INTRODUCTION

Building evidence implicates altered intracellular signal transduction and second messenger function, with attendant alterations in neuroplasticity and cellular resilience, in the pathophysiology of BD.^{1, 2} Among the abnormalities noted, disruption of intracellular calcium (Ca²⁺) homeostasis is a pivotal transition stage in the progression to cell death, whether by apoptosis or necrosis.^{3, 4} Reports of elevated basal and agonist-stimulated intracellular Ca²⁺ concentrations ([Ca²⁺]_B and [Ca²⁺]_S, respectively) in mononuclear leukocytes, platelets and transformed B lymphoblast cell lines (BLCLs) from bipolar I disorder I (BD-I) patients^{5, 6, 7} therefore suggest that alterations of intracellular Ca²⁺ homeostasis play an important role in the pathophysiological changes that are thought to compromise cellular resilience and adaptive response to cellular stress in BD. The mechanism(s) involved in the altered Ca²⁺ homeostasis are still uncertain, but recent observations implicate possible disturbances in one or more of the molecular modules regulating intracellular Ca²⁺ such as mitochondrial Ca²⁺ flux, endoplasmic reticulum (ER) Ca²⁺ storage and release, and store-operated and receptor-operated Ca²⁺ entry (SOCE and ROCE).^{7, 8, 9, 10}

Observations that lithium (Li) treatment impacts Ca²⁺ signaling and homeostatic mechanisms in neuronal and glial cell models, in addition to other nonexcitable cell models, provide yet another line of support for the notion that intracellular Ca²⁺dynamics are disrupted in BD. For example, chronic Li treatment attenuated NMDA receptor-mediated Ca²⁺ influx in neuronal preparations.¹¹ As well, Li treatment decreased both [Ca²⁺]_B and stimulated Ca²⁺ responses in a variety of cell lines including cultured astrocytes,¹² C6 glioma cells¹³ and rat GH₃ pituitary cells.¹⁴ In a more direct clinical paradigm, therapeutic levels of Li significantly lowered [Ca²⁺]_B and [Ca²⁺]_S in platelets from BD patients but not in control subjects.¹⁵ Of additional importance, chronic, but not acute, Li treatment of BLCLs at a therapeutic concentration also attenuated G protein-coupled receptor (lysophosphatidic acid, LPA)-mediated and thapsigargin (TG)-induced (SOCE dependent) Ca²⁺mobilization responses in this surrogate, cellular, pathophysiological model of BD.⁹

While the attenuating effects of Li on NMDA-mediated Ca²⁺ influx are mediated through reduced tyrosine phosphorylation of the NR2B subunit of the NMDA gated Ca²⁺ channel,¹⁶ the effect of Li on intracellular Ca²⁺ dynamics in nonexcitable cell models (eg glial cells, platelets, BLCLs)^{9, 12, 13, 15} suggests additional mechanisms are likely to be involved in its action. This is particularly important given recent reports of altered glial cell morphometrics and gene expression in postmortem brain of BD patients.^{17, 18} Ample evidence supports a role for glial cells, which are nonexcitable cells, in regulating Ca²⁺ homeostasis through glial-neuron interactions, although the mechanisms are not well understood.¹⁹ Unraveling the molecular mechanisms by which Li modifies intracellular Ca²⁺ homeostasis and dynamics in nonexcitable cell models may be equally important to understanding the full spectrum of effects underlying Li's antimanic, mood stabilizing and neuroprotective actions.

Among the molecular mechanisms regulating intracellular Ca²⁺ dynamics that might be disturbed in BD, several recent observations implicate SOCE and ROCE modules. First, TG-induced Ca²⁺ responses, an index of SOCE, were blunted in platelets from BD patients compared with healthy subjects.⁸ Second, mRNA levels of the Ca²⁺ permeable transient receptor potential (TRP) melastatin type 2 (TRPM2) channel were reduced in BLCLs from BD-I patients with high [Ca²⁺]_B compared with BD-I patients showing normal [Ca²⁺]_B.²⁰ Third, chronic treatment of BLCLs from BD and healthy subjects with Li-attenuated LPA and TG-induced Ca²⁺ responses that likely involve SOCE and/or ROCE processes.⁹

Recent research directed at elaborating the molecular components and/or mechanisms mediating SOCE and ROCE highlights the key role of several members of the TRP family of cation permeable channels in these processes in nonexcitable cells, such as lymphocytes and platelets.^{21, 22, 23} This superfamily of proteins is comprised of three branches distinguished by sequence homology and functional similarities: TRPC (canonical), TRPV (vanilloid) and TRPM (melastatin).²⁴ Two subgroups within the canonical branch, (TRPC1,4&5 and TRPC3,6&7, respectively) have been implicated in SOCE and ROCE,^{24, 25} raising the prospect of the involvement of one or more of these TRPC members in the Ca²⁺ abnormalities that have been reported in BD and the in actions of Li in BLCLs highlighted above.

Accordingly, the specific objectives of this study were, first, to determine whether the expression of selected TRPC candidate subtypes, which have been implicated in SOCE and ROCE, is altered in BLCLs from BD-I patients showing disturbed intracellular Ca²⁺ dynamics and second, to evaluate whether they are altered by treatment of BLCLs with Li. We report here the expression in BLCLs and differential regulation of TRPC3, in contrast to TRPC1, in response to chronic Li treatment of cell lines from BD-I patients ex vivo, at a thereapeutically relevant concentration. The lack of attendant changes in TRPC3 mRNA levels suggests this Li-induced effect is likely mediated through post-translational regulation of TRPC3 levels, changes which in turn may contribute to the attenuating effects of chronic Li treatment on LPA- and TG-evoked intracellular Ca²⁺ responses in BLCLs previously reported.⁹

RESULTS

Subject Demographics

Patient (N=15) and healthy (N=10) comparison subjects from whom cell lines were used in this study did not differ in mean age (44. 53.72 years vs 44.44.45, respectively; t=0.013, P>0.05). The proportion of females to males also did not differ significantly between the healthy subject and BD-I groups (Fisher's exact test, P=1.0). At the time blood samples for B lymphocyte transformation were obtained, the majority of BD patients were euthymic (73%), while 27% were depressed. The most prevalent comorbid psychiatric disorder found in the BD patient group was alcohol abuse/dependence (20%). Other comorbidities included eating disorder (1), panic disorder (1), and social phobia (1). There was a positive family history of mood disorders in first-degree relatives in 47% of the patients, whereas 20% gave a negative history; in the remaining patients the history was uncertain or unknown. In all, 40% of BD patients were receiving Li monotherapy and a similar proportion was on anticonvulsant mood-stabilizer monotherapy at the time of blood sampling.

Characterization of TRPC mRNA

Amplification of cDNA reversed transcribed from human brain tissue and BLCL total RNA with TRPC1, TRPC3 and TRPC4 primer sets yielded single PCR products of expected sizes 92 bp for TRPC1 (Figure 1a), 94 bp for TRPC3 (Figure 1b) and 100 bp for TRPC4 (data not shown), respectively. While TRPC6 primer sets yielded a single PCR product of 98 bp in human brain tissues, no detectable product was identified in BLCLs (data not shown). All standard curves from the PCR reactions showed correlation coefficients >0.98 supporting the linear dynamic range (1–10^-4) of the assay. The efficiencies of PCR reactions, as estimated by the slopes of the standard curves (10^(1/-Slope)–1),²⁶ were >98%.

Figure 1.

Ethidium bromide staining of agarose gels of real-time PCR products demonstrating the presence of the TRPC1 (a) and TRPC3 (b) subtypes generated from specific primer sets utilized in the study. PCR products from BLCLs (lane 1) and human brain tissue (cerebellum, lane 2, frontal cortex, lane 3, and temporal cortex, lane 4) were separated on 2% agarose gels. S represents the 100 bp ladder. Representative immunoblots of TRPC1 (C) and TRPC3 (D) in membrane preparations from BLCLS (B), platelet (P) mouse (MC) and rat cerebral tissues (RC) immunodetected with their corresponding antibodies. Representative graphs showing the linear ranges of protein detection and representative immunoblots on which the graphs are based for both TRPC1 (E) and TRPC3 (F), respectively.

Full figure and legend (95K)

Characterization of TRPC Immunolabeling Specificity

Anti-TRPC1 detected a major band of estimated molecular mass of 83 kDa in BLCLs, and the positive control tissues, rat and mouse cerebral cortex (Figure 1c). With respect to TRPC3, anti-TRPC3 antibody detected one major immunoreactive band with apparent molecular weight of 123 kDa in BLCLs and rat cortex but not platelets (negative control) (Figure 1d). Both TRPC1 and TRPC3 immunoreactive signals were linearly related to membrane protein concentration between 2 and 15 g of protein ([r=0.99, P=0.0005] (Figure 1e) and [r=0.99, P=0.0001] (Figure 1f), respectively).

Effect of Chronic Lithium Treatment of BLCLs from BD-I Patients and Healthy Subjects on TRPC1 and TRPC3 mRNA

Table 1 summarizes the results of mRNA levels of TRPC1 and TRPC3 in BLCLs from BD-I patients and control subjects treated chronically with Li or vehicle. The expression level of 18S rRNA did not significantly differ in the samples between the BD and the control groups (P=0.10). Repeated-measures ANOVA revealed no significant effects of subject group or drug-treatment on either TRPC1 mRNA (F=0.001, df=1, 13, P=0.97) or TRPC3 mRNA (F=0.002, df=1, 10, P=0.96), respectively.

Table 1 - TRPC1 and TRPC3 mRNA and immunoreactive levels in acutely- and chronically-Li-treated BLCLs from BD patients and healthy subjects.

Full table

Effect of Acute and Chronic Lithium Treatment of BLCLs from BD-I Patients and Healthy Subjects on TRPC1 and TRPC3 Protein Levels

As shown in Table 1, TRPC1 immunoreactive levels were not significantly different in BLCLs from BD patients treated acutely (F=1.20, df=1, 12, P=0.30) or chronically with Li (F=0.87, df=1, 23, P=0.36) compared with healthy subjects. Similarly, TRPC3 immunoreactive levels were not significantly different in BLCLs treated acutely with Li from BD patients compared with controls (F=1.83, df=1, 12, P =0.20) (see Table 1). In contrast, there was a significant main effect of drug-treatment (F=25.7, df=1, 21, P=0.00005) and interactions between drug-treatment and both diagnostic group (F=4.98, df=1, 21, P=0.037) and sex (F= 4.81, df 1, 21, P=0.04) on TRPC3 immunolabeling in BLCLs chronically treated with Li, from BD patients compared with healthy controls (Figure 2). Analysis of simple effects revealed that TRPC3 immunolabeling decreased significantly and to a greater extent in chronic-Li-treated BLCLs from female BD-I patients as compared with vehicle-treated BLCLs from female BD-I patients (–33 %, P=0.00002) and BLCLs, treated chronically with Li, from healthy females (-27%, P=0.00003). Chronic Li treatment also reduced TRPC3 immunoreactivity in BLCLs from female healthy subjects as compared with vehicle-treated cell lines (–16%, P=0.019). A comparable reduction was also observed in chronic-Li-treated BLCLs from male BD-I patients as compared with vehicle-treated cells from male BD-I patients (–18%, P=0.028). A decrease of similar magnitude in mean TRPC3 immunolabeling of chronic-Li-treated BLCLs from male BD-I patients relative to that in Li-treated BLCLs from healthy male subjects (-19%, P=0.052) fell short of achieving statistical significance, however. In contrast, there was no effect of chronic Li treatment compared to vehicle in BLCLs from healthy male subjects. Finally, there were no significant correlations between the mRNA levels of TRPC1 and TRPC3 and the immunoreactive levels of the corresponding proteins, respectively, in BLCLs from any of the comparison groups (data not shown).

Figure 2.

Immunoreactive levels of TRPC3 in BLCLs from BD-I patients and healthy comparison subjects chronically treated with vehicle (open bars) or 0.75 mM Li (hatched bars) for 7 days. Bars indicate the mean immunoreactive levels of each diagnostic group. Normalized immunolabeling densities were analyzed by repeated-measures ANOVA while group (diagnosis, sex) by Li treatment interaction effects were further evaluated by analysis of simple effects as described in Materials and Methods. ^*Indicates P<0.05 compared with vehicle-treated cell lines within the same sex and ^**Indicates P<0.0005 compared with vehicle-treated BLCLs from female BD-I patients and chronic Li-treated BLCLs from BD-I female patients compared with healthy females. †Indicates P=0.052 compared to vehicle-treated BLCLs from BD-I male subjects.

Full figure and legend (20K)

Relationship between Ca²⁺ Mobilization Measures and, TRPC1 and TRPC3 Protein Levels in BLCLs from BD-I Patients and Healthy Subjects

Given the role of TRPCs in modulating Ca²⁺ influx, we examined whether there were relationships between previously measured parameters of Ca²⁺ homeostasis determined in these cell lines,^{7, 9} and TRPC3 and TRPC1 protein levels in BLCLs from BD-I patients and healthy subjects. TRPC3 protein levels in both vehicle- and Li-treated BLCLs from BD-I and healthy controls did not correlate with either [Ca²⁺]_B, LPA-stimulated [Ca²⁺]_S, or any TG-stimulated SOCE-mediated responses (data not shown). Similarly, there were no statistically significant correlations between TRPC3 levels and the respective TG-stimulated SOCE-mediated responses in the same BLCL samples, both normalized to the vehicle treatment condition. Interestingly, TRPC1 protein levels in vehicle-treated BLCLs from BD-I patients and healthy comparison subjects correlated negatively with [Ca²⁺]_B, (r=-0.58, P=0.05) and positively with TG-stimulated SOCE-mediated responses (TG-sensitive [Ca²⁺]_i accumulation, r=0.57, P=0.04; peak [Ca²⁺]_i influx, r=0.84, P=0.0003, and difference between peak [Ca²⁺]_i influx and [Ca²⁺]_B r=0.82, P=0.0005; data not shown).

DISCUSSION

The principal finding of this study is the demonstration for the first time of a selective effect of chronic treatment at a therapeutically relevant concentration of Li to reduce TPRC3 protein levels in BLCLs from BD-I patients (-29%) as compared with those from healthy subjects (-5%). These observations complement our recent report⁹ that chronic Li treatment of BLCLs blunts agonist- and TG-stimulated Ca²⁺ responses, and implicate these protein changes, in part, in this effect. Collectively, these observations illuminate further the spectrum of actions of Li that may be relevant to its mood stabilizing and antimanic effects. Moreover, the greater magnitude of the TRPC3 reductions in chronically Li-treated BLCLs from BD-I patients is particularly germane to the notion that disrupted intracellular Ca²⁺ homeostasis plays an important pathophysiological role in BD and that the correction of these disturbances is a critical component of Li's mood-stabilizing action.

As the veracity of the findings are contingent upon the specificity and quantitative accuracy of the immunoblot and PCR assays used, some comment on these is merited prior to elaborating further on the nature and basis for the TRPC changes observed. Of the TRPC subtypes screened in BLCLs, only TRPC 1, 3 and 4 mRNA could be detected by real-time PCR with the unique primers used and sequences confirmed. Using epitope specific polyclonal antibodies, TRPC 1 and 3 subtype expression was further confirmed in BLCLs. Briefly, the putative TRPC1 protein band comigrated with immunoreactive bands detected with TRPC1-antiserum in rat and mouse cerebral cortex in which TRPC1 is known to be highly expressed.²⁷ The putative TRPC3 protein band had a similar migratory pattern to that of an immunoreactive protein detected with TRPC3-antiserum in rat cerebral cortex. No immunoreactive TRPC3 band was detected in lysates from platelets, which lacks TRPC3 mRNA (negative control).²⁸ Tissue- and cell-type-dependent expression of various TRPCs subtypes has been reported, but TRPC1 and TRPC3 subtypes have been identified in some hemopoietic cells, and in rat and human brain.^{29, 30} The present findings extend the demonstration of both TRPC1 and TRPC3 expression, based on mRNA and protein levels, to human BLCLs, as well.

Although no differences were found between BD-I patients and healthy comparison subjects in either TRPC3 or TRPC1 protein and mRNA levels in vehicle-treated BLCLs, the perturbation of chronic treatment of BLCLs with Li elicited a differential effect on TRPC3 protein levels in cell lines from BD-I patients as compared with healthy subjects. This was manifest in significant interactions of Li treatment with diagnosis, as well as sex. The analysis of simple effects revealed a statistically significant effect of diagnosis when both treatment and sex were held constant. Tests of contrasts among the entire set of group cell means revealed that the greatest Li-induced reductions in TRPC3 levels occurred in BLCLs from female BD-I patients (-33%); the decrement in BLCLs from male BD-I patients was smaller (-18%). The differential diagnostic effect not with standing, the statistically significant, although smaller magnitude of the reduction in TRPC3 levels in Li-treated BLCLs from healthy female subjects (-16%) compared with vehicle, together with the lack of Li effect in male healthy controls, indicates an enhanced sensitivity of the Li perturbation in females, in general.

The latter findings together with recent evidence of sex-dependent abnormalities in variables such as basal and NaF-stimulated cAMP production in BLCLs from BD-I patients³¹ and in inositol monophosphatase 2 mRNA expression in both BLCLs and postmortem temporal cortex from BD-I patients²⁰ support the notion that sex-related differences in pathophysiology may occur in BD. That said, a caveat is the potential confounding effect of extraneous factors such as Li responsiveness or illness course and pattern specifiers. In this regard, five of the 10 female BD-I patients were receiving Li monotherapy and presumptive Li responders in contrast to only one out of the five male BD-I patients, and half of the female but none of the male BD-I patients met criteria for seasonality. While these attributes are important variables for exploration in their own right, the small sample size of the subject groups at this stage of study limits more detailed analysis of the potential effect of these factors and to explore their predictive potential.

Unlike the reduction in TRPC3 protein, Li treatment did not affect its mRNA levels. While it is tempting to speculate that the latter observations imply that the mechanism(s) underlying the effect of Li treatment involves factors regulating the disposition and clearance of the TRPC3 protein at the post-translational level, the large variance in the mRNA measures (up to 6-fold) in the comparison groups, which likely reflects intersubject and natural genetic variations in the gene expression,^{32, 33, 34} may have obscured changes in mRNA levels. That said, the variance of the mean differences for the within-subject Li treatment effect was considerably smaller (CV=160%) than that for the between-subject comparisons (range of CVs=210–585%). Thus, Li-induced changes in mRNA levels should have been more amenable to detection.

Little is known at the present time of the specific mechanisms by which TRPC3 is targeted for degradation in the cell. These uncertainties aside, the reduction in TRPC3 levels together with the observation that Li attenuates TG-induced Ca²⁺ influx (a measure of SOCE), suggest that Li may modify intracellular Ca²⁺ dynamics through SOCE mechanisms, at least in part, by downregulating TRPC3 at the protein level. While the lack of a correlation between the Li-induced decrements in the latter functional measure and TRPC3 immunolabeling relative to vehicle-treatment seems to conflict with this conclusion, it is not clear yet whether global TG-induced Ca²⁺ mobilization changes in direct proportion to the levels of TRPC3. The functional effects of TRPC3 in modulating SOCE have been shown to vary in relation to its expression levels:²³ at low levels TRPC3 operates as an SOCC, whereas at high expression levels it shows G protein receptor-coupled phospholipase C-operated characteristics.²¹

The notion that pathophysiologically and diagnostically distinctive Ca²⁺ homeostasis abnormalities occur in BD-I disorder has grown out of series of observations of differential changes in indices of Ca²⁺ dynamics measured in surrogate peripheral blood cell and BLCLs models from BD, comparison mood and nonmood disorder patients, and healthy subjects. Elevated [Ca²⁺]_B and enhanced stimulated Ca²⁺ responses are consistent findings in peripheral blood cell (platelet, mononuclear leukocyte and BLCL) preparations from BD patients compared with controls.¹ The notion that these changes are trait-dependent, first suggested by findings that the abnormalities persist after remission,^{35, 36} was further strengthened by the demonstration of similar elevated [Ca²⁺]_B and [Ca²⁺]_S in transformed BLCLs from BD-I patients compared with healthy controls.^{6, 7} Furthermore, Yoon et al²⁰ have also demonstrated reduced TRPM2 mRNA levels in BLCLs from BD-I patients exhibiting high [Ca²⁺]_B compared with those exhibiting normal [Ca²⁺]_B, and with BD-II patients, MDD patients and control subjects. Although only cell lines from BD-I and healthy subjects were compared in the present study, the effect of chronic Li treatment to reduce TRPC3 protein levels differentially in BLCLs from BD-I patients suggests that this effect may be specific to BD-I or a clinical attribute that cosegregates with the diagnostic group. Of additional note, the TRPC3 changes found in this study appear to be a specific effect attributable to Li, as chronic treatment of the same cell lines with a therapeutic concentration of valproate did not lower TRPC3 immunoreactive levels as compared with vehicle in either BD or healthy subjects (Kwan M et al., unpublished data). Finally, the fact that the TRPC3 changes are confined to BLCLs from BD-I patients argues against the possibility that the findings are an epiphenomenon of Li treatment.

In summary, the findings of this study provide the first evidence that the putative SOCC TRPC3 is affected in a disease-specific manner by chronic treatment with a therapeutically relevant concentration of Li. Against the backdrop of building evidence implicating abnormal intracellular Ca²⁺ homeostasis in the pathophysiology of BD and the modulation of intracellular Ca²⁺ dynamics by Li, these disease-specific effects of chronic Li treatment on TRPC3 raise the possibility that they are intimately linked to the pathophysiology of this disorder and represent an important target involved in the mood-stabilizing response. Elaboration of the specific post-translational molecular mechanisms involved in the Li-induced downregulation of TRPC3 protein levels and the functional impact on intracellular Ca²⁺ dynamics will help to illuminate their specific role in the development of BD and in response to Li treatment.

MATERIALS AND METHODS

Subject B-Lymphoblast Cell Lines

BLCLs were selected from both fresh and frozen stocks prepared from samples from BD patients and age- and sex-matched healthy subjects participating in studies of signal transduction disturbances in mood disorders.^{6, 9, 20, 37} Patients had a DSM-IV diagnosis of BD-I as established by the Structured Clinical Interview for DSM-IV (SCID), were physically healthy and had no recent (>3 months) drug or alcohol abuse.⁶ Healthy subjects had no past or current psychiatric illness as determined by SCID-I (nonpatient version) and were physically healthy as determined by systems review and physical examination. All subjects provided informed written consent. This study was approved by Human Subjects Review Board of the University of Toronto.

Establishment of B Lymphoblast Cell Lines

EBV-transformed BLCLs were prepared using standard techniques as previously described.⁶ BLCLs were initially expanded in suspension culture in a 95% air/5% CO₂ humidified incubator at 37°C for 13–16 passages (2–3 days/passage), then assayed for [Ca²⁺]_B,^{6, 7} and either frozen and stored in the vapor phase of liquid nitrogen for later regeneration and use, or, in some cases, expanded further for study.

BLCL Growth, Regrowth and Drug Treatment Protocol

BLCLs, either freshly grown or regrown from liquid nitrogen storage, were passaged minimally to obtain the required cell count (6–12 10⁷ cells). During this phase, B-cell media, consisting of RPMI, 20% FBS, 2 mM L-glutamine, 1 mM pyruvate, 100 g/ml streptomycin, 100 U/ml penicillin, was completely replaced every 2–3 days. Cells were counted using a hemocytometer and viability determined by trypan blue exclusion at each replacement of medium.

Once cells had reached the required cell count and viability (>90%), three equivalent aliquots were transferred to separate Falcon T-75 flasks; one containing B-cell media with 0.75 mM LiCl and the other two containing equivalent volumes of media to which the aqueous vehicle (saline) was added. To maintain a constant Li concentration, media was completely replaced with gentle sedimenting of the cells by centrifugation (50 g, 10 min) followed by resuspension of the cells, every other day for the first 6 days. For the last 24 h of the 7-day treatment period, cells receiving Li chronically were treated with serum-free medium containing LiCl (0.75 mM) in RPMI 1640, whereas cells receiving vehicle treatment were divided into two batches. One batch was resuspended in serum-free medium containing 0.75 mM Li in RPMI 1640 for the final 24 h of incubation (acute Li treatment), whereas the other batch was resuspended in serum-free RPMI-1640 with vehicle.

Isolation of Total Cellular RNA and Synthesis of First-Strand cDNA

Total RNA from 1 10⁷ BLCLs were isolated using an RNeasy kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized in a 20 l mixture containing 1 g of DNase-treated total RNA, 2 l of random decamer primers (50 mM, Ambion, Austin, TX, USA), 5 first-strand buffer (Gibco BRL, Gaitherburg, MD, USA), 1 l of 0.1 M dithiothreitol (DTT) and 10 mM dNTPs, 20 U RNAsin (40 U/l, Promega, Madison, WI, USA), and 200 U Superscript™ RNase H^- reverse transcriptase (Gibco BRL). The reaction mixture was incubated for 1 h at 42°C and the reaction stopped by heating for 15 min at 70°C. The resulting cDNA was diluted in 80 l deionized, reverse osmosis-purified H₂O, aliquoted and stored at -20°C until use. As a control, one reverse transcription reaction was performed in the absence of RNA in all assays. The efficacy of positive reactions and negative controls for DNA contamination was assayed by PCR for -tubulin in a 25 l volume containing 1 QIAGEN PCR buffer (including 1.5 mM MgCl₂, 200 M dNTPs, 1 U QIAGEN HotStarTaq™ DNA polymerase, 1 l first-strand product and the -tubulin primers (100 nM each): 5': CAC CCG TCT TCA GGG CTT CTT GGT TT; 3': CAT TTC ACC ATC TGG TTG GCT GGC TC. Subsequent 1% agarose gel electrophoresis and ethidium bromide staining enabled visualization of PCR products.

Real-Time PCR

Real-time quantitative PCR was performed using an iCycler iQ Detection system (BioRad, Hercules, CA, USA) to assess transcript abundance. The following gene-specific primer pairs were designed for each candidate transcript: (a) TRPC1-5 (5'-TGC TGC CTA GTG CAT CGT-3') and TRPC1-3 (5'-TGG CGC AGT TCG TTT AGA-3') corresponding to nt 2692–2709 and 2766–2783, respectively, of the TRPC1 cDNA (Genbank Accession No. NM003304), and amplifying a 92 bp fragment; (b) TRPC3-5 (5'- GGC CGC ACG ACT ATT TCT -3') and TRPC3-3 (5'- AGC CCC TTG TAG GCA TTG -3') corresponding to nt 570–587 and 652–669, respectively, of the TRPC3 cDNA (Genbank Accession No. HSY13758), and amplifying a 94 bp fragment; (c) TRPC4-5 (5'-CCA GGC TGG AGG AGA AGA-3') and TRPC4-3 (5'-GTG GGC TTT TGG GAG CTA-3') corresponding to nt 1190–1207 and 1281–1298, respectively, of the TRPC4 cDNA (Genbank Accession No. AF421362), and amplifying a fragment of 100 bp; and (d) TRPC6-5 (5'-GAC TCG GAG CTG GGA GAA-3') and TRPC6-3 (5'-CGG TGA GCC AGT CTG TTG-3') corresponding to nt 120–137 and 200–217, respectively, of the TRPC6 cDNA (Genbank Accession No. XM040699), and amplifying a fragment of 98 bp.

Real-time PCR reactions were carried out using the Quantitect SYBR Green PCR kit (Qiagen). Each 25 l PCR mixture contained 12.5 l Quantitect SYBR Green PCR Master Mix, (HotStarTaq DNA polymerase, dNTPs, 5 mM MgCl₂ and fluorescent SYBR Green I dye), 200 nM of TRPC 5' and 3' primer or 200 nM 18S primers (QuantumRNA™ competimer kit, Ambion, Austin, TX), respectively, 1 nM fluorescien dye, 1 l of five-fold diluted first-strand cDNA, and RNase-free H₂O. PCR conditions consisted of initiation at 95°C for 15 min, followed by 55 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 45 s. PCR reactions for subject samples were performed in duplicate with standard and negative control samples in the same 96-well plate. A standard curve plotting threshold cycle (CT) values against input quantity (log scale) was constructed for each amplicon.³⁸ The identities of the PCR products were verified based on their sizes and nucleotide sequences (determined by the DNA Sequencing Facility, Center for Applied Genomics, Hospital for Sick Children, Toronto, ON, Canada), which were compared to the GenBank database using the BLAST search on the National Center for Biotechnology Information web site. The relative concentration of the target gene for subject samples was calculated from a standard curve of threshold cycle vs incremental mRNA concentration derived from pooled RT sample. To correct for sample-to-sample variation in transcript abundance, the amplicon 18S rRNA was also amplified with the target genes. Since transcript abundance values derived for 18S did not differ between drug-treated and diagnostic groups, it served as an internal reference against which TRPC values were normalized. Finally, melting-curve analysis was used to ensure the production of a single PCR product.

B-Lymphoblast Preparation and Western Blotting

B-lymphoblasts and comparison control tissues were prepared as described by Rosado and Sage²² with slight modifications. Briefly, aliquots of BLCLs (2 10⁷ cells), and rat and mouse cerebral cortex (100–300 mg; positive control) were thawed on ice and suspended in 200 l of lysis buffer, pH 7.2, containing 316 mM NaCl, 20 mM Tris, 2 mM EGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 100 g/ml leupeptin, 5 g/ml aprotinin and 10 mM benzamidine. Samples were then sonicated using a Vibra Cell Sonicator for 10 s with 2-s pulses at 80% intensity, placed on ice for 30 min, and then sonicated once again. Insoluble debris was removed by centrifugation (10 000 g, 10 min, 4°C). Supernatants were removed and protein was determined using the Bradford method.³⁹ Aliquots of increasing membrane protein concentration from each sample (4–12 g protein for TRPC1 and 2–8 g protein for TRPC3) were then prepared for SDS-PAGE by the addition of 2 Laemmli buffer, heated (3 min, 95°C) and resolved on 10% polyacrylamide gels. Anti-TRPC4 and anti-TRPC6 antibodies from Alomone and Santa Cruz Inc., respectively, did not elicit any immunoreactive signals in these preparations and, thus, examination of these TRPC subtypes in BLCLs was not pursued further. The subsequent focus of study was directed to characterization of the specificity of detection and quantification of TRPC1 and TRPC3 in BLCLs from BD patients and healthy subjects. For TRPC3 immunoblotting, samples were electrophoretically transferred to PVDF membranes and blocked with 5% egg white albumin (1 h, 4°C). For TRPC1, sample proteins were transferred to nitrocellulose membranes and blocked with 5% bovine serum albumin (1 h, 4°C). For immunodetection, anti-TRPC3 (Alomone) was used at a dilution of 1:200 while anti-TRPC1 (Santa Cruz) was used at a dilution of 1:100. Protein A-labeled HRP was employed as a secondary antibody (1:3000) for anti-TRPC3 detection, while anti-goat IgG (1:5000) was employed for anti-TRPC1. Immunoreactive proteins were detected with ECL+ chemiflourescence system (Amersham Biosciences). For quantitative purposes, assay conditions were established within a linear range of protein concentrations. Immunoreactive signals were normalized against pooled sample run in triplicate on each blot to control for blot-to-blot variation. Signal intensities of TRPC immunoreactive bands were quantified using a Storm phosphorimaging system and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

Statistical Analysis

Normalized transcript abundance and immunolabeling densities were analyzed by repeated-measures ANOVA, with diagnostic grouped sex as between-measures factors and drug treatment as the within-measures factor. Group (diagnosis, sex) by Li treatment interaction effects were further evaluated by analysis of simple effects and tests of contrasts.⁴⁰ Correlations between dependent variables were evaluated using the Pearson Product Moment test. Tests of differences in frequencies of sexes among comparison groups was evaluated using the Fisher Exact Test and of differences in mean ages of groups, using Student's unpaired t-test. All data are expressed as the meanSD. Statistics with P0.05 were taken as significant. Statistical analyses were performed using the SPSS software, version 10 (SPSS Inc.).

Notes

DUALITY OF INTEREST

None declared.

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Acknowledgements

This work was supported by the Canadian Institutes of Health Research (MOP-12851, JJW, and MOP-53323, JJW and PPL), the Ontario Mental Health Foundation (JJW), and a CAMH Postdoctoral Fellowship award (SA). We gratefully acknowledge the contributions of the physicians and research staff of the Mood and Anxiety Programs of the Clarke Institute, Center for Addiction and Mental Health, and University of Toronto who referred potential patient subjects for participation in the studies from which the cell repository was established. Kin Po Siu contributed essential technical assistance in the growth and maintenance of cell lines.