Nitric Oxide-dependent Cilia Regulatory Enzymes Localization in Bovine Bronchial Epithelial Cells

Sarah L. Stout ¹, Todd A. Wyatt ¹, Jennifer J. Adams ¹ and Joseph H. Sisson ^1*

¹ Pulmonary, Critical Care, Sleep & Allergy Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska (SLS,TAW,JHS); Department of Veterans Affairs, Research Service, Omaha, Nebraska (TAW); and Department of Anesthesiology and Critical Care Medicine, Johns Hopkins Hospital, Baltimore, Maryland (JJA)

^* To whom correspondence should be addressed. E-mail: jsisson{at}unmc.edu.

Submitted on September 6, 2006
Accepted on 21 November 2006

	Abstract

Airway epithelial-derived nitric oxide (NO), through the activation of nucleotide cyclases and downstream kinases, stimulates ciliary beating, yet the precise locations of these enzymes are unknown. We hypothesized that these NO-activated enzymes are located within, or closely adjacent to, the ciliary axoneme. Immunohistochemistry of intact ciliated cells revealed that endothelial type nitric oxide synthase (eNOS), the RII isoform of the cyclic-AMP dependent protein kinase (PKA R-II), the type I isoform of the c-GMP dependent protein kinase (PKG-I), and guanylate cyclase (GC-) all co-localized with pericentrin to the basal body. In contrast, the PKA R-I isoform and the PKG-II isoform localized to ciliary axonemes. Western blot analysis of isolated demembranated ciliary preparations detected eNOS, GC- and both isoforms of PKA and PKG; an A-kinase anchoring protein was also detected. Our findings suggest that these enzymes are sequestered close to their points of action into a discreet ciliary metabolon, enabling targeted phosphorylation and efficient upregulation of ciliary beating.

Key Words: cilia, PKA, PKG, nitric oxide, alcohol

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