Aim: 

The G protein-coupled Extracellular Calcium Sensing Receptor (CaSR) is a pleiotropic receptor and, in response to various agonists, it can induce a different intracellular calcium signalling depending on the specific typeof G protein to which is coupled. In this work we studied the signal transduction pathways activated by CaSR in mouse cortical collecting duct cells (MCD4).

Methods: 

MCD4 cells grown on glass cover slips and loaded with Fura-2 AM were used for intracellular [Ca²⁺] measurements in real time video imaging experiments.

Results: 

MCD4 cells were stimulated with three CaSR agonists: [Ca²⁺] 5 mM, Gd³⁺ 300 mM and with the specific allosteric modulator NPS-R 568 (10 mM). The increase in the [Ca²⁺]_o from 1 mM to 5 mM and stimulation with NPS-R 568 induced a rapid peak of [Ca²⁺]_i while stimulation with Gd³⁺ induced transient [Ca²⁺]_i oscillations. U-73122, a PLC inhibitor, completely abolished [Ca²⁺]_i increase after stimulation with the three CaSR agonists. Pretreatment with the permeable Rho inhibitor C3 transferase or with Y27632, selective ROCK inhibitor, blocked only the oscillations induced by Gd³⁺, while the peak induced by [Ca²⁺]_o 5 mM was similar to control. Empting the intracellular calcium stores with Cyclopiazonic acid (CPA), inhibitor of the sarcoplasmic reticulum Ca²⁺-pump, abolished the response to Gd³⁺. Moreover the inhibition of calcium channels with La³⁺ do not alter calcium oscillations.

Conclusion: 

We concluded that in MCD4 cells, CaSR is able to activate two distinct transduction pathways PLC dependents in response to distinct agonists and that the transient [Ca²⁺]_i oscillations produced by Gd³⁺ are due to calcium mobilization from internal stores and are modulated by Rho-Rho kinase signalling.