We have established an in vitro model of airway hyperresponsiveness, using a bovine tracheal smooth muscle cell (BTSMC)-embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates the small G protein RhoA, ATP- and high K+ solution-induced gel contraction was significantly augmented. This was not due to the modulation of Ca2+ mobilizing properties, since ATP- and high K+-induced Ca2+ transients were not significantly different between control and LPA-treated BTSMC. Y-27632, an inhibitor of Rho-kinase, suppressed the LPA-induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (≥ 1 μM) reversed the LPA-induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 μM). We confirmed that theophylline increased the intracellular concentration of cAMP ([cAMP]i) in BTSMC. Elevation of [cAMP]i with dibutyryl cAMP or forskolin also reversed the LPA-induced augmentation of gel contraction. Furthermore, theophylline, as well as dibutyryl cAMP and forskolin, suppressed the LPA-induced membrane translocation of RhoA, indicating that they prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA-induced, RhoA/Rho-kinase-mediated hyperresponsiveness of tracheal smooth muscle cells due to the accumulation of cAMP.
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