Luminal communication between intracellular calcium stores modulated by GTP and the cytoskeleton

The activation properties of inositol 1,4,5-trisphosphate (InsP₃)- sensitive Ca²⁺ stores in permeabilized and intact hepatocytes were investigated by monitoring Mn²⁺ quench of fura-2 compartmentalized within these stores, as Mn²⁺ passed through InsP₃-activated channels in a retrograde manner. In cells permeabilized in suspension the InsP₃-sensitive pool size was dependent on InsP₃ dose, and there was a large unresponsive compartment. By contrast, essentially all of the compartmentalized dye was accessible following activation of a small fraction of the InsP₃ receptors in carefully permeabilized attached cells. After loading the cytosol of intact hepatocytes with Mn²⁺, both submaximal and maximal vasopressin doses caused complete quench of the entire intracellular pool of compartmentalized fura-2. Vasopressin-induced Mn²⁺ quench occurred in a stepwise manner at doses that gave cytosolic Ca²⁺ oscillations, reflecting periodic opening of intracellular Ca²⁺ channels. Pretreatment with thapsigargin to eliminate feedback effects of Ca²⁺ fluxes converted the steps to a continuous quench. The data suggest that Ca²⁺ stores in attached permeabilized and intact hepatocytes are luminally connected, making the entire store accessible to InsP₃. In cells permeabilized in suspension, GTP increased InsP₃-sensitive pool size, and this effect was inhibited by cytochalasin B. GTP did not change the initial rate of Mn²⁺ quench but increased the proportion of slowly accessible stores in the InsP₃-sensitive compartment, apparently by recruitment of InsP₃-insensitive stores. Preincubation on ice or with cytoskeletal inhibitors dissociated slowly accessible compartments from the InsP₃-sensitive stores in both intact and subsequently permeabilized attached hepatocytes. Addition of GTP to permeabilized cells reversed this disruption of store continuity. It is suggested that GTP- and cytoskeleton- dependent luminal communication between Ca²⁺-stores is an important determinant of function, which could modulate the availability of Ca²⁺ for release.