Aberrant 3H in Ehrlich mouse ascites tumor cell nucleotides after in vivo labeling with myo-[2-3H]- and L-myo-[1-3H]inositol: implications for measuring inositol phosphate signaling.
After in vivo radiolabeling of Ehrlich cells for 24h with conventional myo-[2-3H]inositol we previously demonstrated an aberrant 3H-labeling of ATP that interfered in the HPLC analysis of inositol trisphosphates. This aberrant 3H-labeling was accounted for by the extensive kidney catabolism of myo-[2-3H] inositol with delivery of 3H-labeled metabolites to extrarenal tissues. As expected, the aberrant labeling of ATP is markedly reduced with the use of 3H-myo-inositol labeled at L-C1 rather than at C2, reflecting that the 3H at L-C1 disappears in the first step of the myo-inositol catabolism: the oxidative conversion to D-glucuronate. In contrast, with the 3H at C2 of myo-inositol, the 3H-C2 passes into the pentose phosphate conversions with resulting labeling of nucleotides. The extent of catabolism to 3H-labeled water, the cellular accumulation of 3H-myo-inositol, the incorporation into cellular inositol phospholipids, and the labeling pattern of cellular phosphoinositides were all found to be similar for the two labeled myo-inositol moieties. With the use of L-myo-[1-3H]inositol an aberrant 3H-labeling at about 25% remained, for which a presumptive mechanism is proposed. L-myo-[1-3H]Inositol appears nevertheless to be a preferable alternative to myo-[2-3H]inositol for tracing the intact myo-inositol molecule after in vivo labeling, with minimized interference from aberrant 3H-labeling of nucleotides.
Christensen
Søren C
SC
Laboratory for Cellular and Molecular Physiology, August Krogh Institute, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen Ø, Denmark. LOSimonsen@aki.ku.dk
Kolbjørn Jensen
Annelie
A
Simonsen
Lars Ole
LO
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D005260:Female; D007294:Inositol / Q000737:chemistry / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D007553:Isotope Labeling; D051379:Mice; D009711:Nucleotides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D014316:Tritium; D014407:Tumor Cells, Cultured
D007295:Inositol Phosphates; D009711:Nucleotides; D014316:Tritium; D007294:Inositol