Effects of membrane depolarization and changes in intra- and extracellular calcium concentration on phosphoinositide hydrolysis in bovine tracheal smooth muscle.
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 Ca2+ ([Ca2+]i) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release or agonist-mediated Ca2+ influx is able to trigger or augment phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 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)P3 concentration and [3H]-InsPx accumulation have been examined. Reducing [Ca2+]e from 1.8 mM to 6 or 0.8 microM caused a progressive inhibition of agonist-stimulated [3H]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)P3 was completely unaffected by such reductions in [Ca2+]e. Incubation of [3H]inositol-prelabelled BTSM slices with buffer containing 80 mM KCl failed to stimulate [3H]InsPx accumulation, causing instead a small inhibition of carbachol-stimulated [3H]InsPx accumulation with a similar effect seen with respect to Ins(1,4,5)P3 accumulation. Addition of 5 microM ionomycin to BTSM slices similarly did not stimulate Ins(1,4,5)P3 generation and only increased [3H]InsPx 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)P2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P3 was not dependent on [Ca2+]e, a normal plasmalemmal Ca2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P2 hydrolysis.
Department of Medicine (RIE), Rayne Laboratory, City Hospital, Edinburgh, U.K.
E R
ER
Chilvers
B J
BJ
Lynch
G J
GJ
Offer
R A
RA
Challiss
D000818:Animals; D002118:Calcium / Q000032:analysis*; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D005110:Extracellular Space / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006868:Hydrolysis; D015759:Ionomycin / Q000494:pharmacology; D008564:Membrane Potentials; D009130:Muscle, Smooth / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D014132:Trachea / Q000378:metabolism
D010716:Phosphatidylinositols; D015759:Ionomycin; D011189:Potassium Chloride; D006632:Histamine; D002217:Carbachol; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D002118:Calcium