Nitric oxide-dependent cilia regulatory enzyme localization in bovine bronchial epithelial cells.
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 adjacent to, the ciliary axoneme. Immunohistochemistry of intact ciliated cells revealed that endothelial-type nitric oxide synthase (eNOS), the RII isoform of the cAMP-dependent protein kinase (PKA-RII), the type I isoform of the cGMP-dependent protein kinase (PKG-I), and guanylate cyclase beta (GC-beta) all colocalized with pericentrin to the basal body. In contrast, the PKA-RI isoform and the PKG-II isoform localized to ciliary axonemes. Western blot analysis of isolated demembranated ciliary preparations detected eNOS, GC-beta, 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 discrete ciliary metabolon, enabling targeted phosphorylation and efficient upregulation of ciliary beating.
Stout
Sarah L
SL
Pulmonary, Critical Care, Sleep & Allergy Medicine, Dept. of Internal Medicine, University of Nebraska Medical Center 985300, Omaha, NE 68198-5300, USA.
Wyatt
Todd A
TA
Adams
Jennifer J
JJ
Sisson
Joseph H
JH
D000818:Animals; D001980:Bronchi / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D002417:Cattle; D002923:Cilia / Q000201:enzymology / Q000648:ultrastructure; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure
D007527:Isoenzymes; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase