title: Luminal communication between intracellular calcium stores modulated by GTP and the cytoskeleton.
abstract: 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.
authors.affiliation: Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: G
authors.initials: G
authors.lastname: Hajnóczky
authors.forename: C
authors.initials: C
authors.lastname: Lin
authors.forename: A P
authors.initials: AP
authors.lastname: Thomas
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D016257:Fura-2; D006160:Guanosine Triphosphate / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008345:Manganese; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D008345:Manganese; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D002118:Calcium; D016257:Fura-2