Ischemia is associated with a loss of cytosolic
calcium homeostasis. Intracellular stores, particularly in endoplasmic reticulum, are critical for the maintenance of
calcium homeostasis. Recent studies have shown that
ischemia significantly inhibited microsomal
calcium uptake mediated by Mg(2+)/Ca(2+)
ATPase, the major mechanism of endoplasmic reticulum
calcium sequestration. This study was initiated to determine whether the decreased
calcium uptake caused by
ischemia was the result of inhibition of Mg(2+)/Ca(2+)
ATPase activity or an uncoupling of
calcium uptake from
ATP hydrolysis. The microsomal Mg(2+)/Ca(2+)
ATPase specific inhibitor
thapsigargin partially inhibited
ATPase activity and completely inhibited
calcium uptake.
ATPase inhibited by
thapsigargin was considered microsomal Mg(2+)/Ca(2+)
ATPase.
Ischemia from 5 to 60 min had no significant effect on
thapsigargin sensitive
ATPase activity. However, under identical conditions, increasing
ischemia from 5 to 60 min significantly inhibited microsomal
calcium uptake. Comparing
calcium uptake to
ATP hydrolysis as
ischemia increased from 5 to 60 min revealed that the coupling ratio of
calcium molecules sequestered to
ATP molecules hydrolyzed became significantly decreased. The results demonstrated that the effect of
ischemia on microsomal
calcium uptake was mediated by an uncoupling of
calcium transport from Mg(2+)/Ca(2+)
ATPase activity.