The inositol phosphates in WRK1 rat mammary tumour cells

1. A detailed structural survey has been made of the inositol phosphates of unstimulated and vasopressin-stimulated WRK-1 rat mammary tumour cells. Inositol phosphate peaks were separated by h.p.l.c., and structural assignments were made for more than 20 compounds by combinations of: (a) co-chromatography with labelled standards. (b) site-specific enzymic dephosphorylation; (c) complete and partial periodate oxidation. followed by h.p.l.c. of polyols and their stereospecific oxidation by dehydrogenases; and (d) ammoniacal hydrolysis. 2. The 'inositol monophosphates' fraction from unstimulated cells included an uncharacterized peak, probably containing some glycerophosphoinositol, and Ins(1:2-cyclic)P. Stimulation provoked accumulation of both Ins1P and Ins3P, of Ins2P, and of Ins5P and/or the enantiomers Ins4P and Ins6P. The proportions of Ins1P and Ins3P were determined by partial periodate oxidation and enantiomeric identification of the resulting glucitols. 3. Three inositol bisphosphate peaks were detected in unstimulated cells: Ins(1,4)P₂ [this was distinguished chemically from its enantiomer Ins(3,6)P₂], Ins(3,4)P₂ and/or Ins(1,6)P₂, and Ins(4,5)P₂ and/or Ins(5,6)P₂. On stimulation, Ins(1,4)P₂ and Ins(3,4)P₂ [and/or Ins(1,6)P₂] levels increased, and Ins(1:2-cyclic,4)P₂ and Ins(1,3)P₂ were also formed. 4. Three inositol trisphosphate peaks were obtained from unstimulated cells: all increased during stimulation. These were Ins(1,3,4)P₃ [with some Ins(1:2-cyclic,4,5)P₃], Ins(1,4,5)P₃ and Ins(3,4,5)P₃ [and/or Ins(1,5,6)P₃]. During stimulation, another compound, probably Ins(1,4,6)P₃, appeared in the 'Ins(1,4,5)P₃ peak'. The 'Ins(3,4,5)P₃ peak' contained a second trisphosphate, probably Ins(2,4,5)P₃. 5. Three inositol tetrakisphosphates, namely Ins(1,3,4,6)P₄, Ins(1,3,4,5)P₄ and Ins(3.4,5,6)P₄, were present in unstimulated cells, and all accumulated during stimulation. 6. Ins(1,3,4,5,6)P₅, which is the most abundant inositol polyphosphate in these cells, a less abundant inositol pentakisphosphate and inositol hexakisphosphate were all unresponsive to stimulation.