Abstract
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 formationvia an Ins(1,4,5)P3 1-phosphatase, which would represent an additional pathway for [3H]Ins(1,4,5)P3 removal.
Footnotes
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Send reprint requests to: Dr. Edwin R. Chilvers, Respiratory Medicine Unit, Department of Medicine (RIE), Rayne Laboratory, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, U.K.
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↵1 This work was supported by the Chest, Heart and Stroke Association (UK) and National Asthma Campaign. E.R.C. is a Wellcome Trust Senior Research Fellow in Clinical Science. M.M. was the recipient of a British Pharmacological Society intercalated award.
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↵2 Author for correspondence.
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↵3 Personal communication, Dr. R. A. J. Challiss, University of Leicester.
- Abbreviations:
- BTSM
- bovine tracheal smooth muscle
- ASM
- airways smooth muscle
- CCh
- carbachol
- PLC
- phospholipase C
- Ins(P)Px
- inositol (poly)phosphate isomer
- InsP1–6
- D-myo-inositol mono-, bis-, tris-, tetra-, pentakis- and hexakisphosphates with positional isomers, where specified, given in parentheses (e.g., Ins(4,5)P2 represents inositol 4,5-bisphosphate)
- TCA
- trichloroacetic acid
- KH buffer
- Krebs-Henseleit buffer
- HPLC
- high-performance liquid chromatography
- EC50
- drug concentration that causes half-maximal effect
- PKC
- protein kinase C
- Received August 7, 1996.
- Accepted October 21, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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