ABSTRACT
OBJECTIVES: Recent findings implicate the calcium-permeable nonselective ion channels transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) in the pathogenesis of bipolar disorder (BD). These channels are involved in calcium and oxidative stress signaling, both of which are disrupted in BD. Thus, we sought to determine if these channels are differentially affected by oxidative stress in cell lines of BD patient origin. METHODS: B lymphoblast cell lines (BLCLs) from bipolar I disorder (BD-I) patients (n = 6) and healthy controls (n = 5) were challenged with the oxidative stressor rotenone (2.5 µM and 10 µM) or vehicle for acute (24 hours) and chronic (four days) intervals. Cell viability was measured using propidium iodide, while TRPM2- and TRPC3-mediated calcium fluxes were measured in the presence of their respective activators (H(2) O(2) and 1-oleoyl-2-acetyl-sn-glycerol) using Fluo-4. Changes in TRPM2 and TRPC3 expression levels were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: Cell viability decreased with increasing dose and duration of rotenone treatment, with BD-I patient BLCLs more susceptible than controls acutely (p < 0.001). A dose-dependent decrease in TRPC3 protein expression occurred after chronic (24%, p = 0.008) but not acute rotenone treatment. Interestingly, H(2) O(2) -provoked TRPM2-dependent calcium fluxes revealed an interaction between the effects of stressor addition and diagnostic subject group (p = 0.003). CONCLUSIONS: These data support an important role for TRPM2 and TRPC3 in sensing and responding to oxidative stress and in transducing oxidative stress signaling to intracellular calcium homeostasis and cellular stress responses, all of which have been implicated in the pathophysiology of BD.
Subject(s)
B-Lymphocytes/metabolism , Bipolar Disorder/pathology , Oxidative Stress/physiology , TRPC Cation Channels/metabolism , TRPM Cation Channels/metabolism , Adult , B-Lymphocytes/drug effects , Bipolar Disorder/immunology , Calcium/metabolism , Cell Survival , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Insecticides/pharmacology , Male , Oxidative Stress/drug effects , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Rotenone/pharmacology , TRPC Cation Channels/genetics , TRPM Cation Channels/genetics , Time Factors , Young AdultABSTRACT
The canonical transient receptor potential type 3 (TRPC3) channel is a non-selective, voltage-independent cation channel that is expressed in both excitable and non-excitable cells. As little is known regarding its presence in human brain and the influence of age on its expression, we examined TRPC3 protein expression by immunoblotting in postmortem prefrontal cortex and cerebellum obtained from subjects (8 days to 83 years) with no history of psychiatric or neurological disorder. The expression of TRPC3 protein in the prefrontal cortex (Brodmann area A9/A10) of the neonates/infants (<2 y) was significantly higher (25%) than that in the adolescent to adult (11y-83y) age group, whereas cerebellar TRPC3 levels showed no age-related changes. The results indicate that TRPC3 may be developmentally regulated in prefrontal cortex, and its expression in discrete human brain regions throughout the lifespan suggests a physiological role for TRPC3 during postnatal and adult life.
Subject(s)
Cerebellum/growth & development , Cerebellum/metabolism , Prefrontal Cortex/growth & development , Prefrontal Cortex/metabolism , TRPC Cation Channels/metabolism , Adolescent , Adult , Aged, 80 and over , Aging , Analysis of Variance , Blotting, Western , Child, Preschool , Electrophoresis, Polyacrylamide Gel , Female , Humans , Infant , Infant, Newborn , Linear Models , Male , Middle AgedABSTRACT
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.
