Vol. 280, Issue 2, 974-982, 1997

[³H]Inositol Polyphosphate Metabolism in Muscarinic Cholinoceptor-Stimulated Airways Smooth Muscle: Accumulation of [³H]inositol 4,5 Bisphosphate Via a Lithium-Sensitive Inositol Polyphosphate 1-Phosphatase¹

Barbara J. Lynch, Miratul M. K. Muqit, Trevor R. Walker and Edwin R. Chilvers²

Respiratory Medicine Unit, Department of Medicine (RIE), Rayne Laboratory, University of Edinburgh Medical School, Edinburgh, United Kingdom

Agonist-stimulated phosphoinositide hydrolysis is the principal mechanism underlying pharmacomechanical coupling in airways smooth muscle. In bovine tracheal smooth muscle, activation of muscarinic cholinoceptors results in sustained phospholipase C-mediated PtdIns(4,5)P₂ hydrolysis but transient Ins(1,4,5)P₃ accumulation, which implies agonist-stimulated metabolism of Ins(1,4,5)P₃. To investigate the metabolic fate of Ins(1,4,5)P₃ in bovine tracheal smooth muscle, we developed a [³H]inositol-labeling protocol wherein more than 98% of the [³H]inositol polyphosphates that accumulated over a 0 to 30-min incubation with 100 µM carbachol in the presence of 5 mM LiCl were derived from [³H]Ins(1,4,5)P₃ and wherein the Ins(1,4,5)P₃ 3-kinase (EC 2.7.1.127) and 5-phosphatase (EC 3.1.3.56) pathways generated a set of mutually exclusive [³H]inositol polyphosphate isomers. Under these conditions, the 5-phosphatase pathway was shown to be the dominant route for [³H]Ins(1,4,5)P₃ metabolism at all time intervals measured, especially at early times (0-300 sec), where it accounted for more than 85% of [³H]Ins(1,4,5)P₃ metabolism. We also observed accumulation of a novel agonist and LiCl-sensitive [³H]InsP₂ isomer identified as [³H]Ins(4,5)P₂. The presence of a LiCl-sensitive inositol polyphosphate 1-phosphatase (EC 3.1.3.57) was demonstrated, and high LiCl concentrations (30 mM) caused a significant enhancement of [³H]Ins(1,4)P₂ accumulation and a corresponding decline in [³H]Ins4P levels. Because nearly identical bell-shaped LiCl concentration-response curves were obtained for [³H]Ins4P and [³H]Ins(4,5)P₂ accumulation, and [³H]Ins(4,5)P₂ was not generated under conditions expected to stimulate phospholipase D, these data suggest that the most likely precurser of [³H]Ins(4,5)P₂ is [³H]Ins(1,4,5)P₃. This is the first demonstration of Ins(4,5)P₂ accumulation in a non-neuronal cell type, and the foregoing data suggest a novel route of formation via an Ins(1,4,5)P₃ 1-phosphatase, which would represent an additional pathway for [³H]Ins(1,4,5)P₃ removal.