Regulation and identity of intracellular calcium stores involved in membrane cross talk in the early distal tubule of the frog kidney.

Abstract

The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na(+) load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca(2+), originating from an intracellular Ca(2+) store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243-247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca(2+) storage pools in the renal diluting segment. Store Ca(2+) movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca(2+) was required to maintain store Ca(2+) content. Removal of either of these substrates resulted in a passive leak of Ca(2+) from the stores. The uptake of Ca(2+) into the store was sensitive to the SERCA inhibitor 2,5-di(tert-butyl) hydroquinone, whereas Ca(2+) release from the store was stimulated by IP(3) but not cADPR. Store Ca(2+) was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized Deltapsi(m), the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca(2+) following exposure to IP(3). These results suggest that the endoplasmic reticulum is a dominant Ca(2+) store in the frog EDT. A second pool, sensitive to ionomycin but not IP(3), may overlap with the IP(3)-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca(2+) cycling.