Journal: Molecular Pain

Article Title: Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

doi: 10.1186/1744-8069-10-43

Figure Lengend Snippet: TRPC family gene expression in sensory ganglia. (A) TRPC mRNA detection using a RT-PCR screen against TRPC1-7 in adult rat DRG. TRPC1 and TRPC3 are the major subunits expressed in DRG, along with low levels of TRPC6 and little or no signal for TRPC 2, 4, 5 and 7. (B) In situ expression profile of all TRPC family members at the whole DRG and spinal cord level. TRPC1 and TRPC3 are expressed at high levels in DRGs, with minimal detection in the spinal cord. Low but significant TRPC6 expression is also observed at the DRG level. (C) Emulsion staining with cellular resolution clearly shows that TRPC3 mRNA is localized in a subpopulation of small and medium diameter neurons (arrows) in DRG and trigeminal ganglia (TGG). Representative negative small-diameter and large-diameter neurons are also indicated (stars). Scale bar = 50 μm.

Article Snippet: TRPC3-specific shRNA construct (Origene) was used to interfere with the translation of the endogenous TRPC3 subunits as previously described [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Situ, Staining

Journal: Molecular Pain

Article Title: Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

doi: 10.1186/1744-8069-10-43

Figure Lengend Snippet: Contribution of TRPC channels to SOCE in DRG neurons. All traces are representative calcium responses recorded using the Fura-2 ratiometric method. (A) Thapsigargin (Thaps, 1 μM, 7 min) evokes a biphasic SOCE response in cultured rat DRG neurons (control). The first phase reflects the release of calcium from ER stores while the second phase reflects the influx of extracellular calcium, upon calcium re-addition to the perfusion solution (calcium add-back protocol), via Orai and/or TRPC channels. The addition of the Orai blocker Gd 3+ (1 μM, 7 min) resulted in a significant decrease of calcium influx by 50%, suggesting a possible role for other channels including TRPCs in the remaining response (n = 8-19, P < 0.05). (B) SKF96365 (30 μM), a general SOCE and TRPC blocker, completely blocks SOCE (n = 14-23, P < 0.0005). (C) Effect of SKF96365 on TRPC3 activity. Activation of TRPC3 with the DAG analog OAG (50 μM, 3 min), is significantly inhibited by SKF96365 (approximately 75%), indicating a major role for TRPC3 in the TRPC component of the thapsigargin-induced SOCE response observed in DRG neurons (n = 15-18, P < 0.01).

Article Snippet: TRPC3-specific shRNA construct (Origene) was used to interfere with the translation of the endogenous TRPC3 subunits as previously described [ ].

Techniques: Cell Culture, Activity Assay, Activation Assay

Journal: Molecular Pain

Article Title: Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

doi: 10.1186/1744-8069-10-43

Figure Lengend Snippet: TRPC3 participates in SOCE in DRG neurons. (A) TRPC3 shRNA-mediated knockdown was validated in a functional assay to determine the inhibitory effect of the shRNA on OAG-evoked calcium entry in DRG neurons (n = 9-11, P < 0.0001). (B) shRNA-mediated knockdown of TRPC3 in the SOCE assay clearly shows a strong contribution of this channel to the overall calcium influx. A decrease of approximately 42% is observed (n = 13-27, P < 0.05). (C) Heterologous overexpression of TRPC3 (OE) in DRG neurons produced a drastic increase in SOCE-mediated calcium influx of approximately 85% (n = 16-19, P < 0.01). (D) The inhibition of TRPC3 activity by the selective blocker Pyr10 (10 μM, 15 min) also resulted in a strong decrease of SOCE activity, with calcium influx dropping approximately 50% (n = 34-39, P < 0.01). The cumulative inhibition of both Orai and TRPC3 with Gd 3+ and Pyr10, respectively, almost completely abolished SOCE response in DRG (n = 18-21, P < 0.001).

Article Snippet: TRPC3-specific shRNA construct (Origene) was used to interfere with the translation of the endogenous TRPC3 subunits as previously described [ ].

Techniques: shRNA, Functional Assay, Over Expression, Produced, Inhibition, Activity Assay

Journal: Molecular Pain

Article Title: Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

doi: 10.1186/1744-8069-10-43

Figure Lengend Snippet: Functional coupling between TRPC3 and UTP/P2Y2 signaling in DRG neurons. (A) The addition of 100 μM UTP for 7 minutes to the calcium-free perfusion solution activates P2Y2 receptors, which initiate both SOCE and ROCE responses. The addition of Gd 3+ removes the Orai component of the UTP-evoked response, which accounts for approximately 35% of the overall calcium influx (n = 17-19, P < 0.05). (B) The blockade of TRPC3 specifically with Pyr10 (10 μM, 15 min) resulted in a drastic decrease of UTP-evoked calcium entry, resulting in 60% inhibition (n = 25-28, P < 0.001). (C) shRNA-mediated knockdown of TRPC3 induced in similar decrease of P2Y2-mediated calcium entry (n = 34-46, P < 0.0001). (D) Heterologous overexpression of TRPC3 (OE) resulted in 90% increase of calcium influx in the UTP response, indicating a strong link between TRPC3 activity and P2Y2 transduction (n = 24-45, P < 0.001).

Article Snippet: TRPC3-specific shRNA construct (Origene) was used to interfere with the translation of the endogenous TRPC3 subunits as previously described [ ].

Techniques: Functional Assay, Inhibition, shRNA, Over Expression, Activity Assay, Transduction

Journal: Molecular Pain

Article Title: Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

doi: 10.1186/1744-8069-10-43

Figure Lengend Snippet: TRPC3 is linked to proteases/PAR2 transduction in DRG neurons. (A) Addition of 100 μM AC55541 to the calcium-free perfusion solution activates PAR2 receptors, which initiate both SOCE and ROCE responses. Treatment with Gd 3+ removed the Orai component, which produced a small but non-significant decrease in calcium influx (n = 22-24, P > 0.05). (B) The selective inhibition of TRPC3 with Pyr10 (10 μM, 15 min) resulted in a drastic decrease (66%) of AC55541-evoked calcium entry (n = 19-29, P < 0.001). (C) shRNA-mediated knockdown of TRPC3 induced a similar decrease in PAR2-mediated calcium entry of approximately 66% (n = 24-73, P < 0.0001). (D) Heterologous overexpression of TRPC3 (OE) increased by 135% the calcium influx following PAR2 activation, indicating a strong link between TRPC3 activity and PAR2 function (n = 18-26, P < 0.0001).

Article Snippet: TRPC3-specific shRNA construct (Origene) was used to interfere with the translation of the endogenous TRPC3 subunits as previously described [ ].

Techniques: Transduction, Produced, Inhibition, shRNA, Over Expression, Activation Assay, Activity Assay

Journal: Molecular Pain

Article Title: Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

doi: 10.1186/1744-8069-10-43

Figure Lengend Snippet: TRPC3 is involved in peripheral sensitization induced by PAR2 and P2Y2 receptors. Intrinsic excitability of a representative DRG nociceptor recorded sequentially under current-clamp conditions (RMP = -67 mV). (A) Firing of action potentials was consistently triggered by current pulses (200 pA; 50 ms duration) injected at fixed time intervals (n = 25). No firing was detected in conditions of subthreshold current injection (50 pA; 50 ms duration) (B) unless the cells were first sensitized by application of both PAR2 agonist AC55541 (100 μM) and P2Y2 agonist UTP (100 μM) (n = 14/25) (C) . (D) Treatment with the specific TRPC3 antagonist Pyr10 (10 μM) suppressed this sensitization effect in most neurons (n = 6/9). (E) At the end of each recording, cells were tested for viability and displayed normal firing activity after Pyr10 washout (n = 6).

Article Snippet: TRPC3-specific shRNA construct (Origene) was used to interfere with the translation of the endogenous TRPC3 subunits as previously described [ ].

Techniques: Injection, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: Brain-Derived Neurotrophic Factor Regulates TRPC3/6 Channels and Protects Against Myocardial Infarction in Rodents

doi: 10.7150/ijbs.10754

Figure Lengend Snippet: Expression of BDNF/TrkB and downstream TRPC3/6 channels. (A) Representative bands of protein expression of proBDNF and mature BDNF by western blot analysis. (B) Representative bands of protein expression of TrkB. (C, D) Representative bands of protein expression of TRPC3 and 6 channels. Values given are normalized to band intensity of GADPH (anti-GAPDH antibody) used as internal control. *p<0.05, **p<0.01 vs. sham, n = 4 rats each group.

Article Snippet: TRPC3 siRNA (sense: 5′-CCACCCAGUUCACAUGGACAGAAAU-3′; antisense: 5′-AUUUCUGUCCAUGUGAACUGGGUGG-3′) and TRPC6 siRNA (sense: 5′-GUCCAUUCAUGAAGUUCGUTT-3′; antisense: 5′-ACGAACUUCAUGAAUGGACTT-3′) were synthesized by Invitrogen, USA.

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Brain-Derived Neurotrophic Factor Regulates TRPC3/6 Channels and Protects Against Myocardial Infarction in Rodents

doi: 10.7150/ijbs.10754

Figure Lengend Snippet: Effect of BDNF and 2-APB on cell vialibity and apoptosis in NRVMs. (A) Effect of BDNF on cell viability in hypoxia. *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 6 each group. (B) Effect of BDNF (200 ng/ml), 2-APB and TrkB-Fc on cell vialibity in normoxia. (C) mRNA level of TRPC3/TRPC6 in control and TRPC3/TRPC6 siRNA treated cardiomyocytes. (D) Effect of BDNF on cardiomyocytes apoptosis subject to hypoxia by TUNEL staining. Nuclei are stained in blue while apoptotic cells are stained in green, scale bar: 100 μm. (E) TUNEL positive cell (%), *p<0.05 vs. hypoxia, #p<0.05 vs. 200 ng/ml BDNF, &p<0.05 vs. 200 ng/ml BDNF+Si-TRPC3 or 200 ng/ml BDNF +Si-TRPC6, n = 5 each group.

Article Snippet: TRPC3 siRNA (sense: 5′-CCACCCAGUUCACAUGGACAGAAAU-3′; antisense: 5′-AUUUCUGUCCAUGUGAACUGGGUGG-3′) and TRPC6 siRNA (sense: 5′-GUCCAUUCAUGAAGUUCGUTT-3′; antisense: 5′-ACGAACUUCAUGAAUGGACTT-3′) were synthesized by Invitrogen, USA.

Techniques: TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: Human MCs were starved for 24-free medium prior to stimulation with 1 mM (Ctl) or 5 mM [Ca 2+ ] o for 24 h. (A) Real-time PCR experiments showed that the administration of 5 mM [Ca 2+ ] o for 24 h significantly increased the TRPC3 and TRPC6 mRNA levels but did not affect the mRNA levels of TRPC1 or TRPC4 (**p<0.01 vs. Ctl, n = 3). (B) Representative Western blot and corresponding quantitative analysis showing that treatment with 5 mM [Ca 2+ ] o for 24 h increased the TRPC3 and TRPC6 protein expression but did not affect the protein expression of TRPC1 or TRPC4 (**p<0.01 vs. Ctl, n = 3). Asterisks indicate the statistical significance (**p<0.01), with respect to 1 mM [Ca 2+ ] o conditions (Ctl). Data are shown as the means ± SEMs.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: (A, B) Real-time PCR experiments showed that the TRPC3 siRNA (A) and TRPC6 siRNA (B) decreased the mRNA expression of TRPC3 and TRPC6, respectively (*p<0.05 vs. Ctl, n = 3), without affecting other TRPC channels (p>0.05, n = 3). (C, D) Western blot experiments showed that transfection with TRPC3 siRNA (C) and TRPC6 siRNA (D) reduced TRPC3 and TRPC6 protein expression (**p<0.01 vs. Scr, n = 3), respectively, without affecting other TRPC channels (p>0.05, n = 3) compared with transfection with scramble siRNA. Asterisks indicate the statistical significance (**p<0.01). Data are shown as the means ± SEMs.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: [Ca 2+ ] i dynamics were monitored by Fura-3 fluorescence methods. (A, B) Representative traces showing that transfection with TRPC3 siRNA (A) or TRPC6 siRNA (B) significantly inhibit the 3 mM spermine-induced [Ca 2+ ] i increase (p<0.05 vs. Scr, n = 5), respectively, in contrast with cells transfected with scrambled siRNA. (C) Summary of data showing that transfection with TRPC3 siRNA or TRPC6 siRNA significantly inhibits the average value of the plateau of the 3 mM spermine-induced [Ca 2+ ] i increase (**p<0.01 vs. Scr, n = 5), respectively. (D, E) Representative traces showing that transfection with TRPC3 siRNA (D) or TRPC6 siRNA (E) significantly inhibits the 5 mM [Ca 2+ ] o -induced [Ca 2+ ] i increase (p<0.05 vs. Scr, n = 5), respectively, in contrast with cells transfected with scrambled siRNA. (F) Summary of data showing that transfection with TRPC3 siRNA or TRPC6 siRNA significantly inhibits the average value of the plateau of the 5 mM [Ca 2+ ] o -induced [Ca 2+ ] i increase (**p<0.01 vs. Scr, n = 5), respectively. Data are shown as the means ± SEMs. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Fluorescence, Transfection

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: [Ca 2+ ] i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A-D. (A, B) Representative traces (A–B) and summary of data (C) showing that transfection with TRPC3 siRNA (A) or TRPC6 siRNA (B) significantly reduces the 100 µM OAG-induced Ca 2+ influx (p<0.01 vs. Scr, n = 4), respectively, compared with transfection with scrambled siRNA. Data are shown as the means ± SEMs. (D, E) The 5 mM [Ca 2+ ] o (D)- and spermine(E)-induced [Ca 2+ ] i increase is significantly enhanced by pretreatment with 100 µM OAG for 20 min (p<0.05 vs. Ctl, n = 4). The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Fluorescence, Transfection

Journal: PLoS ONE

Article Title: Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

doi: 10.1371/journal.pone.0098777

Figure Lengend Snippet: Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit. After starvation for 24-free medium, cells were incubated in the same medium supplemented with different [Ca 2+ ] o (1–5 mM) in the presence or absence of various inhibitors for 24 h. Untreated cells cultured in medium containing 1 mM Ca 2+ were used as a control. (A) Incubation of cells for 24 h with 3 mM and 5 mM [Ca 2+ ] o increase proliferation of human MCs, respectively, compared with 1 mM [Ca 2+ ] o (Ctl) (*p<0.05, **p<0.01 vs. Ctl, n = 3). (B) Pretreatment with 10 µM NPS2390 significantly reduces the 5 mM [Ca 2+ ] o -induced cell proliferation (**p<0.01 vs. Ctl, # p<0.05 vs. 5 mM [Ca 2+ ] o without NPS2390, n = 3). Incubation with NPS2390 alone do not affect cell proliferation at 1 mM [Ca 2+ ] o (p>0.05, n = 3). (C) The [Ca 2+ ] o -mediated cell proliferation is significantly inhibited by pretreatment with 50 µM SKF96365 or 100 µM 2-APB (**p<0.01 vs. Ctl, #p<0.05 vs. 5 mM [Ca 2+ ] o without inhibitors, n = 3). (D) Transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuate the promotion of proliferation by 5 mM [Ca 2+ ] o (**p<0.01 vs. Scr+1 mM [Ca 2+ ] o , #p<0.05 vs. Scr+5 mM [Ca 2+ ] o ), respectively, compared with scramble siRNA treated with 5 mM [Ca 2+ ] o . Cells were transfected with TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA followed by treatment with 5 mM [Ca 2+ ] o for 24 h. Untransfected cells cultured in medium containing 1 mM Ca 2+ were used as a control. Data are shown as the means ± SEMs.

Article Snippet: Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Transfection