Metabolism of inositol bis-, tris-, tetrakis- and pentakis-phosphates in GH3 cells

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Abstract
Previous studies demonstrated a multiplicity of isomers of inositol phosphates in GH3 rat pituitary tumour cells. In order to determine their origin, we have investigated the metabolism of radiolabelled inositol phosphates (IPn) in GH3 cell homogenates by using h.p.l.c. I(1,4,5)P3 is either phosphorylated to I(1,3,4,5)P4 (in the presence of ATP) or dephosphorylated to I(1,4)P2 (in the absence of ATP). I(1,4)P2 is dephosphorylated to I(4)P (> 95%). I(1,3,4,5)P4 hydrolysis yields two products. By using dual-labelled [32P,3H]I(1,3,4,5)P4 with 32P in either the 3 or the 4, 5 position, we have identified the probable configuration of these isomers. The predominant (> 97%) IP3 formed is I(1,3,4)P3, with a minor I(1,4,5)P3 peak. Subsequent I(1,3,4)P3 hydrolysis yields two IP2 isomers [the major (.apprxeq. 85%) is I(3,4)P2; the minor (.apprxeq. 15%) is I(1,3)P2] and two IP isomers {the major (.apprxeq. 90% is I(3)P [L-I(1)P], the minor I(4)P}. IP5 is very slowly dephosphorylated to an IP4 of undetermined isomeric configuration. We have also examined GH3 cell lipids for the presence of phosphoinositides either more highly phoshorylated than PIP2 (as potential sources of the IP4/IP5 and IP6 in these cells) or phosphorylated in positions other than 1, 4 and 5, and have been unable to find evidence of either. These data suggest two main routes of metabolism for I(1,4,5)P3 in GH3 cells: either (1) phosphorylation to I(1,3,4,5)P4, and the subsequent consecutive dephosphorylation to I(1,3,4)P3, I(3,4)P2 and finally L-I(1)P [D-I(3)P]; or (2) dephosphorylation to I(1,4)P2 and, subsequently, I(4)P.