Formation and metabolism of [3H]inositol phosphates in AR42J pancreatoma cells. Substance P-induced Ca2+ mobilization in the apparent absence of inositol 1,4,5-trisphosphate 3-kinase activity.
After 2 days of incubation of AR42J pancreatoma cells with 400 microM [3H]inositol, the specific radioactivity of [3H]phosphatidylinositol 4,5-bisphosphate and the specific radioactivity of [3H]inositol were similar, indicating that isotopic equilibrium had been achieved. The inositol 1,4,5-trisphosphate (1,4,5-IP3) level in cells was estimated to be approximately 2 microM and was increased by substance P receptor activation to about 25 microM. HPLC analysis of [3H]inositol phosphates indicated that only 1,4,5-IP3, inositol 1,4-bisphosphate, and inositol 4-monophosphate were increased upon receptor activation. There was no increase in inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4), or in any of its metabolites. Incubation of [3H]1,4,5-IP3 with a cell homogenate did not result in the formation of [3H]1,3,4,5-IP4. Therefore, it appears that 1,4,5-IP3 3-kinase is either not present or not functional under these assay conditions. Substance P increased cytosolic calcium levels in fura-2-loaded cells from about 600 nM to 2.5 microM. This increase in Ca2+ was partially attenuated in the absence of extracellular calcium, indicating that in AR42J cells, substance P stimulation appears to activate calcium signaling through both Ca2+ entry and intracellular Ca2+ release. These modes of Ca2+ mobilization occur without an increase in 1,3,4,5-IP4 or any of its metabolites.
Horstman
D A
DA
Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Takemura
H
H
Putney
J W
JW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D007295:Inositol Phosphates / Q000378:metabolism*; D007811:Lanthanum / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013373:Substance P / Q000494:pharmacology*; D013403:Sugar Phosphates / Q000378:metabolism*; D013997:Time Factors
D007295:Inositol Phosphates; D013403:Sugar Phosphates; C028521:lanthanum chloride; D013373:Substance P; D007811:Lanthanum; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium