Masami Shimizu

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M1- and M2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M1- and M2-receptor subtypes through semi-quantitative in vitro autoradiography. [3H]Quinuclidinyl benzilate (QNB) and [3H]pirenzepine (PZ) were used for labeling total mAChR and M1-receptors, respectively. In the cerebral cortex of adult rats, [3H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M1-receptors were diffusely observed in all the layers. This means that M2-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M1- and M2-receptors first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M1-type in the adult rat brain, ontogenic patterns of M1-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M2-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M1-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M1- and M2-receptors show distinct developmental behaviors in the rat brain.

Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ store by antidepressant drugs in cultured neurons of rat frontal cortex

Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca2+ ([Ca2+]i) was examined in primary cultured neurons from rat frontal cortices using the Ca2+‐sensitive fluorescent indicator fura‐2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca2+]i in a concentration‐dependent manner (100 μM to 1 mM). These four AD‐induced [Ca2+]i increases were not altered by the absence of external Ca2+ or by the presence of La3+ (30 μM), suggesting that these ADs provoked intracellular Ca2+ mobilization rather than Ca2+ influx. All four ADs increased inositol 1,4,5‐trisphosphate (IP3) contents by 20–60% in the cultured cells. The potency of the IP3 production by these ADs closely correlated with the AD‐induced [Ca2+]i responses. Pretreatment with neomycin, an inhibitor of IP3 generation, significantly inhibited amitriptyline‐ and imipramine‐induced [Ca2+]i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP3 generation and mobilize the intracellular Ca2+, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline‐induced [Ca2+]i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca2+ from IP3‐sensitive Ca2+ stores and that the responses are pertussis toxin‐insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.

Imipramine stimulates phospholipase C activity in rat brain

We previously demonstrated that antidepressant drugs (ADs) cause Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in cultured neurons of rat frontal cortex. The present study examines the mechanism by which tricyclic ADs activate phospholipase C (PLC) in rat frontal cortex. Using an exogenous substrate to measure PLC activity, we demonstrated that a tricyclic AD, imipramine, stimulated PLC activity of the frontal cortex membrane in a concentration-dependent manner. Two tricyclic ADs, desipramine and amitriptyline, also stimulated PLC activity, while Li+ or pargyline had no effect on PLC activity. Although imipramine did not activate PLC in the membrane in the absence of Ca2+, imipramine synergistically activated PLC in the presence of Ca2+. This result indicates that the mechanism of PLC activation by imipramine is different from its activation by Ca2+. Imipramine stimulated PLC activity in the cytosol of rat frontal cortex as well as in the membrane. Preincubation of the cytosol with anti-PLC-beta 1 antibody prevented the imipramine-mediated activation of PLC. However, preincubation with anti-PLC-gamma 1 or anti-PLC-delta 1 did not prevent activation of PLC. These results suggest that imipramine activates PLC-beta 1 directly without receptor or guanine nucleotide binding protein mediation.

Down‐Regulation of 5‐Hydroxytryptamine7 Receptors by Dexamethasone in Rat Frontocortical Astrocytes

Abstract: Astrocytes derived from rat frontal cortex contain 5‐hydroxytryptamine7 (5‐HT)7 receptors positively coupled to adenylyl cyclase. In the present study, we investigated the effects of glucocorticoids on adenylyl cyclase activity and 5‐HT7 receptor gene expression in astrocytes. Addition of dexamethasone (0.01–100 nM, 12–72 h) to the culture medium decreased cyclic AMP formation induced by 5‐HT in a time‐ and concentration‐dependent manner. Dexamethasone treatment (10 nM, 48 h) reduced maximum responses of cyclic AMP formation induced by 5‐HT and 5‐carboxamidotryptamine without alterations in the EC50 value. In contrast, treatment with the mineralocorticoid aldosterone (48 h) had no significant effects on 5‐HT‐induced cyclic AMP formation with concentrations up to 10 nM. It was observed that dexamethasone treatment (1–100 nM, 3–72 h) also decreased the expression of 5‐HT7 receptor mRNA, using reverse transcription and polymerase chain reaction analysis. A significant reduction in expression of 5‐HT7 mRNA appeared at 3 h of dexamethasone treatment and reached a maximum at 6 h of treatment. On the other hand, dexamethasone treatment (10 nM, 48 h) did not affect basal levels of cyclic AMP and cyclic AMP synthesis stimulated by isoproterenol, N‐ethylcarboxamido‐adenosine, cholera toxin, and forskolin. These results suggest that dexamethasone decreases the expression of the 5‐HT7 receptor gene and, consequently, 5‐HT7 receptor‐mediated signal transduction in frontocortical astrocytes.

Increase in Serotonin 1A Receptors in the Dentate Gyrus as Revealed by Autoradiographic Analysis Following Repeated Electroconvulsive Shock But Not Imipramine Treatment

The effects of repeated treatment with electroconvulsive shock (ECS) and imipramine on [3H]8-OH-DPAT binding to serotonin1A (5-HT1A) receptors in the rat brain were studied by quantitative autoradiographic analysis. A large number of binding sites for [3H]8-OH-DPAT were observed in the hippocampus, especially the dentate gyrus, CA1+CA2 field, dorsal raphe nucleus and septum. Repeated treatment with ECS, but not repeated imipramine treatment, significantly increased [3H]8-OH-DPAT binding sites in the dentate gyrus. These results suggest that the increase in [3H]8-OH-DPAT binding sites in the dentate gyrus may be important in ECS therapy for depressive illness.

Antidepressants inhibit spontaneous oscillations of intracellular Ca2+ concentration in rat cortical cultured neurons.

In rat cortical cultured neurons, spontaneous oscillations in the intracellular Ca2+ concentration ([Ca2+]i) were observed by direct [Ca2+]i measurements using the fluorescent indicator fura-2. These [Ca2+]i responses were eliminated by the removal of extracellular Ca2-, L-type Ca2+ channel blockers or a glutamate receptor antagonist. Three antidepressants (amitriptyline, imipramine and mianserin) suppressed the [Ca2+]i oscillations in the range of 1-50 microM. In addition, amitriptyline inhibited high K(+)-and glutamate-induced [Ca2+]i increases with IC50 values of 19 and 27 microM, respectively. Imipramine and mianserin also inhibited the high K(+)-induced [Ca2+]i increases with IC50 values of 45 and 24 microM, respectively. These results suggest that blocking actions by the antidepressants on voltage-dependent Ca2+ influx and glutamate receptor-mediated Ca2+ influx may be involved in the suppression of the [Ca2+]i oscillations.

Agonist-induced desensitization of adenylyl cyclase activity mediated by 5-hydroxytryptamine7 receptors in rat frontocortical astrocytes

Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)7 receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT7 receptors induced by a treatment with agonists (0.1, 1, and 10 muM, 0.5 to 3.5 h). Maximum responses, but not the EC50 values, in the concentration-response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor alpha-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT7 receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.

Single or repeated treatment with electroconvulsive shock increases number of serotonin uptake binding sites in the frontal cortex

The effects of a single or repeated treatment with electroconvulsive shock (ECS) or imipramine on the central serotonin (5-HT) uptake binding sites were studied in the rat frontal cortex and hippocampus. The selective 5-HT uptake inhibitor citalopram and clomipramine potently inhibited the binding for [3H]paroxetine (5-HT uptake binding sites) in the frontal cortex. The antidepressant drugs imipramine and desipramine inhibited the binding moderately, but the 5-HT-related agents, 5-HT, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), mianserin and ketanserin inhibited it weakly. A single ECS increased the density of [3H]paroxetine binding sites, but did not alter the affinity, after 1 or 24 h, in the frontal cortex. Repeated treatment with ECS, but not with imipramine, increased the density of [3H]paroxetine binding sites in the same region. The hippocampal [3H]paroxetine binding did not change after any of these treatments. These results suggest that a single treatment with ECS causes a rapid increase in the neuronal 5-HT transporter complex and the increase lasts for 14 days in the frontal cortex.

Brain ischemia decreases phosphatidylcholine-phospholipase D but not phosphatidylinositol-phospholipase C in rats.

Background and Purpose Phosphatidylcholine (PC)-phos-pholipase D (PLD) is an important intracellular signaling pathway in response to a variety of agonists, but little is known about the effects of brain ischemia on the PC-PLD system. We thus have examined the effects of global cerebral ischemia on PLD in rats. Methods We have examined the effects of global ischemia (decapitation or four-vessel occlusion) on PLD and PLC activity in the membrane fraction of rat brains. We measured the PLD and PLC activity in detergent-mixed micelle assay systems using 3H-labeled exogenous substrate. Results The results demonstrate that basal PLD activity showed a gradual decrease with increased duration (5 to 30 minutes) of ischemia by decapitation in the hippocampus; after 30 minutes of ischemia, PLD activity was significantly decreased compared with the control. Lineweaver-Burk plots showed that the apparent Vmax value of PLD in ischemia was one half of that in the control without changes in Km value. Ischemia by decapitation significantly decreased PLD activity in the brain stem as well as the hippocampus, whereas in four-vessel occlusion study, ischemia significantly decreased PLD activity in the hippocampus but not in the brain stem. Lowered temperature (30°C and 22°C) during ischemic incubation did not reverse the ischemia-induced PLD activity decrease. In contrast to PLD, ischemia by decapitation had no effect on basal phosphatidylinositol-phospholipase C activity or the amount of phospholipase Cβ1 in the membrane fractions from 30-minute ischemic hippocampus by immunoblots probed with the antibody. Conclusions These results suggest that PC-PLD is one of the target enzymes of ischemia; its decrease may cause a perturbation of PC hydrolysis and/or disorders of intracellular transduction of signals or choline metabolism for acetylcholine formation in brain.

Forskolin and Phorbol Myristate Acetate Inhibit Intracellular Ca2+ Mobilization Induced by Amitriptyline and Bradykinin in Rat Frontocortical Neurons

Abstract— Regulations of the increase in intracellular Ca2+concentration ([Ca2+]i) and inositol 1, 4, 5‐trisphosphate (IP3) production by increasing intracellular cyclic AMP (cAMP) levels or activating protein kinase C (PKC) were studied in rat frontocortical cultured neurons. Amitriptyline (AMI; 1 mM), a trìcyclic antidepressant, and bradykinin (BK; 1 μM) stimulated IP3 production and caused transient [Ca2+]i increases. Pretreatment with forskolin (100mkUM, 15 min) decreased the AMI‐and BK‐induced [Ca2+]i increases by 33 and 48%, respectively. However, this treatment had no effect on the AMI‐and BK‐induced IP3 productions. Dibutyryl‐cAMP (2 mM, 15 min) also decreased the AMI‐and BK‐induced [Ca2+]i increases by 23 and 47%, respectively. H‐8 (30 μM), an inhibitor of protein kinase A (PKA), attenuated the ability of forskolin to inhibit the AMI‐and BK‐induced [Ca2+]i increases, suggesting that the activation of cAMP/PKA was involved in these inhibitory effects of forskolin. On the other hand, forskolin treatment had no effect on 20 mM caffeine‐, 10 μM glutamate‐, or 50 mM K+‐induced [Ca2+]i increases. Pretreatment with phorbol 12‐myristate 13‐acetate (PMA; 100 nM, 90 min) decreased both the AMI‐induced [Ca2+]i increases and the IP3 production by 31 and 25%, respectively. H‐7 (200 μM), an inhibitor of PKC, inhibited the ability of PMA to attenuate the [Ca2+]i increases. PMA also inhibited the BK‐induced IP3 production and the [Ca2+]i increases. Taken together, these results suggest that activation of cAMP/ PKA may inhibit the IP3‐mediated Ca2+ release from internal stores; on the other hand, activation of PKC may inhibit the phosphatidylinositol 4,5‐bisphosphate breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses. PKA and PKC may differently regulate the phosphatidylinositol‐Ca2+ signaling in rat frontocortical cultured neurons.