Evidence for the existence of calcium signaling domains in a renal cortical collecting duct cell line

Abstract

The epithelial sodium channel of the kidney (ENaC) is inhibited by activation of the purinergic P2Y2 receptor in the apical membrane but stimulated by basolateral activation of the P2X4 receptor. We hypothesized that regional regulation of [Ca2+]i is responsible for the polarized effects of ATP on ENaC function. To test this, we analyzed [Ca2+]i responses downstream of purinergic agonists in renal A6 cells. Apically, the P2Y2 agonist UTP caused a rapid, transient increase (15–60 sec) in [Ca2+]i observed by loading cells with fluo4‐AM. The P2X4 receptor agonist 2‐meSATP applied basolaterally significantly increased the open probability of ENaC, an effect which was abolished by pretreatment with the Ca2+chelator EDTA (2 mM). In other epithelial cell types, mitochondria form barriers that compartmentalize Ca2+. To test whether such a barrier exists in renal A6 cells, we loaded cells with MitoTracker Red to visualize mitochondria. In Z‐axis sections, we observed strong bands of mitochondria both apically and basolaterally. Our results suggest that the differing effects of apical vs basolateral ATP on ENaC occur through activation of different purinergic receptors, both of which cause increases in [Ca2+]i but that the Ca2+ is confined to two separate compartments, producing differing effects. This work was supported by NIH grants R37‐DK037963 and T32‐DK07656.