[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-phosphatase.
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 microM 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 [H]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 LiCI-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 [H]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 [H]Ins(1,4,5)P3 removal.
Lynch
B J
BJ
Respiratory Medicine Unit, Department of Medicine (RIE), University of Edinburgh Medical School, United Kingdom.
Muqit
M M
MM
Walker
T R
TR
Chilvers
E R
ER
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002417:Cattle; D002474:Cell-Free System; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D018021:Lithium Chloride / Q000494:pharmacology*; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000502:physiology*; D014132:Trachea / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium
D007295:Inositol Phosphates; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D014316:Tritium; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; C066454:inositol 4,5-bisphosphate; D010744:Phosphoric Monoester Hydrolases; C053087:inositol-1,4-bisphosphate 1-phosphatase; D018021:Lithium Chloride