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We recently reported that adrenomedullary chromaffin cells (AMC) from neonatal rats treated with intermittent hypoxia (IH) exhibit enhanced catecholamine secretion by hypoxia (Souvannakitti D, Kumar GK, Fox A, Prabhakar NR. J Neurophysiol 101: 2837-2846, 2009). In the present study, we examined whether neonatal IH also facilitate AMC responses to nicotine, a potent stimulus to chromaffin cells. Experiments were performed on rats exposed to either IH (15-s hypoxia-5rain normoxia; 8 h/day) or to room air (normoxia; controls) from ages postnatal day 0 (P0) to P5. Quantitative RT-PCR analysis revealed expression of mRNAs encoding [[alpha].sub.3]-, [[alpha].sub.5]-, [[alpha].sub.7]-, and [[beta].sub.2]- and [[beta].sub.4]nicotinic acetylcholine receptor (nAChR) subunits in adrenal medullae from control P5 rats. Nicotine-elevated intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in AMC and nAChR antagonists prevented this response, suggesting that nAChRs are functional in neonatal AMC. In IH-treated rats, nAChR mRNAs were downregulated in AMC, which resulted in a markedly attenuated nicotine-evoked elevation in [[[Ca.sup.2+]].sub.i] and subsequent catecholamine secretion. Systemic administration of antioxidant prevented IH-evoked downregulation of nAChR expression and function. P35 rats treated with neonatal IH exhibited reduced nAChR mRNA expression in adrenal medullae, attenuated AMC responses to nicotine, and impaired neurogenic catecholamine secretion. Thus the response to neonatal IH lasts for at least 30 days. These observations demonstrate that neonatal IH downregulates nAChR expression and function in AMC via reactive oxygen species signaling, and the effects of neonatal IH persist at least into juvenile life, leading to impaired neurogenic catecholamine secretion from AMC. adrenal medullary chromaffin cells; nicotinic cholinergic receptors; recurrent apneas; reactive oxygen species; catecholamine secretion doi: 10.1152/ajpcell.00530.2009.

We previously reported that intermittent hypoxia (IH) augments hypoxic sensory response (HSR) and increases the number of glomus cells in neonatal carotid bodies. In the present study, we tested the hypothesis that recruitment of endothelin-1 (ET-1) signaling by reactive oxygen species (ROS) plays a critical role in IH-evoked changes in neonatal carotid bodies. Experiments were performed on neonatal rats exposed either to I0 days of IH (P0-P10; 8 h/day) or to normoxia. IH augmented HSR of the carotid bodies ex vivo and resulted in hyperplasia of glomus cells. The effects of IH were associated with enhanced basal release of ET-l under normoxia, sensitization of carotid body response to exogenous ET-1, and upregulation of [ET.sub.A] but not an [ET.sub.B] receptor mRNA without altering the ET-1 content. An ETA but not [ET.sub.B] receptor antagonist prevented augmented HSR by IH. ROS levels were elevated in carotid bodies from IH-treated rat pups as evidenced by increased levels of malondialdehyde. Systemic administration of manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 5 mg/kg ip), a scavenger of [O.sub.2.sup.x-], prevented IH-induced elevation of ROS, basal release of ET-1, up-regulation of [ET.sub.A] mRNA, and augmented HSR. In striking contrast, MnTMPyP treatment had no significant effect on IH-induced hyperplasia of glomus cells. These results demonstrate that IH-evoked increase in HSR involve a ROS-mediated increase in basal ET-1 release and upregulation of [ET.sub.A] receptor mRNA. recurrent apneas; premature infants; chemoreflex; arterial chemoreceptors; chronic intermittent hypoxia

Research was conducted to investigate the effects of the different levels of ambient oxygen on the release of dopamine (DA) and norepinephrine (NE) from the pheochromocytoma 12 (PC-12) cells of rats. Microvoltammetry was used to monitor catecholamine release. Results indicate that catecholamines are released during hypoxia from PC-12 cells in a Ca2+-dependent manner and involve the activation of voltage-dependent Ca2+ channels. Findings also suggest that protein kinases generate an increased release of NE during hypoxia.

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. A heightened carotid body activity and the resulting chemosensory reflex mediate increased sympathetic nerve activity by CIH. However, the mechanisms underlying heightened carotid body activity by CIH are not known. An elevation of intracellular calcium ion concentration ([Ca2+]i) in glomus cells, the primary oxygen-sensing cells, is an essential step for carotid body activation by hypoxia. In the present study, we examined the effects of CIH on the glomus cell [Ca2+]i response to hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats or wild-type mice treated with 10 days of either room air (control) or CIH (alternating cycles of 15 s of hypoxia and 5 min of room air; 9 episodes/h; 8 h/day). CIH-treated glomus cells exhibited an enhanced [Ca2+]i response to hypoxia, and this effect was absent in the presence of 2-(4-cyclopropylphenyl)-N-((1R)-1-[5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl]ethyl)acetamide (TTA-A2), a specific inhibitor of T-type Ca2+ channels, and in voltage-gated calcium channel, type 3.2 (CaV3.2), null glomus cells. CaV3.2 knockout mice exhibited an absence of CIH-induced hypersensitivity of the carotid body. CIH increased reactive oxygen species (ROS) levels in glomus cells. A ROS scavenger prevented the exaggerated TTA-A2-sensitive [Ca2+]i response to hypoxia. CIH had no effect on CaV3.2 mRNA levels. CIH augmented Ca2+ currents and increased CaV3.2 protein in plasma membrane fractions of human embryonic kidney-293 cells stably expressing CaV3.2, and either a ROS scavenger or brefeldin-A, an inhibitor of protein trafficking, prevented these effects. These findings suggest that CIH leads to an augmented Ca2+ influx via ROS-dependent facilitation of CaV3.2 protein trafficking to the plasma membrane. [ABSTRACT FROM AUTHOR]

Arterial blood O2 levels are detected by specialized sensory organs called carotid bodies. Voltagegated Ca2+ channels (VGCCs) are important for carotid body O2 sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2 sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1H protein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca2+ concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout (Cacna1h-/-) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2 sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca2+ concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h-/- mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia. [ABSTRACT FROM AUTHOR]

H2S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O2 sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H2S in an O2-sensitive manner and whether endogenous H2S is required for O2 sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE-/- and CSE-/- mice. Physiological levels of hypoxia (PO2 ∼30 mmHg) increased H2S levels in glomus cells, and DL-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE-/- mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H2S donor (50 μM NaHS) increased cytosolic Ca2+ concentration ([Ca2+]i) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca2+]i responses to NaHS and hypoxia were markedly attenuated in the presence of Ca2+-free medium or cadmium chloride, a pan voltagegated Ca2+ channel blocker, or nifedipine, an L-type Ca2+ channel inhibitor, suggesting that both hypoxia and H2S share common Ca2+- activating mechanisms. These results demonstrate that H2S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia. [ABSTRACT FROM AUTHOR]

We previously reported that reactive oxygen species generated by NADPH oxidase 2 (Nox2) induces sensory plasticity of the carotid body, manifested as a progressive increase in baseline sensory activity or sensory long-term facilitation (sLTF). ANG II, a peptide generated within the carotid body, is a potent activator of Nox2. In the present study, we tested the hypothesis that ANG II evokes sLTF of the carotid body via Nox2 activation. Experiments were performed on carotid bodies ex vivo from adult rats and mice. Sensory activity was recorded from the carotid sinus nerve. Repetitive (5 times for 30 s each at 5-min intervals), but not continuous (for 150 s), application of 60 pM ANG II evoked robust sLTF of the carotid body. ACh, ATP, substance P, and KCI, when applied repetitively, stimulated the carotid body but did not evoke sLTF. Reactive oxygen species levels increased in response to repetitive applications of ANG II, and this effect was blocked by apocynin, an inhibitor of Nox2, as well as losartan, an angiotensin type 1 (AT1) receptor antagonist. Losartan, apocynin, and 4-(2-aminoethyl)benzenesulfonyl fluoride prevented ANG lI-induced sLTF. which was absent in mice deficient in gp91phox, the catalytic subunit of the Nox2 complex. These results demonstrate that repetitive application of ANG II induces sLTF of the carotid body via activation of Nox2 by AT1 receptors. [ABSTRACT FROM AUTHOR]

Investigates the effects of preconditioning pheochromocytoma (PC12) cells with intermittent hypoxia (IH) on transmitter release during acute hypoxia. Exposure of cell cultures to either alternating cycles of hypoxia or normoxia; Determination of the amounts of dopamine and acetylcholine released in the medium by high performance liquid chromatography combined with electrochemical detection.

The purpose of the present study was to investigate whether hypoxia influences acetylcholine (ACh) release from the rabbit carotid body and, if so, to determine the mechanism(s) associated with this response. ACh is expressed in the rabbit carotid body (5.6 ± 1.3 pmol/carotid body) as evidenced by electrochemical analysis. Immunocytochemical analysis of the primary cultures of the carotid body with antibody specific to ACh further showed that ACh-like immunoreactivity is localized to many glomus cells. The effect of hypoxia on ACh release was examined in ex vivo carotid bodies harvested from anesthetized rabbits. The basal release of ACh during normoxia (∼150 Torr) averaged 5.9 ± 0.5 fmol·min[sup -1]·carotid body[sup -1]. Lowering the PO[sub 2] to 90 and 20 Torr progressively decreased ACh release by ∼15 and ∼68%, respectively. ACh release returned to the basal value on reoxygenation. Simultaneous monitoring of dopamine showed a sixfold increase in dopamine release during hypoxia. Hypercapnia (21% O[sub 2] + 10% CO[sub 2]) as well as high K[sup +] (100 mM) facilitated ACh release from the carotid body, suggesting that hypoxia-induced inhibition of ACh release is not due to deterioration of the carotid body. Hypoxia had no significant effect on acetylcholinesterase activity in the medium, implying that increased hydrolysis of ACh does not account for hypoxia-induced inhibition of ACh release. In the presence of either atropine (10 µM) or domperidone (10 µM), hypoxia stimulated ACh release. These results demonstrate that glotaus cells of the rabbit carotid body express ACh and that hypoxia overall inhibits ACh release via activation of muscarinic and dopaminergic autoinhibitory receptors in the carotid body. [ABSTRACT FROM AUTHOR]

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. A heightened carotid body activity and the resulting chemosensory reflex mediate increased sympathetic nerve activity by CIH. However, the mechanisms underlying heightened carotid body activity by CIH are not known. An elevation of intracellular calcium ion concentration ([Ca(2+)]i) in glomus cells, the primary oxygen-sensing cells, is an essential step for carotid body activation by hypoxia. In the present study, we examined the effects of CIH on the glomus cell [Ca(2+)]i response to hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats or wild-type mice treated with 10 days of either room air (control) or CIH (alternating cycles of 15 s of hypoxia and 5 min of room air; 9 episodes/h; 8 h/day). CIH-treated glomus cells exhibited an enhanced [Ca(2+)]i response to hypoxia, and this effect was absent in the presence of 2-(4-cyclopropylphenyl)-N-((1R)-1-[5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl]ethyl)acetamide (TTA-A2), a specific inhibitor of T-type Ca(2+) channels, and in voltage-gated calcium channel, type 3.2 (CaV3.2), null glomus cells. CaV3.2 knockout mice exhibited an absence of CIH-induced hypersensitivity of the carotid body. CIH increased reactive oxygen species (ROS) levels in glomus cells. A ROS scavenger prevented the exaggerated TTA-A2-sensitive [Ca(2+)]i response to hypoxia. CIH had no effect on CaV3.2 mRNA levels. CIH augmented Ca(2+) currents and increased CaV3.2 protein in plasma membrane fractions of human embryonic kidney-293 cells stably expressing CaV3.2, and either a ROS scavenger or brefeldin-A, an inhibitor of protein trafficking, prevented these effects. These findings suggest that CIH leads to an augmented Ca(2+) influx via ROS-dependent facilitation of CaV3.2 protein trafficking to the plasma membrane., (Copyright © 2016 the American Physiological Society.)

Arterial blood O2 levels are detected by specialized sensory organs called carotid bodies. Voltage-gated Ca(2+) channels (VGCCs) are important for carotid body O2 sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2 sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1H protein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca(2+) concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout (Cacna1h(-/-)) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2 sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca(2+) concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h(-/-) mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia., (Copyright © 2015 the American Physiological Society.)

The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung microvascular endothelial cells exposed to IH-consisting alternating cycles of 1.5% O2 for 30s followed by 20% O2 for 5 min. IH decreased transendothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus dependent and reversible after reoxygenation. IH-exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins, and zona occludens-1 junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) were phosphorylated in IH-exposed cells. Inhibiting either ERK or JNK prevented the IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels, and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant, prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to reorganization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.

H(2)S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O(2) sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H(2)S in an O(2)-sensitive manner and whether endogenous H(2)S is required for O(2) sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE(+/+) and CSE(-/-) mice. Physiological levels of hypoxia (Po(2) ∼30 mmHg) increased H(2)S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE(-/-) mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H(2)S donor (50 μM NaHS) increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca(2+)](i) responses to NaHS and hypoxia were markedly attenuated in the presence of Ca(2+)-free medium or cadmium chloride, a pan voltage-gated Ca(2+) channel blocker, or nifedipine, an L-type Ca(2+) channel inhibitor, suggesting that both hypoxia and H(2)S share common Ca(2+)-activating mechanisms. These results demonstrate that H(2)S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia.

Central and peripheral neurons as well as neuroendocrine cells express a variety of neurotransmitters/modulators that play critical roles in regulation of physiological systems. The synthesis of several neurotransmitters/modulators is regulated by O(2)-requiring rate-limiting enzymes. Consequently, hypoxia resulting from perturbations in O(2) homeostasis can affect neuronal functions by altering neurotransmitter synthesis. Two broad categories of hypoxia are frequently encountered: continuous hypoxia (CH) and intermittent hypoxia (IH). CH is often seen during high altitude sojourns, whereas IH is experienced in sleep-disordered breathing with recurrent apneas (i.e., brief, repetitive cessations of breathing). This article presents what is currently known on the effects of both forms of hypoxia on neurotransmitter levels and neurotransmitter synthesizing enzymes in the central and peripheral nervous systems.

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.