Open AccessEffect of the Aminosteroid, U73122, on Ca 2+ Uptake and Release Properties of Rat Liver Microsomes
Author(s) -
Moel Mariëlle P.,
Put Frans H. M. M.,
Vermegen Tino M. J. A.,
Pont JanJoep H. H. M.,
Willems Peter H. G. M.
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.626_b.x
Subject(s) - thapsigargin , endoplasmic reticulum , phospholipase c , ic50 , biophysics , intracellular , inositol , microsome , atpase , endocrinology , chemistry , biology , medicine , biochemistry , signal transduction , receptor , in vitro , enzyme
The putative phospholipase C inhibitor, U73122, transiently increases the cytosolic free Ca 2+ concentration in rabbit pancreatic acinar cells by stimulating the release of Ca 2+ from intracellular stores [Willems, Van de Put, Engbersen, Bosch, Van Hoof & De Pont (1994) Pflügers Arch, 427 , 233–243]. In order to elucidate the exact mechanism of action of U73122 we studied its effects on both Ca 2+ ‐stimulated Mg 2+ ‐dependent ATPase activity and Ca 2+ ‐stimulated ATP‐dependent Ca 2+ uptake in rat liver microsomes. In addition, we studied its effects on Ca 2+ release from steady‐state loaded microsomes. The effects of U73122 were compared with those of thimerosal, described in the literature as inhibiting Ca 2+ ‐ATPases and sensitizing inositol 1,4,5‐trisphosphate‐operated Ca 2+ release channels, and thapsigargin, a specific inhibitor of sarcoplasmic and endoplasmic reticulum Ca 2+ ‐ATPases. Both U73122 (IC 50 = 9 μM) and thimerosal (IC 50 = 11 μM) dose‐dependently inhibited Ca 2+ ‐stimulated Mg 2+ ‐dependent ATPase activity, without significantly affecting Mg 2+ ‐stimulated ATPase activity. Similarly, both U73122 (IC 50 = 9 μM) and thimerosal (IC 50 = 14 μM) dose‐dependently inhibited ATP‐dependent Ca 2+ uptake. At concentrations beyond 20 μM, U73122 stimulated Ca 2+ release from steady‐state loaded microsomes at a rate considerably higher than obtained with a maximally inhibitory concentration of thapsigargin (1 μM). This observation, which was not reached with equally inhibitory concentrations of thimerosal, demonstrates that higher U73122 concentrations cause an additional increase of the passive Ca 2+ leak. The data presented demonstrate that U73122 stimulates the release of actively stored Ca 2+ primarily through inhibition of the internal Ca 2+ pump.