SIGNAL-TRANSDUCTION PATHWAYS IN MODULATION OF CILIARY BEAT FREQUENCY BY METHACHOLINE

The release of endogenous neurotransmitters plays an important role in the airway mucosal defense system, We studied the in vitro effect of methacholine, a beta-methyl ester of acetylcholine, on the ciliary bea t frequency (CBF) of human adenoid explants and its mechanism of actio n. Tissue explants were cultured at 35 degrees C and covered with 1.0 mi, of culture medium: minimum essential Eagle's medium (MEM) containi ng L-arginine (1.2 x 10(-3) mol/L). Methacholine was added to the cult ured tissue at concentrations of 10(-10) 10(-8), and 10(-6) mol/L. The CBF was determined by phase contrast microscopy and microphotometry. Methacholine increased CBF in a dose-dependent manner with a maximum i ncrease of 23.0% +/- 1.8% (p <.001). Atropine (10(-6) mol/L) significa ntly inhibited the ciliostimulatory effects of methacholine (p <.0007) . The role of endogenous prostaglandins in methacholine-induced cilios timulation was determined by treating specimens with a cyclooxygenase inhibitor (diclofenac sodium). Diclofenac (10(-6) mol/L) significantly inhibited the ciliostimulatory effects of methacholine (p <.0007). To determine if nitric oxide (NO) acts as an intermediary in ciliostimul ation by methacholine, endogenous NO production was inhibited by treat ing specimens with an L-arginine analog, N-G-nitro L-arginine methyl e ster (L-NAME), prior to addition of methacholine. L-NAME (10(-6) mol/L ) inhibited the effects of methacholine in L-arginin-free MEM (p <.008 ), and this inhibition was reversed by L-arginine (10(-3) mol/L). To f urther examine the actions of NO in methacholine-induced ciliostimulat ion, a cyclic guanosine 3'5'-monophosphate (cGMP) kinase inhibitor (KT -5823) was used, prior to the addition of methacholine. KT-5823 (10(-6 ) mol/L) significantly inhibited the effects of methacholine (p <.0001 ). Ciliostimulation by methacholine in human upper airway mucosa invol ves both prostaglandin and NO second messengers and activation of a cG MP-dependent kinase.