EFFECTS OF MEMBRANE DEPOLARIZATION AND CHANGES IN INTRACELLULAR AND EXTRACELLULAR CALCIUM-CONCENTRATION ON PHOSPHOINOSITIDE HYDROLYSIS IN BOVINE TRACHEAL SMOOTH-MUSCLE

Agonist-stimulatd 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 Ca2+ ([Ca2+](i)) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3)-induced Ca2+ release or agonist-mediated Ca2+ influx is able to trigger or augment phosphaticdylinositol 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 Ca2+ ([Ca2+](e)) chelation, and addition of ionomycin to elevate [Ca2+](i) on basal and agonist-stimulated Ins(1,4,5)P-3 concentration and [H-3]-InsP(x) accumulation have been examined. Reducing [Ca2+](e) from 1.8 mM to 6 or 0.8 mu M caused a progressive inhibition of agonistic-stimulated [H-3]inositol polyphosphate accumulation over 30 min with the histamine-stimulated response being significantly more sensitive to [Ca2+](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 [Ca2+](e). Incubation of [H-3]inositol-prelabelled BTSM slices with buffer containing 80 mM KCl failed to stimulat [H-3]InsP(x) accumulation, causing instead a small inhibition of carbachol-stimulated [H-3]InsP(x) accumulation with a similar effect seen with respect to Ins(1,4,5)P-3 accumulation. Addition of 5 mu M ionomycin to BTSM slices similarily did not stimulate Ins(1,4,5)P-3 generation and only increased [H-3]InsP(x) accumulation after prolonged stimulation in the presence of high (mM) [Ca2+](e). These data indicated that in ASM, membrane depolarization or physiological increases in [Ca2+](i) did not result in either independent activation of PIC or augmentation of initial agonist-stimulated PtdIns(4,5)P-2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P-3 was not dependent on [Ca2+](e), a normal plasmalemmal Ca2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P-2 hydrolysis.