Determination of mass changes in phosphatidylinositol 4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle.
Stimulation of muscarinic receptors in bovine tracheal smooth muscle (BTSM) causes a sustained increase in muscle tone, but a transient increase in the second messenger Ins(1,4,5)P3. To examine whether this brief increase in Ins(1,4,5)P3 mass results from transient formation or is due to agonist-stimulation of Ins(1,4,5)P3 metabolism, we have studied the relationship between mass changes in PtdIns(4,5)P2 and Ins(1,4,5)P3 accumulation, and changes in [3H]InsP3, [3H]PtdIns, [3H]PtdInsP1 and [3H]PtdInsP2 in carbachol-stimulated myo-[3H]inositol-prelabelled BTSM slices. Carbachol (0.1 mM) caused a rapid transient increase in Ins(1,4,5)P3 concentration (basal, 12.9 +/- 0.8 pmol/mg of protein; 5 s carbachol treatment, 27.1 +/- 1.5 pmol/mg of protein), with values returning to basal levels by 30 s, but a sustained accumulation of total [3H]InsP3s, with [3H]Ins(1,3,4)P3 being the predominant isomer present at later time points. In contrast, PtdIns(4,5)P2 mass, determined by radioreceptor assay of Ins(1,4,5)P3 in desalted alkaline hydrolysates of acidified chloroform/methanol tissue extracts, declined rapidly (basal, 941 +/- 22 pmol/mg of protein; 120 s carbachol, 365 +/- 22 pmol/mg of protein; t1/2 14 s) and remained at this new steady-state level for at least 20 min in the continued presence of carbachol. Addition of 10 microM-atropine 2 min after carbachol caused a prompt return of PtdIns(4,5)P2 concentration to prestimulated values (t1/2 210 s). Ongoing resynthesis of PtdIns(4,5)P2 after carbachol stimulation was demonstrated in [3H]inositol-labelled tissue by observing a persistent increase in the specific radioactivity of [3H]PtdInsP2, shown to be exclusively [3H]PtdIns(4,5)P2, over a 10 min period. These findings strongly suggest the occurrence of persistent receptor-mediated increases in PtdIns(4,5)P2 hydrolysis and Ins(1,4,5)P3 formation which, in conjunction with the transient accumulation of Ins(1,4,5)P3 observed, provide evidence that regulation of the metabolism of Ins(1,4,5)P3 is a major determinant of Ins(1,4,5)P3 concentration in this tissue under agonist-stimulated conditions.
Chilvers
E R
ER
Department of Pharmacology and Therapeutics, University of Leicester, U.K.
Batty
I H
IH
Challiss
R A
RA
Barnes
P J
PJ
Nahorski
S R
SR
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014132:Trachea / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium
D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D014316:Tritium; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol