Stimulation of ciliary cells through muscarinic receptors leads to a strong biphasic enhancement of ciliary beat frequency (CBF). The main goal of this work is to delineate the chain of molecular events that lead to the enhancement of CBF induced by acetylcholine (ACh). Here we show that the Ca2+, cGMP, and cAMP signaling pathways are intimately interconnected in the process of cholinergic ciliary stimulation. ACh induces profound time-dependent increase in cGMP and cAMP concentrations mediated by the calcium–calmodulin complex. The initial strong CBF enhancement in response to ACh is mainly governed by PKG and elevated calcium. The second phase of CBF enhancement induced by ACh, a stable moderately elevated CBF, is mainly regulated by PKA in a Ca2+-independent manner. Inhibition of either guanylate cyclase or of PKG partially attenuates the response to ACh of Ca2+i, but completely abolishes the response of CBF. Inhibition of PKA moderately attenuates and significantly shortens the responses to ACh of both Ca2+i and CBF. In addition, PKA facilitates the elevation in Ca2+i and cGMP levels induced by ACh, whereas an unimpeded PKG activity is essential for CBF enhancement mediated by either Ca2+ or PKA.
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