IMMUNOCYTOCHEMICAL LOCALIZATION OF O-2-SENSING PROTEIN (NADPH OXIDASE) IN CHEMORECEPTOR CELLS

A potential candidate for an oxygen-sensing protein in chemoreceptor c ells is a heme-linked multicomponent NADPH oxidase, originally describ ed in neutrophils. The postulated function for the oxidase in chemorec eptor 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) pro duction may affect the gating properties of the O-2-sensitive K+ chann els. We have previously reported immunohistochemical localization of g p91 glycoprotein component of the oxidase to the plasma membrane of pu lmonary neuroepithelial body (NEB) cells. In this study we have invest igated the immunocytochemical localization of the other polypeptide co mponents of the oxidase in NEB cells and in the glomus cells of the ca rotid body. Cultures of dissociated fetal rabbit NEB cells and newborn rat glomus cells were immunostained with specific antibodies recogniz ing the various polypeptide subunits of the oxidase using indirect imm unofluorescence methods. Immunostaining with the anti-oxidase antibodi es revealed strong positive reaction in both NEB and glomus cell clust ers while other cells were unstained. The positive reaction product wa s localized to the plasma membrane and/or cytoplasm and no nuclear sta ining was observed. Live cell labelling studies with anti-p22 antibody showed positive immunofluorescence on the surface of NEB cells, sugge sting that this component of the oxidase is also associated with the p lasma membrane. In glomus cells, similar strongly positive immunofluor escence signal was observed for p22 and gp91 in paraformaldehyde-fixed cultures, regardless whether they were permeabilized or not. Taken to gether, our findings of cell surface localization of gp91 and p22 comp onents of the oxidase in chemoreceptive cells suggests that the heme-l inked cytochrome b(558) component is associated with the plasma membra ne. This association allows for direct interaction with the O-2-sensit ive K+ channel thus forming the molecular complex of membrane bound O- 2 sensor. (C) 1997 Wiley-Liss, Inc.