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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 intracellular Ca2+ release.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. (A) Representative traces showing that 3 mM spermine induces an increase in [Ca2+]i even in the absence of extracellular Ca2+(left). Cells were bathed in Ca2+-free solution followed by addition of 3 mM spermine. In the presence of extracellular 1 mM Ca2+, addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i (right). (B) Depletion of internal Ca2+ stores by 1 µM thapsigargin (TG) abolishes the 3 mM spermine-induced [Ca2+]i signaling in the absence of extracellular Ca2+. Cells bathed in Ca2+-free solution were stimulated with 1 µM TG, and then, 3 mM spermine was added to the bath solution. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
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pone-0098777-g003: CaSR activation induces intracellular Ca2+ release.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. (A) Representative traces showing that 3 mM spermine induces an increase in [Ca2+]i even in the absence of extracellular Ca2+(left). Cells were bathed in Ca2+-free solution followed by addition of 3 mM spermine. In the presence of extracellular 1 mM Ca2+, addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i (right). (B) Depletion of internal Ca2+ stores by 1 µM thapsigargin (TG) abolishes the 3 mM spermine-induced [Ca2+]i signaling in the absence of extracellular Ca2+. Cells bathed in Ca2+-free solution were stimulated with 1 µM TG, and then, 3 mM spermine was added to the bath solution. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.

Mentions: Because Ca2+ mobilization from intracellular stores by CaSR agonists has been shown in many cell types, we investigated whether similar effects of CaSR agonists occur in human MCs. Cells were stimulated by spermine in the absence of extracellular Ca2+. As shown in Fig. 3A, 3 mM spermine induced an increase in [Ca2+]i in Ca2+-free solutions. Accordingly, no Ca2+ signal was ever observed after store depletion by 1 µM thapsigargin (TG), an endoplasmic reticulum Ca2+-ATPase inhibitor, further indicating that CaSR agonists stimulate Ca2+ release from intracellular stores (Fig. 3B). Interestingly, as shown in Fig. 3A, the spermine-induced [Ca2+]i increase in the absence of extracellular Ca2+ was smaller than that observed in the presence of 1 mM [Ca2+]o and had no subsequent sustained increase in [Ca2+]i, suggesting that extracellular Ca2+ influx is most likely involved in the increase of [Ca2+]i by CaSR agonists in human MCs.


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 intracellular Ca2+ release.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. (A) Representative traces showing that 3 mM spermine induces an increase in [Ca2+]i even in the absence of extracellular Ca2+(left). Cells were bathed in Ca2+-free solution followed by addition of 3 mM spermine. In the presence of extracellular 1 mM Ca2+, addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i (right). (B) Depletion of internal Ca2+ stores by 1 µM thapsigargin (TG) abolishes the 3 mM spermine-induced [Ca2+]i signaling in the absence of extracellular Ca2+. Cells bathed in Ca2+-free solution were stimulated with 1 µM TG, and then, 3 mM spermine was added to the bath solution. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048219&req=5

pone-0098777-g003: CaSR activation induces intracellular Ca2+ release.[Ca2+]i dynamics were monitored by Fura-3 fluorescence methods. (A) Representative traces showing that 3 mM spermine induces an increase in [Ca2+]i even in the absence of extracellular Ca2+(left). Cells were bathed in Ca2+-free solution followed by addition of 3 mM spermine. In the presence of extracellular 1 mM Ca2+, addition of 3 mM spermine evokes a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i (right). (B) Depletion of internal Ca2+ stores by 1 µM thapsigargin (TG) abolishes the 3 mM spermine-induced [Ca2+]i signaling in the absence of extracellular Ca2+. Cells bathed in Ca2+-free solution were stimulated with 1 µM TG, and then, 3 mM spermine was added to the bath solution. The results were from at least three independent experiments, and each experiment measured 20 to 40 cells.
Mentions: Because Ca2+ mobilization from intracellular stores by CaSR agonists has been shown in many cell types, we investigated whether similar effects of CaSR agonists occur in human MCs. Cells were stimulated by spermine in the absence of extracellular Ca2+. As shown in Fig. 3A, 3 mM spermine induced an increase in [Ca2+]i in Ca2+-free solutions. Accordingly, no Ca2+ signal was ever observed after store depletion by 1 µM thapsigargin (TG), an endoplasmic reticulum Ca2+-ATPase inhibitor, further indicating that CaSR agonists stimulate Ca2+ release from intracellular stores (Fig. 3B). Interestingly, as shown in Fig. 3A, the spermine-induced [Ca2+]i increase in the absence of extracellular Ca2+ was smaller than that observed in the presence of 1 mM [Ca2+]o and had no subsequent sustained increase in [Ca2+]i, suggesting that extracellular Ca2+ influx is most likely involved in the increase of [Ca2+]i by CaSR agonists in human MCs.

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