Immunocytochemical localization of O2-sensing protein (NADPH oxidase) in chemoreceptor cells

Abstract

A potential candidate for an oxygen‐sensing protein in chemoreceptor cells is a heme‐linked multicomponent NADPH oxidase, originally described in neutrophils. The postulated function for the oxidase in chemoreceptor cells is to signal changes in oxygen levels (either in the blood or in the airway lumen) via changes in oxygen metabolite production. An alteration in either superoxide (or dismuted hydrogen peroxide) production may affect the gating properties of the O2‐sensitive K+ channels. We have previously reported immunohistochemical localization of gp91 glycoprotein component of the oxidase to the plasma membrane of pulmonary neuroepithelial body (NEB) cells. In this study we have investigated the immunocytochemical localization of the other polypeptide components of the oxidase in NEB cells and in the glomus cells of the carotid body. Cultures of dissociated fetal rabbit NEB cells and newborn rat glomus cells were immunostained with specific antibodies recognizing the various polypeptide subunits of the oxidase using indirect immunofluorescence methods. Immunostaining with the anti‐oxidase antibodies revealed strong positive reaction in both NEB and glomus cell clusters while other cells were unstained. The positive reaction product was localized to the plasma membrane and/or cytoplasm and no nuclear staining was observed. Live cell labelling studies with anti‐p22 antibody showed positive immunofluorescence on the surface of NEB cells, suggesting that this component of the oxidase is also associated with the plasma membrane. In glomus cells, similar strongly positive immunofluorescence signal was observed for p22 and gp91 in paraformaldehyde‐fixed cultures, regardless whether they were permeabilized or not. Taken together, our findings of cell surface localization of gp91 and p22 components of the oxidase in chemoreceptive cells suggests that the heme‐linked cytochrome b558 component is associated with the plasma membrane. This association allows for direct interaction with the O2‐sensitive K+ channel thus forming the molecular complex of membrane bound O2 sensor. Microsc. Res. Tech. 37:101–106, 1997. © 1997 Wiley‐Liss, Inc.

Combined Antisense and Pharmacological Approaches Implicate hTASK as an Airway O2 Sensing K+Channel

“…O 2 -sensitive currents in both NEB and H146 cells are 4-aminopyridine-insensitive, weakly TEA-sensitive, blockable by quinidine, and have a significant calcium-independent component (7,8,39,41). Thus, the present study provides direct functional evidence, through the use of pharmacology and an antisense knock-down/knockout approach, that hTASK channels are oxygen-sensitive in a native cellular system.…”

NADPH oxidase-derived superoxide anion mediates angiotensin II-enhanced carotid body chemoreceptor sensitivity in heart failure rabbits

“…Gao, et al found that Ang II-NADPH oxidase-derived radical stress in autonomic areas of the brain was involved in the sympathoexcitation in rabbits with pacing-induced CHF [19], the same animal model as in the present study. Furthermore, a significant body of evidence indicates that the components of NADPH oxidase are present in the peripheral chemoreceptors including pulmonary neuroepithelial bodies and CB [20][21][22].…”

“…Further studies are needed to define the involvement of NADPH oxidase-derived superoxide anion on the mod- NADPH oxidase is a complex enzyme consisting of two membrane-bound components (gp91 phox and p22 phox ) and three cytosolic components (p67 phox , p47 phox , and p40 phox ) [28], which was first found in phagocytic cells [29]. Studies have shown that gp91 phox , p22 phox , p67 phox , and p47 phox subunits of the NADPH oxidase exist in the CB glomus cells in rats [20,21]. However, we found the mRNA of gp91 phox , p47 phox , and p40 phox but not p22 phox and p67 phox are expressed in the rabbit CBs (Fig.…”

“…Ang II activates NADPH oxidase via the AT 1 receptor [13], which serves as a significant source of intracellular reactive oxygen species (ROS) in many tissues [14][15][16][17][18][19]. Furthermore, a significant body of evidence indicates that the components of the NADPH oxidase are present in peripheral chemoreceptor sites including pulmonary neuroepithelial bodies and CBs [20][21][22]. We hypothesized that NADPH oxidase-derived superoxide anion mediates the Ang II-enhanced CB chemoreceptor sensitivity in rabbits with CHF.…”

Functional facets of the pulmonary neuroendocrine system

“…Incidentally, it was recently proposed that voltage-gated ion channels and the membrane 'exitability' seen in SCLCs might also accelerate metastatic progression. 113,114 The studies by Youngson et al 112,115 revealed that NEB cells possess an oxygen-binding protein cytochrome b, a NAPDH oxidase located in cellular membranes. Proof that NAPDH oxidase acts as the O 2 receptor came from oxidase-deficient mice.…”