Characterization of ectonucleotidase expression in the rat carotid body: regulation by chronic hypoxia.

The carotid body (CB) chemoreflex maintains blood Po2 and Pco2/H+ homeostasis and displays sensory plasticity during exposure to chronic hypoxia. Purinergic signaling via P1 and P2 receptors plays a pivotal role in shaping the afferent discharge at the sensory synapse containing catecholaminergic chemoreceptor (type I) cells, glial-like type II cells, and sensory (petrosal) nerve endings. However, little is known about the family of ectonucleotidases that control synaptic nucleotide levels. Using quantitative PCR (qPCR), we first compared expression levels of ectonucleoside triphosphate diphosphohydrolases (NTPDases1,2,3,5,6) and ecto-5'-nucleotidase (E5'Nt/CD73) mRNAs in juvenile rat CB vs. brain, petrosal ganglia, sympathetic (superior cervical) ganglia, and a sympathoadrenal chromaffin (MAH) cell line. In whole CB extracts, qPCR revealed a high relative expression of surface-located members NTPDase1,2 and E5'Nt/CD73, compared with low NTPDase3 expression. Immunofluorescence staining of CB sections or dissociated CB cultures localized NTPDase2,3 and E5'Nt/CD73 protein to the periphery of type I clusters, and in association with sensory nerve fibers and/or isolated type II cells. Interestingly, in CBs obtained from rats reared under chronic hypobaric hypoxia (~60 kPa, equivalent to 4,300 m) for 5-7 days, in addition to the expected upregulation of tyrosine hydroxylase and VEGF mRNAs, there was a significant upregulation of NTPDase3 and E5'Nt/CD73 mRNA, but a downregulation of NTPDase1 and NTPDase2 relative to normoxic controls. We conclude that NTPDase1,2,3 and E5'Nt/CD73 are the predominant surface-located ectonucleotidases in the rat CB and suggest that their differential regulation during chronic hypoxia may contribute to CB plasticity via control of synaptic ATP, ADP, and adenosine pools.

Age-related changes in immunoreactivity for dopamine ß-hydroxylase in carotid body glomus cells in spontaneously hypertensive rats.

The purpose of this study was to investigate immunoreactivity for dopamine ß-hydroxylase (DBH) and tyrosine hydroxylase (TH) in carotid body (CB) glomus cells in spontaneously hypertensive rats (SHR/Izm) at 4 (prehypertensive stage), 8 (early stage of developmental hypertension), 12 (later stage of developmental hypertension), and 16weeks of age (established hypertensive stage). Age-matched Wistar Kyoto rats (WKY/Izm) were used as controls. Staining properties for TH were similar between both strains at each age. Regarding DBH immunostaining, although some glomus cells showed intense DBH immunoreactivity at 4weeks of age, these cells were rarely observed at 8, 12, and 16weeks of age in WKY/Izm. In SHR/Izm, intense DBH immunoreactivity was observed in some glomus cells at 4weeks of age, these cells were also observed at 8 and 12weeks of age, and their number increased at 16weeks of age. An image analysis showed that the percentage of DBH-immunopositive glomus cells in WKY/Izm was approximately 30% at 4weeks of age and significantly decreased to approximately 10% at 8, 12, and 16weeks of age (p<0.05). This percentage in SHR/Izm was approximately 40% at each age. The gray scale intensity for DBH immunoreactivity in DBH-immunopositive glomus cells was similar in both strains at 4weeks of age, but became significantly lower in WKY/Izm and higher in SHR/Izm with increase in age (p<0.05). These results suggest that noradrenaline in glomus cells plays an important role in the regulation of neurotransmission between CB and afferent nerves during developmental hypertension.

Effect of Systemic Application of 5-Hydroxytryptamine on Hypoglossal Nerve Discharge in Anesthetized Rats.

The aim of this study is to investigate whether systemic 5-hydroxytryptamine (5-HT) can promote long-lasting form of respiratory plasticity in vivo via 5-HT2AR-activated protein kinase C (PKC) mechanism. The frequency and peak amplitude of hypoglossal nerve discharges in anesthetized rats were compared before and after intravenous injections of different treatments, including saline, 5-HT, ketanserin tartrate, or staurosporine. The administration of 5-HT at a systemic bolus imposed an initial ephemeral inhibition subsequently followed by striking facilitation, which demonstrates a biphasic manner of hypoglossal nerve output in anesthetized adult rats. The facilitatory stage conformed to the reinforced hypoglossal activity that lasted for more than 60 min after drug administration. The 5-HT evoked biphasic manner of hypoglossal output and hypoglossal nerve activity LTF (hLTF) were 5-HT2A receptor-dependent and coupled to PKC activation. The initial inhibition of hypoglossal activity was associated with nodose ganglion, and the subsequent facilitation was associated with carotid body. The reactive oxygen species (ROS) formation was triggered in the systemic 5-HT2-dependent hLTF model in vivo. The expressions of immunofluorescent histochemistry provide morphological evidence of a 5-HT/5-HT2A receptor coupled to PKC mechanism. In conclusion, systemic 5-HT challenge contributes to long-lasting form of respiratory plasticity and to elicit hLTF or elevated hLTF in animals, which with decreased or even with inhibited peripheral inhibitory activations. The effect of systemic 5-HT was regulated by a 5-HT2AR-activated PKC mechanism.

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat.

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X3 purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.

Development and aging are oxygen-dependent and correlate with VEGF and NOS along life span.

During development and aging, vascular remodeling represents a critical adaptive response to modifications in oxygen supply to tissues. Hypoxia inducible factor (HIF) has a crucial role and is modulated by oxygen levels, with an age-dependent response in neonates, adult, and aged people. ROS are generated under hypoxic conditions and the accumulation of free radicals during life reduces the ability of tissues to their removal. In this immunohistochemical study we investigated the presence and localization of VEGF and iNOS in human carotid bodies (CB) sampled at autopsy from three children (mean age - 2 years), four adult young subjects (mean age - 44.3 years), and four old subjects (mean age - 67.3 years). VEGF immunoreactivity was significantly enhanced in CB tissues from the children (7.2 ± 1.2%) and aged subjects (4.7 ± 1.7%) compared with the young adults (1.4 ± 0.7%). On the other hand, iNOS immunoreactivity was enhanced in CB tissues from the children (0.4 ± 0.04%) and young adult subjects (0.3 ± 0.02%) compared with the old subjects (0.2 ± 0.02%). Prevention of oxygen desaturation, reducing all causes of hypoxemia from neonatal life to aging would decrease the incidence of diseases in the elderly population with lifespan extension.

Carbon monoxide (CO) and hydrogen sulfide (H(2)S) in hypoxic sensing by the carotid body.

Carotid bodies are sensory organs for monitoring arterial blood oxygen (O(2)) levels, and the ensuing reflexes maintain cardio-respiratory homeostasis during hypoxia. This article provides a brief update of the role of carbon monoxide (CO) and hydrogen sulfide (H(2)S) in hypoxic sensing by the carotid body. Glomus cells, the primary site of O(2) sensing in the carotid body express heme oxygenase-2 (HO-2), a CO catalyzing enzyme. HO-2 is a heme containing enzyme and has high affinity for O(2). Hypoxia inhibits HO-2 activity and reduces CO generation. Pharmacological and genetic approaches suggest that CO inhibits carotid body sensory activity. Stimulation of carotid body activity by hypoxia may reflect reduced formation of CO. Glomus cells also express cystathionine γ-lyase (CSE), an H(2)S generating enzyme. Exogenous application of H(2)S donors, like hypoxia, stimulate the carotid body activity and CSE knockout mice exhibit severely impaired sensory excitation by hypoxia, suggesting that CSE catalyzed H(2)S is an excitatory gas messenger. Hypoxia increases H(2)S generation in the carotid body, and this response was attenuated or absent in CSE knockout mice. HO inhibitor increased and CO donor inhibited H(2)S generation. It is proposed that carotid body response to hypoxia requires interactions between HO-2-CO and CSE-H(2)S systems.

Increased total volume and dopamine ß-hydroxylase immunoreactivity of carotid body in spontaneously hypertensive rats.

Under hypertension, it has been reported that the carotid body (CB) is enlarged and noradrenaline (NA) content in CB is increased. Therefore, it is hypothesized that morphological and neurochemical changes in CB are induced in hypertensive animal models. In the present study, we examined the morphological features and dopamine ß-hydroxylase (DBH) immunoreactivity in CB of spontaneously hypertensive rats (SHR/Izm) and Wistar Kyoto rats (WKY/Izm). The CB of SHR/Izm was elongated in terms of the cross section of center and was enlarged in the reconstructed images compared with that of WKY/Izm, and the total volume of CB in SHR/Izm (0.048 ± 0.004 mm³) was significantly (p<0.05) increased compared with the value in WKY/Izm (0.032 ± 0.006 mm³). By immunohistochemistry, immunoreactivity for tyrosine hydroxylase in CB was mainly observed in glomus cells and the immunostaining properties were similar between WKY/Izm and SHR/Izm. On the other hand, DBH immunoreactivity was mainly observed in nerve fibers around blood vessels and observed in a few glomus cells in CB of WKY/Izm. The number of glomus cells with strong DBH immunoreactivity was increased in SHR/Izm compared with that in WKY/Izm. In conclusion, the present study exhibited the enlargement of CB as three-dimensional image and revealed the enhanced immunoreactivity for DBH of glomus cells in SHR/Izm. These results suggest that the morphology of CB is affected by the effect of sympathetic nerve and that the signal transduction from CB is regulated by NA in glomus cells under hypertensive conditions.

NO modulation of carotid body chemoreception in health and disease.

Nitric oxide (NO), at physiological concentrations, is a tonic inhibitory modulator of carotid body (CB) chemosensory discharges. NO modulates the chemoreception process by several mechanisms, indirectly by modifying the vascular tone and oxygen delivery, and directly through the modulation of the excitability of glomus cells and petrosal neurons. In addition to the inhibitory effect, at high concentrations NO has a dual dose-dependent effect on CB chemoreception that depends on the P(O(2)). In hypoxic conditions, NO is primarily an inhibitory modulator of CB chemoreception, while in normoxia NO increases the chemosensory discharges. In this review, we will examine new evidence supporting the idea that NO is involved in the CB chemosensory potentiation induced by congestive heart failure (CHF) and chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea (OSA). Evidence from patients and experimental animal models indicates that CHF and OSA, as well as CIH, potentiate the carotid hypoxic chemoreflexes, contributing to enhance the sympathetic tone. Moreover, animals exposed to CIH or to pacing-induced CHF showed enhanced baseline CB discharges in normoxia and potentiated chemosensory responses to acute hypoxia. Several molecules and pathways are altered in CHF, OSA and CIH, but the available evidence suggests that a reduced NO production in the CB plays an essential role in both diseases, contributing to enhance the CB chemosensory discharges.

Differential expression of pro-inflammatory cytokines, endothelin-1 and nitric oxide synthases in the rat carotid body exposed to intermittent hypoxia.

The enhanced carotid body (CB) chemosensory response to hypoxia induced by chronic intermittent hypoxia (CIH) has been attributed to oxidative stress, which is expected to increase the expression of chemosensory modulators including chemoexcitatory pro-inflammatory cytokines in the CB. Accordingly, we studied the time-course of the changes in the immunohistological expression of TNF-α, IL-1ß, IL-6, ET-1, iNOS, eNOS and 3-nitrotyrosine in the CB, along with the progression of enhanced CB chemosensory responses to acute hypoxia in male Sprague-Dawley rats exposed to CIH (5%O2, 12 times/h per 8h) for 7, 14 and 21 days. Exposure to CIH for 7 days resulted in a sustained potentiation of CB chemosensory responses to acute hypoxia, which persisted until 21 days of CIH. The chemosensory potentiation was paralleled by an increased 3-nitrotyrosine expression in the CB. On the contrary, CIH produced a transient 2-fold increase of ET-1 immunoreactivity at 7 days, a decrease in eNOS immunoreactivity, and a delayed but progressive increase of TNF-α, IL-1ß and iNOS immunoreactivity, which was not associated with changes in systemic plasma levels or immune cell invasion within the CB. Thus, present results suggest that the local expression of chemosensory modulators and pro-inflammatory cytokines in the CB may have different temporal contribution to the CB chemosensory potentiation induced by CIH.

Short-term hypoxia increases tyrosine hydroxylase immunoreactivity in rat carotid body.

Neurochemical and morphological changes in the carotid body are induced by chronic hypoxia, leading to regulation of ventilation. In this study, we examined the time courses of changes in immunohistochemical intensity for tyrosine hydroxylase (TH) and cellular volume of glomus cells in rats exposed to hypoxia (10% O(2)) for up to 24 hr. Grayscale intensity for TH immunofluorescence was significantly increased in rats exposed to hypoxia for 12, 18, and 24 hr compared with control rats (p<0.05). The transectional area of glomus cells was not significantly different between experimental groups. The TH fluorescence intensity of the glomus cells exhibited a strong negative correlation with the transectional area in control rats (Spearman's rho = -0.70). This correlation coefficient decreased with exposure time, and it was lowest for the rats exposed to hypoxia for 18 hr (rho = -0.18). The histogram of TH fluorescence intensity showed a single peak in control rats. The peaks were gradually shifted to the right and became less pronounced in hypoxia-exposed rats, suggesting that a hypoxia-induced increase in TH immunoreactivity occurred uniformly in glomus cells. In conclusion, this study demonstrates that short-term hypoxia induces an increase in TH protein expression in rat carotid body glomus cells.

Modulation of chronic hypoxia-induced chemoreceptor hypersensitivity by NADPH oxidase subunits in rat carotid body.

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.

A role for succinate dehydrogenase genes in low chemoresponsiveness to hypoxia?

The detection of hypoxia by the carotid bodies elicits a ventilatory response of utmost importance for tolerance to high altitude. Germline mutations in three genes encoding subunit B, C and D of succinate dehydrogenase (SDHB, SDHC and SDHD) have been associated with paragangliomas of the carotid body. We hypothesized that SDH dysfunction within the carotid body could result in low chemoresponsiveness and intolerance to high altitude. The frequency of polymorphisms of SDHs, hypoxia-inducible factor type 1 (HIF1alpha) and angiotensin converting enzyme (ACE) genes was compared between 40 subjects with intolerance to high altitude and a low hypoxic ventilatory response at exercise (HVRe < or = 0.5 ml min(-1) kg(-1); HVR- group) and 41 subjects without intolerance to high altitude and a high HVRe (> or = 0.80 ml min(-1) kg(-1); HVR+). We found no significant association between low or high HVRe and (1) the allele frequencies for nine single nucleotide polymorphisms (SNPs) in the SDHD and SDHB genes, (2) the ACE insertion/deletion polymorphism and (3) four SNPs in the HIF1alpha gene. However, a marginal significant association was found between the synonymous polymorphism c.18A>C of the SDHB gene and chemoresponsiveness: 8/40 (20%) in the HVR- group and 3/41 (7%) in the HVR+ group (p = 0.12). A principal component analysis showed that no subject carrying the 18C allele had both high ventilatory and cardiac response to hypoxia. In conclusion, no clear association was found between gene variants involved in oxygen sensing and chemoresponsiveness, although some mutations in the SDHB and SDHD genes deserve further investigations in a larger population.

Altered expression of adenosine A1 and A2A receptors in the carotid body and nucleus tractus solitarius of adult male and female rats following neonatal caffeine treatment.

Neonatal caffeine treatment (adenosine receptor antagonist, 15 mg/kg/day, between postnatal days 3 and 12) affects respiratory patterns in adult male but not female rats as shown by an increase in the respiratory frequency in the early phase of response to hypoxia and an increase in the tidal volume in the late phase of response. Here, we tested the hypothesis that these changes are correlated with modified expression of adenosine receptors in the chemoreflex pathway. Carotid bodies, nucleus tractus solitarii, and superior cervical ganglia were collected from 3-month-old male and female rats that were either naive (not manipulated during the neonatal period) or treated with caffeine (NCT) or water (NWT) between postnatal days 3 and 12 by gavage. Western blot analysis was used to assess the expression of adenosine A(1) and A(2A) receptors and tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis. In male rats, there was a 37% increase in the level of A(2A) receptor and a 17% decrease in tyrosine hydroxylase in the carotid body of NCT (p<0.001) as compared to NWT rats. In the nucleus tractus solitarius, we found a 13% and 19% decrease in A(1) receptor expression in NWT and NCT rats (p<0.01), respectively, compared to naive rats. In the superior cervical ganglion, there was no change in A(1) receptor, A(2A) receptor, and tyrosine hydroxylase expression. In female rats, the only changes observed were decreases of 12% and 15% in A(1) receptor levels in the nucleus tractus solitarius of NWT and NCT rats (p<0.01), respectively, compared to naive rats. We conclude that NCT induces long-term changes in the adenosine receptor system. These changes may partially explain the modifications of the respiratory pattern induced by NCT in adults. The increased expression of the adenosine A(2A) receptor (specific to male rats), combined with the decreased tyrosine hydroxylase expression in the carotid body, suggests that NCT affects adenosine-dopamine interactions regulating chemosensory activity.

Hydrogen sulfide inhibits human BK(Ca) channels.

Hydrogen sulfide (H(2)S) is produced endogenously in many types of mammalian cells. Evidence is now accumulating to suggest that H(2)S is an endogenous signalling molecule, with a variety of molecular targets, including ion channels. Here, we describe the effects of H(2)S on the large conductance, calcium-sensitive potassium channel (BK(Ca)). This channel contributes to carotid body glomus cell excitability and oxygen-sensitivity. The experiments were performed on HEK 293 cells, stably expressing the human BK(Ca) channel alpha subunit, using patch-clamp in the inside-out configuration. The H(2)S donor, NaSH (100microM-10 mM), inhibited BK(Ca) channels in a concentration-dependent manner with an IC(50) of ca. 670microM. In contrast to the known effects of CO donors, the H(2)S donor maximally decreased the open state probability by over 50% and shifted the half activation voltage by more than +16mV. In addition, although 1 mM KCN completely suppressed CO-evoked channel activation, it was without effect on the H(2)S-induced channel inhibition, suggesting that the effects of CO and H(2)S were non-competitive. RT-PCR showed that mRNA for both of the H(2)S-producing enzymes, cystathionine-beta-synthase and cystathionine-gamma-lyase, were expressed in HEK 293 cells and in rat carotid body. Furthermore, immunohistochemistry was able to localise cystathionine-gamma-lyase to glomus cells, indicating that the carotid body has the endogenous capacity to produce H(2)S. In conclusion, we have shown that H(2)S and CO have opposing effects on BK(Ca)channels, suggesting that these gases have separate modes of action and that they modulate carotid body activity by binding at different motifs in the BK(Ca)alphasubunit.

Functional characterization of phosphodiesterases 4 in the rat carotid body: effect of oxygen concentrations.

The non-specific cAMP phosphodiesterase (PDE) inhibitor isobutyl- methylxanthine (IBMX) has been used to manipulate cAMP levels in carotid body (CB) preparations but the characterization of different PDE isoforms in CB has never been performed. PDE4 is one of the PDE families that uses cAMP as a specific substrate and changes its activity and affinity for drug inhibitors according to the degree of its phosphorylation. We investigated the effects of hypoxia on cAMP accumulation induced by different PDE4 inhibitors in the CB based on the hypothesis that acute changes in O(2) could interfere with their affinity.Concentration-response curves for the effects of the PDE4 selective inhibitors, rolipram and Ro 20-1724 and IBMX on cAMP were obtained in CBs, removed from rats and incubated in normoxia (20%O(2)) or hypoxia (5%O(2)).No differences were found between cAMP concentrations in normoxic and hypoxic conditions in the absence of PDE inhibitors. In both conditions, the E(max) calculated for IBMX was similar to that of the specific PDE4 inhibitors. Hypoxia shifted the concentration response curves to the left with the following rank order of potency IBMX> RO 20-1724=rolipram and increased E(max) by about 25%.This pharmacological approach supports the hypothesis that there is PDE4 activity in CBs that is enhanced by acute hypoxia although the low potency of the PDE4 inhibitors to increase cAMP do not support an important role for PDE4 activation in the O(2)-sensing machinery at the CB.

Bicarbonate-regulated soluble adenylyl cyclase (sAC) mRNA expression and activity in peripheral chemoreceptors.

UNLABELLED: Peripheral arterial chemoreceptors in the carotid body (CB) are modulated by pH/CO(2). Soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate ions (HCO(3)). Because CO(2)/HCO(3) mediates depolarization in chemoreceptors, we hypothesized that sAC mRNA would be expressed in the CB, and its expression and function would be regulated by CO(2)/HCO(3).Sprague-Dawley rats at postnatal days 16-17 were used to compare sAC mRNA gene expression between CB and non-chemosensitive tissues: superior cervical (SCG), petrosal (PG) and nodose ganglia (NG) by quantitative real time-PCR. Rat sAC gene expression was standardized to the expression of GAPDH (housekeeping gene) and the data were analyzed with the Pfaffl method. Gene and protein expression, and sAC regulation in the testis was used as a positive control. To determine the regulation of sAC mRNA expression and activity, all tissues were exposed to increasing concentrations of bicarbonate (0, 24, 44 mM, titrated with CO(2) and maintained a constant pH of 7.40). RESULTS: sAC mRNA expression was between 2-11% of CB expression in the SCG, PG and NG. Furthermore, only in the CB did HCO(3) upregulate sAC gene expression and increase cAMP levels. CONCLUSION: sAC mRNA and protein expression is present in peripheral arterial chemoreceptors and non-chemoreceptors. In the CB, CO(2)/HCO(3) not only activated sAC but also regulated its expression, suggesting that sAC may be involved in the regulation of cAMP levels in response to hyper/hypocapnia.

NADPH oxidase is required for the sensory plasticity of the carotid body by chronic intermittent hypoxia.

Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a approximately 12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT(2) receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H(2)O(2) generated from O(2).(-) contributes to sLTF. Priming with H(2)O(2) elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Aging and expression of heme oxygenase-1 and endothelin-1 in the rat carotid body after chronic hypoxia.

Hypoxia transiently increases transcription of the gene encoding heme oxygenase-1 (HO-1) and potently activates production of endothelin-1 (ET-1), the latter of which plays a central role in cellular adaptation to hypoxia. The ventilatory response to hypoxia attenuates with aging, and decreased responsiveness to hypoxia is seen in the aged vs. young rats, suggesting that the functionality of the oxygen-sensitive mechanism is age-dependent. In the present study, we examined the effects of aging on the expression of HO-1 and ET-1 in the carotid body, which is a small cluster of chemoreceptors and supporting cells that measure changes in the composition of arterial blood flowing through it. Our results revealed that HO-1 and ET-1 were expressed in carotid bodies of both young and old rats, although less so in the old ones. Exposure to chronic intermittent hypoxia significantly increased both HO-1 and ET-1 immunoreactivity in both young and old carotid body tissues, with the persisting age-dependent inequality to the disadvantage of old age. Considering that ET-1 is capable of enhancing intermittent hypoxia-induced chemosensory responses by the carotid body, our results suggest that decreased induction of ET-1 and HO-1 during aging could form the basis for age-related reductions in chemosensory discharge.

Exposure to cyclic intermittent hypoxia increases expression of functional NMDA receptors in the rat carotid body.

Although large quantities of glutamate are found in the carotid body, to date this excitatory neurotransmitter has not been assigned a role in chemoreception. To examine the possibility that glutamate and its N-methyl-d-aspartate (NMDA) receptors play a role in acclimatization after exposure to cyclic intermittent hypoxia (CIH), we exposed male Sprague-Dawley rats to cyclic hypoxia or to room air sham (Sham) for 8 h/day for 3 wk. Using RT-PCR, Western blot analysis, and immunohistochemistry, we found that ionotropic NMDA receptors, including NMDAR1, NMDAR2A, NMDAR2A/2B, are strongly expressed in the carotid body and colocalize with tyrosine hydroxylase in glomus cells. CIH exposure enhanced the expression of NMDAR1 and NMDAR2A/2B but did not substantially change the level of NMDAR2A. We assessed in vivo carotid sinus nerve activity (CSNA) at baseline, in response to acute hypoxia, in response to infused NMDA, and in response to infused endothelin-1 (ET-1) with and without MK-801, an NMDA receptor blocker. Infusion of NMDA augmented CSNA in CIH rats (124.61 +/- 2.64% of baseline) but not in sham-exposed rats. Administration of MK-801 did not alter baseline activity or response to acute hypoxia, in either CIH or sham animals but did reduce the effect of ET-1 infusion on CSNA (CSNA after ET-1 = 160.96 +/- 8.05% of baseline; ET-1 after MK-801 = 118.56 +/- 9.12%). We conclude that 3-wk CIH exposure increases expression of NMDA functional receptors in rats, suggesting glutamate and its receptors may play a role in hypoxic acclimatization to CIH.