The Role Of TRPC3/6 Channels In Calcium Sensing Receptor-induced Ca²⁺ Signaling In Mesangial Cells

Objects: Calcium-sensing receptor?CaSR?,a cell-surface protein,is highly expressed in tissues and cells involved in systemic calcium homeostasis,including the parathyroid gland,kidney,and bone.However,CaSR is also expressed in many other tissues and cells that are not primarily involved in extracellular calcium homeostasis,such as in the brain,skin,lungs,suggesting that this receptor plays additional physiological roles in the regulation of cell functions,such as cellular proliferation,differentiation and apoptosis.In the kidney,CaSR is well known to regulate calcium excretion and absorption in the renal tubules.There is increasing evidence indicate that CaSR is localized not only in the renal tubules but also in the glomeruli.A previous study showed that CaSR was expressed in mouse glomerular mesangial cells,and stimulation of CaSR induced cell proliferation.However,nothing is currently known about the signaling cascades initiated by CaSR activation in mesangial cells.Furthermore,several studies indicated that canonical transient receptor potential channels?TRPC channels?are involved in the CaSR stimulation-induced calcium influx in some cell types,such as salivary ductal cells,MCF-7 breast cancer cells,aortic smooth muscle cells.In this experiment,we investigated the role of TRPC3 and TRPC6 in CaSR activation-induced Ca²⁺ signaling and cell proliferation in cultured human mesangial cells.Methods:?1?Using reverse transcription PCR,Western Blot and immunofluorescence to detect the m RNA and protein expression of CaSR in mesangial cells.?2?Fluo-3/AM was used as a calcium indicator and intracellular Ca²⁺concentration was monitored by using a laser confocal scanning microscope afterexposure to CaSR agonists or TRPC channel blockers.?3?Using Real-time PCR and Western Blot to detect the m RNA and protein expression of TRPC1,TRPC3,TRPC4 and TRPC6 in mesangial cells under normal or high extracellular Ca²⁺ concentration.?4?Cell proliferation was measured by using a Cell Proliferation ELISA Brd U kit in mesangial cells under normal or high extracellular Ca²⁺ concentration.Results:?1?CaSR is expressed in cultured human mesangial cells.?2?CaSR activation induces both intracellular Ca²⁺ release and TRPC-dependent Ca²⁺ influx in mesangial cells.?3?CaSR activation upregulates the m RNA and protein levels of TRPC3 and TRPC6,but not TRPC1 or TRPC4.?4?TRPC3 and TRPC6 mediate calcium influx in a store-independent manner in response to CaSR stimulation.?5?TRPC3 and TRPC6 contribute to high [Ca²⁺]o-mediated mesangial cells proliferation.Conclusions:?1?CaSR is functionally expressed in cultured human mesangial cells.?2?CaSR activation induces both intracellular Ca²⁺ release and TRPC-dependent Ca²⁺ influx.?3?CaSR activation upregulates the m RNA and protein levels of TRPC3 and TRPC6.?4?TRPC3 and TRPC6 contribute to CaSR activation-mediated mesangial cells proliferation.