Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.
The identity of 3H-labelled material ascribed to Ins(1,4,5)P3 in resting or bombesin-stimulated myo-[3H]inositol-labelled AR4-2J cells was investigated by determining its ability to serve as substrate for partially purified Ins(1,4,5)P3/Ins(1,3,4,5)-P4 5-phosphatase and Ins(1,4,5)P3 3-kinase. This 3H-labelled material was metabolized by these two enzymes at rates which were indistinguishable from those for an internal [32P]Ins(1,4,5)P3 standard, establishing its identity as authentic Ins(1,4,5)P3. In addition, and in contrast with findings in earlier studies utilizing substance P as an agonist, prolonged stimulation with bombesin resulted in an increase in an InsP4 which was degraded by Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. These findings serve to confirm the previous estimate of Horstman, Takemura & Putney [(1988) J. Biol. Chem. 263, 15297-15303] for the intracellular concentrations of Ins(1,4,5)P3 in resting (2 microM) and agonist-stimulated (25 microM) AR4-2J cells. The implications of these findings for the physiological regulation of intracellular Ca2+ through this intracellular messenger are discussed.
Nogimori
K
K
Calcium Regulation Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.
Menniti
F S
FS
Putney
J W
JW
D001839:Bombesin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D010190:Pancreatic Neoplasms / Q000032:analysis*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D015544:Inositol 1,4,5-Trisphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; C035215:inositol triphosphate phosphomonoesterase; D010744:Phosphoric Monoester Hydrolases; D001839:Bombesin