TY - JOUR
T1 - Calcium mobilization in permeabilized fibroblasts
T2 - Effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate
AU - Muldoon, Leslie L.
AU - Jamieson, Gordon A.
AU - Villereal, Mitchel L.
PY - 1987/1
Y1 - 1987/1
N2 - Utilizing a digitonin‐permeabilized cell system, we have studied the release of calcium from a non‐mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (±6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3‐sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP‐dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protrein kinase C activator 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down‐regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.
AB - Utilizing a digitonin‐permeabilized cell system, we have studied the release of calcium from a non‐mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (±6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3‐sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP‐dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protrein kinase C activator 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down‐regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.
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U2 - 10.1002/jcp.1041300106
DO - 10.1002/jcp.1041300106
M3 - Article
C2 - 3492499
AN - SCOPUS:0023135769
SN - 0021-9541
VL - 130
SP - 29
EP - 36
JO - Journal of Cellular Physiology
JF - Journal of Cellular Physiology
IS - 1
ER -