Alterations in DNA methylation rates of brain-derived neurotrophic factor in patients with schizophrenia

Background and objectives: Schizophrenia (SZ) is one of the most disabling mental illness and the elucidation of diagnostic and therapeutic biomarkers are required. Recent studies investigating the brain morphology, the gene expression profile, and the genetic epidemiology have suggested the involvement of Brain-derived neurotrophic factor (BDNF) and its epigenetic regulation in the pathophysiology of SZ. The current study was conducted to determine the association of DNA methylation of the BDNF gene with the diagnosis or with the characteristics of patients with SZ.MethodsWe analyzed genomic DNA from peripheral blood of 22 patients with SZ and 22 healthy subjects. The DNA methylation rates (DMRs) of the CpG island at the promoter of exón I of the BDNF gene were measured using EpiTYPER® and the MassARRAY® system (Agena Biosciences). We examined the validity of the methylation profiles as a diagnostic biomarker for SZ by clustering analyses, differences in DMRs between patients and healthy controls, and the relationship between DMRs and patient characteristics.ResultsThe clustering analysis failed to distinguish between healthy controls and patients with SZ, though the DMRs of 4 CpG units were significantly different between these two groups. Whereas the DMR of one CpG (CpG 28) was significantly correlated with the amount of daily antipsychotics, there was no influence of age, severity, or duration of illness on the DMRs of the BDNF gene.ConclusionDespite the small number of subjects, our results suggest the involvement of the changes in DMRs of the BDNF gene in the pathophysiology of SZ. (AU)

Studies of extraction and transport system for highly charged ion beam of 18 GHz superconducting electron cyclotron resonance ion source at Research Center for Nuclear Physics.

An 18 GHz superconducting electron cyclotron resonance ion source is installed to increase beam currents and to extend the variety of ions especially for highly charged heavy ions which can be accelerated by cyclotrons of Research Center for Nuclear Physics (RCNP), Osaka University. The beam production developments of several ions from B to Xe have been already done [T. Yorita, K. Hatanaka, M. Fukuda, M. Kibayashi, S. Morinobu, H.Okamura, and A. Tamii, Rev. Sci. Instrum. 79, 02A311 (2008) and T. Yorita, K. Hatanaka, M. Fukuda, M. Kibayashi, S. Morinobu, H.Okamura, and A. Tamii, Rev. Sci. Instrum. 81, 02A332 (2010)] and the further studies for those beam extraction and its transport have been done in order to increase the beam current more. The plasma electrode, extraction electrode, and einzel lens are modified. Especially extraction electrode can be applied minus voltage for the beam extraction and it works well to improve the extracted beam current. The extraction voltage dependences of transmission and emittance also have been studied for beam current improvement which is injected into azimuthally varying field cyclotron at RCNP.

Transport and emittance study for 18 GHz superconducting-ECR ion source at RCNP.

As the upgrade program of the azimuthally varying field (AVF) cyclotron is at the cyclotron facility of the RCNP, Osaka University for the improvement of the quality, stability, and intensity of accelerated beams, an 18 GHz superconducting (SC) ECR ion source has been installed to increase beam currents and to extend the variety of ions, especially for highly charged heavy ions which can be accelerated by RCNP AVF cyclotron. The production development of several ions such as B, O, N, Ne, Ar, Ni, Kr, and Xe has been performed by Yorita et al. [Rev. Sci. Instrum. 79, 02A311(2008); 81, 02A332 (2010)]. Further studies for the beam transport have been done in order to improve the beam current more for injection of cyclotron. The effect of field leakage of AVF main coil is not negligible and additional steering magnet has been installed and then beam transmission has been improved. The emittance monitor has also been developed for the purpose of investigating correlation between emittance of beam from ECR ion sources and injection efficiency. The monitor consists with BPM82 with rotating wire for fast measurement for efficient study.

Long-term prognosis of patients with major depression and silent cerebral infarction.

OBJECTIVE: Many studies have examined the effects of cerebrovascular changes on treatment response in geriatric depression. However, few such studies have examined the relationship between cerebrovascular changes and long-term prognosis. We examined the effects of cerebrovascular changes on the course of geriatric depressive symptoms, dementia rates, and mortality over a follow-up period of approximately 10 years. METHOD: Participants were 84 patients with major depression (age of onset over 50 years); patients suffering from strokes, neurological disorders, and other psychiatric disorders were excluded. Magnetic resonance imaging findings were used to classify all patients into silent cerebral infarction (SCI)-positive (n = 37) or SCI-negative groups (n = 47). Prognoses were ascertained using a review of clinical charts and mailed questionnaires. RESULTS: Only 5% of patients with SCI were able to maintain remission whereas 36% of patients without SCI were able to do so. Total duration of depressive episodes was significantly longer in the SCI-positive group than in the SCI-negative group. SCI was also associated with a higher risk of dementia. CONCLUSION: The results of this long-term follow-up study demonstrate that the presence of SCI is associated with a relatively poor prognosis in geriatric depression.

Neonatal tactile stimulation reverses the effect of neonatal isolation on open-field and anxiety-like behavior, and pain sensitivity in male and female adult Sprague-Dawley rats.

It is well known that early life events induce long-lasting psychophysiological and psychobiological influences in later life. In rodent studies, environmental enrichment after weaning prevents the adulthood behavioral and emotional disturbances in response to early adversities. We compared the behavioral effect of neonatal isolation (NI) with the effect of NI accompanied by tactile stimulation (NTS) to determine whether NTS could reverse or prevent the effects of NI on the adulthood behavioral and emotional responses to environmental stimuli. In addition, we also examined the sex difference of the NTS effect. Measurements of body weights, an open-field locomotor test, an elevated plus maze test, a hot-plate test, and a contextual fear-conditioning test were performed on postnatal day 60. As compared with rats subjected to NI, rats subjected to NTS showed significantly higher activity and exploration in the open-field locomotor test, lower anxiety-like behavior in the elevated plus maze test, and significantly prolonged latencies in the hot-plate test, and this effect was equal among males and females. In the contextual fear-conditioning test, whereas NTS significantly reduced the enhanced freezing time due to NI in females, no significant difference in the freezing time between NI and NTS was found in males. These findings indicate that adequate tactile stimulation in early life plays an important role in the prevention of disturbances in the behavioral and emotional responses to environmental stimuli in adulthood induced by early adverse experiences.

Glucocorticoid receptor activation is involved in producing abnormal phenotypes of single-prolonged stress rats: a putative post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder caused by traumatic experience, and presents with characteristic symptoms, such as intrusive memories, a state of hyperarousal, and avoidance, that endure for years. Single-prolonged stress (SPS) is one of the animal models proposed for PTSD. Rats exposed to SPS showed enhanced inhibition of the hypothalamo-pituitary-adrenal (HPA) axis, which has been reliably reproduced in patients with PTSD, and increased expression of glucocorticoid receptor (GR) in the hippocampus. In this study, we characterized further neuroendocrinologic, behavioral and electrophysiological alterations in SPS rats. Plasma corticosterone recovered from an initial increase within a week, and gross histological changes and neuronal cell death were not observed in the hippocampus of the SPS rats. Behavioral analyses revealed that the SPS rats presented enhanced acoustic startle and impaired spatial memory that paralleled the deficits in hippocampal long-term potentiation (LTP) and depression. Contextual fear memory was enhanced in the rats 1 week after SPS exposure, whereas LTP in the amygdala was blunted. Interestingly, blockade of GR activation by administering 17-beta-hydroxy-11-beta-/4-/[methyl]-[1-methylethyl]aminophenyl/-17-alpha-[prop-1-ynyl]estra-4-9-diene-3-one (RU40555), a GR antagonist, prior to SPS exposure prevented potentiation of fear conditioning and impairment of LTP in the CA1 region. Altogether, SPS caused a number of behavioral changes similar to those described in PTSD, which marks SPS as a putative PTSD model. The preventive effects of a GR antagonist suggested that GR activation might play a critical role in producing the altered behavior and neuronal function of SPS rats.

Glutathione S-transferase gene polymorphisms in Japanese patients with rheumatoid arthritis.

OBJECTIVE: To investigate the role of polymorphisms of the glutathione S-transferase M1 (GSTM1), GSTT1, and GSTP1 genes in determining susceptibility to rheumatoid arthritis (RA) and association with the clinical features. METHODS: Polymorphisms of the GSTM1, GSTT1, and GSTP1 genes in 108 Japanese patients with RA and in 143 healthy controls were analyzed by polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism. RESULTS: The frequency of the GSTM1 null genotype was significantly higher among RA patients than among control subjects (60.2% and 44.1%, respectively. P = 0.011). Moreover, the female patients with GSTM1 homozygous null genotype showed significantly higher serum MMP-3 level than the female patients with non-null genotype (P = 0.030). Frequencies of the GSTT1 and GSTP1 gene polymorphism were not different between RA patients and controls. CONCLUSION: The GSTM1 homozygous null genotype could be a genetic factor that determines susceptibility to RA and may have influence on the disease process.

Reduction in levels of amphiphysin 1 mRNA in the hippocampus of aged rats subjected to repeated variable stress.

Various neurobiological studies of aging indicate that elevated levels of circulating glucocorticoids lead to hippocampal vulnerability to stress, though little is known about the molecular mechanism underlying stress vulnerability in the elderly. We have compared the gene expression profiles in the hippocampus of aged (20 months) and adult (3 months) rats in response to repeated variable stress (RVS) for 4 days, using a cDNA array technique and real-time quantitative PCR, to identify putative genes involved in the mechanism of stress vulnerability in the elderly. We found a significant decrease in the levels of amphiphysin 1 mRNA in aged rats subjected to RVS compared with treated and untreated adult rats or to untreated aged rats. Similarly, we found a significant decrease in hippocampal levels of amphiphysin 1 mRNA in aged rats subjected to RVS for 8 days, but not in those subjected to a single VS. These findings suggest that the decrease in the hippocampal levels of amphiphysin 1 mRNA in response to repeated stress may be involved in the stress vulnerability in the elderly, and may lead to the disturbance of learning and memory under stressful conditions in the elderly.

Lithium, but not valproate, induces the serine/threonine phosphatase activity of protein phosphatase 2A in the rat brain, without affecting its expression.

Protein phosphatase 2A (PP2A) regulates protein kinase cascades and thus plays an important role in the regulation of cell growth, gene expression and development. We examined the influence of lithium and valproate on the expression of PP2A and its serine/threonine phosphatase activity in the rat frontal cortex and hippocampus. Western blot and immunohistochemical analyses demonstrated that neither lithium nor valproate treatment had an effect on the levels of PP2A immunoreactivity in these brain regions. However, administration of lithium for 1 or 14 days significantly upregulated the activity of PP2A in the frontal cortex. Similarly, lithium administration tended to increase the activity of PP2A in the hippocampus. In contrast, neither a single nor repeated administration of valproate affected the activity of PP2A in these brain regions. These findings indicate that lithium, but not valproate, upregulated the activity of PP2A in the rat brain. It is suggested that the changes in neuronal functions induced by PP2A may be, at least in part, associated with the therapeutic action of lithium.

Attenuated left prefrontal activation during a verbal fluency task in patients with depression.

Functional neuroimaging studies on patients with depression have found abnormal activity in the left prefrontal and anterior cingulate cortex compared with healthy controls. Other studies have shown that these regions become active in healthy subjects during verbal fluency tasks, while patients with depression show impaired performance on such tasks. We used functional magnetic resonance imaging to investigate changes in cerebral blood oxygenation associated with a verbal fluency task in depressed patients and healthy volunteers. In contrast to 10 age- and sex-matched healthy control subjects who activated the left prefrontal cortex and the anterior cingulate cortex during word generation, 10 depressed subjects showed attenuated activation in the left prefrontal cortex and did not show significant activation in the anterior cingulate cortex. These findings suggest that impaired performance during verbal fluency task in depressed patients is associated with abnormal neural responses within these regions.

Differential changes in messenger RNA expressions and binding sites of neuropeptide Y Y1, Y2 and Y5 receptors in the hippocampus of an epileptic mutant rat: Noda epileptic rat.

The anti-convulsive effects of neuropeptide Y have been suggested in several animal models of epilepsy. We have found the sustained increase of neuropeptide Y contents and the seizure-induced elevation of hippocampal messenger RNA in a novel spontaneous epileptic mutant rat: Noda epileptic rat. In the present study, we investigated the change of neuropeptide Y Y1 and Y2 receptor messenger RNA expressions and binding sites in the hippocampus following a spontaneous generalized tonic-clonic seizure of Noda epileptic rat. Furthermore, the binding sites of a more recently isolated receptor subtype, neuropeptide Y Y5 receptors, were also evaluated by receptor autoradiography. A marked elevation of neuropeptide Y immunoreactivity in the mossy fiber, and Y2-receptor up-regulation in the dentate gyrus were observed in the hippocampus of Noda epileptic rat, which coincided with the previous results of the other epileptic models. In contrast, Y1-receptor down-regulation was not found after a spontaneous seizure of Noda epileptic rat while this occurs in kindling and after kainic acid-induced seizures. [125I][Leu31, Pro34]peptide YY/BIBP 3226-insensitive (Y5 receptor) binding sites in CA1 stratum radiatum were significantly decreased following a spontaneous seizure of Noda epileptic rat. The present results suggest that a spontaneous seizure of Noda epileptic rat induces significant changes in neuropeptide Y-mediated transmission in the hippocampus via Y2 and Y5 receptors, but not Y1 receptors. Therefore, specific subset of neuropeptide Y receptor subtypes might be involved in the epileptogenesis of Noda epileptic rat.

Meige syndrome with apraxia of lid opening after the discontinuation of sulpiride treatment.

We report a case of Meige syndrome with apraxia of lid opening that lasted for about seven months after discontinuation of sulpiride treatment. To our knowledge, this is the first report demonstrating that Meige syndrome with apraxia of lid opening is induced by sulpiride, and that the condition persists.

Cognitive dysfunction in recovered depressive patients with silent cerebral infarction.

In this study, we characterized cognitive functioning in patients with major depression and silent cerebral infarction (SCI), as detected by magnetic resonance imaging (MRI), after they had recovered from depression. Thirty-five patients with unipolar depression who experienced the onset of depression after the age of 50 underwent MRI and were classified as SCI(+) (n = 17) or SCI(-) (n = 18). The Wechsler Adult Intelligence Scale-Revised (WAIS-R) and the Uchida-Kraepelin psychodiagnostic test were administered after the patients had recovered from depression. In addition, the intelligence quotient (IQ) and mental speed of the patients in the two groups were compared. The total, verbal and performance IQ scores, as determined by the WAIS-R, were significantly lower in the SCI(+) group than in the SCI(-) group. The mental speed of patients in the SCI(+) group, as assessed by the Uchida-Kraepelin psychodiagnostic test, was almost half that of the SCI(-) group. Our findings provide further evidence that a comprehensive impairment of cognitive functioning, especially a severe reduction in mental speed, remains after recovery from depression in patients with major depression and SCI.

Clinical features and treatment response of patients with major depression and silent cerebral infarction.

Previously, we reported a relationship between silent cerebral infarction (SCI), as detected by magnetic resonance imaging (MRI), and late onset major depression. In the present study, we clarify the clinical features of the depressive phase of patients with major depression and SCI, and their response to antidepressant pharmacotherapy. Using clinical charts, we retrospectively examined patients with depression, who were first admitted for antidepressant pharmacotherapy. All patients were classified according to the MRI findings and the age on admission (older or younger than 50 years) into either the young SCI(-) group (n = 23), the elderly SCI(-) group (n = 27) or the elderly SCI(+) group (n = 20).The characteristics of the clinical features were evaluated at the time of admission, after 2 weeks of treatment and at the time of discharge using the Hamilton rating scale for depression (HAMD). These data were compared between each patient group. No differences in the clinical features, as evaluated by HAMD, were observed between the three groups at the time of admission. However, the mean length of treatment was significantly longer and the treatment response, as evaluated by the total HAMD score, was significantly worse in the elderly SCI(+) group than in the other two groups, when examined after 2 weeks of treatment and at the time of discharge. The elderly SCI(+) group demonstrated higher scores in feelings of guilt, suicide, retardation and hypochondriasis than the young SCI(-) group and the elderly SCI(-) group after two weeks of treatment, and higher scores in early insomnia, late insomnia, somatic anxiety and hypochondriasis at the time of discharge. Our findings suggest that while the presence of SCI does not affect the clinical features observed at the time of admission, it does affect the treatment response to antidepressant pharmacotherapy.

Chronic lithium treatment increases the expression of brain-derived neurotrophic factor in the rat brain.

RATIONALE: Lithium is the most widely prescribed mood stabilizer, but the precise mechanism of lithium is unresolved. OBJECTIVE: We examine the effects of the administration of therapeutically relevant concentrations of lithium on the expression of brain-derived neurotrophic factor (BDNF) and its receptor, Trk B, as well as glia-derived neurotrophic factor (GDNF) and its receptors, RET and GDNFR-alpha, in the rat brain. In addition, we also examined the effect of another well-prescribed mood stabilizer, valproate, on the expression of BDNF and GDNF. METHODS: Rats were kept on a 0.2% lithium carbonate-containing diet for 1, 7, 14, or 28 days or treated with valproate (400 mg/kg per day i.p.) for 1 or 14 days. After the brains were rapidly removed, the levels of BDNF, GDNF, and their receptors were measured by ELISA or western blot analysis. RESULTS: Chronic lithium treatment for 14 and 28 days significantly increased the expression of BDNF in the hippocampus and temporal cortex. In addition, chronic lithium treatment for 14 days significantly increased the expression of BDNF in the frontal cortex. In contrast, acute or chronic dietary lithium treatment did not alter GDNF expression in these brain regions. In addition, acute or chronic lithium treatments did not change the levels of Trk B, RET, or GDNFR-alpha immunoreactivity. As well as lithium, repeated administration of valproate also increased the expression of BDNF in the frontal cortex and hippocampus. CONCLUSIONS: Our results suggest that the chronic administration of mood stabilizers may produce a neurotrophic effect mediated by the upregulation of BDNF in the rat brain.

Antidepressant drug treatments induce glial cell line-derived neurotrophic factor (GDNF) synthesis and release in rat C6 glioblastoma cells.

Modulation of neurotrophic factors to protect neurons from damage is proposed as a novel mechanism for the action of antidepressants. However, the effect of antidepressants on modulation of glial cell line-derived neurotrophic factor (GDNF), which has potent and widespread effects, remains unknown. Here, we demonstrated that long-term use of antidepressant treatment significantly increased GDNF mRNA expression and GDNF release in time- and concentration-dependent manners in rat C6 glioblastoma cells. Amitriptyline treatment also increased GDNF mRNA expression in rat astrocytes. GDNF release continued for 24 h following withdrawal of amitriptyline. Furthermore, following treatment with antidepressants belonging to several different classes (amitriptyline, clomipramine, mianserin, fluoxetine and paroxetine) significantly increased GDNF release, but which did not occur after treatment with non-antidepressant psychotropic drugs (haloperidol, diazepam and diphenhydramine). Amitriptyline-induced GDNF release was inhibited by U0126 (10 microM), a mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H-89 (1 microM), a protein kinase A inhibitor, calphostin C (100 nM), a protein kinase C inhibitor and PD 169316 (10 microM), a p38 mitogen-activated protein kinase inhibitor. These results suggested that amitriptyline-induced GDNF synthesis and release occurred at the transcriptional level, and may be regulated by MEK/MAPK signalling. The enhanced and prolonged induction of GDNF by antidepressants could promote neuronal survival, and protect neurons from the damaging effects of stress. This may contribute to explain therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.

Modulation of serotonin2A receptor function in rats after repeated treatment with dexamethasone and L-type calcium channel antagonist nimodipine.

1. It has been conceivable that the hypothalamic-pituitary-adrenal (HPA) axis hyperactivity plays an important role in the pathophysiology of depression. In the present study, we have investigated the effect of repeated treatment with dexamethasone on serotonin (5-HT) 1A, 5-HT2A and alpha1-adrenergic receptors in the rat frontal cortex. Moreover, several studies have suggested the effectiveness of L-type calcium channel antagonist nimodipine for the treatment of depression. We also investigated the effect of repeated treatment with nimodipine on 5-HT2A receptor in rats with repeated dexamethasone treatment. 2. Repeated treatment with dexamethasone (1 mg/kg/day for 14 days) increased the density of 5-HT2A receptor, but not 5-HT1A and alpha1-adrenergic receptors in the rat frontal cortex. 3. The density of 5-HT2A receptor in the rat frontal cortex was significantly increased 1 day after repeated treatment with dexamethasone, but was not increased 7 or 14 days after repeated treatment. Wet dog shakes (WDS) induced by (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI), a 5-HT2A receptor agonist, in rats were significantly enhanced 1, 7 and 14 days after repeated treatment with dexamethasone, although the frequency of WDS gradually decreased after repeated treatment. 4. Repeated treatment with nimodipine (5 mg/kg/day for 14 days) attenuated DOI-induced WDS enhanced by repeated treatment with dexamethasone (1 mg/kg/day for 14 days), however, it did not change the density of 5-HT2A receptor. Repeated treatment with dexamethasone decreased locomotor activity and body weight, but repeated treatment with nimodipine did not recover these parameters. 5. The results of the present study suggest that repeated treatment with dexamethasone may selectively increase the 5-HT2A receptor in the rat frontal cortex and affect 5-HT2A receptor-mediated signal transduction. In addition, the intracellular calcium homeostasis by blocking calcium influx through L-type calcium channel may play an important role in the regulation of the 5-HT2A receptor function by dexamethasone.

Characterization of 5-HT2A receptor desensitization and the effect of cycloheximide on it in C6 cells.

Effect of prolonged pretreatment with serotonin (5-HT) on 5-HT2A receptor desensitization was examined by the measurement of intracellular calcium ([Ca2+]i) mobilization in C6 cells. 5-HT-induced desensitization of [Ca2+]i mobilization was in a time and dose dependent manner and reached a plateau after 3 hr. After 1 and 3 hr 5-HT pretreatment, 5-HT concentration in the medium little changed. 5-HT pretreatment with cycloheximide, a protein synthesis inhibitor, produced an enhancement of the desensitization for 3 and 6 hr pretreatment. However, 5-HT pretreatment for 3 and 6 hr caused no marked change in the 5-HT2A receptor mRNA level or Galphaq/11 protein in this study, suggesting that 5-HT may decrease 5-HT-induced [Ca2+]i mobilization independent of 5-HT2A receptor mRNA or G-proteins. Endothelin-1-induced [Ca2+]i mobilization did not alter after 5-HT and/or cycloheximide pretreatment. These results showed that activation of the 5-HT2A receptor induced homologous desensitization and pretreatment with 5-HT and/or cycloheximide did not change the efficacy of the second messenger pathway from Gq to a [Ca2+]i rise.

Nucleotide sequence analysis of the binding site on the inositol 1,4,5-trisphosphate type-1 receptor in bipolar disorder -- a negative study.

Pharmacological studies of bipolar disorder suggest that dysfunction of calcium mobilization via phosphatidylinositol-mediated transduction may be involved in its pathogenesis. The present study tests the hypothesis that dysfunction of calcium mobilization in bipolar disorder is due to the mutation of the nucleotide sequence in the FKBP12 binding site on the inositol 1,4,5-trisphosphate type-1 receptor (IP(3)R1). Nucleotide sequence analysis of the FKBP12 binding site on IP(3)R1 was performed using reverse transcription-polymerase chain reaction and DNA sequencing. The nucleotide sequence in this region was preserved in all subjects. This finding suggests that IP(3)R1 dysfunction through the FKBP12 binding site is not involved in the pathogenesis of bipolar disorder.

Effect of beta-estradiol on voltage-gated Ca(2+) channels in rat hippocampal neurons: a comparison with dehydroepiandrosterone.

We investigated the effects of beta-estradiol, dehydroepiandrosterone and dehydroepiandrosterone sulfate on intracellular calcium concentration ([Ca(2+)](i)) increases induced by gamma-aminobutyric acid (GABA), high K(+) and N-methyl-D-aspartate acid (NMDA) in cultured hippocampal neurons. Acute treatment with beta-estradiol, dehydroepiandrosterone and dehydroepiandrosterone sulfate inhibited the GABA-induced [Ca(2+)](i) increases to the similar extent. Tamoxifen, an estrogen receptor antagonist, did not block the inhibitory effects of beta-estradiol. On the other hand, GABA type A (GABA(A)) receptor antagonists, picrotoxin and bicuculline, blocked the GABA-induced [Ca(2+)](i) increases. Previously, we demonstrated that GABA- and high K(+)-induced [Ca(2+)](i) increases were commonly mediated by voltage-gated calcium channels (VGCCs). Therefore, we examined the effects of these steroids on the high K(+)-induced [Ca(2+)](i) increases. The inhibitory effect of beta-estradiol on the high K(+)-induced [Ca(2+)](i) increases was much greater than that of dehydroepiandrosterone and dehydroepiandrosterone sulfate. beta-Estradiol inhibited the NMDA-induced [Ca(2+)](i) increases with an IC(50) of 51.8 microM and NMDA responses were reduced to half in the presence of 10 micro M nifedipine, indicating that the NMDA-induced [Ca(2+)](i) increases also involved VGCCs. Further, we examined the inhibitory effect of beta-estradiol on the high K(+)-induced [Ca(2+)](i) increases in the presence of a N-type VGCCs antagonist, 1 microM omega-conotoxin, or a L-type VGCCs antagonist, 10 microM nifedipine. The IC(50) value of beta-estradiol alone (45.5 microM) was similar to that of omega-conotoxin (33.1 microM), while the value combined with nifedipine was reduced to 2.2 microM. beta-Estradiol also abolished the positive modulatory effect of L-type VGCCs agonist, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]pyridine-3-carboxylic acid methyl ester (Bay K 8644). Our results showed that the inhibitory mechanism of beta-estradiol is different from that of dehydroepiandrosterone and dehydroepiandrosterone sulfate and beta-estradiol may act primarily at L-type VGCCs.