Effects of membrane depolarization and changes in intra- and extracellular calcium concentration on phosphoinositide hydrolysis in bovine tracheal smooth muscle

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Agonist-stimulated phosphoinositide metabolism plays a central role in pharmacomechanical coupling in airways smooth muscle (ASM). In many other tissues and cells, most noteably excitable cells, membrane depolarization or an increase in intracellular Ca 2+ ([Ca 2+] i) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3)-induced Ca 2+ release or agonist-mediated Ca 2+influx is able to trigger or augment phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) hydrolysis and/or initiate PtdIns4P/ PtdIns hydrolysis by direct stimulation of PIC. To assess the importance of these mechanisms in ASM the effects of KCl-induced membrane depolarization, extracellular Ca 2+([Ca 2+] e) chelation, and addition of ionomycin to elevate [Ca 2+] i on basal and agonist-stimulated Ins(1,4,5)P 3 concentration and [ 3H]-InsP x accumulation have been examined. Reducing [Ca 2+] e from 1.8 mM to 6 or 0.8μM caused a progressive inhibition of agonist-stimulated [ 3H]inositol polyphosphate accumulation over 30 min with the histamine-stimulated response being significantly more sensitive to [Ca 2+] e chelation than the response to carbachol. In contrast, the initial accumulation of Ins(1,4,5)P 3 was completely unaffected by such reductions in [Ca 2+] e. Incubation of [ 3H]inositol-prelabelled BTSM slices with buffer containing 80 mM KC1 failed to stimulate [ 3H]InsP x accumulation, causing instead a small inhibition of carbacholstimulated [ 3H]InsP x accumulation with a similar effect seen with respect to Ins(1,4,5)P 3 accumulation. Addition of 5 μM ionomycin to BTSM slices similarily did not stimulate Ins(1,4,5)P 3 generation and only increased [ 3H]InsP x accumulation after prolonged stimulation in the presence of high (mM) [Ca 2+] e. These data indicated that in ASM, membrane depolarization or physiological increases in [Ca 2+] i did not result in either independent activation of PIC or augmentation of initial agoniststimulated PtdIns(4,5)P 2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P 3 was not dependent on [Ca 2+] e, a normal plasmalemmal Ca 2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P 2 hydrolysis.