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On the peripheral and central chemoreception and control of breathing: an emerging role of ATP
http://www.100md.com 《生理学报》 2005年第21期
     1 Department of Physiology, Royal Free and University College London Medical School, London NW3 2PF, UK

    Abstract

    Peripheral and central respiratory chemoreceptors are ultimately responsible for maintenance of constant levels of arterial PO2, PCO2 and [H+], protecting the brain from hypoxia and ensuring that the breathing is always appropriate for metabolism. The aim of this discussion is to shed some light on the potential mechanisms of chemosensory transduction – the process which links chemosensory mechanisms to the central nervous mechanisms controlling breathing. Recent experimental data suggest that the purine nucleotide ATP acts as a common mediator of peripheral and central chemosensory transduction (within the carotid body and the medulla oblongata, respectively). In response to a decrease in PO2 (hypoxia) oxygen-sensitive glomus cells of the carotid body release ATP to activate chemoafferent fibres of the carotid sinus nerve which transmit this information to the brainstem respiratory centres. In response to an increase in PCO2/[H+] (hypercapnia) chemosensitive structures located on the ventral surface of the medulla oblongata rapidly release ATP, which acts locally within the medullary respiratory network. The functional role of ATP released at both sites is similar – to evoke adaptive enhancement in breathing. Understanding the mechanisms of ATP release in response to chemosensory stimulation may prove to be essential for further detailed analysis of cellular and molecular mechanisms underlying respiratory chemosensitivity.
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    Seventy-five years ago, during a meeting of The Physiological Society, Corneille Heymans communicated for the first time his landmark discovery of the chemosensory function of the carotid bodies (Heymans & Bouckaert, 1930). Although the existence of peripheral chemoreceptors was first suggested by the work of Pagano in 1900 (Pagano, 1900), elegant systematic observations by Heymans triggered extensive studies designed to determine the mechanisms of peripheral chemosensitivity and the role played by the arterial chemoreceptors in the respiratory and cardiovascular responses evoked by changes in arterial PO2, PCO2 and pH (reviewed in depth by Heymans & Neil (1958) and later by Michael de Burgh Daly in his Monograph for the Physiological Society (Daly M de B, 1997)).
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    In the 1960s studies by the groups led by Hans Loeschcke and Robert Mitchell identified the sites of CO2 chemosensitivity within the central nervous system. First, in accord with classical reaction theory and experimental evidence, it was postulated that changes in the [H+] (pH) of the extracellular fluid that follow changes in PCO2 represent the adequate and the main stimulus for the central chemosensors (for review see Loeschcke, 1982). Then, Loeschcke and Mitchell found that the primary set of CO2 chemoreceptors was localized at, or in close proximity to, the ventral surface of the medulla oblongata (Mitchell et al. 1963). Recent evidence suggests an important role of intracellular pH changes in triggering responses of central chemoreceptors to CO2 (Putnam et al. 2004). There are also data indicating that, in addition to the ventral medullary surface chemoreceptors, functional respiratory chemoreceptors may also be located in several other sites throughout the brainstem (Nattie, 1999, 2000).
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    Experiments conducted in animals in which the carotid and aortic bodies have been denervated indicated different functional roles of peripheral and central chemoreceptors. A large number of studies, impossible to list here, demonstrated clearly that under these conditions hypoxia fails to stimulate ventilation centrally, suggesting that carotid and aortic bodies are the primary O2-sensitive sites (Daly M de B, 1997). By contrast, although peripheral chemoreceptors are sensitive to changes in PCO2 and pH, the ventilatory response to CO2 is largely preserved in animals after denervation of peripheral chemoreceptors. Heeringa et al. (1979) estimated that up to 80% of the CO2-evoked response is mediated by the action of CO2 at the chemosensitive sites located in the brain.
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    In 2000 I joined the laboratory of Professor K. Michael Spyer in the Department of Physiology, University College London to investigate the role of purinoceptors in the mechanisms of CO2/H+ sensitivity of respiratory neurones that constitute the medullary respiratory network. This network contains neurones responsible for generation of the respiratory rhythm and pattern as well as premotor neurones responsible for transmitting this rhythm to spinal motoneurones controlling the diaphragm and intercostal muscles (Richter & Spyer, 2001; Feldman et al. 2003).
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    By this time it was already firmly established that, in addition to its known role as an intracellular energy source, ATP also functions as an extracellular signalling molecule in the central and peripheral nervous system and many peripheral tissues (Burnstock, 1997). Several subtypes of ionotropic (P2X) and metabotropic (P2Y) purinoceptors have been cloned and characterized (Ralevic & Burnstock, 1998; North, 2002); some of them were found to be [H+] sensitive and therefore could potentially confer this sensitivity onto the medullary respiratory neurones which express these receptors.
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    Indeed, many medullary neurones with respiratory-related activity are sensitive to changes in external pH (Kawai et al. 1996). The hypothesis that purinoceptors may be responsible for this chemosensitivity was proposed when Teresa Thomas demonstrated that blockade of ATP receptors within the ventral respiratory column of the medulla oblongata (microinjection of suramin) decreased resting respiratory activity and attenuated respiratory responses induced by inhaled CO2 (Thomas et al. 1999). Furthermore, the ATP receptor antagonists suramin or pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) reduced baseline firing and blocked CO2-induced increases in the activity of pre-inspiratory and inspiratory neurones (Thomas & Spyer, 2000).
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    Among different subtypes of P2 receptors, ionotropic P2X2 receptors are quite unique in terms of their high sensitivity to changes in external pH within the physiological range 7.4–7.1 (King et al. 1997; Wildman et al. 1997). We suggested therefore that if purinoceptors are indeed responsible for central respiratory chemosensitivity, they have to be of the P2X2 receptor subtype (either homomeric P2X2 receptors or heteromeric receptors containing P2X2 subunits).

, http://www.100md.com     When P2X2 receptor knockout (P2X2–/–) mice (Cockayne et al. 2005) became available for our studies, they were expected to provide quick and definitive answers regarding the role of P2X receptors in central chemosensory process. However, this was not the case. In conscious freely moving mice we recorded ventilation using whole-body plethysmography and found that resting ventilation and ventilatory responses to rising levels of CO2 in the inspired air were normal in P2X2–/– mice, and were normal also in P2X3 knockout (P2X3–/–) and in P2X2/P2X3 double receptor knockout (P2X2/3Dbl–/–) mice (Fig. 1A; Rong et al. 2003). These results suggested that either these transgenic animals effectively compensate for the loss of P2X2 (and P2X3) receptor subunits, or other subtypes of P2 receptors are involved in central CO2/H+ chemoreception.
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    A, changes in ventilation during hypercapnia (3 and 6% CO2 in the inspired air) in conscious P2X2 and P2X3 receptor double knockout (P2X2/3Dbl–/–) and wildtype (P2X2/3Dbl+/+) mice. B, changes in ventilation during hypoxia (15, 10 and 7.5% O2 in the inspired air) in conscious P2X2/3Dbl–/– and P2X2/3Dbl+/+ mice. The resting ventilation during normocapnia/normoxia was identical in the wildtype and knockout animals. The ventilatory response to hypoxia in the P2X3–/– mice was not significantly different from that in P2X3+/+ mice, while in the P2X2–/– mice it was markedly reduced and was similar to that in the P2X2/3Dbl–/–. VE, minute ventilation (respiratory rate x tidal volume). Data are presented as means ± S.E.M. Numbers in parentheses indicate sample sizes. *Significant difference, P < 0.05. Data redrawn with permission from Rong et al. (2003). Copyright 2003 by the Society for Neuroscience.
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    However, an unexpected role for ATP became apparent when P2X2–/– mice were exposed to hypoxic conditions. We observed that the increase in ventilation during hypoxia was markedly reduced in the P2X2–/– and P2X2/3Dbl–/– mice (Fig. 1B), but not in the P2X3–/– mice (Rong et al. 2003). At 7.5% oxygen in the breathing air a profound depression of respiration was observed in mice deficient in the P2X2 receptor subunit – ventilation fell well below the baseline (Fig. 1B). These data indicated that the P2X2 receptor subunit is essential for the development of the normal ventilatory response to hypoxia and suggested that peripheral chemoreceptor function is compromised in these animals.
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    In adult mammals type I (glomus) cells of the carotid body are the main peripheral O2 sensors (Gonzalez et al. 1994; Prabhakar, 2000; Lahiri et al. 2001; Williams et al. 2004). Upon stimulation (e.g. by hypoxia) type I cells release neurotransmitters (for a recent review see Nurse, 2005) to activate afferent nerve fibres of the carotid sinus nerve, which in turn relays this information to the CNS respiratory centres to evoke adaptive changes in breathing. The data we obtained subsequently in collaboration with Weifang Rong and Geoff Burnstock using a superfused in vitro carotid body–carotid sinus nerve preparation indicated that ATP may act as a key transmitter released in the carotid body by the oxygen-sensing type I cells. Our data confirmed the results of an earlier pharmacological studies conducted by Colin Nurse and colleagues using co-cultures of rat type I cells and petrosal neurones (Zhang et al. 2000; Prasad et al. 2001; Buttigieg & Nurse, 2004; Zhang & Nurse, 2004).
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    First, in studies using in vitro preparations taken from the wildtype mice we found that application of ATP evokes a dramatic increase in the carotid sinus nerve chemoafferent discharge (Rong et al. 2003), the effect originally described by Jarisch more than 50 years ago (Jarisch et al. 1952). Although, Jarisch and co-authors did not know about ‘purinergic signalling’, their conclusion about ‘nerve endings in the carotid bifurcation’ as the site of ATP action was quite right.
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    When the baseline carotid sinus nerve discharge in preparations taken from the wildtype and knockout animals was compared, a tonic influence of ATP on the sinus nerve chemoafferent discharge was revealed. Resting chemoafferent activity was found to be significantly lower in the P2X2–/–, P2X2/3Dbl–/–, as well as in P2X3–/– mice compared with that in their wildtype counterparts (Fig. 2). A further reduction in resting carotid sinus nerve discharge was observed in the presence of P2 receptor antagonists PPADS or 2' (or 3')-O-(trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) (Fig. 2).
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    The plot of average baseline firing rates of the carotid sinus nerve recorded in the superfused in vitro carotid body–carotid sinus nerve preparations taken from the P2X2–/–, P2X3–/–, P2X2/3Dbl–/– and wildtype mice. Note that TNP-ATP is a significantly more potent antagonist at homomeric P2X3 receptors (and heteromeric receptors which contain P2X3 subunit) in comparison to other P2X receptor subtypes. Accordingly, in the presence of TNP-ATP, resting carotid sinus nerve discharge in preparations taken from the wildtype mice is reduced to the level recorded in the P2X3–/– mice, and in preparations taken from the P2X2–/– mice, to the level seen in the P2X2/3Dbl–/– mice. Data are presented as means ± S.E.M. *Significant difference compared with the level of baseline discharge in the preparations taken from the wildtype mice, P < 0.05. Significant effect of TNP-ATP on the carotid sinus nerve discharge in preparations taken from the wildtype and P2X2–/– mice, P < 0.05. (A. V. Gourine, W. Rong, D. A. Cockayne, A. P. Ford, G. Burnstock & K. M. Spyer, unpublished observations).
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    Hypoxia-induced increases in the discharge of single chemoafferent fibres of the carotid sinus nerve were found to be dramatically reduced in the P2X2–/– mice (by 80%) and even further in the P2X2/3Dbl–/– mice (by 85%, Fig. 3) (Rong et al. 2003). PPADS, in a dose-dependent manner, reduced hypoxia-evoked activation of carotid chemoafferents in the preparations taken from the wildtype animals. Interestingly, afferent responses to a decrease in PO2 were not affected by the P2X3 receptor subunit deficiency (Fig. 3), an observation that correlates well with the results obtained using whole-body plethysmography and mentioned above. These data indicated that receptors of the P2X2 subtype are essential and sufficient to mediate the effect of hypoxia on the activity of the carotid sinus nerve chemoafferents. Nevertheless, reduced resting sinus nerve discharge in P2X3–/– mice (and in the presence of the potent P2X3 receptor antagonist TNP-ATP), and significantly smaller hypoxia-induced chemoafferent responses in P2X2/3Dbl–/– mice compared with that in P2X2–/– mice suggested a role for the P2X3 receptor subtype as well (Rong et al. 2003).
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    The plot of average hypoxia-induced peak firing rates of single chemoafferent fibres of the carotid sinus nerve recorded in the superfused in vitro carotid body–carotid sinus nerve preparations taken from the P2X2–/–, P2X3–/–, P2X2/3Dbl–/– and respective wildtype mice. Note that hypoxia-induced increase in the chemoafferent discharge in the P2X2/3Dbl–/– mice was significantly smaller in comparison to that in the P2X2–/– mice. Data are presented as means ± S.E.M. Numbers in parentheses indicate sample sizes. *Significant difference, P < 0.05. Data from Rong et al. (2003).
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    Finally, in accord with previous studies in rats (Prasad et al. 2001), we observed that in the wildtype mice both P2X2 and P2X3 receptor subunits are expressed in the carotid body (Rong et al. 2003). In collaboration with Michael Duchen we examined staining patterns of the P2X2 and P2X3 receptor subunit immunoreactivities using confocal microscopy and found that both subunits were confined to the afferent terminals of the sinus nerve surrounding individual glomus cells or their clusters.
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    Thus, ATP can now be considered as one of the key mediators of peripheral chemosensory transduction in the carotid body, playing a pivotal role in transmitting information about arterial PO2 levels. We propose that during hypoxia O2-sensitive glomus cells release ATP to activate the peripheral terminals of the carotid sinus nerve via interaction with P2X receptors that contain the P2X2 subunit, with or without the P2X3 subunit.

    The evidence that P2X2–/– mice can mount a normal respiratory response to an increase in the level of inspired CO2 was considered insufficient to rule out the role of purinergic signalling in the central chemosensory process. As mentioned above, there was always a possibility remaining that the P2X2 receptor subtype plays an important role in medullary CO2/H+ chemoreception, but knockout mice effectively compensate for its loss with increased expression of other subunit(s) or up-regulation of an as yet unknown parallel mechanism. Alternatively, even if the P2X2 subtype has no role at all, other P2 receptors expressed in the medulla oblongata (Yao et al. 2000; Thomas et al. 2001) could as well mediate the effect of CO2 on breathing.
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    Simultaneously with the carotid body work, in collaboration with Jim Deuchars and Lucy Atkinson (University of Leeds) we investigated the extent of P2X2 receptor subunit expression among the physiologically identified (using in vivo rat models) respiratory neurones of the medullary ventral respiratory column (Gourine et al. 2003). The P2X2 receptor subunit was detected in 50% of expiratory neurones and in 20% of neurones with inspiratory-related discharge: pre-inspiratory and inspiratory (Gourine et al. 2003). Importantly, a substantially larger proportion of the respiratory neurones increased their discharge in response to microionophoretic application of ATP: 80% of expiratory neurones and 30% of neurones with inspiratory-related discharge were found to be rapidly excited by ATP (Gourine et al. 2003). These data suggested that, in addition to the P2X2 receptor subtype, other P2X receptors are expressed by the medullary respiratory neurones. Furthermore, significant numbers of respiratory neurones (30% of all tested units) were excited strongly by the P2Y receptor agonist uridine 5'-triphosphate (UTP), indicating that these neurones express functional metabotropic P2Y receptors (A. V. Gourine & K. M. Spyer, unpublished observations).
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    The ventral medullary respiratory network which generates breathing activity is located just above the classical CO2/H+ chemosensitive areas which were identified on the ventral surface of the medulla oblongata in the pioneering studies by Loeschcke and Mitchell (Mitchell et al. 1963). We have found that some of the medullary respiratory neurones which express the P2X2 receptor subunit have long processes projecting towards the ventral medullary surface chemosensitive areas (Gourine et al. 2003). Thus, ATP could act on these ventrally projecting dendrites of respiratory neurones to activate the respiratory network and evoke changes in breathing. This would suggest that ATP may still have a role in the central CO2 chemosensory process; nevertheless, definite evidence for and understanding of this role were missing.
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    To advance this problem further we established in our laboratory a surgical approach to the ventral surface of the medulla oblongata in anaesthetized and artificially ventilated rats. Meanwhile in the late 2002, our long-standing collaborators Nicholas Dale and Enrique Llaudet from the University of Warwick invented enzyme-based microelectrode ATP biosensors which allowed real-time measurements of changes in extracellular concentration of ATP with unprecedented resolution and sensitivity (Llaudet et al. 2003, 2005; Dale et al. 2005).
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    In the very first experiment, ATP biosensors placed in direct contact with the ventral surface CO2/H+ chemosensitive areas of the medulla recorded an almost immediate release of ATP in response to systemic hypercapnia induced by an increase in the level of inspired CO2 (Gourine et al. 2005a). This CO2-induced ATP release was observed in normally breathing (isocapnic eupnoea) rats as well as in animals in which hypocapnic apnoea had been evoked (by mechanical hyperventilation to reduce blood and brain levels of PCO2). In both cases the hypercapnia-induced release of ATP from the ventral surface chemosensitive areas preceded (by 20 s) the increase in respiratory activity (Fig. 4A) (Gourine et al. 2005a). Further experiments revealed that ATP release from the surface chemosensitive areas mirrors the CO2 stimulus, is site specific (it occurs on the ventral but not on the dorsal surface of the medulla) and does not require inputs from the peripheral chemoreceptors (i.e. the amount of hypercapnia-induced ATP released in sino-aortically denervated and vagotomized rats was similar to that of the control animals) (Gourine et al. 2005a). Using miniature (125 or 250 μm in diameter) disk biosensors we were able to demonstrate that during hypercapnia ATP is released on the ventral surface of the medulla in discrete locations that correspond very closely to the classical CO2 chemosensitive areas described in the pioneering studies by Loeschcke and Mitchell (Loeschcke, 1982) (Fig. 5A; Gourine et al. 2005a).
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    A, representative raw data illustrating changes in respiratory activity (phrenic nerve discharge) and concentration of ATP on the ventral surface of the medulla in response to an increase in the level of inspired CO2 in the anaesthetized and artificially ventilated rat. To determine the temporal relationship between changes in ATP levels and CO2-evoked enhancement in the respiratory activity, hypocapnic apnoea was induced in this animal by mechanical hyperventilation so that PCO2 in the arterial blood and end-tidal levels of CO2 were below the apnoeic threshold. B, representative raw data illustrating changes in the respiratory activity and concentration of ATP on the ventral surface of the medulla during systemic hypoxia (10% O2 in the inspired air). Biosensors (> 1 mm in length; 100 μm in diameter) were placed in direct contact with a significant portion of the ventral surface CO2 chemosensitive areas. A dual recording configuration of ATP sensor placed upon one side of the medulla along with a control (null) sensor that was placed in an equivalent position on the other side was used. The null sensor lacked the essential enzymes and thus served as a control to determine whether any ‘non-specific’ electroactive interferents were released and could confound ATP measurements. Arrow indicates the moment at which the concentration of ATP starts to increase above the baseline. PNG, integrated phrenic nerve activity (arbitrary units). ABP, arterial blood pressure. Data in A redrawn from Gourine et al. (2005a) and data in B redrawn with permission from Gourine et al. (2005b). Copyright 2003 by the Society for Neuroscience.
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    A, schematic drawing of the ventral aspect of the rat medulla oblongata showing sites that exhibited release of ATP in response to CO2 (). No ATP release was detected in locations depicted by . Miniature (125 or 250 μm in diameter) disk biosensors were used to map the sites of CO2-induced ATP production. B, representative raw data illustrating CO2/H+-induced release of ATP from the ventral medullary surface in vitro. CO2-induced acidification of the incubation media from pH 7.4 to 7.0 evoked marked release of ATP from the most ventral slice of the medulla oblongata, which contained surface chemosensitive areas. C, summary data (means ± S.E.M.) of peak CO2/H+-induced release of ATP from horizontal slices of the medulla. ATP release occurs predominantly within 400 μm of the ventral surface. The ‘Net ATP’ trace represents the difference in signal between ATP and null sensors. PNG, integrated phrenic nerve activity (arbitrary units). 7n, relative position of the facial nucleus; XII, hypoglossal nerve roots. Data reproduced from Gourine et al. (2005a).
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    To dissect the sites of CO2-induced ATP production further, I spent several weeks at the University of Warwick preparing together with Nick Dale horizontal slices of the rat medulla oblongata in order to isolate and study medullary chemosensitive structures in vitro. This approach proved to be surprisingly difficult as most of the medullary slices cut in our initial experiments exhibited no ATP release in response to an in vitro analog of hypercapnia (CO2-induced acidification of the incubation media from pH 7.4 to 7.0). However, when the brainstem dissection and slicing techniques were refined to preserve the surface layers we consistently recorded marked CO2/H+-induced release of ATP from the most ventral slice, which contained surface chemosensitive areas (Fig. 5B and C; Gourine et al. 2005a). Interestingly, more dorsal slices in the sequence never exhibited significant ATP release in response to a decrease in pH (Fig. 5B and C). These results indicated that during hypercapnia ATP is released from the sources located at the very surface of the medulla, as preservation of the surface layers during slice preparation was essential to observe release of ATP in response to CO2/H+.
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    As yet, the functional role of ATP released during hypercapnia from the ventral surface chemosensitive areas remained unclear and back in London, using in vivo rat models, we investigated whether ATP applied to the ventral surface of the medulla could mimic the effect of CO2 on breathing. Indeed, we found that application of exogenous ATP to the medullary surface chemosensitive areas (which release ATP in response to CO2) induces an almost immediate increase in respiratory activity (Fig. 6A) (Gourine et al. 2005a). This effect of ATP was not affected by the adenosine receptor blockade but was markedly reduced in the presence of the P2 receptor antagonist suramin (Gourine et al. 2005a).
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    A, ATP and UTP applied (30 μl droplet) to the ventral medullary surface enhance respiratory activity. The plot of peak ATP- and UTP-induced changes in the amplitude of the phrenic nerve discharge. Note that changes in the phrenic nerve discharge induced by ATP and UTP were equivalent in magnitude. However, the effect of UTP was delayed (time to peak of response 239 ± 41 s) compared with that of ATP (time to peak 27 ± 4 s). B, summary data showing significant increase in mean threshold level of end-tidal CO2 required to induce breathing from hypocapnic apnoea in the presence of P2 receptor antagonists PPADS or TNP-ATP on the ventral surface of the medulla oblongata. Data are presented as means ± S.E.M. Numbers in parentheses indicate sample sizes. *Significant difference, P < 0.05. Data redrawn from Gourine et al. (2005a).
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    The functional role of CO2-induced ATP release was finally revealed when we observed that blockade of ATP receptors within the ventral surface chemosensitive areas reduced the respiratory response to systemic hypercapnia (Fig. 6B). The P2 receptor antagonists PPADS or TNP-ATP when applied to the ventral surface of the medulla reduced to a similar extent both the sensitivity and the gain of the respiratory responses to rising levels of inspired CO2 (Gourine et al. 2005a). Considering the pharmacological profiles of PPADS and TNP-ATP, speculations concerning the subtype of the P2 receptors which may mediate the effect of ATP on breathing are possible. For example, to activate the respiratory network ATP may act at P2X2 receptors (PPADS and TNP-ATP are equipotent antagonists of the P2X2 receptors) located on the dendrites of medullary respiratory neurones which project ventrally, towards the source of the ATP release (Gourine et al. 2003). However, taking into the account data obtained in the P2X2–/– mice we now consider this issue to be of a secondary importance as multiple P2 receptors may be involved. This point is further strengthen by the fact that UTP applied on the ventral surface of the medulla had a delayed effect (but equivalent to ATP in magnitude, Fig. 6A) on respiratory activity, suggesting that metabotropic P2Y receptors may in part mediate the action of ATP on breathing (Gourine et al. 2005a).
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    These findings indicated that an increase in CO2/H+ induces immediate release of ATP from the chemosensitive structures located on the ventral surface of the medulla. After being released ATP is acting locally (presumably on the distal dendrites of the ventral respiratory column neurones that project close to the ventral surface) to evoke adaptive enhancement in breathing and therefore can be considered as one of the key mediators of central chemosensory transduction.
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    However, when the hypoxic stimulus was applied in the same experimental setting we obtained results which were difficult at a first glance to reconcile with the proposed model of central chemosensory transduction which involves ATP as the key mediator. It was found that during systemic hypoxia (10% O2 in the inspired air) ATP is also released from the ventral surface chemosensitive areas of the medulla (Fig. 4B) (Gourine et al. 2005b). Further experiments conducted both in vivo and in vitro revealed that the amount of the hypoxia-induced ATP released from the ventral medullary surface is very similar to that released in response to CO2 (Gourine et al. 2005a, b). In addition, ATP release was also present in sino-aortically denervated and vagotomized rats, in which hypoxia induces only depression of respiration (Gourine et al. 2005b).
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    Since hypoxia fails to stimulate ventilation centrally, ATP release from the ventral surface chemosensitive areas in these conditions seemed to present a problem for our model of central chemosensory transduction which proposes that the release of ATP constitutes a key initial event. There was, however, one major difference between hypercapnia- and hypoxia-induced release of ATP on the ventral medullary surface. As mentioned above, hypercapnia-induced release of ATP from the ventral surface chemosensitive areas always preceded (by 20 s) the increase in respiratory activity (Fig. 4A) (Gourine et al. 2005a). Conversely, during hypoxia ATP release was delayed and occurred 25 s after the initiation of enhanced respiratory activity (Fig. 4B) (Gourine et al. 2005b).
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    Interestingly, the secondary hypoxia-induced slowing of the respiratory rhythm was significantly augmented following blockade of ATP receptors in the ventral medulla (microinjection of PPADS) (Gourine et al. 2005b), revealing the functional role of ATP release during hypoxia – to maintain respiratory activity in conditions when hypoxia-induced slowing of respiration occurs. This conclusion is further supported by our data obtained in P2X2–/– mice which in contrast to their wild-type counterparts displayed profound respiratory depression at 7.5% oxygen in the breathing air (Fig. 1B) (Rong et al. 2003). Thus, the role of ATP released in the ventral medulla during the period of hypoxia-induced depression of ventilation appears to be similar to that during hypercapnia, i.e. to stimulate breathing. However, the influence of ATP is clearly not sufficient to fully counteract other powerful mechanisms responsible for hypoxia-induced respiratory depression (Bissonnette, 2000).
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    The sources and the mechanisms of ATP release from the ventral surface chemosensitive areas as yet remain unknown. The evidence that both hypercapnia and hypoxia induce release of ATP which is similar in magnitude (albeit with different time courses) may be very useful for further detailed analysis of the underlying cellular and molecular mechanisms. Hypoxia has been found to induce a delayed(!) extracellular acidification in the medullary chemosensitive areas (Xu et al. 1992), while neurones in the ventrolateral medulla exposed to anoxia display a decrease in intracellular pH similar to that evoked by CO2 (Chambers-Kersh et al. 2000; Putnam et al. 2004). Therefore, studies of the relationships between ATP release and intracellular pH in neurones and glial cells from chemosensitive and non-chemosensitive regions of the medulla could be a good starting point.
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    Further experiments are also needed to reconcile our data with the existing ‘cholinergic’ hypothesis of central chemosensory transduction (Loeschcke, 1982) as well as with the recent evidence indicating that both chemosensitive serotonergic neurones of the midline raphé (Richerson, 2004; Richerson et al. 2005) and glutamatergic neurones of the retrotrapezoid nucleus (Mulkey et al. 2004; Guyenet et al. 2005) can function as central respiratory chemoreceptors. Interestingly, we observed CO2-induced ATP release from the ventral medullary surface sites located in close proximity to the basilar artery and lateral to the pyramidal tracts (Fig. 5A), where chemosensitive serotonergic medullary neurones are concentrated (Bradley et al. 2002). Studies designed to establish whether during hypercapnia these populations of medullary chemosensitive neurones are the sources of ATP release or the targets of ATP action could yield potentially interesting and important results.
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    In summary, the data obtained over the last 5 years have revealed the importance of ATP-mediated purinergic signalling in the mechanisms responsible for the control of respiration. It emerges that ATP acts as a common mediator of peripheral and central chemosensory transduction. In the carotid body ATP transmits information about oxygen levels in the arterial blood, while in the medulla oblongata it mediates the action of CO2/H+ on breathing. Thus, at both sites the functional role of ATP is similar – to link chemoreception to the central nervous mechanisms controlling respiratory activity (Fig. 7). The fact that the same transmitter is employed within one physiological system to mediate afferent transduction in two distinct sites, one in the periphery and one within the central nervous system, seems very interesting from both physiological and evolutionary points of view.
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    In the carotid body, a decrease in PO2 or an increase in PCO2/[H+] activates glomus cells which release ATP as the main transmitter to stimulate afferent terminals of the carotid sinus nerve via interaction with P2X receptors that contain the P2X2 subunit, with or without P2X3 subunit. On the ventral surface of the medulla an increase in PCO2/[H+] activates primary chemosensors which release ATP to act via P2 receptors on ventrally projecting dendrites of more dorsally located secondary chemosensitive neurones and/or respiratory neurones. The activity of these neurones feeds into the respiratory network and evokes adaptive increases in breathing. The cellular sources of ATP release during hypercapnia as yet remain unknown. Other putative chemosensory transduction mechanisms (involving ACh, serotonin and others) are not shown for presentation purposes. Inset, the drawing shows a reconstruction of a representative pre-inspiratory neurone in the ventral respiratory column which was strongly activated during systemic hypercapnia. Long dendrites of this particular neurone have been found to reach the ventral surface of the medulla (A. V. Gourine & K. M. Spyer, unpublished observations). NTS, nucleus of the solitary tract.
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    Footnotes

    This Wellcome Prize Lecture was given on 20 December 2004 at King's College London.

    References

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    Acknowledgements

    I am most grateful to Professor K. Michael Spyer and Professor Nicholas Dale who have given me essential support and encouragement over the years and have made an invaluable contribution to the studies described above. I would like also to thank Enrique Llaudet, co-inventor of ATP biosensors (together with Nicholas Dale), with whom we performed all the ATP measurements; Dr Weifang Rong, with whom we conducted experiments using superfused carotid body preparations; Drs Jim Deuchars and Lucy Atkinson, who helped me with the P2X2 immunohistochemistry; Professor Geoffrey Burnstock, who supported carotid body work; Drs Debra Cockayne and Anthony Ford from Roche Bioscience, who supplied P2X knockout mice; Professor Michael Duchen, who helped us with confocal microscopy; and Dr Zhenghua Xiang, who assisted with detection of the P2X2 and P2X3 immunoreactivities in the carotid bodies.

    I am also most grateful to the members of the Physiological Society prize committee who nominated me for this award and The Wellcome Trust for the prize and the medal.

    The experimental work described in this paper was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust., 百拇医药(Alexander V Gourine)