NA/CA EXCHANGE IN THE BASOLATERAL MEMBRANE OF THE A6 CELL MONOLAYER - ROLE IN CA-I HOMEOSTASIS

The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Ca-i) fluctuations and the Ca-45 fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na+ from the medium produced a transient increase in Ca-i followed by a regulatory phase returning Ca-i to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent K-m of approximately 5 mM Na+). The Ca-i increase was only found with the Na+-free medium on the basolateral side of the cell monolayer. A twofold increase in the Ca-45 influx was observed under these conditions. In Ca2+- depleted cells, the initial Ca-i increase after Ca2+ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na+-free medium). Ca-45 effluxes through the blm of the monolayer were greatly and transiently increased by a Na+-free medium on the serosal side and blocked by orthovanadate (1 mM). The Ca-i increase induced by a hypo-osmotic shock was greater in cells bathed in a Na+-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Ca-i homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca2+ pump also located on the blm and Ca2+ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Cai homeostasis.