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

Abstract

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⁺, 300µM Gd³⁺ and with 10µM 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.