Calcium control on InsP3-induced discharge of calcium from permeabilised hepatocyte pools

The control exerted by intralumenal and cytosolic Ca2+ on InsP3-induced release of Ca2+ from intracellular Ca2+ pools in suspensions of saponin-permeabilised rat hepatocytes was investigated by combined Quin-2 and 45Ca2+ measurements at 20 degrees C. We failed to detect a major effect of intralumenal Ca2+ in regulating this release, as various manipulations in which the load of the Ca2+ pools was varied by a factor of two did not significantly affect the apparent relative efficiency of InsP3 in releasing Ca2+; these manipulations included loading the Ca2+ pools up to various steady state levels by preliminary equilibration at various external free Ca2+ concentrations, as well as emptying them progressively through the blockade of pump-mediated Ca2+ uptake. As regards Ca2+ on the cytosolic side, in contrast with recent results obtained with other systems, we found that, at maximal doses, InsP3-induced Ca2+ release was not stimulated by raising Ca2+ from very low to submicromolar or micromolar concentrations, and that only relatively high concentrations of free Ca2+ inhibited this release (half-maximal inhibition was between 3 and 15 microM). Such elevated Ca2+ concentrations reduced the size of the InsP3-sensitive Ca2+ pool. We also noted that the apparent cooperativity of InsP3 activation of release at pCa 5 was noticeably less than that observed at pCa 7. As a result, at low InsP3 concentrations, a rise in cytosolic Ca2+ from pCa 7 to pCa 5 stimulated InsP3-mediated Ca2+ release. These results are discussed in the context of the current speculations about tissue specificity, heterogeneity, quantal release, oscillations, and the several different mechanisms that may control InsP3-induced Ca2+ release.