TY - JOUR
T1 - Luminal communication between intracellular calcium stores modulated by GTP and the cytoskeleton
AU - Hajnóczky, György
AU - Lin, Chi
AU - Thomas, Andrew P.
N1 - Copyright:
Copyright 2005 Elsevier B.V., All rights reserved.
PY - 1994/4/8
Y1 - 1994/4/8
N2 - 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.
AB - 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.
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M3 - Article
C2 - 8144609
AN - SCOPUS:0028264160
SN - 0021-9258
VL - 269
SP - 10280
EP - 10287
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -