 Cells

Abstract: We previously reported that growth‐associated protein‐43 (GAP‐43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP‐43. In the present study, to clarify the functional significance of GAP‐43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG‐G11 cells by transfection of GAP‐43 cDNA into NG108‐15 cells. NG‐G11 cells expressed GAP‐43 mRNA at levels approximately twice that in nontransfected or vector‐transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu‐cAMP) or 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) plus diBu‐cAMP. Neurite outgrowth after addition of diBu‐cAMP was greater in NG‐G11 than in control cells. In NG‐G11 cells, neurites elongated by treatment with diBu‐cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu‐cAMP for 24 h induced neurite outgrowth in NG‐G11 cells, although control cells required 72 h. Depolarization by 50 mM KCI induced ACh release in both NG‐G11 and control cells treated with diBu‐cAMP or TPA/diBu‐cAMP. Although removal of the drugs following diBu‐cAMP treatment reversed ACh release to nontreated levels in control cells, a high‐K+‐induced level of ACh release remained in NG‐G11 cells after removal of diBu‐cAMP. ACh release induced by TPA plus diBu‐cAMP for 24 h was further enhanced after removal of the drugs in NG‐G11 cells, but it was not seen in control cells. These results suggest that levels of GAP‐43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP‐43 may be involved in neuronal differentiation in NG108‐15 cells.

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.

Serotonin stimulates both cytosolic and membrane-bound guanylate cyclase in NG108-15 cells

Abstract: The cyclic GMP (cGMP) content was rapidly (>30 s) increased by serotonin [5‐hydroxytryptamine (5‐HT)] (EC50= 10 μM), and the increase lasted for > 10 min in NG108–15 cells. The 5‐HT‐induced elevation of cGMP level (EC50= 10 μM) at 20 s (“fast” elevation) was inhibited by ICS 205–930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5‐HT effect at 10 min (“slow” elevation) was not inhibited by several antagonists for 5‐HT receptors of the IA, IB, IC., ID, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5‐HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin‐induced cGMP accumulation as shown in the fast response to 5‐HT. ANP. an activator of membrane‐associated guanylate cyclase (mGC), slowly (∼60 s) increased the cGMP content (EC50= 10 nAf), a result lasting for >10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5‐HT. 5‐HT and ANP did not induce formation of inositol phosphates. These results suggest that (a) the fast effects of 5‐HT on cGMP level elevation are mediated by 5‐HT3 receptors, which activate cytosolic guanylate cyclase through Ca2+ entry via ion channels other than voltage‐sensitive Ca24 channels, and (b) the slow effects seem to be due to an unidentified subtype of 5‐HT receptor that activates ANP‐sensitive mGC.

Inhibitory effects of KN-62, a specific inhibitor of Ca/calmodulin-dependent protein kinase II, on serotonin-evoked Cl− current and 36Cl− 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.

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.