[H-3] INOSITOL POLYPHOSPHATE METABOLISM IN MUSCARINIC CHOLINOCEPTOR-STIMULATED AIRWAYS SMOOTH-MUSCLE - ACCUMULATION OF [H-3]INOSITOL-4,5 BISPHOSPHATE VIA A LITHIUM-SENSITIVE INOSITOL POLYPHOSPHATE 1-PHOSPHATASE

Agonist-stimulated phosphoinositide hydrolysis is the principal mechan ism underlying pharmacomechanical coupling in airways smooth muscle. I n bovine tracheal smooth muscle, activation of muscarinic cholinocepto rs results in sustained phospholipase C-mediated PtdIns(4,5)P-2 hydrol ysis but transient Ins(1,4,5)P-3 accumulation, which implies agonist-s timulated metabolism of Ins(1,4,5)P-3. To investigate the metabolic fa te of Ins(1,4,5)P-3 in bovine tracheal smooth muscle, we developed a [ H-3]inositol-labeling protocol wherein more than 98% of the [H-3]inosi tol polyphosphates that accumulated over a 0 to 30-min incubation with 100 mu M carbachol in the presence of 5 mM LiCl were derived from [H- 3]Ins(1,4,5)P-3 and wherein the Ins(1,4,5)P-3 3-kinase (EC 2.7.1.127) and 5-phosphatase (EC 3.1.3.56) pathways generated a set of mutually e xclusive [H-3]inositol polyphosphate isomers. Under these conditions, the 5-phosphatase pathway was shown to be the dominant route for [H-3] Ins(1,4,5)P-3 metabolism at all time intervals measured, especially at early times (0-300 sec), where it accounted for more than 85% of [H-3 ]Ins(1,4,5)P-3 metabolism. We also observed accumulation of a novel ag onist and LiCl-sensitive [H-3]InsP(2) isomer identified as [H-3]Ins(4, 5)P-2. The presence of a LiCl-sensitive inositol polyphosphate 1-phosp hatase (EC 3.1.3.57) was demonstrated, and high LiCl concentrations (3 0 mM) caused a significant enhancement of [H-3]Ins(1,4)P-2 accumulatio n and a corresponding decline in [H-3]Ins4P levels. Because nearly ide ntical bell-shaped LiCl concentration-response curves were obtained fo r [H-3]Ins4P and [H-3]Ins(4,5)P-2 accumulation, and [H-3]Ins(4,5)P-2 w as not generated under conditions expected to stimulate phospholipase D, these data suggest that the most likely precurser of [H-3]Ins(4,5)P -2 is [H-3]Ins(1,4,5)P-3. This is the first demonstration of lns(4,5)P -2 accumulation in a non-neuronal cell type, and the foregoing data su ggest a novel route of formation via an lns(1,4,5)P-3 1-phosphatase, w hich would represent an additional pathway for [H-3]Ins(1,4,5)P-3 remo val.