Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice. Am J Physiol Cell Physiol 282: C27-C33, 2002.-Various hemecontaining proteins have been proposed as primary molecular O2 sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and

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... iodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91 phox ) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O2-sensitive cells in other species, and the incidence of tyrosine hydroxylaseimmunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K ϩ currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca 2ϩ concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91 phox gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O2 sensor in arterial chemoreception. hypoxia; reactive oxygen species; sensory transduction; chemoreceptor IT IS WIDELY HELD that O 2 chemoreception in the mammalian carotid body is initiated by specialized chemosensory type I cells that respond to hypoxia with depolarization and release of multiple neurotransmitter agents (9, 11). The cascade of molecular and cellular events involved in O 2 chemotransduction has been the subject of intense scrutiny in recent years. Numerous laboratories (5, 14, 21, 22, 24, 27) have reported that low PO 2 inhibits the conductance of a variety of voltagesensitive and voltage-insensitive K ϩ channels in type I cells, yet the molecular mechanisms underlying the modulation of these currents remain uncertain and controversial. Various heme proteins have also been proposed as the primary O 2 sensors (11), and one set of data in particular suggests the involvement of a multicomponent cytochrome b-containing NADPH oxidase that may be similar, if not identical, to an enzyme commonly found in phagocytic cells (1, 2). According to this hypothesis, the oxidase generates reactive oxygen species (ROS) in proportion to available O 2 , which then modulates K ϩ channels via the formation of H 2 O 2 from ROS by the action of superoxide dismutase. Immunoreactivity for multiple subunits of the phagocytic form of the enzyme, including p22 phox , gp91 phox , p47 phox , and p67 phox , has been localized to type I cells (17) , and an inhibitor of the oxidase, diphenyleneiodonium (DPI), alters carotid body chemoreceptor activity evoked by hypoxia (17) . Neuroepithelial bodies (NEBs) located in lung airways have also been proposed as O 2 chemoreceptors, and like the carotid body, they consist of specialized cells that are thought to release neuroactive agents in response to hypoxia (18) (19) (20) . NEB cells also express O 2 -sensitive K ϩ channels, and immunocytochemical studies indicate that these cells contain components of the phagocytic NADPH oxidase (29, 32). A possible functional link between the oxidase and K ϩ channels in NEB cells was established in gp91 phox gene knockout (KO) mice in which the K ϩ currents were inhibited by H 2 O 2 but not by hypoxia or DPI, suggesting that the ROS-producing enzyme is an essential component of the transduction machinery (10). In contrast, other investigators (3) using gp91 phox KO animals reached opposite conclusions in studies of pulmonary arterial smooth muscle cells (PASMCs) that showed that K ϩ currents displayed normal sensitivity to hypoxia. In the present study, we have used the gp91 phox gene KO strain of animals to examine the hypothesis that