Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors

O2 chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2 sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2 sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2 sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine beta-synthase and cystathionine gamma-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2 dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a PO2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar approximately 10-mV depolarization. These studies are consistent with H2S involvement in O2 sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive, yet simple, O2 sensor in a variety of tissues.     

Regulation of oxygen sensing by ion channels.

O(2) sensing is of critical importance for cell survival and adaptation of living organisms to changing environments or physiological conditions. O(2)-sensitive ion channels are major effectors of the cellular responses to hypoxia. These channels are preferentially found in excitable neurosecretory cells (glomus cells of the carotid body, cells in the neuroepithelial bodies of the lung, and neonatal adrenal chromaffin cells), which mediate fast cardiorespiratory adjustments to hypoxia. O(2)-sensitive channels are also expressed in the pulmonary and systemic arterial smooth muscle cells where they participate in the vasomotor responses to low O(2) tension (particularly in hypoxic pulmonary vasoconstriction). The mechanisms underlying O(2) sensing and how the O(2) sensors interact with the ion channels remain unknown. Recent advances in the field give different support to the various current hypotheses. Besides the participation of ion channels in acute O(2) sensing, they also contribute to the gene program developed under chronic hypoxia. Gene expression of T-type calcium channels is upregulated by hypoxia through the same hypoxia-inducible factor-dependent signaling pathway utilized by the classical O(2)-regulated genes. Alteration of acute or chronic O(2) sensing by ion channels could participate in the pathophysiology of human diseases, such as sudden infant death syndrome or primary pulmonary hypertension.     

NADPH oxidase does not account fully for O2-sensing in model airway chemoreceptor cells

A key feature of O2 sensing by chemoreceptor tissues is the hypoxic inhibition of K+ channels. However, mechanisms coupling a fall of pO2 to channel closure differ between tissues: O2 regulation of K+ channels in chemoreceptive neuroepithelial bodies and their immortal counterparts, H146 cells, involves altered reactive oxygen species generation by NADPH oxidase. In contrast, this enzyme complex is not involved in O2 sensing by the carotid body and pulmonary vasculature. Here, we provide pharmacological evidence to support a role for NADPH oxidase in hypoxic inhibition of K+ currents in H146 cells. Two structurally unrelated NADPH oxidase inhibitors, diphenylene iodonium and phenylarsine oxide, suppressed hypoxic inhibition of K+ currents recorded using the patch-clamp technique. Most importantly, however, neither inhibitor fully blocked this response. Our findings provide the first evidence that multiple mechanisms may coexist within a specific cell type to account for hypoxic suppression of K+ channel activity.     

Mitochondrial oxygen sensing of acute hypoxia in specialized cells - Is there a unifying mechanism?

Acclimation to acute hypoxia through cardiorespiratory responses is mediated by specialized cells in the carotid body and pulmonary vasculature to optimize systemic arterial oxygenation and thus oxygen supply to the tissues. Acute oxygen sensing by these cells triggers hyperventilation and hypoxic pulmonary vasoconstriction which limits pulmonary blood flow through areas of low alveolar oxygen content. Oxygen sensing of acute hypoxia by specialized cells thus is a fundamental pre-requisite for aerobic life and maintains systemic oxygen supply. However, the primary oxygen sensing mechanism and the question of a common mechanism in different specialized oxygen sensing cells remains unresolved. Recent studies unraveled basic oxygen sensing mechanisms involving the mitochondrial cytochrome c oxidase subunit 4 isoform 2 that is essential for the hypoxia-induced release of mitochondrial reactive oxygen species and subsequent acute hypoxic responses in both, the carotid body and pulmonary vasculature. This review compares basic mitochondrial oxygen sensing mechanisms in the pulmonary vasculature and the carotid body.     

Does AMP-activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2-sensing cells?

Specialized O2-sensing cells exhibit a particularly low threshold to regulation by O2 supply and function to maintain arterial pO2 within physiological limits. For example, hypoxic pulmonary vasoconstriction optimizes ventilation-perfusion matching in the lung, whereas carotid body excitation elicits corrective cardio-respiratory reflexes. It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O2-sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca2+ signaling mechanisms remains elusive, and investigation of previous hypotheses has generated contrary data and failed to unite the field. We propose that a rise in the cellular AMP/ATP ratio activates AMP-activated protein kinase and thereby evokes Ca2+ signals in O2-sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with alpha1 beta2 gamma1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-alpha1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP-activated protein kinase-alpha1 to be located throughout the cytoplasm of pulmonary arterial myocytes. In contrast, it was targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMP-activated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca2+ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca2+ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca2+ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.     

颈动脉体对化学信号的换能作用

颈动脉体可以将低氧和血液中其它刺激信号（可能包括低血糖）转换成不同强度的传入神经放电,沿心肺和神经内分泌反射的传入途径进入中枢,形成反射环路。低氧可抑制颈动脉体Ⅰ型细胞中的多种K＋通道,这种作用可能有种属差异; K＋通道的抑制使膜电位去极化,启动电压依赖性Ca2＋内流,最后导致神经分泌和传入放电。离子通道埘低氧的反应可能是通过间接途径发生的,因此近期的工作集中在研究颈动脉体Ⅰ型细胞中在低氧感受中起关键作用的其它蛋白质。虽然有人认为来源于线粒体和／或NADPH的活性氧（reactive oxygen species,ROS）起一定作用,但是它们在颈动脉体中转导低氧信号的证据还不足。目前正在对另外两种假设进行检验。第一种假设是血红素加氧酶2（haemoxygenase 2,HO-2）通过信号分子CO控制特殊K＋通道的活动,而CO的生成量与氧分压高低有关。第二种假设是认为细胞能量感受器腺苷酸活化蛋白激酶（AMP- activated protein kinase,AMPK）起作用;低氧时AMP／ATP比值升高,激活AMPK,从而抑制Ⅰ型细胞的K＋通道,传入放电增加。颈动脉体的细胞上具有丰富的对氧敏感的K＋通道,低氧感受这个重要的细胞活动可以通过多条途径进行,在总反应中每种蛋白质也可能起不同的作用,例如不同蛋白质对氧的亲合力不同等。关于颈动脉体感受低血糖的机制尚不清楚,但最近有证据提示,它并非由K＋通道关闭引起的,因此感受低血糖的机制和感受低氧的机制是不同的。     

Oxygen and glucose sensing by carotid body glomus cells

Carotid body glomus cells sense hypoxia through the inhibition of plasmalemmal K(+) channels, which leads to Ca(2+) influx and transmitter release. Although the mechanism of O(2) sensing remains enigmatic, it does not seem to depend on cellular redox status or inhibition of mitochondrial electron transport. Hypoxia inducible factors appear to be necessary for the expression of the O(2) sensor and carotid body remodeling in chronic hypoxia, but are not directly involved in acute O(2) sensing. Glomus cells are also rapidly activated by reductions of glucose concentration due to inhibition of K(+) channels. These cells function as combined O(2) and glucose sensors that help to prevent neuronal damage by acute hypoxia and/or hypoglycemia.     

Peripheral O2 chemoreceptors mediate humoral catecholamine secretion from fish chromaffin cells

This study addressed the hypothesis that the secretion of catecholamines from trout (Oncorhynchus mykiss) chromaffin cells, during hypoxia, is triggered by stimulation of O(2) chemoreceptors located within the gills. Sodium cyanide was administered into the inspired water (external cyanide) or injected into the gill circulation (internal cyanide) to pharmacologically stimulate external (water sensing) or internal (blood sensing) O(2) chemoreceptors, respectively. Both of these treatments caused an elevation of circulating catecholamine levels. The response to external, but not internal, cyanide was abolished by removal of the first gill arch. Hypoxia produced an increase in circulating catecholamine levels that was unaffected by removal of the first gill arch or by denervation of the pseudobranch. Cyanide and hypoxia both caused the well-documented cardiorespiratory reflexes normally observed in this species. This study demonstrates, for the first time, that gill O(2) chemoreceptors can initiate the reflex that leads to catecholamine release from the chromaffin cells and that stimulation of internally oriented O(2) receptors on all gill arches appears to be the physiologically important mechanism for initiating release.     

An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems

Despite much recent interest in the biochemistry of reactive oxygen species, the mechanisms by which hydrogen peroxide (H2O2) functions in mammalian cells remain poorly defined. Proposed mechanisms for sensing H2O2 in mammalian cells include inactivation of protein tyrosine phosphatases and dual specificity phosphatases as well as inactivation of peroxiredoxins. In this critical review, proteins proposed to serve as sensors for H2O2 in mammals will be compared to peroxidases, catalases, and the bacterial H2O2 sensor OxyR for their ability to react with H2O2, in the context of our current knowledge concerning the concentrations of H2O2 present in cells.     

Functional mitochondria are required for O2 but not CO2 sensing in immortalized adrenomedullary chromaffin cells

Catecholamine (CAT) release from adrenomedullary chromaffin cells (AMC) in response to stressors such as low O(2) (hypoxia) and elevated CO(2)/H(+) is critical during adaptation of the newborn to extrauterine life. Using a surrogate model based on a v-myc immortalized adrenal chromaffin cell line (i.e., MAH cells), combined with genetic perturbation of mitochondrial function, we tested the hypothesis that functional mitochondria are required for O(2) sensing. Wild-type MAH cells responded to both hypoxia and increased CO(2) (hypercapnia) with K(+) current inhibition and membrane depolarization. Additionally, these stimuli caused a rise in cytosolic Ca(2+) and CAT secretion, determined by fura-2 spectrofluorimetry and carbon fiber amperometry, respectively. In contrast, mitochondria-deficient (rho(0)) MAH cells were hypoxia insensitive, although responses to hypercapnia and expression of several markers, including carbonic anhydrase II, remained intact. Rotenone (1 microM), a mitochondrial complex I blocker known to mimic and occlude the effects of hypoxia in primary AMC, was effective in wild-type but not rho(0) MAH cells. These data demonstrate that functional mitochondria are involved in hypoxia-sensing by adrenal chromaffin cells. We also show for the first time that, like their neonatal chromaffin cell counterparts, MAH cells are CO(2) sensors; however, this property is independent of functional mitochondria.     

Adenosine A₂a receptors and O₂ sensing in development

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O? sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5'-nucleotidase and the resulting activation of adenosine A(?A) receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A(?A) receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A(?A) receptors mediate hypoxic inhibition of breathing and rapid eye movements. A(?A) receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A(?A) receptors play virtually no role in O? sensing by the carotid bodies, but brain A(?A) receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A(?A) receptors have been implicated in O? sensing by carotid glomus cells, while central A(?A) receptors likely blunt hypoxic hyperventilation. In conclusion, A(?A) receptors are crucially involved in the transduction mechanisms of O? sensing in fetal carotid bodies and brains. Postnatally, central A(?A) receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O? sensing in carotid chemoreceptors, particularly in developing lambs.     

The oxygen sensing signal cascade under the influence of reactive oxygen species

Structural and functional integrity of organ function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of organ function. Particular reductions in oxygen availability (hypoxia) caused by respiratory or blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply by anaerobic glycolysis. Complex cellular oxygen sensing systems have evolved to tightly regulate oxygen homeostasis. In response to variations in oxygen partial pressure (PO2), these systems induce adaptive and protective mechanisms to avoid or at least minimize tissue damage. These various responses might be based on a range of oxygen sensing signal cascades including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low PO2 affinity cytochrome c oxidase (aa3) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl-hydroxylase and HIF asparaginyl hydroxylase called factor-inhibiting HIF (FIH-1). Thus, specific oxygen sensing cascades involving reactive oxygen species as second messengers may by means of their different oxygen sensitivities, cell-specific and subcellular localization help to tailor various adaptive responses according to differences in tissue oxygen availability.     

O2 exchange between blood and brain tissues studied with 18O2 indicator-dilution technique

A technique has been developed to record 18O2 dilution curves of an organ in vivo by use of 51Cr-labeled erythrocytes as a reference tracer. The technique employs anaerobic sampling of venous outflow following an intraarterial injection of tracer-laden blood and off-line determination of [18O2] and [51Cr] profiles in the venous outflow. O2 and reference indicator-dilution curves of cerebral circulation were recorded in eight experiments with six halothane-anesthetized dogs. Autologous blood labeled with the tracers was injected into a carotid artery, and brain venous outflow was sampled from the sagittal sinus. The total net extraction of O2 tracer was equal to the extraction of elemental O2. Instantaneous extraction of 18O2 along the outflow curve fell linearly with time, from an initial value of 0.6-0.7 to very small or even negative values toward the end of a pulse. This indicates that O2 undergoes a flow-limited distribution. In all experiments, the mean transit time of unmetabolized 18O2 was longer than the mean transit time of the Cr tracer. An index of the tissue O2 dilution space, hence the mean tissue PO2, is calculated from this data with the use of a modified central volume principle. This estimate of mean tissue PO2 increases as a linear function of sagittal sinus PO2 with a slope of 0.97. The method may provide an index of the critical PO2 of venous blood, the PO2 below which O2 diffusion from blood to tissue may limit its rate of metabolic uptake.     

Cellular oxygen sensing by mitochondria: old questions, new insight.

Hypoxia elicits a variety of adaptive responses at the tissue level, at the cellular level, and at the molecular level. A physiological response to hypoxia requires the existence of an O(2) sensor coupled to a signal transduction system, which in turn activates the functional response. Although much has been learned about the signaling systems activated by hypoxia, no consensus exists regarding the nature of the underlying O(2) sensor or whether multiple sensors exist. Among previously considered mechanisms, heme proteins have been suggested to undergo allosteric modification in response to O(2) binding or release at different PO(2) levels. Other studies suggest that ion channels may change conductance as a function of PO(2), allowing them to signal the onset of hypoxia. Still other studies suggest that NADPH oxidase may decrease its generation of reactive O(2) species (ROS) during hypoxia. Recent data suggest that mitochondria may function as O(2) sensors by increasing their generation of ROS during hypoxia. These oxidant signals appear to act as second messengers in the adaptive responses to hypoxia in a variety of cell types. Such observations contribute to a growing awareness that mitochondria do more than just generate ATP, in that they initiate signaling cascades involved in adaptive responses to hypoxia and that they participate in the control of cell death pathways.     

Detection of hypoxia-evoked ATP release from chemoreceptor cells of the rat carotid body

The carotid body (CB) is a chemosensory organ that detects changes in chemical composition of arterial blood and maintains homeostasis via reflex control of ventilation. Thus, in response to a fall in arterial PO(2) (hypoxia), CB chemoreceptors (type I cells) depolarize, and release neurotransmitters onto afferent sensory nerve endings. Recent studies implicate ATP as a key excitatory neurotransmitter released during CB chemoexcitation, but direct evidence is lacking. Here we use the luciferin-luciferase bioluminescence assay to detect ATP, released from rat chemoreceptors in CB cultures, fresh tissue slices, and whole CB. Hypoxia evoked an increase in extracellular ATP, that was inhibited by L-type Ca(2+)channel blockers and reduced by the nucleoside hydrolase, apyrase. Additionally, iberiotoxin (IbTX; 100 nM), a blocker of O(2)-sensitive Ca(2+)-dependent K(+) (BK) channels, stimulated ATP release and largely occluded the effect of hypoxia. These data strongly support a neurotransmitter role for ATP in carotid body function.     

siRNA knock-down of gamma-glutamyl transpeptidase does not affect hypoxic K+ channel inhibition

Large conductance, Ca(2+)-sensitive potassium (BK) channels are critical components of the O(2) signalling cascade in a number of cells, including the carotid body and central neurones. Although the nature of the BK channel O(2) sensor is still unknown, evidence suggests redox modulators might form part of the O(2) sensing channel complex. By metabolising glutathione, gamma-glutamyl transpeptidase (gammaGT) could act as such an O(2) sensor. Western blotting and immunocytochemistry revealed high gammaGT expression in HEK293 cells expressing the alpha- and beta-subunits of human recombinant BK and gammaGT co-immunoprecipitated with BKalpha. Acivicin blockade of gammaGT reversibly inhibited BK channels, suggesting that this BKalpha protein partner contributes to tonic channel activity. However, knock-out of gammaGT using siRNA had no effect on hypoxic BK channel inhibition. Together, these data indicate that gammaGT is a BKalpha protein partner, that its activity regulates BK channels but that it is not the BK O(2) sensor.     

H2O2 and cGMP may function as an O2 sensor in the pulmonary artery

The effects of O2 tension on force in precontracted isolated pulmonary arterial smooth muscle from calf lungs was characterized to investigate the mechanism of O2 tension sensing. These arteries display a decrease in force with increasing O2 tension that is antagonized via inhibition of soluble guanylate cyclase activation by 10 microM methylene blue or inactivation of catalase by pretreatment with 50 mM 3-amino-1,2,4-triazole for 30 min. O2 tension-dependent relaxation is associated with an increase in intracellular H2O2 metabolism through catalase (detected as the peroxide-dependent inactivation of tissue catalase activity by aminotriazole) and cyclic guanosine 5'-monophosphate (cGMP), known mediators of relaxation in calf pulmonary arteries. Thus a recently reconstructed mechanism of activation of soluble guanylate cyclase involving the metabolism of H2O2 by catalase appears to function as an O2 tension sensor in pulmonary arteries.     

Oxygen sensing by ion channels and chemotransduction in single glomus cells

We have monitored cytosolic [Ca2+] and dopamine release in intact fura- 2-loaded glomus cells with microfluoroimetry and a polarized carbon fiber electrode. Exposure to low PO2 produced a rise of cytosolic [Ca2+] with two distinguishable phases: an initial period (with PO2 values between 150 and approximately 70 mm Hg) during which the increase of [Ca2+] is very small and never exceeds 150-200 nM, and a second phase (with PO2 below approximately 70 mm Hg) characterized by a sharp rise of cytosolic [Ca2+]. Secretion occurs once cytosolic [Ca2+] reaches a threshold value of 180 +/- 43 nM. The results demonstrate a characteristic relationship between PO2 and transmitter secretion at the cellular level that is comparable with the relation described for the input (O2 tension)output (afferent neural discharges) variables in the carotid body. Thus, the properties of single glomus cells can explain the sensory functions of the entire organ. In whole-cell, patch- clamped cells, we have found that in addition to O2-sensitive K+ channels, there are Ca2+ channels whose activity is also regulated by PO2. Ca2+ channel activity is inhibited by hpoxia, although in a strongly voltage-dependent manner. The average hypoxic inhibition of the calcium current in 30% +/- 10% at -20 mV but only 2% +/- 2% at +30 mV. The differential inhibition of K+ and Ca2+ channels by hypoxia helps to explain why the secretory response of the cells is displaced toward PO2 values (below approximately 70 mm Hg) within the range of those normally existing in arterial blood. These data provide a conceptual framework for understanding the cellular mechanisms of O2 chemotransduction in the carotid body.     

Graded response of K+ current,membrane potential,and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle

Many studies indicate that hypoxic inhibition of some K+ channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+ current (I(k)), resting membrane potential (E(m)), and intracellular Ca2+ concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing PO2 levels gradually inhibited steady-state I(k) of rat resistance PASMCs and depolarized the cell membrane. The block of I(k) by hypoxia was voltage dependent in that low O2 tensions (3 and 0% O2) inhibited I(k) more at 0 and -20 mV than at 50 mV. As expected, the hypoxia-sensitive I(k) was also 4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]i as PO2 levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in I(k) and E(m) and an increase of [Ca2+]i.