The role of cGMP in the regulation of rabbit airway ciliary beat frequency

Abstract

The involvement of cyclic guanosine 3′,5′-monophosphate (cGMP) and cGMP-dependent protein kinase (PKG) and their interaction with the Ca²⁺-dependent mechanisms in the regulation of ciliary activity are not well understood. To investigate how cGMP regulates ciliary activity, changes in ciliary beat frequency (CBF) and intracellular calcium concentration ([Ca²⁺]_i) of rabbit tracheal ciliated cells in response to 8-bromo-cGMP (Br-cGMP) were simultaneously quantified using digital, high-speed phase-contrast and fluorescence imaging. Br-cGMP induced a response in ciliary activity that could be separated into two parts. Firstly, Br-cGMP induced a concentration-dependent increase in the basal CBF that occurred without increasing the [Ca²⁺]_i. This response was not affected by excessively buffering the [Ca²⁺]_i with BAPTA but was abolished by KT5823, a PKG inhibitor. Secondly, Br-cGMP induced a series of transient increases in CBF that were superimposed on the sustained increases in CBF. These transient increases in CBF correlated with the stimulation of a series of transient increases in [Ca²⁺]_i and were abolished by BAPTA, but were unaffected by KT5823. The magnitude of the transient increases in CBF and [Ca²⁺]_i were not dependent on the concentration of Br-cGMP. The Ca²⁺-dependent changes in CBF induced by ionomycin or ATP were not affected by KT5823. From these results, we propose that cGMP increases CBF in two ways: firstly through a Ca²⁺-independent mechanism involving PKG, and secondly through a Ca²⁺-dependent mechanism following the stimulation of changes in [Ca²⁺]_i. In addition, we suggest that the Ca²⁺-dependent stimulation of rabbit airway ciliary activity does not initially require PKG activation.