Differential modulation of intracellular Ca2+ responses associated with calcium-sensing receptor activation in renal collecting duct cells

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 [Ca2+] measurements performed with real time video imaging revealed that CaR stimulation with 5mM Ca2+, 300 mu M Gd3+ and with 10 mu M of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca2+](i) although displaying different features. Specifically, Ca2+ as well as stimulation with NPS-R 568 induced a rapid peak of [Ca2+](i) while stimulation with Gd3+ induced transient intracellular Ca2+ oscillations. PLC inhibition completely abolished any [Ca2+](i) increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca2+](i) oscillations induced by Gd3+, while the peak induced by high Ca2+ was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd3+. 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 Ca2+ oscillations produced by Gd3+ are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca2+ in response to 5mM [Ca2+](o) is mainly due to PLC/IP3 pathway activation.