MODULATION OF AGONIST-INDUCED PHOSPHOINOSITIDE METABOLISM, CA2-MUSCLEBY CYCLIC-AMP-DEPENDENT MECHANISMS( SIGNALING AND CONTRACTION OF AIRWAY SMOOTH)

1 The effects of increased cellular cyclic AMP levels induced by isopr enaline, forskolin and 8-bromo-adenosine 3':5'-cyclic monophosphate (8 -Br-cyclic AMP) on phosphoinositide metabolism and changes in intracel lular Ca2+ elicited by methacholine and histamine were examined in bov ine isolated tracheal smooth muscle (BTSM) cells. 2 Isoprenaline (pD(2 ) (-log(10) EC(50)) = 6.32 +/- 0.24) and forskolin (pD(2) = 5.6 +/- 0. 05) enhanced cyclic AMP levels in a concentration-dependent fashion in these cells, while methacholine (pD(2) = 5.64 +/- 0.12) and histamine (pD(2) = 4.90 +/- 0.04) caused a concentration-related increase in [H -3]-inositol phosphates (IF) accumulation in the presence of 10 mM LiC l. 3 Preincubation of the cells (5 min, 37 degrees C) with isoprenalin e (1 mu M), forskolin (10 mu M) and 8-Br-cyclic AMP (1 mM) did not aff ect the IP accumulation induced by methacholine, but significantly red uced the maximal IP production by histamine (1 mM). However, the effec t of isoprenaline was small (15.0 +/- 0.6% inhibition) and insignifica nt at histamine concentrations between 0.1 and 100 mu M. 4 Both methac holine and histamine induced a fast (max, in 0.5-2 s) and transient in crease of intracellular Ca2+ concentration ([Ca2+](i)) followed by a s ustained phase lasting several minutes. EGTA (5 mM) attenuated the sus tained phase, indicating that this phase depends on extracellular Ca2. 5 Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 mu M), forskolin (10 mu M) and 8-Br-cyclic AMP (1 mu M) significan tly attenuated both the Ca2+-transient and the sustained phase generat ed at equipotent IP producing concentrations of 1 mu M methacholine an d 100 mu M histamine (approx. 40% of maximal methacholine-induced IP r esponse), but did not affect changes in [Ca2+](i) induced by 100 mu M methacholine (95.2 +/- 3.5% of maximal methacholine-induced IP respons e). 6 Significant correlations were found between the isoprenaline-ind uced inhibition of BTSM contraction and inhibition of Ca2+ mobilizatio n or influx induced by methacholine and histamine, that were similar f or each contractile agonist.7 These data indicate that (a) cyclic AMP- dependent inhibition of Ca2+ mobilization in BTSM cells is not primari ly caused by attenuation of IP production, suggesting that cyclic AMP induced protein kinase A (PKA) activation is effective at a different level in the [Ca2+](i) homeostasis, (b) that attenuation of intracellu lar Ca2+ concentration plays a major role in beta-adrenoceptor-mediate d relaxation of methacholine- and histamine-induced airway smooth musc le contraction, and (c) that the relative resistance of the muscarinic agonist-induced contraction to beta-adrenoceptor agonists, especially at (supra) maximal contractile concentrations is largely determined b y its higher potency in inducing intracellular Ca2+ changes.