We investigated the effects of cultured epithelial cells and supernatants on resting membrane potential and excitatory neuroeffector transmission in smooth muscle cells of dog trachea and bronchioles. The mean resting membrane potential of the mucosa-free tracheal smooth muscle cells was -59.5 +/- 1.5 mV (+/- SD). Application of cultured epithelial cells (> 2.5 x 10(5) cells/ml) hyperpolarized the membrane, resulting in a potential of -64.5 +/- 1.7 mV. The supernatant of the cultured epithelial cells also increased the resting membrane potential of the mucosa-free tracheal smooth muscle cells by 4 to 9 mV. These hyperpolarizing actions were not modified by indomethacin (10(-5) M), l-NG-nitroarginine (10(-5) M), or oxyhemoglobin (10(-5) M), but were inhibited by glibenclamide (10(-6) M). The supernatants of the cultured epithelial cells completely or partially suppressed the contractile response of epithelium-denuded bronchioles to electrical field stimulations and suppressed the amplitude of excitatory junction potentials of the trachealis evoked by electrical field stimulations. Indomethacin prevented the inhibitory effect of supernatants on the amplitude of twitch contractions and excitatory junction potentials and markedly suppressed supernatant-associated inhibition of the excitatory neuroeffector transmission. Furthermore, indomethacin with AA861, a lipoxygenase inhibitor, completely suppressed this effect. Our findings suggest that cultured airway epithelial cells spontaneously release at least two factors. One factor selectively modulates the resting membrane potential, and the other inhibits the excitatory neuroeffector transmission.