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The extracellular calcium-sensing receptor (CaSR) located in either luminal or basolateral cell membranes of various types of renal tubules including proximal tubules, Henle's loop and collecting ducts has been thought to play a fundamental role in electrolyte metabolism. To further identify the physiological roles of the CaSR, we examined the effects of Ca(2+) and calcimimetics neomycin (Neo), gentamicin and gadolinium chloride (Gd(3+)) on the intracellular pH (pHi) of in vitro microperfused mouse medullary thick ascending limb (mTAL) cells of Henle's loop, by loading the cells with fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and measuring the ratio of fluorescence emission at 530 nm after exciting the dye at 490 and 440 nm. In a steady-state condition in Hepes-buffered solution, the pHi in the mTALs was 7.29 +/- 0.04 (n = 9). A concentration of 200 micromol/l Neo in the basolateral side decreased the pHi after 1 min by -0.13 +/- 0.02 (n = 34, p &lt; 0.0001). The other calcimimetics showed similar effects on pHi, whereas none of these calcimimetics in the lumen affected pHi. Na(+) removal or the inhibition of Na(+) and proton transport with amiloride, bumetanide, or bafilomycin did not eliminate the effect of Neo on pHi. On the other hand, Cl(-) removal clearly eliminated the Neo-induced pHi decrease (-0.06 +/- 0.01 vs -0.00 +/- 0.05 in Cl(-) removal, n = 4, p &lt; 0.003). Thus, we have demonstrated for the first time that the CaSR is involved in the regulation of the pHi in the mTAL and requires Cl(-) to exert its effect.

Chloride-Dependent Intracellular pH Regulation via Extracellular Calcium-Sensing Receptor in the Medullary Thick Ascending Limb of the Mouse Kidney