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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 agonists mediate Ca2+ influx in store-independent manner.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–B. (A) In the presence of 1 µM TG, a change in [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i, and then, addition of 3 mM spermine evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx induced by 3 mM spermine was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). (B) A restoration of [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i in the presence of 1 µM TG, and then, a change in [Ca2+]o from 0.5 to 5 mM evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx due to 5 mM [Ca2+]o was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
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pone-0098777-g009: CaSR agonists mediate Ca2+ influx in store-independent manner.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–B. (A) In the presence of 1 µM TG, a change in [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i, and then, addition of 3 mM spermine evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx induced by 3 mM spermine was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). (B) A restoration of [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i in the presence of 1 µM TG, and then, a change in [Ca2+]o from 0.5 to 5 mM evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx due to 5 mM [Ca2+]o was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Mentions: To further demonstrate that calcium influx stimulated by CaSR agonists does not require the release of calcium stores, we depleted stores with TG before CaSR stimulation. TG blocks Ca2+-ATPase located in the membrane of the endoplasmic reticulum (ER) and other intracellular vesicular store compartments. As shown in Fig. 9A, in the presence of 1 µM TG, a restoration of extracellular calcium from 0 to 0.5 mM induced an expected rise in [Ca2+]i, which was due to SOCE, and 3 mM spermine evoked an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores were already depleted. However, the additional substantial increase in [Ca2+]i was blocked by NSP2390, a CaSR antagonist. Similar results were obtained with a 5 mM [Ca2+]o-induced calcium influx (Fig. 9B). These results suggest that TRPC3- and TRPC6-mediated calcium influx by CaSR activation does not require the release of calcium stores.


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 agonists mediate Ca2+ influx in store-independent manner.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–B. (A) In the presence of 1 µM TG, a change in [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i, and then, addition of 3 mM spermine evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx induced by 3 mM spermine was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). (B) A restoration of [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i in the presence of 1 µM TG, and then, a change in [Ca2+]o from 0.5 to 5 mM evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx due to 5 mM [Ca2+]o was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). 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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getmorefigures.php?uid=PMC4048219&req=5

pone-0098777-g009: CaSR agonists mediate Ca2+ influx in store-independent manner.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. Representative traces are shown in A–B. (A) In the presence of 1 µM TG, a change in [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i, and then, addition of 3 mM spermine evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx induced by 3 mM spermine was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). (B) A restoration of [Ca2+]o from 0 to 0.5 mM induces a rise in [Ca2+]i in the presence of 1 µM TG, and then, a change in [Ca2+]o from 0.5 to 5 mM evokes an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores are already depleted. The store-independent calcium influx due to 5 mM [Ca2+]o was blocked by pretreatment with 10 µM NPS2390 (p<0.05 vs. Ctl, n = 5). The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
Mentions: To further demonstrate that calcium influx stimulated by CaSR agonists does not require the release of calcium stores, we depleted stores with TG before CaSR stimulation. TG blocks Ca2+-ATPase located in the membrane of the endoplasmic reticulum (ER) and other intracellular vesicular store compartments. As shown in Fig. 9A, in the presence of 1 µM TG, a restoration of extracellular calcium from 0 to 0.5 mM induced an expected rise in [Ca2+]i, which was due to SOCE, and 3 mM spermine evoked an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores were already depleted. However, the additional substantial increase in [Ca2+]i was blocked by NSP2390, a CaSR antagonist. Similar results were obtained with a 5 mM [Ca2+]o-induced calcium influx (Fig. 9B). These results suggest that TRPC3- and TRPC6-mediated calcium influx by CaSR activation does not require the release of calcium stores.

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