Teruo Hayashi

γ-Aminobutyric acid increases intracellular Ca2+ concentration in cultured cortical neurons: role of Cl− transport

The effect of gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) in cultured prenatal rat cortical neurons was investigated using fluorescence imaging. GABA or muscimol, but not baclofen, increased [Ca2+]i in a dose-dependent manner. The GABAA receptor antagonists, bicuculline and picrotoxin, inhibited the GABA response. Furosemide, an inhibitor of the Na+/K+/2Cl- cotransporter, inhibited the GABA response in a noncompetitive manner. Ethacrynic acid, an inhibitor of an ATP-dependent Cl- pump, also inhibited the GABA-induced increased in [Ca2+]i. These results suggest a role for Cl- transport processes in the GABA response. The coapplication of GABA and high K+ led to a non-additive increase in the GABA response. The GABA response was also inhibited by nifedipine, a voltage-gated Ca2+ channel blocker, and abolished by the absence of extracellular Ca2+. Results indicate that the GABA response shares a common pathway of Ca2+ movement with the high K(+)-induced response. These observations suggest that the stimulation with GABA results in Ca2+ influx through voltage-gated Ca2+ channels, and that these effects are dependent on Cl- transport systems.

Conversion of psychological stress into cellular stress response: roles of the sigma-1 receptor in the process

Psychiatrists empirically recognize that excessive or chronic psychological stress can result in long‐lasting impairments of brain functions that partly involve neuronal cell damage. Recent studies begin to elucidate the molecular pathways activated/inhibited by psychological stress. Activation of the hypothalamic–pituitary–adrenal axis under psychological stress causes inflammatory oxidative stresses in the brain, in part due to elevation of cytokines. Psychological stress or neuropathological conditions (e.g., accumulation of β‐amyloids) trigger ‘cellular stress responses’, which promote upregulation of molecular chaperones to protect macromolecules from degradation. The unfolded protein response, the endoplasmic reticulum (ER)‐specific cellular stress response, has been recently implicated in the pathophysiology of neuropsychiatric disorders and the pharmacology of certain clinically used drugs. The sigma‐1 receptor is an ER protein whose ligands are shown to exert antidepressant‐like and neuroprotective actions. Recent studies found that the sigma‐1 receptor is a novel ligand‐operated ER chaperone that regulates bioenergetics, free radical generation, oxidative stress, unfolded protein response and cytokine signaling. The sigma‐1 receptor also regulates morphogenesis of neuronal cells, such as neurite outgrowth, synaptogenesis, and myelination, which can be perturbed by cellular stress. The sigma‐1 receptor may thus contribute to a cellular defense system that protects nervous systems against chronic psychological stress. Findings from sigma receptor research imply that not only cell surface monoamine effectors but also intracellular molecules, especially those at the ER, may provide novel therapeutic targets for future drug developments.

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

SummaryDantrolene has been known to affect intracellular Ca2+ concentration ([Ca2+]i) by inhibiting Ca2+ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca2+]i affected by the NMDA receptor ligands, KCl, L-type Ca2+ channel blocker nifedipine, and two other intracellular Ca2+-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca2+]i was also examined. Dantrolene in μM concentrations dose-dependently inhibited the increase in [Ca2+]i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca2+]i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca2+]i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca2+]i caused by NMDA or KCl. At 30 μM, dantrolene partially inhibited caffeine-induced increase in [Ca2+]i whereas it has no effect on the bradykinin-induced change in [Ca2+]i. The spontaneous oscillation of [Ca2+]i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 μM dantrolene. Our results indicate that dantrolenes mobilizing effects on intracellular Ca2+ stores operate independently from the influxed Ca2+ and that a component of the apparent increase in [Ca2+]i elicited by NMDA or KCl represents a dantrolene-sensitive Ca2+ release from intracellular stores. Results also suggest that dantrolene does not affect the IP3-gated release of intracellular Ca2+ and that the spontaneous Ca2+ oscillation is, at least partially, under the control of Ca2+ mobilization from internal stores.

Life-threatening dysphagia following prolonged neuroleptic therapy

We report the cases of two patients with complaints of dysphagia following long-term neuroleptic therapy. Esophageal contrast radiography revealed that one patient suffered disruption of the normal swallowing activity of the pharyngoesophagus due to tardive dyskinesia. Her dysphagia disappeared following changes in her neuroleptic medications and the administration of clonazepam. The other patient demonstrated severe rabbit syndrome involving the glossopharynx. This 3-Hz rhythmic movement disorder resolved following injection of an anticholinergic agent. Thereafter, the addition of oral trihexyphenidyl to her medication regimen improved her dysphagia. It should be emphasized that the differential diagnosis of neuroleptic-associated dysphagia subtypes is important because therapeutic strategies differ depending on the subtype of this life-threatening illness.

First Japanese family with primary familial brain calcification due to a mutation in the PDGFB gene: An exome analysis study

Primary familial brain calcification (PFBC) is a rare disorder characterized by abnormal deposits of calcium in the basal ganglia and cerebellum. PFBC can present with a spectrum of neuropsychiatric symptoms resembling those seen in dementia and schizophrenia. Mutations in a few genes have been identified as causing PFBC: namely, the SLC20A2 gene that codes for the sodium‐dependent phosphate transporter and the PDGFRB gene that codes for the platelet‐derived growth factor receptor β (PDGF‐Rβ). A recent study identified mutations in PDGFB coding for PDGF‐B, the main ligand for PDGF‐Rβ, in six families with PFBC. Here we report the first Japanese family with PFBC carrying a mutation in PDGFB, which causes the substitution of an arginine with a stop codon at amino acid 149 of the PDGF‐B protein (p. Arg149*).

Possible Mechanism of Dantrolene Stabilization of Cultured Neuroblastoma Cell Plasma Membranes

Abstract: Some reports have suggested that dantrolene interacts directly with the membrane bilayer. We investigated effects of dantrolene on changes in membrane properties induced by compound 48/80 (C48/80), a membrane stimulator. The addition of C48/80 for 1 min elicited a rapid, dose‐dependent Ca2+ influx, which was reduced to 14% by the absence of external Ca2+. Dantrolene inhibited the C48/80‐induced increase in Ca2+ permeability of plasma membranes in a concentration‐dependent manner (0.33–10 µM, IC50 value was 5 µM). We next examined C48/80‐induced changes in structural and dynamic membrane properties by electron spin resonance (ESR). The ratio h0/h−1 was determined to evaluate membrane fluidity. C48/80 increased the membrane fluidity in a concentration‐dependent manner (0.1–0.56 mg/ml). Dantrolene (10 µM) itself did not change the membrane fluidity, but it significantly reduced the C48/80‐induced increase in membrane fluidity (0.56 mg/ml). Moreover, the C48/80‐induced increase in fluidity was dependent on extracellular Ca2+. We conclude that dantrolene protects neuroblastoma cell plasma membrane from C48/80‐induced membrane perturbation, which causes Ca2+ influx and an increase in membrane fluidity. These findings strongly suggest that dantrolene directly stabilizes the neuronal plasma membrane.

Cyclic GMP generation mediated by 5-HT-2 receptors via nitric oxide-dependent pathway and its effect on the desensitization of 5-HT-2 receptors in C6 glioma cells

Serotonin (5-HT)-2 receptor-mediated cGMP generation was investigated in comparison with calcium (Ca2+) mobilization in C6 glioma cells. 5-HT enhanced cGMP generation, and risperidone and ketanserin potently blocked the response. These results indicate that 5-HT-2 receptors are responsible for the cGMP generation. 5-HT-induced cGMP production was completely abolished by BAPTA, an intracellular Ca2+ chelating agent, or N g -monomethyl-L-arginine (NMMA), a nitric oxide synthase (NOS) inhibitor, suggesting that 5-HT-induced cGMP generation was through nitric oxide (NO)-dependent pathway. 5-HT (10 μM)-elicited Ca2+ mobilization and cGMP generation were reduced to 40 and 15 % after pretreatment with 10 μM 5-HT for 4 hours. NMMA did not modify 5-HT-induced desensitization of either Ca2+ mobilization or cGMP generation, suggesting that NO pathway is independent of the desensitization. The present study has demonstrated the nature of 5-HT-2 receptormediated cGMP generation in C6 glioma cells.

Prevalence of and risk factors for respiratory dyskinesia

We explored the prevalence of respiratory dyskinesia (RD), diagnosed objectively using a spirograph, and the major risk factors for tardive dyskinesia (TD) and RD. A total of 258 inpatients treated with neuroleptics was interviewed, and TD was evaluated using the Abnormal Involuntary Movement Scale (AIMS). Movement of the chest and respiratory regularity were assessed on clinical examination. Spirographs of patients with suspected RD were recorded, and RD was diagnosed based on spirographic data and the concurrence of two investigators. The prevalence of TD in this study was 22.1% (57 of 258). Aging and organic brain damage (OBD) were confirmed as risk factors; female gender, mood disorders, and the duration of neuroleptic exposure were not. Ten of 28 patients suspected of having RD were diagnosed with RD on the basis of persistent respiratory irregularities without other physiologic causes. The overall prevalence of RD was 3.9% (10 of 258) and was 17.5% (10 of 57) among the TD patient population. Four of these patients complained of dyspnea, and three demonstrated grunting. RD was more highly associated with aging and OBD than with TD itself. The identification of risk factors for RD is not only helpful in planning prophylactic strategies, but also facilitates the understanding of the pathogenesis of this syndrome.

Involvement of the α2-adrenergic system in polydipsia in schizophrenic patients: a pilot study

Abstract Animal studies have suggested the involvement of the adrenergic system in drinking behavior. The present study investigated the involvement of the α2-adrenergic system in the polydipsia of patients with chronic schizophrenia by use of an α2 agonist and an antagonist. Four patients with schizophrenic disorders accompanied by intermittent hyponatremia and polydipsia were the subjects of, and completed, this study. Drinking behavior was assessed by calculating the percent of maximum weight gain [PMWG: (maximum diurnal weight – standard weight) × 100/standard weight]. Standard weight was defined as body weight after 8 h of water restriction. Clonidine (75, 150, and 225 mg/day) increased the PMWG in a dose-dependent manner in the four subjects. In contrast, in three of the subjects, mianserin (30, 60, and 90 mg/day) decreased PMWG, and the severe polydipsia disappeared almost completely. These findings indicate clearly that the α2-adrenergic system is involved in the drinking behavior of schizophrenic patients. Mianserin appears to be clinically useful in treating such patients with polydipsia.

Effect of citalopram on the desensitization of serotonin-2A receptor-mediated calcium mobilization in rat glioma cells

1. The authors have investigated the effect of citalopram, an effective antidepressant drug with selective serotonin (5-HT) uptake inhibition, on 5-HT-2A receptor-mediated intracellular calcium (Ca2+) rise in C6 cultured cells. 2. Citalopram, at concentrations of 10 and 30 mu M, did not significantly reduce the Ca2+ mobilization induced by 10 mu M 5-HT, indicating that citalopram has little affinity for 5-HT-2A receptors. 3. Citalopram did not alter a subsequent response to 5-HT after citalopram was pre-applied to the cells. 4. However, citalopram inhibited the desensitization of 5-HT-2A receptors. When the cells were pretreated with citalopram and 5-HT, the subsequent response to 5-HT was significantly greater than that obtained following pretreatment with 5-HT alone. 5. To investigate the mechanism of action of citalopram on the desensitization of 5-HT-2A receptors, NaF-induced cGMP generation was measured. Citalopram inhibited the generation of cGMP induced by NaF in C6 cells as well as W-7. 6. These results indicate that citalopram antagonized the desensitization of 5-HT-2A receptor-mediated Ca2+ mobilization and this antagonism may be mediated by a calmodulin-dependent pathway in C6 glioma cells.