Integrative and Comparative Biology Advance Access originally published online on July 20, 2007
Integrative and Comparative Biology 2007 47(4):592-600; doi:10.1093/icb/icm076
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Respiratory chemoreceptor function in vertebrates—comparative and evolutionary aspects
*Department of Zoophysiology, Göteborg University, Box 463, SE-405 30 Göteborg, Sweden; Department of Biological Sciences, University of North Texas, PO Box 305220, Denton, TX 76203-5220, USA; Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3.900, Ribeirão Preto, SP, Brazil; Department of Pediatrics, Laval University, Chairholder of the Canada Research chair in Respiratory Neurobiology, Canada; ¶Department of Animal Morphology and Physiology, São Paulo State University - FCAV at Jaboticabal, SP, Brazil; ||Department of Neuroscience, Cell Biology, and Physiology, Wright State University, Boonshoft School of Medicine, 3640 Colonel Glenn Highway, Dayton, Ohio, 45435, USA; #Institute of Physiology, University Duisburg-Essen Hufelandstr, Germany; **Department of Physiology, The Medical School, University of Birmingham, B15 2TT, UK
Correspondence: 1E-mail: mlglass{at}rfi.fmrp.usp.br
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The sensing of blood gas tensions and/or pH is an evolutionarily conserved, homeostatic mechanism, observable in almost all species studied from invertebrates to man. In vertebrates, a shift from the peripheral O2-oriented sensing in fish, to the central CO2/pH sensing in most tetrapods reflects the specific behavioral requirements of these two groups whereby, in teleost fish, a highly O2-oriented control of breathing matches the ever-changing and low oxygen levels in water, whilst the transition to air-breathing increased the importance of acid–base regulation and O2-related drive, although retained, became relatively less important. The South American lungfish and tetrapods are probably sister groups, a conclusion backed up by many similar features of respiratory control. For example, the relative roles of peripheral and central chemoreceptors are present both in the lungfish and in land vertebrates. In both groups, the central CO2/pH receptors dominate the ventilatory response to hypercarbia (60–80%), while the peripheral CO2/pH receptors account for 20–30%. Some basic components of respiratory control have changed little during evolution. This review presents studies that reflect the current trends in the field of chemoreceptor function, and several laboratories are involved. An exhaustive review on the previous literature, however, is beyond the intended scope of the article. Rather, we present examples of current trends in respiratory function in vertebrates, ranging from fish to humans, and focus on both O2 sensing and CO2 sensing. As well, we consider the impact of chronic levels of hypoxia—a physiological condition in fish and in land vertebrates resident at high elevations or suffering from one of the many cardiorespiratory disease states that predispose an animal to impaired ventilation or cardiac output. This provides a basis for a comparative physiology that is informative about the evolution of respiratory functions in vertebrates and about human disease. Currently, most detail is known for mammals, for which molecular biology and respiratory physiology have combined in the discovery of the mechanisms underlying the responses of respiratory chemoreceptors. Our review includes new data on nonmammalian vertebrates, which stresses that some chemoreceptor sites are of ancient origin.
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Our main theme is to present systems for regulation of acid–base status and oxygen demand in various groups of vertebrates including fish, lissamphibians, lungfish, and mammals (Dejours 1981
, Gray et al. 2004). Lungfish and land vertebrates have similar regulatory systems that point to a very ancient origin of the control of pulmonary function (Amin-Naves et al. 2007), but most of the information on the chemoreceptors and neurons involved stems from mammals, although there has been much recent progress with lissamphibians. In the tradition of Anaximander, who about 2500 years ago stated: "In the beginning man was another animal perhaps a fish" our purpose is to present part of the growing progress in chemoreceptor research.
Sensing low oxygen
The O2 chemoreflex in fish
The aquatic environment is both spatially and temporally unstable with respect to O2, which leaves fish apt to be exposed to hypoxia. The primary sites of O2 chemoreceptors in water-breathing fish are located in the gills (Perry and Gilmour 2002; Sundin and Nilsson 2002); these became the primary peripheral chemoreceptors in all other vertebrates (Milsom 2002). Current belief is that the neuroepithelial cells (NECs) are the O2 sensitive receptors and that their location within the gill tissue allows monitoring of oxygen levels in both water and blood (Sundin et al. 1998; Jonz et al. 2004; Burleson et al. 2006). It is likely that these branchial NECs are homologous to the O2-sensing glomus cells found in the carotid body of mammals and birds. Selective denervation of cranial nerves (IX and X) to the gill arches and various branches of cranial nerve V and VII to the oro-branchial cavity, in combination with receptor stimulants, have revealed that different respiratory reflex modalities (i.e., increases in frequency and amplitude) are elicited by different oxygen receptor groups (Sundin et al. 1999, 2000; Milsom et al. 2002).
Recently, it has been shown that each cardiorespiratory reflex appears to have a designated terminal field (sensory area) of their afferents in the medulla that integrates oxygen-receptor information (Sundin et al. 2003b). Furthermore, since application of kynurenic acid (a broad-spectrum ionotropic glutamate receptor antagonist) into the sensory area abolished all hypoxia-induced respiratory increase in the channel catfish, Ictalurus punctatus, this integration process is mediated by ionotropic glutamate receptors (Sundin et al. 2003a). From work on the shorthorn sculpin, Myoxocephalus scorpious, it became clear that N-methyl-D-aspartate (NMDA) receptors mediate the hypoxia-induced increase in ventilation frequency (Turesson and Sundin 2003), as fish pretreated with MK-801 (a specific NMDA receptor antagonist) blocked the rise in ventilatory frequencies while the amplitude did not change. As it had been shown that kynurenate blocked all hypoxia elicited ventilatory changes in channel catfish (Sundin et al. 2003a), it was hypothesized that the hypoxic MK801-insensitive increase in ventilation in shorthorn sculpins was mediated via the 
-amino-3-OH-5-methyl-4-isoxazole-propionic-acid (AMPA) or the kainate receptor. Consequently, increasing doses of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (AMPA/kainate receptor antagonist) were introduced into the fourth ventricle prior to exposures to hypoxia in shorthorn sculpins. This treatment reduced, in a dose-dependent fashion, the hypoxia-induced increases in amplitude, indicating that AMPA and/or kainate receptors are essential (Turesson and Sundin, unpublished manuscript). To determine if the AMPA receptor is involved in the amplitude response, sculpins were also pretreated with GYKI 52466 (a specific AMPA receptor antagonist, applied systemically). The hypoxia-induced amplitude response remained unchanged, however; which indicates that the kainate receptor must be the mediator of the augmented breathing amplitude (Turesson and Sundin, unpublished manuscipt). Thus, it appears that each reflex modality is operating through a specific glutamate-receptor type.
Effects of acclimatization to hypoxia on cardioventilatory control in fish
The effects of acute hypoxia (min to h) on cardiovascular, ventilatory, blood-gas, and acid–base parameters in fishes have been extensively studied but long-term (days to weeks) studies are rare. According to the Krogh principle, fish should be the better model organism for studying the effects of long-term hypoxia. (see Powell et al. 1998 for review).
While there has been significant progress towards understanding the effects of chronic hypoxia and how it alters cardioventilatory control in vertebrates, the mechanisms responsible for various changes remain elusive. Acclimatization of Ictalurus punctatus for 1 week under moderate aquatic hypoxia (PO2 75 mmHg, 50% air saturation) increases the ventilatory sensitivity to hypoxia. Heart rate was significantly higher in hypoxic-acclimatized fish but the sensitivity of the cardiac response to hypoxia (bradycardia) was not affected (Burleson et al. 2002). Thus, chronic hypoxia appears to have different effects on cardiovascular and ventilatory sensitivity to hypoxia in channel catfish.
More recently, Vulesevic et al. (2006) studied the effects of a longer period of acclimatization (28 days) to lower PO2 (30 mmHg) on cardioventilatory control in the zebrafish, Danio rerio. In contrast to channel catfish, zebrafish acclimatized to hypoxia do not show increased ventilatory sensitivity to either hypoxia or cyanide. The number of putative O2-sensitive chemoreceptor cells in the gills (5-HT immunopositive NECs) did not change in hypoxia-acclimatized zebrafish, but chronic hypoxia (60 days at 35 mmHg) did increase the number of neuroepithelial cells that lack 5-HT (Jonz et al. 2004). The roles of 5-HT and NECs in the response to hypoxia, both chronic and acute, remain unresolved.
O2 sensing mechanisms in the carotid bodies of mammals
The mammalian carotid body is a peripheral chemoreceptor that senses a number of blood-borne stimuli, including hypoxia and hypercapnia, transducing them into neural discharges that initiate a number of cardiorespiratory reflexes (Gonzalez et al. 1994). These include hyperventilation, but an elevated chemoafferent discharge can also induce direct cardiac and vascular reflexes such as bradycardia, decreased cardiac output and peripheral vasoconstriction, as well as augmented adrenomedullary output. These direct effects may, however, be obscured by secondary feedbacks via thoracic afferents and baroreceptors, and the precise pattern of reflexes often depends upon the degree of hyperventilation (de Burgh Daly et al. 1978).
The type-I cells of the carotid body contain the chemosensory elements and are presynaptic to the afferent nerve endings of the carotid sinus, with the Ca2+ dependent release of neurotransmitter believed to be consequent to voltage-gated Ca2+ entry through L-type channels. The necessary membrane depolarization is believed to occur subsequent to the closure of plasmalemmal K-channels (Lopez-Barneo et al. 2001). Although there is, as yet, no consensus on the precise K-channels inactivated or the mechanism of their inactivation, a role for voltage-gated calcium entry is largely accepted and much interest is now focused upon identifying the hypoxia sensor or sensors. Currently, key roles for the "energy sensor" enzyme, AMP-activated kinase (Evans et al. 2005) and for hemoxygenase-2 (Kemp 2005) have been proposed for the sensing of hypoxia, with both seemingly able to link cellular O2 lack with K-channel inactivation. Not all elevation of Ca2+, however, appears to be voltage-dependent and even when cells were voltage clamped around their resting membrane potential, some Ca2+ elevation in type-I cells was still observed during anoxia (Buckler 1997). An alternate route for stimulus-induced Ca2+ entry is therefore suggested and the transient receptor potential (TRP) superfamily of ion channels may provide this.
The TRP channel family mediates Na+ and Ca2+ entry in a wide variety of tissues and cells in response to G-protein activation and/or intracellular Ca2+-store depletion. These ion channels are evolutionarily well conserved and consist of six related protein families (including the classical or "canonical" TRPC family) that are arranged in tetramers to form relatively nonselective cation channels. These channels appear to be involved in the cellular sensing of a number of modalities and in a number of organisms, from yeast to mammals (Ramsey et al. 2006).
Interestingly, these modalities include temperature and osmolarity, which are also adequate stimuli for the carotid body (Kumar et al. 2007). The identification and location of TRPC channels has recently been determined in the type I cell by immunostaining (Buniel et al. 2003) and a functional role for this voltage-independent pathway is presently being investigated. Thus, whereas Ca2+-free + EGTA perfusate is known to abolish the carotid body's chemoafferent response to hypoxia, nifedipine, as an antagonist for L-type calcium channels, blocked ca. 45% of the response. In addition, 2-aminoethoxydiphenyl borate (2-APB), a commonly used, albeit relatively nonselective, antagonist for TRP channels, effectively reversed hypoxia/hipercapnia-induced chemoafferent discharge in the carotid body of the rat in vitro. Depletion of internal calcium stores induced a significant increase in afferent discharge frequency that was less sensitive to nifedipine, but was prevented by preincubating the carotid body with 2-APB. By transfecting type-I cells with a variety of TRPC antibodies we determined that TRPC1 and TRPC3 could, independently, inhibit the chemoafferent response to hypoxia.
Hypoxic ventilatory depression
Unlike adults, most newborn mammals (including those of humans) respond to hypoxia in a biphasic manner. That is, an initial increase in minute ventilation is followed by a depression in respiratory activity that can go below baseline (normoxic) level (Mortola 2001). In mature animals, however, the sharp increase in ventilation observed at the onset of hypoxia is usually followed by a respiratory depression, but the minute ventilation remains well above baseline level (Powell et al. 1998). The physiological significance of this hypoxic ventilatory depression is not clear, but may be part of an adaptive strategy that minimizes energy expenditure during hypoxia. While this chemoreflex is relatively well described in late fetal and postnatal stages, its maturation and emergence are not well understood owing, in part, to technical limitations associated with in utero measurements during early fetal stages. Bullfrogs, therefore, constitute a valuable alternative for such exploration of the development of respiratory control, because animals of all developmental stages are easily accessible and can be used for electrophysiological investigation using in vitro brainstem preparations.
Hypoxic ventilatory depression is well conserved amongst vertebrates, as exposing adult frogs to hypoxia leads to ventilatory depression (Rose and Drotman 1967). Furthermore, a recent study using in vitro brainstem preparations from Rana catesbeiana has shown that reducing O2 levels of the artificial cerebrospinal fluid (aCSF) superfusing the preparation decreases frequency of fictive lung ventilation (Winmill et al. 2005). As this preparation is completely devoid of peripheral (sensory) inputs, this chemoreflex must be of central origin. Moreover, the use of brainstems from various developmental stages enabled to show that in bullfrogs, this chemoreflex emerges over the course of development because hypoxic ventilatory depression becomes evident only following metamorphosis.
Several mechanisms have been proposed to explain hypoxic ventilatory depression, and much data indicate that an area in, and around, the Locus coeruleus (LC) in the rostral pons underlies this hypoxic respiratory depression (Bisonnette 2000). In newborn lambs, for instance, brainstem transection and focal cooling eliminating LC modulation of the respiratory network prevents ventilatory depression during hypoxia (Dawes et al. 1983; Moore et al. 1996). In the bullfrog, several in vitro experiments indicate that noradrenergic modulation, which likely originates from the LC, is necessary for full expression of the central hypoxic chemoreflex. First, application of noradrenaline onto brainstem preparations from different developmental stages elicits responses in lung-burst frequency similar to those observed during hypoxia: a small increase in premetamorphic stages and a robust depression in more mature (postmetamorphic) stages (Fournier and Kinkead 2006). Secondly, inactivation of 1-adrenoceptors by application of prazosine prior to hypoxia eliminates the lung-burst frequency response in all stages. Finally, results from preliminary experiments suggest that brainstem transection that eliminates LC neurons prevents the lung-burst response to hypoxia, thereby suggesting that LC neurons are necessary for the full manifestation of this reflex. The mechanisms underlying maturation of this reflex are still unclear, but we are currently testing the hypothesis that the change in the response (from excitatory in premetamorphic tadpoles to inhibitory in adult frogs) is related to the maturation of GABAergic neurotransmission, which likely acts as an intermediate in this chemoreflex.
Taken together, these data suggest that activation of the oxygen chemoreflex in vertebrates is highly complex. In fish, it encompasses several oxygen-receptor groups with different central projection areas and specific glutamate-receptor types for each reflex modality. In mammals, the transduction mechanism whereby blood O2 levels are translated into afferent neural impulses is becoming increasingly understood with new data implicating a number of ion channels that may be modulated either directly or indirectly by alterations in the local Po2 and/or the cellular metabolic status. The effects of chronic hypoxia in fishes and land vettebrates are probably time-dependent and complex, since they involve several physiological systems and responses, e. g., metabolic, cardiovascular, ventilatory, and autonomic. Therefore, exposure to chronic hypoxia on cardioventilatory control vary among different species as do the effects of acute hypoxia. Regardless, chronic hypoxia can be used as another tool for determining the mechanisms of O2-chemoreception and cardioventilatory control in vertebrates.
Sensing CO2/pH
Respiratory control and pulmonary ventilation in lungfish compared to land vertebrates
Sarcopterygians (the lobed-finned fish) gave rise to the land vertebrates (Tetrapoda), lungfish (Dipnoi) and Latimeria (Crossopterygii). Three genera of lungfish exist: Lepidosiren (South America), Protopterus (Africa), and Neoceratodus (Australia). Lungs and central chemoreceptors evolved within two lines: if lungfish are the sister group of tetrapods (Zardoya et al. 1998), many common features of pulmonary control would be explained. The toad Chaunus schneideri (earlier Bufo paracnemis) and the South American lungfish (Lepidosiren paradoxa) have lungs of very similar diffusing capacity (Bassi et al. 2005). In addition, both have central and peripheral H+/CO2–receptors that control acid–base status of the cerebrospinal fluid and blood (Amin-Naves et al. 2006). Chronic hypercarbia (48 h) caused large increases of pulmonary ventilation in Lepidosiren (Sanchez et al. 2005). Moreover, Lepidosiren increased pulmonary ventilation in response to gas phase hypoxia, whereas aquatic hypoxia had no effect on pulmonary ventilation (Sanchez et al. 2001a). By comparison, the gill ventilation of teleost fish adjusts to the ambient O2-levels of the water. A large number of teleost fish possess air breathing organs, but CO2-oriented ventilatory responses are usually absent. Instead, acid–base regulation is accomplished by ion exchanges.
Chemoreceptor function in lungfish has been studied to date only by means of superfusion of the ventral part of the medulla (Sanchez et al. 2001b). A reduction in the pH of the superfusate from 8.00 to 7.40 caused a 3-fold increase in pulmonary ventilation. Moreover, the CO2/H+-receptors produced hyperventilation in response to aquatic and/or gas phase hypercarbia over 48 h, while active plasma [HCO3–] was absent (Sanchez et al. 2005) in spite of the often hypercarbic and/or hypoxic habitat of the animal (Harder et al. 1999). Wilson et al. (2000) demonstrated pH-dependent frequency changes in fictive in vitro air breathing, using the preparations of the ray-finned, air-breathing fish Lepisosteus osseus. This is particularly interesting, since such measurements are inconclusive for modern teleost fish.
The relative contributions of peripheral and central CO2/H+-receptors are remarkably similar within tetrapods. The central drive is dominant with 70–80% of the total CO2/H+-receptor drive. The peripheral drive contributes 25% in a bird (Milsom et al. 1981) and 24% in a toad (Branco and Wood 1994). The corresponding value for Lepidosiren is 20% (Amin-Naves et al. 2007).
Lepidoserinid lungfish (Protopterus and Lepidosiren) and lissamphibians have very different acid–base status. At 25°C, Lepidosiren has PaCO2 = 21 mmHg/pHa = 7.53 (Bassi et al. 2005), while Chaunus schneideri has a PaCO2 of 12.4 mmHg/pHa 7.79 (Fernandes et al. 2005). At higher temperature (35°C) the values for the lungfish are similar to those of humans, with a PaCO2 of mmHg/pHa = 7.39 (Bassi et al. 2005).
Locus coeruleus as a chemoreceptive site in lissamphibians
The chemoreceptors of the ventral part of the medulla were the first to be located, but many sites are now under investigation. An example is Locus coeruleus, which has been described both in amphibians and mammals, suggesting an ancient origin.
Central CO2/pH neurons that can increase ventilation in mammals were earlier considered to be located close to the surface of the ventral medulla only. More recent studies suggest that central CO2/pH receptors are actually widespread within the brainstem (Coates et al. 1993) and sites have been identified in the ventrolateral medulla, nucleus of the solitary tract, ventral respiratory group, LC, caudal medullary raphe, and fastigial nucleus of the cerebellum (Nattie 2001). The specific contribution of each of these sites to the CO2 drive to breathing, however, is still debated (Mitchell 2004). LC neurons are of particular interest in CO2 challenge since >80% of neurons are found to be chemosensitive, responding to hypercapnia with an increased firing rate (Pineda and Aghajanian 1997; Oyamada et al. 1998; Filosa et al. 2002).
In lissamphibians, LC is located in the isthmic region, which lies at the rostral end of the hindbrain and is considered to be homologous to the LC of mammals (Marin et al. 1996). Recently, Noronha-de-Souza et al. (2006) documented several interesting features indicating that the LC plays an important role in CO2/pH drive to breathing in toads. First, a marked increase in c-fos positive cells in the LC was induced after breathing a hypercarbic gas mixture. Second, selective chemical lesions of LC catecholaminergic neurons attenuated the increase of the ventilatory response to CO2. Third, microinjection of acidic solutions into the LC increased pulmonary ventilation, indicating that LC neurons are intrinsically pH-sensitive. Collectively, these data provide evidence that the area in the toad brainstem homologous to the LC of mammals is chemosensitive, suggesting that nonmammalian vertebrates also may possess multiple sites of central chemosensitivity. Very likely, the transition from water-breathing to air-breathing was associated with demands for a more flexible and sensitive CO2 control system in tetrapods. Our findings further emphasize the similarities between anuran and mammalian LC, and support the proposed homology of this nucleus in both groups.
Chemosensitive signaling in CO2-sensitive neurons from the rat Locus coeruleus
A number of sites of chemosensitivity have been identified and differential responses of neurons from these sites have been described (for a review, see Putnam et al. 2004). For example, while nearly all neurons within the LC are chemosensitive, their response to hypercapnic acidosis is relatively small. In contrast, while less than half of neurons from the solitary tract nucleus are chemosensitive, they respond strongly to hypercapnic acidosis. Two of the remaining major questions pertain to what is/are the chemical signal(s) to which chemosensitive neurons respond and where on the neurons are the signals being sensed. Filosa et al. (2002) exposed LC neurons to a variety of acid stimuli in such a way that the neurons experienced an increase in CO2 or a decrease in extracellular pH (or both), to which they correlated the increased firing rate of these neurons. When CO2 was increased or when extracellular pH decreased, intracellular pH also decreased. In fact, it was the intracellular acidification that most strongly correlated with the increased firing rate.
Because there were at least two variables changing in the experiments by Filosa et al. (2002), Hartzler et al. (2007) developed a technique by which intracellular pH could be held constant during exposure to hypercapnic acidosis in spontaneously active neurons. Neurons exposed to hypercapnic acidosis with intracellular pH clamped still elicited an increased firing rate (1.0 ± 0.2 Hertz versus 1.9 ± 0.2 Hertz, P < 0.01, n = 6).
It is possible that the chemosensitive response reported here was influenced by chemical or electrical input from neighboring neurons. Preliminary experiments in which the intracellular pH is clamped while synapses and gap junctions are blocked suggest that a decrease of intracellular pH is a sufficient—although not necessary—signal for the chemosensitive response (Hartzler et al. unpublished results). Changes in intracellular pH have usually been measured in the soma of chemosensitive neurons; however, it is possible that the targets for these signals are not located in the soma, but are rather on the dendrites of these neurons. Local acidification of dendrites of LC neurons elicited no change in firing rate, in contrast to near-maximal increases upon somal acidification, indicating the soma to be the site of chemosensitivity (Ritucci et al. 2005).
The role of sodium/proton exchanger type-3 for central chemosensitivity in mammals
Antiporters are integral membrane proteins involved in active transport of ions in opposite directions across the plasma membrane, and one type, NHE3, is crucial to respiratory regulation in mammals. The sodium proton exchanger type 3 (NHE3) is a major membrane-bound antiporter in the proximal tubules of the kidney and in intestinal epithelia. It serves for the (re)uptake of considerable amounts of Na+ in exchange for protons. Mice lacking NHE3 are viable and largely compensate this malfunction by an increase in aldosterone-stimulated Na+ uptake (Schultheis et al. 1998). Nevertheless, these mice show signs of diarrhea and mild acidosis. On the other hand, NHE3 is important for the central control of breathing.
In the medulla, it is found in neurons of the ventrolateral and raphe region that have prevalence for central chemosensitivity (Ma and Haddad 1997; Wiemann et al. 1999; Kiwull Schöne et al. 2001). The role of NHE3 for neurons was first studied in organotypic cultures derived from coronal sections of the rat medulla (obex region). In this preparation, neurons retained their chemosensitivity, i.e., they increased firing rate upon hypercapnia. Moreover, they responded very similarly when intracellular pH (pHi) was lowered by 0.05–0.1 pH units, regardless whether this was achieved by hypercapnia, bicarbonate withdrawal, or ammonium prepulses (Wiemann et al. 1998). This led to the hypothesis that the pHi largely determines firing frequency of medullary chemosensitive neurons (Wiemann and Bingmann 2001). NHE3 inhibitors (S1611, S3226) reduced steady state pHi and, consequently, increased firing frequency of chemosensitive ventrolateral neurons, while neurons that were not excited by CO2/H+ failed to respond (Wiemann et al. 1999).
Effects of NHE3 inhibition on breathing and chemosensitivity were further studied in rabbits and rats. The selective brain permeant NHE3 inhibitor (S8218) applied to vagotomized and anesthetized animals increased alveolar ventilation and shifted the apnoeic threshold PCO2 to lower values (Kiwull-Schöne et al. 2001). Also, in a rat in situ model, which allows the study of the effects of drugs in the absence of anesthetics, S11599 (synonym: AVE1599, an enantiomer of S8218) reversibly increased phrenic-nerve discharges (Piechatzek et al. 2003). In anesthetized rats, infusion of AVE1599 increased the number of c-fos expressing neurons within the parapyramidal region of the brainstem, as did hypercapnia (Ribas-Salgueiro et al. 2006). Together, these findings provide convincing evidence for a CO2-mimetic effect of NHE3 inhibitors and strongly suggest that the NHE3-mediated pHi regulation is of biological significance in regard to the central control of breathing.
Interestingly, quantitative studies of RT-PCR revealed that brainstem NHE3 mRNA levels varied by more than one order of magnitude in rabbits (Wiemann et al. 2005). The different NHE3 mRNA levels inversely correlated with the individual ventilation of resting, awake rabbits: high levels of NHE3 were accompanied by low alveolar ventilation and high arterial PCO2 (Wiemann et al. 2005). Since NHE3 expression was not influenced by PaCO2 (Kiwull-Schöne et al. 2003), the level of NHE3 expression (which might reflect NHE3 activity) may be causally related to the set point of respiration in awake animals. Factors underlying the variability of NHE3 expression are still incompletely known.
In brainstem tissue, prolonged metabolic acidosis increased NHE3 mRNA within the limits found in nontreated animals (Kiwull-Schöne et al. 2006). In human brainstem tissue, NHE3 expression was also highly variable and initial data suggest that the level of NHE3 expression may become important under pathophysiological conditions such as sudden infant death. In line with this hypothesis, the threshold for the laryngeal reflex, a piglet model of sudden infant death, was elevated by NHE3 inhibition (Abu Shaweesh et al. 2002).
In conclusion, the lungfish and the land vertebrates share many components of respiratory control. Styloichthys lived about 417 million years ago and was the last common ancestor of Dipnoi and Tetrapoda (Zhu and Xu 2002). The many common features of respiratory control originated at a very early. As stated by Perry et al. (2001): "Fewer changes are required to conserve a respiratory mechanism than to completely eliminate it, to temporarily replace it and then to re-evolve structures in the same position."
The experiments described above support the multiple-factors hypothesis for central chemosensitivity, whereby multiple signals can each be sufficient to elicit the chemosensitive response. In addition, the data described emphasize the similarities between anuran and mammalian LC and support the proposed homology of this nucleus in both groups. The cellular mechanisms that allow LC neurons to respond to hypoxia remain to be investigated to determine whether mechanisms of O2 chemodetection share elements with the ability to detect CO2/H+. Based on the abundant evidence indicating that the function of these neurons is highly conserved amongst species, lissamphibian are an attractive model system for such investigations, especially when developmental issues are considered. Finally, NHE3 appears to be critically involved in the control of breathing in mammals.
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Teleost fish match an O2-oriented ventilation to ambient O2-levels, while CO2-sensing is secondary. Moreover, the gill receptors monitor blood and water, and provide information to sensory areas in the medulla. Tetrapods and lungfish have central and peripheral H+/CO2 receptors that control the acid–base status of CSF and the arterial blood. The African and South American lungfish share very distinct CO2-sensitive stretch receptors with tetrapods. Likewise, type-1 cells of the carotid body in mammals are probably identical to the O2-sensing cells in teleost fish gills. The LC is present in toads and mammals and provides a CO2/pH drive for lung ventilation, suggesting an early origin of key components for respiratory control. A fuller understanding of the similarities and the differences in peripheral and central chemosensing in both fish and land vertebrates is important for an appreciation of the evolutionary process of cardiorespiratory control and acid–base regulation and will certainly involve an integrated approach, including molecular biology, neurobiology, and respiratory physiology, which is in the spirit of the meeting that originated this text.
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This paper summarizes one of the 22 symposia that constituted the "First International Congress of Respiratory Biology" held August 14–16, 2006, in Bonn, Germany.
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