Inositol phosphates in rat atria and the importance of the extraction procedure.
The nature of the inositol phosphates present in adult rat left atria have been examined. Trichloroacetic acid extracts of [3H] inositol-labelled left atria contained the 1 (or 3)-, 2- and 4-isomers of inositol monophosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-trisphosphate. Addition of noradrenaline increased all of these isomers. However, even in the presence of noradrenaline, there was no evidence for the presence of inositol-1,3,4,5-tetrakisphosphate or its dephosphorylation products including inositol-1,3,4-trisphosphate and the 1,3- and 3,4-isomers of inositol bisphosphate. Total accumulation of inositol phosphates in the presence of LiCl was 98% accounted for by dephosphorylation of inositol-1,4,5-trisphosphate and its cyclic isomer. These data indicate that, in intact left atria, metabolism of inositol-1,4,5-trisphosphate is by dephosphorylation and that significant activity of the phosphorylation pathway is not present. When extractions were performed using either acidified chloroform-methanol or perchloric acid, other [3H] inositol-labelled compounds were detected in atrial extracts. One of these chromatographed with ATP and might be mistaken for inositol-1,3,4-trisphosphate if co-chromatography with ATP was used to identify that compound. Another compound had a similar, but not identical chromatographic profile to inositol-1,3,4,5-tetrakisphosphate and is thus likely to be mistaken for that compound. In addition, perchloric acid extraction caused phosphate group migration generating extra isomers of the inositol phosphates and other unidentified [3H]-labelled compounds. Such extraction artifacts are likely especially problematic in studies of intact heart tissue because of the time required to effectively homogenize the tissue. These findings demonstrate the need for caution in interpreting data relating to estimations of inositol phosphates in intact heart tissue.
Baker Medical Research Institute, Prahran, Victoria, Australia.
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EA
Woodcock
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KE
Anderson
D000818:Animals; D016477:Artifacts; D002725:Chloroform; D006325:Heart Atria / Q000737:chemistry* / Q000187:drug effects; D007295:Inositol Phosphates / Q000302:isolation & purification*; D000432:Methanol; D009638:Norepinephrine / Q000494:pharmacology; D010472:Perchlorates; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014238:Trichloroacetic Acid
D007295:Inositol Phosphates; D010472:Perchlorates; D014238:Trichloroacetic Acid; D002725:Chloroform; D009638:Norepinephrine; D000432:Methanol