Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of 3H]inositol into the phosphoinositides and the formation of 3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with 3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of 3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of 3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on 3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of 3H]inositol and 3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on 3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer Ca2+] increased the incorporation of 3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the 3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the 3H]InsP response completely. Labelling muscle slices with 3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated 3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the 3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on 3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting 3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.