Differential Modulation of Intracellular Ca2+ Responses Associated with Calcium-Sensing Receptor Activation in Renal Collecting Duct Cells

摘要

In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Casup2+/sup] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Casup2+/sup, 300µM Gdsup3+/sup and with 10µM of specific allosteric modulator NPS-R 568, all resulted in an increase in [Casup2+/sup]subi/sub although displaying different features. Specifically, Casup2+/sup as well as stimulation with NPS-R 568 induced a rapid peak of [Casup2+/sup]subi/sub while stimulation with Gdsup3+/sup induced transient intracellular Casup2+/sup ioscillations/i. PLC inhibition completely abolished any [Casup2+/sup]subi/sub increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Casup2+/sup]subi/sub oscillations induced by Gdsup3+/sup, while the peak induced by high Casup2+/sup was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gdsup3+/sup. On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Casup2+/sup ioscillations/i produced by Gdsup3+/sup are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Casup2+/sup in response to 5mM [Casup2+/sup]subo/sub is mainly due to PLC/IP3 pathway activation.