Previously we demonstrated that chronic hypoxia (CH) induces an inflammatory condition characterized by immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. It is well established that chronic inflammatory pain induces the expression of acid-sensitive ion channels (ASIC) in primary sensory neurons, where they contribute to hyperalgesia and allodynia. The present study examines the effect of CH on ASIC expression in petrosal ganglion (PG), which contains chemoafferent neurons that innervate oxygen-sensitive type I cells in the carotid body. Five isoforms of ASIC transcript were increased ∼1.5-2.5-fold in PG following exposure of rats to 1, 3, or 7 days of hypobaric hypoxia (380 Torr). ASIC transcript was not increased in the sympathetic superior cervical ganglion (SCG). In the PG, CH also increased the expression of channel-interacting PDZ domain protein, a scaffolding protein known to enhance the surface expression and the low pH-induced current density mediated by ASIC3. Western immunoblot analysis showed that CH elevated ASIC3 protein in PG, but not in SCG or the (sensory) nodose ganglion. ASIC3 transcript was likewise elevated in PG neurons cultured in the presence of inflammatory cytokines. Increased ASIC expression was blocked in CH rats concurrently treated with the nonsteroidal anti-inflammatory drug ibuprofen (4 mg·kg(-1)·day(-1)). Electrophysiological recording of carotid sinus nerve (CSN) activity in vitro showed that the specific ASIC antagonist A-317567 (100 μM) did not significantly alter hypoxia-evoked activity in normal preparations but blocked ∼50% of the hypoxic response following CH. Likewise, a high concentration of ibuprofen, which is known to block ASIC1a, reduced hypoxia-evoked CSN activity by ∼50% in CH preparations. Our findings indicate that CH induces inflammation-dependent phenotypic adjustments in chemoafferent neurons. Following CH, ASIC are important participants in chemotransmission between type I cells and chemoafferent nerve terminals, and these proton-gated channels appear to enhance chemoreceptor sensitivity.

Experiments in recent years have revealed labile electrophysiological and neurochemical phenotypes in primary afferent neurons exposed to specific stimulus conditions associated with the development of chronic pain. These studies collectively demonstrate that the mechanisms responsible for functional plasticity are primarily mediated by novel neuroimmune interactions involving circulating and resident immune cells and their secretory products, which together induce hyperexcitability in the primary sensory neurons. In another peripheral sensory modality, namely the arterial chemoreceptors, sustained stimulation in the form of chronic hypoxia (CH) elicits increased chemoafferent excitability from the mammalian carotid body. Previous studies which focused on functional changes in oxygen-sensitive type I cells in this organ have only partially elucidated the molecular and cellular mechanisms which initiate and control this adaptive response. Recent studies in our laboratory indicate a unique role for the immune system in regulating the chemo-adaptive response of the carotid body to physiologically relevant levels of hypoxia.

Previous studies in our laboratory established that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) facilitate the open state of a subset of K+ channels in oxygen-sensitive type I cells of the carotid body. Thus pharmacological inhibition of NOX or deletion of a NOX gene resulted in enhanced chemoreceptor sensitivity to hypoxia. The present study tests the hypothesis that chronic hypoxia (CH)-induced hypersensitivity of chemoreceptors is modulated by increased NOX activity and elevated levels of ROS. Measurements of dihydroethidium fluorescence in carotid body tissue slices showed that increased ROS production following CH (14 days, 380 Torr) was blocked by the specific NOX inhibitor 4-(2-amino-ethyl)benzenesulfonyl fluoride (AEBSF, 3 microM). Consistent with these findings, in normal carotid body AEBSF elicited a small increase in the chemoreceptor nerve discharge evoked by an acute hypoxic challenge, whereas after 9 days of CH the effect of the NOX inhibitor was some threefold larger (P<0.001). Evaluation of gene expression after 7 days of CH showed increases in the isoforms NOX2 (approximately 1.5-fold) and NOX4 (approximately 3.8-fold) and also increased presence of the regulatory subunit p47phox (approximately 4.2-fold). Involvement of p47phox was further implicated in studies of isolated type I cells that demonstrated an approximately 8-fold and an approximately 11-fold increase in mRNA after 1 and 3 days, respectively, of hypoxia in vivo. These findings were confirmed in immunocytochemical studies of carotid body tissue that showed a robust increase of p47phox in type I cells after 14 days of CH. Our findings suggest that increased ROS production by NOX enzymes in type I cells dampens CH-induced hypersensitivity in carotid body chemoreceptors.

CH in rat carotid body induces an inflammatory response which is characterized by immune cell invasion and the expression of pro‐inflammatory cytokines. In the present study, we have investigated the role of ET‐1 and ET‐A receptors in the development of CH‐induced inflammation. Following 7 days of CH (380 Torr), immunocytochemical (ICC) studies demonstrated elevated levels of ET‐1 and ET‐A receptors in O2‐sensitive type I cells. In addition, double‐label ICC showed that following CH, ET‐A receptors were also expressed on invasive CD45+ immune cells distributed in tissue surrounding chemosensory cell lobules. Concurrent treatment with the ET‐A/B receptor antagonist, bosentan (200mg/kg/day), blocked ED‐1+ immune cell invasion and the up‐regulation of cytokines, including interleukin‐1β (IL‐1β), IL‐6 and tumor necrosis factorα (TNFα). Moreover, bosentan treatment blocked CH‐induced increased expression of acid sensitive ion channels (ASICs) in chemoafferent neurons in the petrosal ganglion (PG), and drug treatment significantly reduced chemoreceptor hypersensitivity. Our findings are consistent with the hypothesis that CH‐induced inflammation involves the up‐regulation and release of ET‐1 from type I cells; ET‐1 acts in an autocrine/paracrine mechanism via ET‐A receptors on chemosensory type I cells, and immune cells to promote an inflammatory response. USPHS Grants NS 12636 and NS 07938.

sGC in oxygen‐sensitive carotid body type I cells is activated by the diffusible gas, nitric oxide (NO). In rats exposed to CH, sGC is down‐regulated, with a corresponding drop in NO‐stimulated production in cGMP. Moreover, CH induces an invasion of activated macrophages and the production of inflammatory cytokines in chemosensory tissue. The present study investigates the relationship between inflammation and the down‐regulation of sGC in type I cells. Quantitative PCR (qPCR) assays of carotid bodies from 7‐day CH (380 Torr) rats demonstrated a 50–500% increase in expression of cytokines interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNFα). CH (7 days) elicited a >90% reduction in the expression of sGC. In contrast, animals concurrently treated with anti‐inflammatory drugs, ibuprofen (4mg/kg/day) or bosentan (200mg/kg/day), expressed normal levels of the sGC gene. sGC expression was reduced by 50% in normal type I cells cultured for 48 hrs in the presence of TNFα (50 ng/ml). However, exposure to TNFα did not alter the expression of the gene for tyrosine hydroxylase (TH). Our data indicate that a CHinduced increase in cytokine production directly influences the expression of sGC, a signaling molecule which mediates type I cell inhibition. Inflammation appears to be an important factor in the readjustment of chemoreceptor sensitivity following CH. USPHS Grants NS 12636 and NS 07938.

Exposure to CH elicits a marked increase in chemosensory excitability in the mammalian CB. In chronic inflammatory pain unique hyperexcitable phenotypes emerge in primary afferent neurons as a consequence of invasive activated immune cells and their secretory cytokine products. The present study examines the effect of CH on the expression of inflammatory cytokines in mouse CB, and the effect of IL-6 gene knockout (KO) on CH-induced chemoreceptor hyperexcitability. Real-time quantitative PCR showed that 1, 3 or 7 days of hypobaric hypoxia (BP = 380 Torr) elicited significant increases (4–12 fold) in the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, the chemokine, monocyte chemoattractant protein-1 (MCP-1), was increased 6-, 31- and 41-fold, on days 1, 3, and 7 of CH, respectively. In vitro recordings of carotid sinus nerve (CSN) activity showed that following 10–12 days of CH, responses evoked by an acute hypoxic challenge were increased by approximately 30% in normal mice. Responses to acute hypoxia were similar to normal in IL-6 KO preparations, but following CH they were not elevated. The data are in agreement with our previous findings that CH elicits an inflammatory condition in rat CB, and they further suggest that increased cytokine production is critical for adaptation and the development of hypersensitivity. USPHS Grants NS 12636 and NS 07938.

Multiple studies have shown that chronic hypoxia (CH) elicits a time-dependent upregulation of carotid body chemoreceptor sensitivity in mammals. In the present study, we demonstrate that enhanced excitation is accompanied by a parallel increase of nitric oxide (NO)-dependent inhibition, which acts via a CH-induced modification of the normal mechanism in O(2)-sensitive type I cells. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), elicits a progressively larger increase in carotid sinus nerve (CSN) chemoreceptor activity following incremental increases in CH exposure lasting 1-16 days. The inhibitory effect of the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), on CSN activity is enhanced following CH. However, the activation of soluble guanylate cyclase (sGC) by SNAP, assessed via production of cGMP, is impaired, along with decreased expression of sGC mRNA transcript. Inhibition of hypoxia-evoked Ca(2+) responses by SNAP is mediated via a cGMP/protein kinase G (PKG)-dependent mechanism in normal type I cells that is sensitive to the PKG inhibitor KT-5823, but following CH, inhibitory responses are minimally sensitive to PKG inhibition. The data are consistent with the hypothesis that CH hampers cGMP-mediated inhibition of type I cells in favor of an alternative mechanism.

The current study examines the hypothesis that ASICs are involved in chronic hypoxia (CH, 3–14 days @ BP = 380 Torr)-induced hypersensitivity in rat carotid body. Expression of ASIC1a/1b, ASIC2a and ASIC3 genes were significantly elevated (~1.3–2.5-fold) in the PG following 3 or 7 days of CH. Western immunoblot showed increased expression of ASIC3 protein in PG, but not in nodose ganglion (NG) following CH. Exposure to pH 6.0 solution elicited a moderate increase in [Ca2+] in primary cultures of normal PG neurons. However, following 5–6 daysI of CH, exposure to low pH evoked a significantly enhanced [Ca2+]I-response in many small diameter neurons. The ASICs antagoinist, ibuprofen (200 μM), which has been shown to directly block currents mediated by ASIC3 and ASIC3/2b hetero-multimers, marginally reduced chemoreceptor nerve activity evoked by acute hypoxia in superfused carotid body preparations from normal animals. However following 8–10 days of CH, ibuprofen markedly inhibited evoked nerve activity. Finally, concurrent treatment with ibuprofen (4 mg/kg/day), an agent known to depress ASICs expression associated with chronic inflammation, prevented the CH-induced enhancement of chemosensitivity. The data suggest that low pH and acid metabolites may contribute to depolarization of carotid body afferent terminals in CH adapted animals. USPHS Grants NS 12636 and NS 07938.

O2-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca2+ entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O2-sensitivity of these cells currently focus on the coupling between local PO2 and the open-closed state of K+-channels. Amongst multiple competing hypotheses is the notion that K+-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing PO2. In O2-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that EM and K+-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O2 facilitate cell repolarization via specific subsets of K+-channels.

O2-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells, where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca2+ entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O2-sensitivity of these cells have focused primarily on the relationship between PO2 and the activity of K+-channels. An important hypothesis developed by Acker and his colleagues suggests that coupling between local PO2 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 PO2, and a subset of K+-channels which control the EM, should close as ROS levels decrease. In O2-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).

Recent studies indicate that chemoafferent nerve fiber excitation in the rat carotid body is mediated by acetylcholine and ATP, acting at nicotinic cholinergic receptors and P2X2 purinoceptors, respectively. We previously demonstrated that, after a 10- to 14-day exposure to chronic hypoxia (CH), the nicotinic cholinergic receptor blocker mecamylamine no longer inhibits rat carotid sinus nerve (CSN) activity evoked by an acute hypoxic challenge. The present experiments examined the effects of CH (9-16 days at 380 Torr) on the expression of P2X2 purinoceptors in carotid body and chemoafferent neurons, as well as the effectiveness of P2X2 receptor blocking drugs on CSN activity evoked by hypoxia. In the normal carotid body, immunocytochemical studies demonstrated a dense plexus of P2X2-positive nerve fibers penetrating lobules of type I cells. In addition, type I cells were lightly stained, indicating P2X2 receptor expression. After CH, the intensity of P2X2 receptor immunostaining was maintained in chemosensory type I cells and in the soma of chemoafferent neurons. P2 receptor expression on type I cells was confirmed by demonstrations of ATP-evoked increased intracellular Ca2+; this response was modulated by simultaneous exposure to hypoxia. In normal preparations, CSN activity evoked by hypoxia in vitro was 65% inhibited in the presence of specific P2X2 receptor antagonists. However, unlike the absence of mecamylamine action after CH, P2X2 antagonists remained effective against hypoxia-evoked activity after CH. Our findings indicate that ATP acting at P2X2 receptors contributes to adjusted chemoreceptor activity after CH, indicating a possible role for purinergic mechanisms in the adaptation of the carotid body in a chronic low-O2 environment.

Recent studies in our laboratory suggest that chemoreceptor adaptation in carotid body is mediated by immune cell invasion and cytokine expression. In chronic neuropathic pain immune cell recruitment is triggered by production of inflammatory cytokines in Schwann cells distal to a nerve lesion (Shamash, et al., J. Neurosci. 22:3052,2002), leading to primary sensory neuron hyperexcitability and hyperalgesia. The present study uses quantitative RT‐PCR (qPCR) to compare the time‐courses of inflammatory cytokine production versus immune cell invasion in carotid bodies exposed to hypobaric hypoxia (380 Torr). IL‐6 expression increased 2‐fold after 24h, and 8‐fold and 12‐fold, respectively, following 3 and 7 days of CH. IL‐1β and tumor necrosis factor‐α were not changed following 24h, but were significantly increased at 3 and 7 days of low O2. Markers for immune cell invasion, including CD14 and the gp91phox, were not changed following 24h of hypoxia. However, expression of these genes was increased 2–3‐fold in carotid body at 3 and 7 days of CH. Likewise, the expression of the chemokine, monocyte chemoattractant protein‐1 (MCP‐1), increased at 3 (2‐fold) and 7 (4.5‐fold) days, but not following 1 day of hypoxia. Present data are consistent with the hypothesis that prolonged hypoxia‐induced depolarization of type I cells elicits IL‐6 production which activates a cytokine network resulting in MCP‐1 expression, recruitment of circulating leukocytes, and enhanced cytokine activity.

Current views suggest that oxygen sensing in the carotid body occurs in chemosensory type I cells, which excite synaptically apposed chemoafferent nerve terminals in the carotid sinus nerve (CSN). Prolonged exposure in a low-oxygen environment [i.e., chronic hypoxia (CH)] elicits an elevated stimulus-evoked discharge in chemoreceptor CSN fibers (i.e., increased chemosensitivity). In the present study, we evaluated cholinergic chemotransmission in the rat carotid body in an effort to test the hypothesis that CH enhances ACh-mediated synaptic activity between type I cells and chemoafferent nerve terminals. Animals were exposed in a hypobaric chamber (barometric pressure = 380 Torr) for 9-22 days before evaluation of chemoreceptor activity using an in vitro carotid body/CSN preparation. Nerve activity evoked by ACh was significantly larger (P < 0.01) after CH, suggesting increased expression of cholinergic receptors. Approximately 80% of the CSN impulse activity elicited by ACh (100- or 1,000-microg bolus) in both normal and CH preparations was blocked by the specific nicotinic receptor antagonist mecamylamine (100 microM). CSN activity elicited by acute hypoxia or hypercapnia in normal preparations was likewise blocked (> or =80%) in the presence of 100 muM mecamylamine, but after CH the enhanced CSN activity elicited by acute hypoxia or hypercapnia was not reduced in the presence of 100 or 500 microM mecamylamine. A muscarinic receptor antagonist, atropine (10 microM), and a specific nicotinic receptor alpha7 subunit antagonist, methyllycaconatine (50 nM), blocked approximately 50% of the hypoxia-evoked activity in normal preparations but were ineffective after CH. Prolonged exposure to hypoxia appears to dramatically alter chemotransmission in the carotid body, and may induce alternative neurotransmitter mechanisms and/or electrical coupling between type I cells and chemoafferent nerve terminals.

Membrane potential in oxygen-sensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K(+) channels. Coupling of Po(2) to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing Po(2) and a subset of K(+) channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47(phox) gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 microM) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formyl-Met-Leu-Phe (1 microM), arachidonic acid (10 microM), and cytochalasin B (5 microg/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 microM). KO neutrophils did not respond; however, azide (5 microM) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K+ current and increased intracellular Ca2+ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca2+ and enhanced the depression of K+ current in low O(2). Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K+ channel activity in hypoxia.

Exposure of the carotid body to hypoxia elicits increased neural activity in the carotid sinus nerve (CSN), and reflex cardio-pulmonary adjustments which mitigate the adverse effects of hypoxemia. Increased carotid body activity occurs at relatively moderate arterial P02, in contrast to the severe hypoxia required to elicit metabolic and functional adjustments in non-02 sensing tissues (S.J.Fidone et al. 1997). Chemosensory type I cells derived from neuroectoderm are responsible for this exquisite sensitivity, and numerous laboratories have reported that low P02 inhibits the conductance of a variety of voltage sensitive and voltage-insensitive K+-channels in these cells. Yet the molecular mechanism underlying the P02 modulation of cell currents remains uncertain and controversial (H.Acker et al 1994, A.M.Riesco-Fagundo et al2001). Various heme proteins have been proposed as primary O2 sensors, and one set of data in particular suggests the involvement of a multi-component cytochrome b-containing NADPH oxidase which may be similar if not identical to the superoxide generating enzyme commonly found in phagocytic cells (H.Acker et al. 1994) (H.Acker et al. 1994), but the relationship between PO2 and ROS levels in type I cells has not been firmly established. In other cells and tissues hypoxia can increase or decrease ROS production in either mitochondria or via NADPH oxidase (I.O’Kelly et al. 2000, G.B Waypa et al. 2001). In addition, the target of ROS in type I cells is an unknown and critical factor in determining the effect of NADPH oxidase on cell activity. Recent studies have indicated that voltage-sensitive K+-channels in type I cells are modulated by hypoxia via a mechanism independent of soluble factors such as ROS (A.M. Riesco-Fagundo et al. 2001). Thus ROS do not appear to be necessary for cell activation. On the other hand, if hypoxia enhances NADPH oxidase activity, elevated ROS levels may increase the open probability of K+-channels thus facilitating cell repolarization. Such a scheme is consistent with elevated CSN activity in p47phox-gene deleted animals. Clarification of these issues must await future measurements of the effect of hypoxia on NADPH oxidase activity, and evaluation of the interaction of ROS with the chemotransduction machinery in type I cells.

Reactive oxygen species generated from NADPH oxidase(s) in airway smooth muscle cells and pulmonary artery smooth muscle cells are important signaling intermediates. Nox4 appears to be the predominant gp91 homologue in these cells. However, expression of NADPH oxidase components is dependent on phenotype, and different homologues may be expressed during different functional states of the cell. NADPH oxidase(s) appear to be important not only for mitogenesis by these cells, but also for O2 sensing. The regulation of NADPH oxidase(s) in airway and pulmonary artery smooth muscle cells has important implications for the pathobiochemistry of asthma and pulmonary vascular diseases.