thapsigargin and Bipolar-Disorder

Irregularities of intracellular calcium (Ca2+) homeostasis have been implicated in the pathophysiology of bipolar disorder (BD). Findings that chronic ex-vivo treatment with lithium modifies lysophosphatidic acid (LPA)-stimulated Ca2+ responses in B lymphoblast cell lines (BLCLs) from BD-I patients and healthy controls, and differentially decreases levels of the type-3 canonical transient receptor potential Ca2+-permeable channel in BLCLs from BD-I patients, support the view that the amelioration of these abnormalities is important in the therapeutic action of lithium. To determine whether other clinically efficacious mood stabilizers share these effects, LPA (100 mum)- and thapsigargin (TG, 200 nm)-stimulated Ca2+ responses were determined in BLCLs from BD-I patients and healthy controls treated acutely (24 h) and chronically (7 d) ex vivo with therapeutically relevant concentrations of lithium (0.75 mm), valproate (0.5 mm), lamotrigine (15 mum) or respective vehicles. Chronic treatment with valproate significantly attenuated LPA-stimulated Ca2+ responses ([downward arrow]8%: F's=9.1-9.4, d.f.=1, 9, p's<0.05) compared to vehicle in BLCLs from BD-I patients and healthy controls, similar to chronic lithium treatment ([downward arrow]8%: F=6.2, d.f.=1, 21, p<0.05), but also attenuated TG-evoked Ca2+ responses ([downward arrow]10% to [downward arrow]19%: F's=5.5-15.5, d.f.=1, 12, p's<0.05). However, chronic lamotrigine treatment did not affect LPA- or TG-stimulated Ca2+ responses. These results suggest that chronic lithium and valproate treatments act differently from lamotrigine in respect of modulation of receptor- and/or capacitance-mediated Ca2+ flux. These differential effects on Ca2+ responses may be relevant to the distinctive clinical profiles of these mood stabilizers.

Topics: Adult; Antimanic Agents; B-Lymphocytes; Bipolar Disorder; Calcium; Cell Line, Transformed; Drug Interactions; Enzyme Inhibitors; Female; Humans; Intracellular Fluid; Lipopolysaccharides; Lithium Chloride; Male; Middle Aged; Thapsigargin; Valproic Acid

Impaired endoplasmic reticulum (ER) stress response has been suggested as a possible pathophysiological mechanism of bipolar disorder (BD). The expression of ER stress-related genes, spliced form or unspliced form of XBP1, GRP78 (HSPA5), GRP94 (HSP90B1), CHOP (DDIT3), and calreticulin (CALR), were examined in lymphoblastoid cells derived from 59 patients with BD and 59 age- and sex-matched control subjects. Basal mRNA levels and induction by 4 h or 12 h of treatment with two ER stressors, thapsigargin or tunicamycin, were examined using real-time quantitative reverse transcription-polymerase chain reaction. Induction of the spliced form of XBP1 as well as total XBP1 by thapsigargin was significantly attenuated in patients with BD. Induction of GRP94 by thapsigargin was also decreased in the BD group. A haplotype of GRP94, protective against BD, exhibited significantly higher GRP94 expression upon ER stress. This report confirms and extends earlier observations of impaired ER stress response in larger samples of lymphoblastoid cell lines derived from BD patients. Altered ER stress response may play a role in the pathophysiology of BD by altering neural development and plasticity.

Topics: Adult; Bipolar Disorder; Cells, Cultured; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Female; Gene Expression Regulation; Genotype; HSP70 Heat-Shock Proteins; Humans; Lymphocytes; Male; Membrane Proteins; Middle Aged; Protein Folding; Psychiatric Status Rating Scales; Reverse Transcriptase Polymerase Chain Reaction; RNA; Stress, Physiological; Thapsigargin; Tunicamycin

Substantial evidence implicates abnormalities of intracellular calcium (Ca2+) dynamics in the pathophysiology of bipolar disorder (BD). However, the precise mechanisms underlying such disturbances are poorly understood. To further elaborate the nature of altered intracellular Ca2+ signalling dynamics that occur in BD, we examined receptor- and store-operated Ca2+ responses in B lymphoblast cell lines (BLCLs), which have been found in earlier studies to 'report' BD-associated disturbances. Basal Ca2+ concentrations ([Ca2+]B), and lysophosphatidic acid (LPA)- and thapsigargin-stimulated Ca2+ responses were determined in BLCLs from 52 BD-I patients and 30 healthy comparison subjects using fura-2, and ratiometric fluorometry. ANOVA revealed a significant effect of diagnosis, but not gender, on [Ca2+]B (F1,63=4.4, p=0.04) and the rate of rise (F1,63=5.2, p=0.03) of LPA-stimulated Ca2+ responses in BLCLs from patients compared with those from healthy subjects. A significant genderxdiagnosis interaction on the LPA-induced rate of rise (F1,63=4.6, p=0.03) was accounted for by a faster rate of rise (97%) in BLCLs from BD-I males compared with healthy males but not in those from female patients compared with healthy females. A genderxdiagnosis interaction in thapsigargin-evoked Ca2+ influx (F1,61=3.8, p=0.05) resulted from a significantly higher peak [Ca2+]influx (24%) in BLCLs from female compared with male patients. The results suggest more rapid LPA-stimulated Ca2+ responses occur in BLCLs from BD-I patients compared with controls, which are probably mediated, in part, by canonical transient receptor potential type 3 (TRPC3)-like channels. Additionally, this study highlights sex-dependent differences that can occur in the pathophysiological disturbances involved in BD.

Topics: Adult; B-Lymphocytes; Bipolar Disorder; Calcium; Calcium Signaling; Case-Control Studies; Cell Line, Transformed; Diglycerides; Endoplasmic Reticulum; Enzyme Inhibitors; Female; Humans; Ion Channel Gating; Kinetics; Lysophospholipids; Male; Middle Aged; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sex Factors; Thapsigargin; TRPC Cation Channels

Disturbed intracellular calcium (Ca(2+)) homeostasis has been implicated in bipolar disorder, which mechanisms may be involved in the dysregulation of protein kinase C (PKC) and calmodulin systems. In this study, we investigated a transient intracellular Ca(2+) increase induced by thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase pump (SERCA), and a capacitative Ca(2+) entry followed by addition of extracellular Ca(2+), in the presence or absence of PKC/calmodulin modulators in the platelets of healthy subjects in order to elucidate the role of SERCA in Ca(2+) homeostasis and to assess how both PKC and calmodulin systems regulate the two Ca(2+) responses. Moreover, we also examined the thapsigargin-elicited transient Ca(2+) increase and capacitative Ca(2+) entry in patients with mood disorders. PKC and calmodulin systems have opposite regulatory effects on the transient Ca(2+) increase and capacitative Ca(2+) entry in the platelets of normal subjects. The inhibitory effect of PKC activation on capacitative Ca(2+) entry is significantly increased and the stimulatory effect of PKC inhibition is significantly decreased in bipolar disorder compared to major depressive disorder and normal controls. These results suggest the possibility that increased PKC activity may activate the inhibitory effect of capacitative Ca(2+) entry in bipolar disorder. However, this is a preliminary study using a small sample, thus further studies are needed to examine the PKC and calmodulin modulators on the capacitative Ca(2+) entry in a larger sample.

Topics: Adult; Bipolar Disorder; Blood Platelets; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Calmodulin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Homeostasis; Humans; Indoles; Male; Maleimides; Protein Kinase C; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Tetradecanoylphorbol Acetate; Thapsigargin

Altered 1-oleoyl-lysophosphatidic acid (LPA, 100 microM)-stimulated calcium responses occur in B-lymphoblast cell lines from bipolar disorder patients, but the mechanism(s) involved is uncertain. Lysophosphatidic acid shares a structurally similar fatty acid side chain with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of subtypes 3, 6 and 7 of the canonical transient receptor potential (TRPC) cation channel subfamily. Accordingly, the objective of this study was to determine whether the LPA-stimulated calcium response in B-lymphoblasts is mediated, in part, through this TRPC channel subfamily. Divalent cation selectivity in response to thapsigargin, LPA and OAG were used to distinguish TRPC-like character of the responses to these agents in BLCLs. The sensitivity to gadolinium, an inhibitor of capacitative calcium channels, was used to determine the store-operated nature of the responses. The TRPC isoforms that are present in BLCLs as identified by immunoblotting and/or PCR include TRPC1, 3 and 5. Minimal barium influx in calcium-free buffer was observed following thapsigargin stimulation. However, LPA stimulated barium influx of a magnitude similar to that induced by OAG. Thapsigargin-provoked calcium influx was completely inhibited by gadolinium (10 microM), whereas LPA and OAG-stimulated responses were partially inhibited and potentiated, respectively. The results suggest that 100 microM LPA stimulates calcium entry through channels with characteristics similar to TRPC3, as TRPC6 and 7 are absent in B-lymphoblasts.

Topics: B-Lymphocytes; Barium; Bipolar Disorder; Calcium Signaling; Cell Line; Diglycerides; Humans; Immunoblotting; Lysophospholipids; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thapsigargin; TRPC Cation Channels

Elevated basal intracellular calcium (Ca(2+)) levels ([Ca(2+)](B)) in B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) patients implicate altered Ca(2+) homeostasis in this illness. Chronic lithium treatment affects key proteins modulating intracellular Ca(2+) signaling. Thus, we sought to determine if chronic exposure to therapeutic lithium concentrations also modifies intracellular Ca(2+) homeostasis in this surrogate cellular model of signal transduction disturbances in BD. BLCLs from BD-I (N=26) and healthy subjects (N=17) were regrown from frozen stock and incubated with 0.75 mM lithium or vehicle for 24 h (acute) or 7 days (chronic). [Ca(2+)](B), lysophosphatidic acid (LPA)-stimulated Ca(2+) mobilization ([Ca(2+)](S)), and thapsigargin-induced store-operated Ca(2+) entry (SOCE) were determined using ratiometric fluorometry with Fura-2. Compared with vehicle, chronic lithium exposure resulted in significantly higher [Ca(2+)](B) (F=8.47; p=0.006) in BLCLs from BD-I and healthy subjects. However, peak LPA-stimulated [Ca(2+)](S) and SOCE were significantly reduced (F=11.1, p=0.002 and F=8.36, p=0.007, respectively). Acute lithium exposure did not significantly affect measured parameters. In summary, the effect of chronic lithium to elevate [Ca(2+)](B) in BLCLs while attenuating both receptor-stimulated and SOCE components of intracellular Ca(2+) mobilization in BLCLs suggests that modulation of intracellular Ca(2+) homeostasis may be important to the therapeutic action of lithium.

Topics: Adult; B-Lymphocytes; Bipolar Disorder; Calcium; Case-Control Studies; Cell Count; Cell Line; Cell Transformation, Viral; Chi-Square Distribution; Drug Administration Schedule; Enzyme Inhibitors; Female; Fura-2; Herpesvirus 4, Human; Homeostasis; Humans; Intracellular Space; Lithium; Lysophospholipids; Male; Multivariate Analysis; Thapsigargin; Time Factors

Altered Ca2+ signalling has been reported in the platelets and lymphoblastoid cells of patients with bipolar disorder. Recent genetic studies have suggested possible pathophysiological roles for mitochondria and endoplasmic reticulum, both of which are essential for the regulation of intracellular Ca2+ signalling. The goal of this study was to determine molecular mechanisms of altered intracellular Ca2+ signalling in bipolar disorder. Lymphoblastoid cell lines were established from patients with bipolar I disorder (n=13) and controls (n=11). Using Ca2+ indicators, cytosolic and mitochondrial Ca2+ responses to the following three reagents were examined: platelet-activating factor; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler that abolishes mitochondrial Ca2+ uptake; and thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. The 10-5 M thapsigargin-induced cytosolic Ca2+ response was significantly higher in patients with bipolar disorder (p&0.05). Such difference was not seen when the effects of Ca2+ influx from outside the plasma membrane was eliminated using Ca2+-free measurement buffer. On the other hand, response to 10-7 M thapsigargin tended to be higher in patients with bipolar disorder when at the Ca2+-free conditions. CCCP-induced Ca2+ responses differed significantly between mitochondrial DNA 5178/10398 haplotypes (p=0.001) that had been previously reported to be associated with bipolar disorder. These results suggest that all components, i.e. the store-operated calcium channel (SOCC), endoplasmic reticulum, and mitochondria, somehow contribute to the altered Ca2+ signalling in bipolar disorder.

Topics: Adult; Bipolar Disorder; Calcium; Calcium Channels; Cell Line, Transformed; Cytosol; DNA-Binding Proteins; DNA, Mitochondrial; Endoplasmic Reticulum; Female; Haplotypes; Humans; Lymphocytes; Male; Middle Aged; Mitochondria; Signal Transduction; Thapsigargin

A number of investigators have reported finding elevated basal and stimulated intracellular calcium levels in the platelets or lymphocytes of bipolar disorder patients.. Intracellular calcium was measured by a micro fura-2 fluorometric method in the platelets and lymphocytes of 30 affective disorder patients and 14 control subjects.. We observed significantly elevated basal calcium concentrations in bipolar patient platelets and lymphocytes compared to control subjects. Bipolar patient platelet calcium responses to thrombin, serotonin, and thapsigargin were also significantly greater than control subjects. The peak calcium levels of lymphocytes of bipolar patients were greater than control subjects only when stimulated by thapsigargin. There were significant differences between bipolar and unipolar patients in basal and thapsigargin-stimulated calcium measures but not between bipolar I and bipolar II patients. Unmedicated versus medicated calcium measures were not significantly different. We also found little correlation between calcium measures and the severity of mood rating.. Using this method, we were able to confirm and extend the work of others, indicating altered intracellular calcium homeostasis in the blood cells of bipolar disorder patients. In addition, our data suggest that storage operated calcium channels may be the source of the elevated intracellular calcium in platelets and lymphocytes of bipolar patients.

Topics: Adult; Basal Metabolism; Bipolar Disorder; Blood Platelets; Calcium; Calcium Channels; Enzyme Inhibitors; Equipment Design; Female; Fluorometry; Humans; Ion Transport; Lymphocytes; Male; Platelet Activation; Prospective Studies; Retrospective Studies; Thapsigargin; Time Factors