8-Bromo-cCAMP decreases the Ca2+ sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase : Rho GTPases in lung physiology and disease

To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca 2+ sensitivity of airway smooth muscle contraction, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTP γ S)-, 4β-phorbol 12,13-dibutyrate (PDBu)-, and leukotriene D 4 (LTD 4 )-induced Ca 2+ sensitization in a-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-BrcAMP and Y-27632 to a similar extent. However, GTP γ S-induced smooth muscle contraction was resistant to 8-BrcAMP. In the presence of a saturating concentration'of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-BrcAMP, GTP γ S-induced Ca 2+ sensitization was also reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTP γ S-treated smooth muscle under myosin light chain kinase-inhibited conditions. In human bronchus, LTD 4 -induced smooth muscle contraction was inhibited by both 8-BrcAMP and Y-27632. We conclude that cAMP/PKA-induced Ca 2+ desensitization contains at least two mechanisms: I) inhibition of the muscarinic receptor signaling upstream from Rho activation and 2) cAMP/PKA's preferential reversal of PKC-mediated Ca 2+ sensitization in airway smooth muscle.