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Detecting and reacting to acute perturbation in the partial pressure of atmospheric oxygen (pO), particularly hypoxia, is a fundamental adaptive mechanism which is conserved throughout the animal kingdom. In mammals, a number of cellular systems respond, often co-operatively as oxygen availability becomes compromised, with the express aim of maximising oxygen uptake by the lungs and of optimising its delivery to the metabolically most active tissues. Thus, during hypoxia, ventilation rate and depth are increased to maximize air flow across the gaseous exchange surface, local lung perfusion rates become rapidly matched to local alveolar ventilation and systemic arteriolar dilatation ensures that tissue and cerebral blood flow become swiftly optimized.

Pulmonary arteries constricts in response to hypoxia and thereby aid ventilation-perfusion matching in the lung. Although O-sensitive mechanisms independent of mitochondria may also play a role, it is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation and that this underpins, at least in part, cell activation. Despite this consensus, the mechanism by which inhibition of mitochondrial oxidative phosphorylation couples to Ca-dependent vasoconstriction has remained elusive. To date, the field has focussed on the role of the cellular energy status (ATP), reduced redox couples and reactive oxygen species respectively, but investigation of these hypotheses has delivered conflicting data and failed to unite the field.

O-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells, where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O-sensitivity of these cells have focused primarily on the relationship between PO and the activity of K-channels. An important hypothesis developed by Acker and his colleagues suggests that coupling between local PO and the open-closed state of K- channels is mediated by reactive oxygen species (ROS) generated by a phagocytic-like multisubunit enzyme, NADPH oxidase (Nox)(1). According to this scheme, ROS production will occur in proportion to the prevailing PO, and a subset of K-channels which control the EM, should close as ROS levels decrease. In O-sensitive cells contained in lung neuroepithelial bodies (NEB), experiments have confirmed that ROS levels decrease in hypoxia, and that EM and K-channel activity are indeed controlled by ROS produced by an Nox isoform similar, if not identical to the enzyme expressed in phagocytic cells that use ROS as part of an extracellular killing mechanism activated in response to invading micro-organisms(8; 15).

All nucleated cells depend on heme for their survival, as heme senses or uses oxygen. In fact, heme is a prosthetic moiety of various hemoproteins such as hemoglobin, myoglobin, and cytochromes. Accordingly, heme must be synthesized and degraded within an individual cell, because heme cannot be recycled among different cells, except for senescent erythrocytes, which are phagocytosed by macrophages in the reticuloendothelial system (for review, Shibahara, 2003). Heme, derived from hemoproteins, is broken down by heme oxygenase, which catalyzes the oxidative breakdown of heme, generating biliverdin, carbon monoxide (CO), and iron (Fig. 1). These heme degradation products are important bioactive molecules (For review, Shibahara, 2003 and references therein). Bilirubin functions as a chain-breaking antioxidant. CO represents a direct marker for heme catabolism, and binds to hemoglobin to form carboxyhemoglobin, which is transported to the lungs and is excreted in exhaled air. CO has received much attention because of its physiological functions similar to those of NO. Iron is transported to the entire tissues, especially bone marrow, and is reutilized for erythropoiesis and heme biosynthesis.

The carotid body plays a central role in initiating cardiovascular and respiratory responses to hypoxia. Previous work from this laboratory has demonstrated that hypoxia inhibits TASK-like K -channels in isolated neonatal rat carotid body type I cells (Buckler et al., 2000). The consequent reduction in background K-current leads to type-1 cell membrane depolarisation (Buckler, 1997), voltage-gated calcium entry and thus excitation of the carotid body. The mechanisms by which hypoxia modulates these K channels is still unknown, however the effects of hypoxia are mimicked by a wide range of inhibitors of oxidative phosphorylation. Uncouplers, electron transport inhibitors (e.g. cyanide, rotenone & myxothiazol) and inhibitors of ATP synthase (oligomycin) are all potent stimulants of the carotid body (Anichkov & Belen‘kii, 1963; Gonzalez et al., 1994) and potent inhibitors of background K-current (Wyatt & Buckler, 2004). Moreover background K-current sensitivity to hypoxia is lost in the presence of metabolic inhibitors (Wyatt & Buckler, 2004) suggesting that ATP synthesis may be a prerequisite for the expression of oxygen sensitivity. Further support for the idea that background K-channel activity may be dependent upon cellular ATP levels comes from the observation that following patch excision (from the cell attached to the inside out configuration) there is an abrupt rundown of background K-channel activity which can be partially reversed by the addition of mM levels of ATP to the intracellular solution (Williams & Buckler, 2004). In this study we have further investigated the action of ATP on background K-channels in order to evaluate the potential role for ATP in modulating channel activity and to identify general mechanisms by which ATP might act.

Exogenously administered dopamine (DA) is liable to penetrate into the carotid body, which, as opposed to the brain, has no endothelial barrier. DA is stored in the secretory vesicles of chemoreceptor cells. The vesicles are reminiscent of micelle-like entities and the hydrophilic properties of DA molecules make it dubious that DA could be packed and stay sustained in such an environment. The possible problems with the intravesicular arrangement of DA molecules may be one reason for thecomplex, often erratic, and as yet not full well understood DA action in the chemosensing process. DA displays a spate of varying effects, from stimulation to inhibition, on carotid chemosensory discharge and ventilation, depending on the species, the dose, and the presynaptic or postsynaptic dopamine D receptor it interacts with (see for review Gonzales et al., 1994).

Excitatory effects on carotid body (CB) chemotransduction have been described for both adenosine and ATP. Adenosine when applied exogenously increases carotid sinus nerve (CSN) discharges in the cat, (McQueen and Ribeiro, 1983) and (Runold et al., 1990). Administration of adenosine and drugs that increase its endogenous levels stimulate ventilation in rats, an effect abolished by the section of CSN and mediated by A receptors (Monteiro and Ribeiro, 1987, 1989; Ribeiro and Monteiro, 1991). In humans, the intravenous infusion of adenosine causes hyperventilation and dyspnoea, an effect attributed to the activation of CB (Watt and Routledge, 1985, Watt et al., 1987; Maxwell et al., 1986; 1987, Uematsu et al., 2000). The excitatory effect of ATP at the CB described by Zhang et al. (2000) in co-cultures of type I cells with petrosal neurons was further supported by the finding that mice deficient in P2X showed a markedly attenuated ventilatory response to hypoxia (Rong et al., 2003) and by the detection of hypoxia- evoked ATP release from chemoreceptor cells of the rat carotid body (Buttigieg and Nurse, 2004).

Hypoxic modulation of K channels is now firmly established in a variety of tissue types (Lopez-Barneo et al., 1988; Weir and Archer, 1998; Franco- Obregon et al., 1995; Youngson et al., 1993; Hool, 2001; Jiang and Haddad, 1994; Rychkov et al., 1998), and hypoxic modulation of specific channel types can also be reproduced in recombinant expression systems (Fearon et al., 2000; Lewis et al., 2001; Lewis et al., 2002; Williams et al., 2004), providing an opportunity to probe the molecular mechanism(s) of O sensing by ion channels (see e.g. Kemp et al., this volume). In addition, the consequences for cell function of hypoxic ion channel modulation are fairly well established. In most cases, an appropriate response to hypoxia (such as systemic vasodilation, pulmonary vasoconstriction or carotid body glomus cell transmitter release – see (Lopez-Barneo et al., 2001) for review) involves modulation of [Ca]i and this occurs primarily via modulation of Ca influx (but in the lung vasculature this is contentious - see e.g. (Evans and Dipp, 2002)). Ca influx can be regulated either through control of membrane potential via modulation of K channel activity (Buckler and Vaughan-Jones, 1994; Wyatt et al., 1995; Osipenko et al., 1997; Weir and Archer, 1998), or via a direct effect on Ca channels (Franco- Obregon et al., 1995; Hool, 2001).

The carotid bodies are the primary peripheral chemoreceptors. They respond to a fall in blood pO, a rise in blood pCO and consequent fall in pH by releasing neurotransmitters. These increase the firing frequency of the carotid sinus nerves which then correct the pattern of breathing via an action at the brainstem. It is now generally accepted that the type 1 or glomus cells are the chemosensory element within the carotid body. However, the precise mechanism by which a fall in pO excites the neurotransmitter rich type 1 cells has been the subject of hearty debate for decades now.

Exposure to chronic hypoxia (CH) initiates cellular responses designed to counteract this deleterious stimulus, providing a physiological response to low oxygen. However, long-term exposure to CH, such as that which occurs in cardiorespiratory diseases such as ischaemic stroke, can also have pathological consequences. In many cases, CH alters the transcription of genes encoding numerous proteins, secondary to accumulation of the transcriptional activator hypoxia inducible factor-1 (HIF-1) (Schofield and Ratcliffe, 2004). In contrast, we recently reported that hypoxic regulation of the plasma membrane expression of L-type Ca channel α subunits occurred in a post-transcriptional manner due to the trafficking of these subunits towards, and / or their retention within, the plasma membrane (Scragg et al., 2004). This process involved the altered production of amyloid β peptides (A Ps), since it was inhibited by selective inhibitors of the secretases involved in the production of these peptides, and mimicked by exogenous AβP. This regulation of the functional membrane expression of a voltage-gated Ca channel may contribute to the Ca dyshomeostasis seen in Alzheimer’s disease, a prevalent disorder in which hypoxia / ischaemia is a predisposing factor (Moroney et al., 1996).