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Calcium sensing receptor modulates extracellular calcium entry and proliferation via TRPC3/6 channels in cultured human mesangial cells.

Meng K, Xu J, Zhang C, Zhang R, Yang H, Liao C, Jiao J - PLoS ONE (2014)

Bottom Line: Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx.Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase.Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China.

ABSTRACT
Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.

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CaSR activation induces TRPC-dependent Ca2+ signaling.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–D. (A) The 5 mM [Ca2+]o-induced [Ca2+]i increase is inhibited in the presence of TRPC channel blockers (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by a change in [Ca2+]o from 1 to 5 mM. (B) In the presence of TRPC channel blockers, the 3 mM spermine-induced [Ca2+]i increase is inhibited (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by addition of 3 mM spermine. (C) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 5 mM [Ca2+]o-induced [Ca2+]i signaling (p>0.05, n = 5). (D) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 3 mM spermine-induced [Ca2+]i increase (p>0.05, n = 5). All other additions are indicated in the figure. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
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pone-0098777-g004: CaSR activation induces TRPC-dependent Ca2+ signaling.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–D. (A) The 5 mM [Ca2+]o-induced [Ca2+]i increase is inhibited in the presence of TRPC channel blockers (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by a change in [Ca2+]o from 1 to 5 mM. (B) In the presence of TRPC channel blockers, the 3 mM spermine-induced [Ca2+]i increase is inhibited (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by addition of 3 mM spermine. (C) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 5 mM [Ca2+]o-induced [Ca2+]i signaling (p>0.05, n = 5). (D) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 3 mM spermine-induced [Ca2+]i increase (p>0.05, n = 5). All other additions are indicated in the figure. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Mentions: Because TPRC channels, voltage-gated calcium channels and Na+/Ca2+ exchangers are the main pathways for Ca2+ influx in MCs [18], we examined the role of these pathways in CaSR agonist-induced [Ca2+]i increase. As shown in Fig. 4A and 4B, pretreatment with SKF96365 (50 µM) or 2-APB (100 µM), nonselective TRPC channel blockers [19], significantly inhibited the 5 mM [Ca2+]o- and 3 mM spermine-induced [Ca2+]i increase, whereas pretreatment with efonidipine (10 µM, a voltage-gated calcium blocker) or SN-6 (10 µM, an specific inhibitor of NCX) had no apparent effect (Fig. 4C and 4D). These data indicate that TRPC-dependent Ca2+ entry is involved in the CaSR agonist-induced [Ca2+]i increase. Therefore, in the following experiments, we focus on TRPC channels that contribute to [Ca2+]i signaling in response to CaSR stimulation


Calcium sensing receptor modulates extracellular calcium entry and proliferation via TRPC3/6 channels in cultured human mesangial cells.

Meng K, Xu J, Zhang C, Zhang R, Yang H, Liao C, Jiao J - PLoS ONE (2014)

CaSR activation induces TRPC-dependent Ca2+ signaling.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–D. (A) The 5 mM [Ca2+]o-induced [Ca2+]i increase is inhibited in the presence of TRPC channel blockers (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by a change in [Ca2+]o from 1 to 5 mM. (B) In the presence of TRPC channel blockers, the 3 mM spermine-induced [Ca2+]i increase is inhibited (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by addition of 3 mM spermine. (C) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 5 mM [Ca2+]o-induced [Ca2+]i signaling (p>0.05, n = 5). (D) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 3 mM spermine-induced [Ca2+]i increase (p>0.05, n = 5). All other additions are indicated in the figure. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
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Related In: Results  -  Collection

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pone-0098777-g004: CaSR activation induces TRPC-dependent Ca2+ signaling.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–D. (A) The 5 mM [Ca2+]o-induced [Ca2+]i increase is inhibited in the presence of TRPC channel blockers (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by a change in [Ca2+]o from 1 to 5 mM. (B) In the presence of TRPC channel blockers, the 3 mM spermine-induced [Ca2+]i increase is inhibited (p<0.05 vs. Ctl, n = 5). Cells were pretreated with 50 µM SKF96365 or 100 µM 2-APB for 20 min in medium containing 1 mM Ca2+ followed by addition of 3 mM spermine. (C) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 5 mM [Ca2+]o-induced [Ca2+]i signaling (p>0.05, n = 5). (D) Pretreatment of the cells with efonidipine (10 µM) or SN-6 (10 µM) for 20 min had no apparent effect on the 3 mM spermine-induced [Ca2+]i increase (p>0.05, n = 5). All other additions are indicated in the figure. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
Mentions: Because TPRC channels, voltage-gated calcium channels and Na+/Ca2+ exchangers are the main pathways for Ca2+ influx in MCs [18], we examined the role of these pathways in CaSR agonist-induced [Ca2+]i increase. As shown in Fig. 4A and 4B, pretreatment with SKF96365 (50 µM) or 2-APB (100 µM), nonselective TRPC channel blockers [19], significantly inhibited the 5 mM [Ca2+]o- and 3 mM spermine-induced [Ca2+]i increase, whereas pretreatment with efonidipine (10 µM, a voltage-gated calcium blocker) or SN-6 (10 µM, an specific inhibitor of NCX) had no apparent effect (Fig. 4C and 4D). These data indicate that TRPC-dependent Ca2+ entry is involved in the CaSR agonist-induced [Ca2+]i increase. Therefore, in the following experiments, we focus on TRPC channels that contribute to [Ca2+]i signaling in response to CaSR stimulation

Bottom Line: Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx.Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase.Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China.

ABSTRACT
Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.

Show MeSH
Related in: MedlinePlus