Calmodulin and protein kinases A/G mediate ciliary beat response in the human nasal epithelium.

Abstract

Mucociliary clearance of the airway epithelium is an essential function for mucosal defense. We recently proposed a hypothetical mechanism of ciliary beat regulation, in which the pannexin-1 (Panx1)-P2X7 unit serves as an oscillator generating a periodic increase in intracellular Ca2+ ([Ca2+ ]i ). In the present study, we examined the localization of Panx1 and P2X7 at the ultrastructural level, and investigated the regulatory pathway subsequent to [Ca2+ ]i increase. The inferior turbinate mucosa was collected from patients with chronic hypertrophic rhinitis during endoscopic sinonasal surgery. The mucosa was examined by transmission immunoelectron microscopy for Panx1 and P2X7. Alternatively, the mucosa was cut into thin strips, and ciliary beat frequency (CBF) was measured under a phase-contrast light microscope with a high-speed digital video camera. In immunoelectron microscopy, immunoreactivities for Panx1 and P2X7 were localized along the plasma membrane of the entire length of the cilia. CBF was significantly increased by stimulation with 100 µM acetylcholine (Ach). The Ach-induced CBF increase was significantly inhibited by calmidazolium (calmodulin antagonist), SQ22536 (adenylate cyclase inhibitor), ODQ (guanylate cyclase inhibitor), KT5720 (protein kinase A inhibitor), and KT5823 (protein kinase G inhibitor). Fluorodinitrobenzene (creatine kinase inhibitor) completely inhibited the ciliary beat in a time- and dose-dependent manner. These results indicate that Panx1 and P2X7 coexist at the cilia of the human nasal epithelial cells and that the ciliary beat is regulated by calmodulin, adenylate/guanylate cyclases and protein kinases A/G, and crucially depends on creatine kinase.