The Ca2+-sensing receptor in the rabbit cortical thick ascending limb (CTAL) is functionally not coupled to phospholipase C

The recently cloned rabbit kidney Ca2+-sensing receptor (RabCaR) was functionally characterized in microperfused rabbit cortical thick ascending limb (CTAL) segments. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed that this nephron segment contains mRNAs coding for the RabCaR. Elevation of the extracellular Ca2+ concentration ([Ca2+](e)) from 1 to 5 mmol l(-1) induced an increase in the fluorescence emission ratio (R), thus reflecting an increase in intracellular Ca2+ activity ([Ca2+](i)). This increase was inhibited by verapamil, nifedipine and SKF 96365, and potentiated by a previous application of Bay K 8644. Neither verapamil nor Bay K 8644 modified the resting [Ca2+](i). This suggests that the basolateral Ca2+ influx induced by a high [Ca2+](e) occurs via verapamil- and dihydropyridine-sensitive Ca2+ channels, which are not open under resting conditions. In contrast to that evoked by antidiuretic hormone (ADH), the [Ca2+](i) increase induced by a high [Ca2+](e) did not result from an accumulation of inositol phosphates. Neomycin, Gd3+, Mg2+, commonly used agonists of the Ca2+-sensing receptor, did not increase the [Ca2+](i). In the presence of verapamil, ADH still produced a transient [Ca2+](i) increase that was not observed in the presence of an increased [Ca2+](e). These results suggest that the RabCaR in rabbit CTAL cells is not functionally coupled to phospholipase C. In conclusion, the high [Ca2+](e)-induced [Ca2+](i) increase involves verapamil- and dihydropyridine-sensitive Ca2+ channels and is independent of phosphoinositide metabolism. Whether these channels are activated by the RabCaR remains to be elucidated.