Ca²⁺ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca²⁺ in permeabilized cells

Abstract

LOW concentrations of inositol 1,4,5-trisphosphate (InsP₃) evoke a very rapid mobilization of intracellular Ca²⁺ stores in many cell types, which can be followed by a further, much slower efflux^1–8. Two explanations have been suggested for this biphasic release. The first proposes that the Ca²⁺ stores vary in their sensitivity to InsP₃, and each store releases either its entire contents or nothing^2,5,7 (all-or-none release); the second proposes instead that the stores are uniformly sensitive to the effects of InsP₃, but that they can release only a fraction of their Ca²⁺ before their sensitivity is somehow attenuated^6,8–11(steady-state release). Experiments using purified InsP₃ receptor molecules reconstituted into lipid vesicles have shown heterogeneity of the receptors in their response to InsP₃ under conditions in which the total Ca²⁺ level at both sides of the receptor is held constant⁷. We now report that in permeabilized A7r5 smooth-muscle cells incubated in Ca²⁺-free medium, the amount of ⁴⁵Ca²⁺ remaining in the stores after the rapid transient phase of release is independent of their initial Ca²⁺ levels, indicating that partially depleted stores are less sensitive to InsP₃. Moreover, if the stores are reloaded with ⁴⁰Ca²⁺ after the first stimulus, reapplication of the same low concentration of InsP₃ will release further ⁴⁵Ca²⁺. This recovery of InsP₃ sensitivity is almost complete. Under these conditions, Ca²⁺ release must thus occur by a steady-state mechanism, in which the decreasing Ca²⁺ content of the stores slows down further release.