General redox environment and carotid body chemoreceptor function.

Carotid body (CB) chemoreceptor cells detect physiological levels of hypoxia and generate a hyperventilation, homeostatic in nature, aimed to minimize the deleterious effects of hypoxia. Intimate mechanisms involved in oxygen sensing in chemoreceptor cells remain largely unknown, but reactive oxygen species (ROS) had been proposed as mediators of this process. We have determined glutathione levels and calculated glutathione redox potential (E(GSH); indicator of the general redox environment of cells) in rat diaphragms incubated in the presence of oxidizing agents of two types: nonpermeating and permeating through cell membranes; in the latter group, unspecific oxidants and inhibitors of ROS-disposing enzymes were used. Selected concentrations of oxidizing agents were tested for their ability to modify the normoxic and hypoxic activity of chemoreceptor cells measured in vitro as their rate of release of neurotransmitters. Results evidence variable relationships between E(GSH) and the activity of chemoreceptor cells. The independence of chemoreceptor cell activity from the E(GSH) would imply that the ability of the CB to play its homeostatic role is largely preserved in any pathological or toxicological contingency causing oxidative stress. Consistent with this suggestion, it was also found that CB-mediated hypoxic hyperventilation was not altered by treatment of intact animals with agents that markedly decreased the E(GSH) in all tissues assayed.

Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia.

Reactive oxygen species (ROS) are oxygen-containing molecular entities which are more potent and effective oxidizing agents than is molecular oxygen itself. With the exception of phagocytic cells, where ROS play an important physiological role in defense reactions, ROS have classically been considered undesirable byproducts of cell metabolism, existing several cellular mechanisms aimed to dispose them. Recently, however, ROS have been considered important intracellular signaling molecules, which may act as mediators or second messengers in many cell functions. This is the proposed role for ROS in oxygen sensing in systems, such as carotid body chemoreceptor cells, pulmonary artery smooth muscle cells, and erythropoietin-producing cells. These unique cells comprise essential parts of homeostatic loops directed to maintain oxygen levels in multicellular organisms in situations of hypoxia. The present article examines the possible significance of ROS in these three cell systems, and proposes a set of criteria that ROS should satisfy for their consideration as mediators in hypoxic transduction cascades. In none of the three cell types do ROS satisfy these criteria, and thus it appears that alternative mechanisms are responsible for the transduction cascades linking hypoxia to the release of neurotransmitters in chemoreceptor cells, contraction in pulmonary artery smooth muscle cells and erythropoietin secretion in erythropoietin producing cells.

Molecular identification of Kvalpha subunits that contribute to the oxygen-sensitive K+ current of chemoreceptor cells of the rabbit carotid body.

Rabbit carotid body (CB) chemoreceptor cells possess a fast-inactivating K+ current that is specifically inhibited by hypoxia. We have studied the expression of Kvalpha subunits, which might be responsible for this current. RT-PCR experiments identified the expression of Kv1.4, Kv3.4, Kv4.1 and Kv4.3 mRNAs in the rabbit CB. There was no expression of Kv3.3 or Kv4.2 transcripts. Immunocytochemistry with antibodies to tyrosine hydroxylase (anti-TH) and to specific Kv subunits revealed the expression of Kv3.4 and Kv4.3 in chemoreceptor cells, while Kv1.4 was only found in nerve fibres. Kv4.1 mRNA was also found in chemoreceptor cells following in situ hybridization combined with anti-TH antibody labelling. Kv4.1 and Kv4.3 appeared to be present in all chemoreceptor cells, but Kv3.4 was only expressed in a population of them. Electrophysiological experiments applying specific toxins or antibodies demonstrated that both Kv3.4 and Kv4.3 participate in the oxygen-sensitive K+ current of chemoreceptor cells. However, toxin application experiments confirmed a larger contribution of members of the Kv4 subfamily. [Ca2+]i measurements under hypoxic conditions and immunocytochemistry experiments in dispersed CB cells demonstrated the expression of Kv3.4 and Kv4.3 in oxygen-sensitive cells; the presence of Kv3.4 in the chemoreceptor cell membrane was not required for the response to low PO2. In summary, three Kv subunits (Kv3.4, Kv4.1 and Kv4.3) may be involved in the fast-inactivating outward K+ current of rabbit CB chemoreceptor cells. The homogeneous distribution of the Kv4 subunits in chemoreceptor cells, along with their electrophysiological properties, suggest that Kv4.1, Kv4.3, or their heteromultimers, are the molecular correlate of the oxygen-sensitive K+ channel.