Detection of sustained mass increases in inositol 1,3,4,5-tetrakisphosphate in agonist-stimulated airway smooth muscle.

Abstract

The initial contractile response in airway smooth muscle (ASM) to spasmogen agonists occurs independently to changes in membrane potential and appears to involve receptor-mediated hydrolysis of PtdIns(4,5)P2 with Ins(1,4,5)P3 formation and consequent release of Ca2+ from internal stores [I]. Previous studies in bovine tracheal smooth muscle (BTSM) have indicated that, despite initiating a sustained contractile response, the muscarinic receptor agonist carbachol causes a transient accumulation of Ins(1,4,5)P, despite evidence, albeit indirect, for ongoing PtdIns(4,5)P2 hydrolysis [2]. Furthermore, studies in [ 3H]inositol pre-labelled BTSM have failed to identify any significant early accumulation of [3H]InsP, in response to carbachol [3]. We report the use of novel radioreceptor assays to allow quantitation of changes in Ins(1,3,4,5)P4 and PtdIns(4,5)P2 mass in response to agonist stimulation. Incubations using BTSM slices were performed as described previously [2,3]. Ins(1,3,4,5)P4 and Ins(1,4,5)P3 mass were determined in neutralized acid-extracts as in [4,5]. ified by measurement of Ins(1,4,5)P3 [4] in desalted, alkaline hydrolysates (1 M KOH, 100°C, 30 min) of acidified ch1oroform:methanol extracts from acid precipitated tissue pellets. This procedure converts PtdIns(4,5)P2 to Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 in an experimentally determined fixed molar ratio of 66:20:14. The effect of carbachol (0.1 mM) on Ins(1,4,5)P3, Ins(1,3,4,5)P4 and PtdIns(4,5)P2 mass in BTSM is shown in Fig. 1. The addition of atropine 2 min after agonist addition caused a prompt return of PtdIns(4,5)P concentration to pre-stimulation values. Although preliminary experiments using [ 3H] inositol pre-labelled slices have identified [ 'H]Ins(1,3,4,6)P4 as the major ['HJInsP, accumulating under conditions of prolonged (30 min) maximal carbachol stimulation (data not shown), the specificity of the I11s(1,3~~,5)P, mass assay [51 (IC5,, for displacement of [ 1ns(1,3,4,5)P4: 3 nM; and by Ins(1,3,4,6)P4: 800 nM) indicates that the presence of Ins(1,3,4,6)P4 in BTSM extracts would not significantly influence the values for Ins(1,3,4,5)P4 concentration presented here. Likewise, although basal and carbachol-stimulated Ins(1,4,5)P3 concentrations are approx. 11and 22fold higher respectively than Ins(1,3,4,5)P4 (Fig. l), Ins(1,4,5/P affinity for the [ [5] and hence does not affect Ins(1,3,4,5)P4 values. These data represent the first report of mass measurements of Ins(1,3,4,5)P in ASM. The rapid and sustained carbachol-stimulated accumulation of Ins(1,3,4,5)P4, together with the ongoing, receptordependent decrease in PtdIns(4,5)P2 mass is further evidence suggesting that muscarinic-receptor stimulation in BTSM results in persistent phosphoinositidase C-mediated PtdIns(4,5)P2 hydrolysis, with the PtdIns(4,5)P2 was quant-