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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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Activation of CaSR induces an increase in [Ca2+]i in human MCs.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods with a laserconfocal scanning microscope. Representative traces are shown in A and C-H. (A) An increase in [Ca2+]o from 1 to 5 mM evokes a rapid peak and a subsequent sustained elevated level of [Ca2+]i. (B) Summary of data showing a concentration-dependent relationship for the effect of [Ca2+]o stimulation or peak [Ca2+]i responses. Human MCs were stimulated with different concentrations of [Ca2+]o (0.5–20 mM). Data are shown as the means ± SEs of 40–50 cells. (C) In the presence of extracellular Ca2+ (1 mM), addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. (D) Dose-dependence of the spermine-induced increase in [Ca2+]i. Cells were stimulated by different concentrations of spermine (1–5 mM) in medium containing 1 mM Ca2+. (E, F) Pretreatment with 10 µM NPS2390 for 20 min inhibits the 5 mM [Ca2+]o(E)- or 3 mM spermine(F)-induced [Ca2+]i increase (p<0.01 vs. NPS2390(-), n = 6), respectively. (G, H) Treatment with 5 mM [Ca2+]o(G) or 3 mM spermine(H) induced an increase in [Ca2+]i, and this increase is inhibited by pretreatment with 30 µM U73122 for 20 min (p<0.01 vs. U73122(-), n = 6), respectively. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
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pone-0098777-g002: Activation of CaSR induces an increase in [Ca2+]i in human MCs.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods with a laserconfocal scanning microscope. Representative traces are shown in A and C-H. (A) An increase in [Ca2+]o from 1 to 5 mM evokes a rapid peak and a subsequent sustained elevated level of [Ca2+]i. (B) Summary of data showing a concentration-dependent relationship for the effect of [Ca2+]o stimulation or peak [Ca2+]i responses. Human MCs were stimulated with different concentrations of [Ca2+]o (0.5–20 mM). Data are shown as the means ± SEs of 40–50 cells. (C) In the presence of extracellular Ca2+ (1 mM), addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. (D) Dose-dependence of the spermine-induced increase in [Ca2+]i. Cells were stimulated by different concentrations of spermine (1–5 mM) in medium containing 1 mM Ca2+. (E, F) Pretreatment with 10 µM NPS2390 for 20 min inhibits the 5 mM [Ca2+]o(E)- or 3 mM spermine(F)-induced [Ca2+]i increase (p<0.01 vs. NPS2390(-), n = 6), respectively. (G, H) Treatment with 5 mM [Ca2+]o(G) or 3 mM spermine(H) induced an increase in [Ca2+]i, and this increase is inhibited by pretreatment with 30 µM U73122 for 20 min (p<0.01 vs. U73122(-), n = 6), respectively. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Mentions: To evaluate if the expression of CaSR protein is associated with the presence of functional receptors, Fluo-3/AM-loaded human MCs were stimulated by known CaSR agonists. As shown in Fig. 2A, a change in [Ca2+]o from 1 to 5 mM evoked a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. The increase in the [Ca2+]i due to the [Ca2+]o occurred in a concentration-dependent manner, as shown in Fig. 2B. The half maximal effective concentration (EC50) of [Ca2+]o that was necessary to achieve the [Ca2+]i response in the human MCs was approximately 4.93 mM. A similar effect was observed with the use of 3 mM spermine, another CaSR agonist (Fig. 2C), indicating that the observed effect of CaSR activation was not agonist-specific. Additionally, the increase in [Ca2+]i induced by spermine was dose-dependent (Fig. 2D). Both the 5 mM [Ca2+]o- and 3 mM spermine-induced [Ca2+]i increases were significantly inhibited by pretreatment with 10 µM NPS2390, an antagonist of CaSR (Fig. 2E and 2F). To evaluate whether the increase in [Ca2+]i induced by CaSR activation involves a PLC-dependent pathway, cells were stimulated with CaSR agonists in the presence of the PLC inhibitor U73122. The [Ca2+]i increase induced by 5 mM [Ca2+]o or 3 mM spermine was significantly inhibited by pretreatment with 30 µM U73122, as shown in Fig. 2G and 2H, respectively. Taken together, these data confirm that CaSR protein is functionally expressed in human MC and activates a PLC-dependent [Ca2+]i increase.


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)

Activation of CaSR induces an increase in [Ca2+]i in human MCs.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods with a laserconfocal scanning microscope. Representative traces are shown in A and C-H. (A) An increase in [Ca2+]o from 1 to 5 mM evokes a rapid peak and a subsequent sustained elevated level of [Ca2+]i. (B) Summary of data showing a concentration-dependent relationship for the effect of [Ca2+]o stimulation or peak [Ca2+]i responses. Human MCs were stimulated with different concentrations of [Ca2+]o (0.5–20 mM). Data are shown as the means ± SEs of 40–50 cells. (C) In the presence of extracellular Ca2+ (1 mM), addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. (D) Dose-dependence of the spermine-induced increase in [Ca2+]i. Cells were stimulated by different concentrations of spermine (1–5 mM) in medium containing 1 mM Ca2+. (E, F) Pretreatment with 10 µM NPS2390 for 20 min inhibits the 5 mM [Ca2+]o(E)- or 3 mM spermine(F)-induced [Ca2+]i increase (p<0.01 vs. NPS2390(-), n = 6), respectively. (G, H) Treatment with 5 mM [Ca2+]o(G) or 3 mM spermine(H) induced an increase in [Ca2+]i, and this increase is inhibited by pretreatment with 30 µM U73122 for 20 min (p<0.01 vs. U73122(-), n = 6), respectively. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone-0098777-g002: Activation of CaSR induces an increase in [Ca2+]i in human MCs.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods with a laserconfocal scanning microscope. Representative traces are shown in A and C-H. (A) An increase in [Ca2+]o from 1 to 5 mM evokes a rapid peak and a subsequent sustained elevated level of [Ca2+]i. (B) Summary of data showing a concentration-dependent relationship for the effect of [Ca2+]o stimulation or peak [Ca2+]i responses. Human MCs were stimulated with different concentrations of [Ca2+]o (0.5–20 mM). Data are shown as the means ± SEs of 40–50 cells. (C) In the presence of extracellular Ca2+ (1 mM), addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. (D) Dose-dependence of the spermine-induced increase in [Ca2+]i. Cells were stimulated by different concentrations of spermine (1–5 mM) in medium containing 1 mM Ca2+. (E, F) Pretreatment with 10 µM NPS2390 for 20 min inhibits the 5 mM [Ca2+]o(E)- or 3 mM spermine(F)-induced [Ca2+]i increase (p<0.01 vs. NPS2390(-), n = 6), respectively. (G, H) Treatment with 5 mM [Ca2+]o(G) or 3 mM spermine(H) induced an increase in [Ca2+]i, and this increase is inhibited by pretreatment with 30 µM U73122 for 20 min (p<0.01 vs. U73122(-), n = 6), respectively. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
Mentions: To evaluate if the expression of CaSR protein is associated with the presence of functional receptors, Fluo-3/AM-loaded human MCs were stimulated by known CaSR agonists. As shown in Fig. 2A, a change in [Ca2+]o from 1 to 5 mM evoked a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. The increase in the [Ca2+]i due to the [Ca2+]o occurred in a concentration-dependent manner, as shown in Fig. 2B. The half maximal effective concentration (EC50) of [Ca2+]o that was necessary to achieve the [Ca2+]i response in the human MCs was approximately 4.93 mM. A similar effect was observed with the use of 3 mM spermine, another CaSR agonist (Fig. 2C), indicating that the observed effect of CaSR activation was not agonist-specific. Additionally, the increase in [Ca2+]i induced by spermine was dose-dependent (Fig. 2D). Both the 5 mM [Ca2+]o- and 3 mM spermine-induced [Ca2+]i increases were significantly inhibited by pretreatment with 10 µM NPS2390, an antagonist of CaSR (Fig. 2E and 2F). To evaluate whether the increase in [Ca2+]i induced by CaSR activation involves a PLC-dependent pathway, cells were stimulated with CaSR agonists in the presence of the PLC inhibitor U73122. The [Ca2+]i increase induced by 5 mM [Ca2+]o or 3 mM spermine was significantly inhibited by pretreatment with 30 µM U73122, as shown in Fig. 2G and 2H, respectively. Taken together, these data confirm that CaSR protein is functionally expressed in human MC and activates a PLC-dependent [Ca2+]i increase.

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