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

Abstract

(JJA) S U M M A R Y 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 b (GC-b) 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-b, 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. (J Histochem Cytochem 55:433-442, 2007)

Long-Term Cigarette Smoke Exposure in a Mouse Model of Ciliated Epithelial Cell Function

“…Specifically, PKA and PKG activation were shown to increase CBF, resulting in increased mucociliary clearance (12,31). Protein kinase C activation was also demonstrated to reduce CBF, thus impeding mucociliary clearance (13,15).…”

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

“…Unlike PKA isozymes, the more N-terminal cN-binding site in PKGI isozymes has higher affinity for cGMP than does the more C-terminal site (Reed et al, 1996. PKGI isozymes are found in particular subcellular membrane fractions, in complex with certain cytosolic proteins, and as free cytosolic proteins; they are also reported to reversibly translocate among cellular compartments after changes in cGMP level (Pryzwansky et al, 1990(Pryzwansky et al, , 1995Cornwell et al, 1991;Wyatt et al, 1991;Surks et al, 1999;Yuasa et al, , 2000bGeiselhöringer et al, 2004;Fiedler et al, 2006;Antl et al, 2007;Stout et al, 2007;Zhang et al, 2007a;Casteel et al, 2008;Sharma et al, 2008;Wilson et al, 2008;Takimoto et al, 2009). The current consensus is that cGMP concentration may vary substantially in different cellular compartments as a result of a variety of factors (e.g., selective localization of the proteins that provide for cGMP synthesis, breakdown, or extrusion) that confine certain pools of cGMP to particular areas of the cell.…”

RACK1, a PKC Targeting Protein, Is Exclusively Localized to Basal Airway Epithelial Cells

“…IHC localization experiments using airway epithelial cells were performed similar to published methods (Jurczyk et al 2004;Stout et al 2007). Briefly, BBECs or BEAS-2B cells were either permeabilized for 30 sec with 80 mM PIPES, pH 6.8, 5 mM EDTA, 1 mM MgCl 2 and 0.5% Triton X-100 (permeabilization buffer) and fixed for 15 min in absolute methanol at 2 20C (Figures 1A-1F, Figure 5) or fixed for 15 min in absolute methanol and permeabilized with Triton X-100 containing buffer ( Figures 1G and 1H).…”