Modulation of agonist-induced phosphoinositide metabolism, Ca2+ signalling and contraction of airway smooth muscle by cyclic AMP-dependent mechanisms.

1. The effects of increased cellular cyclic AMP levels induced by isoprenaline, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cyclic AMP) on phosphoinositide metabolism and changes in intracellular Ca2+ elicited by methacholine and histamine were examined in bovine isolated tracheal smooth muscle (BTSM) cells. 2. Isoprenaline (pD2 (-log10 EC50) = 6.32 +/- 0.24) and forskolin (pD2 = 5.6 +/- 0.05) enhanced cyclic AMP levels in a concentration-dependent fashion in these cells, while methacholine (pD2 = 5.64 +/- 0.12) and histamine (pD2 = 4.90 +/- 0.04) caused a concentration-related increase in [3H]-inositol phosphates (IP) accumulation in the presence of 10 mM LiCl. 3. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 mM) did not affect the IP accumulation induced by methacholine, but significantly reduced the maximal IP production by histamine (1 mM). However, the effect of isoprenaline was small (15.0 +/- 0.6% inhibition) and insignificant at histamine concentrations between 0.1 and 100 microM. 4. Both methacholine and histamine induced a fast (max. in 0.5-2 s) and transient increase of intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained phase lasting several minutes. EGTA (5 mM) attenuated the sustained phase, indicating that this phase depends on extracellular Ca2+. 5. Preincubation of the cells (5 min, 37 degrees C) with isoprenaline (1 microM), forskolin (10 microM) and 8-Br-cyclic AMP (1 microM) significantly attenuated both the Ca(2+)-transient and the sustained phase generated at equipotent IP producing concentrations of 1 microM methacholine and 100 microM histamine (approx. 40% of maximal methacholine-induced IP response), but did not affect changes in [Ca2+]i induced by 100 microM methacholine (95.2 +/- 3.5% of maximal methacholine-induced IP response). 6. Significant correlations were found between the isoprenaline-induced inhibition of BTSM contraction and inhibition of Ca2+ mobilization or influx induced by methacholine and histamine, that were similar for each contractile agonist. 7. These data indicate that (a) cyclic AMP-dependent inhibition of Ca2+ mobilization in BTSM cells is not primarily 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 intracellular Ca2+ concentration plays a major role in beta-adrenoceptor-mediated relaxation of methacholine- and histamine-induced airway smooth muscle 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 by its higher potency in inducing intracellular Ca2+ changes.