Lack of Nitric Oxide Involvement in Cholinergic Modulation of Ovine Ciliary Beat Frequency

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Abstract
Ciliary beat frequency (CBF) is regulated, at least in part, by the cytoplasmic calcium concentration ([Ca2+]i). Because Ca2+ can stimulate nitric oxide (NO) production by nitric oxide synthase (NOS) and NO has been implicated in the regulation of CBF in some species, we examined whether NOS is present in cultured ovine ciliated epithelial cells and whether NO plays a role in the Ca2+-mediated muscarinic stimulation of CBF. Dissociated ovine tracheal epithelial cells were grown in culture for 2 to 14 days. Frequency from a single cilium was measured by on-line Fourier transform methods using video microscopy. [Ca2+]i was determined with fura-2 using fluorescence ratio imaging from the same single cells. Ciliated cells contained NOS in culture as indicated by NADPH-diaphorase staining. Acetylcholine (ACh) increased CBF and [Ca2+]i transiently as previously shown. Measurements with 2′,7-′dichlorofluorescin diacetate indicated that reactive oxygen/nitrogen species were produced in these cells on ACh exposure. NOS inhibitors NG-nitro-L-arginine methyl ester (≤10 mM), NG-nitro-L-arginine (≤10 mM), and 7-nitro indazole (1 μM) were unable to block the CBF or [Ca2+]i response to ACh. Furthermore, the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine (≤1 mM) did not change CBF or [Ca2+]i. Above these concentrations, they both lead to a reversible decrease in CBF. The membrane-permeable cyclic guanosine monophosphate analogue 8-bromo-cyclic guanosine monophosphate had no effect on CBF, whereas 8-bromo-cyclic adenosine monophosphate stimulated CBF. Taken together, these results suggest that NO does not play a role in mediating the ACh-induced increase in CBF through [Ca2+]i. The role and targets for NO in ovine ciliated cells remains to be determined.