The carotid body chemoreceptors, the major hypoxia sensory organs for the respiratory system, undergo a significant increase in their hypoxia responsiveness in the postnatal period. This is manifest by a higher level of afferent nerve activity for a given level of arterial oxygen tension. The mechanism for the enhanced sensitivity is unresolved, but most work has focused on the glomus cell, a secretory cell apposed to the afferent nerve ending and believed to be the site of hypoxia transduction. The glomus cell secretory response to hypoxia increases postnatally, and this is correlated with an enhanced calcium rise in response to hypoxia and an increase in oxygen-sensitive potassium currents. These changes are sensitive to the level of hypoxia in the postnatal period, and significant impairment of organ function is observed with postnatal hypoxia as well as postnatal hyperoxia. Although many questions remain, especially with regard to the coupling of glomus cells to nerve endings, the use of cellular and molecular techniques should offer resolution in the near future.
- receptor properties
the carotid body is a small sensory organ located at the bifurcation of the common carotid artery into the internal and external carotid arteries. Its mission is to respond to decreases in the level of arterial oxygen tension by increasing the spontaneous spiking activity on some sinus nerve afferent fibers. These fibers join the main trunk of the glossopharyngeal nerve with central terminations in the vicinity of respiratory and cardiovascular control nuclei in the dorsal brain stem and, when stimulated, evoke arousal, increased blood pressure, and increased breathing (appropriate physiological responses to counter the decrease in arterial oxygen tension).
The relationship between afferent nerve activity and arterial oxygen tension is hyperbolic in much the same way that hemoglobin saturation is a hyperbolic function of oxygen (20). The reason for this is not immediately obvious because the carotid body primarily responds to arterial oxygen tension and not oxygen content, so that carbon monoxide poisoning fails to excite afferent activity (40). Nevertheless, the relationship provides a useful reference, and one may anticipate a half-maximal nerve response at oxygen levels that reduce saturation by 50%, ∼35 Torr.
CHANGES IN OXYGEN RESPONSIVENESS IN THE NEWBORN PERIOD
In contrast to recordings in the mature animal, recordings from fetal or newborn animals showed little chemoreceptor activity to be present on the sinus nerve (5, 36). What little activity was present responded poorly to hypoxia but could be increased by strong stimuli, such as an intra-arterial injection of carbon dioxide-saturated blood (4). However, several days to weeks after birth, normal chemoreceptor activity could be recorded, indicating that a significant developmental increase in activity had occurred during this newborn period (12, 43).
The trigger for the increase in hypoxia sensitivity appears to be the increase in arterial oxygen that occurs at birth, increasing from ∼25 to ∼50 Torr and then to 100 Torr. If this rise in oxygen is prevented by birthing into a low-oxygen environment (e.g., 12% oxygen), then chemoreceptor maturation is seemingly inhibited. The ventilatory response to hypoxia maintains an immature pattern (22); the nerve response to acute hypoxia may be reduced (18), and the normal stimulus interaction between hypoxia and hypercapnia fails to develop (41). Similarly, exposure to hyperoxia in utero can apparently initiate the maturation process. Chemoreceptor activity appears to be more abundant in the fetus after hyperoxia to the ewe (6).
Any understanding of the mechanism for the postnatal increase in hypoxia sensitivity depends on an understanding of how the mature organ functions. At first glance, this should be a relatively easy task because the anatomy appears relatively simple. When they enter the organ, the afferent nerve fibers bifurcate up to 20 times (Fig.1, line 7) and terminate in complicated synapses on glomus cells (Fig. 1). These secretory cells occur in clusters and are surrounded by a supporting glial-like cell. On the basis of the anatomic relationship, a reasonable and best-accepted model is that hypoxia is transduced by the glomus cell, which leads to afferent nerve excitation, probably through the release of an excitatory transmitter (Fig. 1, line 4). Release of the (purported) excitatory transmitter is triggered by a rise in glomus cell intracellular calcium (Fig. 1, line 3), which is dependent on glomus cell depolarization. The secretory vesicles (see Fig. 1) contain catecholamines (primarily dopamine) and may contain other neurotransmitters. There is strong evidence that maturational changes occur in each of these steps, but, as one works back from measurements of nerve activity, the importance of any given step becomes less certain. Hence, let us consider the maturational changes in reverse of normal, i.e., from changes in nerve activity to changes in glomus cell membrane potential. This view may also be considered to be from least speculative to more speculative.
DEVELOPMENTAL CHANGES WITHIN OUR TRANSDUCTION SCHEMA
As stated above, it is best established that chemoreceptor nerve activity increases in the newborn period in vivo. At least part of this maturational change is independent of circulatory factors, since an increase in hypoxia responsiveness is observed using isolated carotid bodies in vitro, which eliminates humoral and circulatory variables (37). The peak nerve activity to a strong hypoxic stimulus increases about fourfold between 1 and 30 days of age in the rat, with most of this maturation occurring in the first week of life (37).
The increase in nerve activity is related to at least two changes. First, the number of nerve terminals per parent afferent axon increases about fourfold during the postnatal period (39). However, the electrophysiological properties of the afferent axon (rheobase, input resistance) do not significantly change during this period, so the increase in spiking is not due to an increase in afferent neuron excitability (15). Second, the magnitude of hypoxia-induced catecholamine secretion is enhanced about fourfold during the newborn period (Fig. 1, line 4) (17). These changes are also correlated with changes in catecholamine-synthesizing enzymes and some dopamine receptors. Quantitative in situ hybridization histochemistry and RT-PCR indicate that the message in the carotid body for the dopamine D2 receptor increases during the postnatal period in rats (26) and rabbits (1). D2 receptors on the glomus cell may serve as autoreceptors for secreted catecholamines (Fig. 1, line 6). Similarly, the message for D2 receptors is increased in the petrosal ganglia during the postnatal period, perhaps reflecting an increase in postsynaptic dopamine receptors on the afferent nerve endings (Fig. 1, line 5) (1). Northern analysis and RT-PCR have also demonstrated the presence of a dopamine D1 receptor message in the carotid body and petrosal ganglia, but developmental changes have not been addressed (2). In contrast, the message for tyrosine hydroxylase decreases during the postnatal period (26), probably reflecting a net decrease in catecholamine turnover after birth as the partial pressure of oxygen in arterial blood rises (33).
Interpretation of the catecholamine results is not immediately clear. Catecholamines (primarily dopamine) are stored in dense-cored secretory granules of glomus cells, and secretion is temporally linked to changes in afferent nerve activity (16). However, exogenous administration of dopamine is generally inhibitory to ongoing nerve activity, and administration of dopamine antagonists generally leads to an increase (not the expected decrease) in spontaneous spiking activity (20, 53). This leads to the postulate that endogenous catecholamines are inhibitory and may serve as an inhibitory modulator of chemoreceptor activity. However, as suggested by Gonzalez et al. (27), exogenous administration of catecholamine agonists or antagonists may primarily target autoreceptors, leaving the important postsynaptic receptors on the afferent nerves relatively untouched. A resolution to this controversy is beyond the present scope and, indeed, beyond our present ken. At present, we will consider it axiomatic that afferent nerve activity is initiated by glomus cell secretion, but it is unclear whether catecholamines are primarily responsible for nerve excitation or whether other costored or separately stored neurotransmitters are responsible for nerve excitation.
Glomus cell secretion, as assayed by catecholamine release, is likely caused by a rise in intracellular calcium concentration ([Ca2+]i), since both occur in close temporal association in isolated glomus cells (44). Like the nerve activity, the increase in [Ca2+]i is a hyperbolic function of environmental Po 2, with a rapid increase below 30 Torr (3, 51). The magnitude of the calcium response is a strong function of developmental age (51). The responses to an anoxic stimulus or cyanide are three- to fivefold greater in cells harvested from adult rabbits compared with newborn rabbits (49). Not only is the magnitude of the response greater but the sensitivity to hypoxia appears to increase. In rat cells, the point of 50% maximal calcium response increased from ∼1 Torr in cells from 1-day-old fetal rats to ∼6 Torr in cells from 11- to 21-day-old rats (51).
The rise in calcium is dependent on glomus cell depolarization and activation of voltage-dependent calcium channels (9). Voltage clamping at the resting membrane potential or administration of calcium channel blockers reduces or ablates the hypoxia-induced rise in [Ca2+]i or the catecholamine secretory response to hypoxia (9, 45). However, calcium channels do not appear to undergo a major maturational change, since the magnitude of change in [Ca2+]i in response to increased extracellular potassium is the same across development (51).
Activation of these calcium currents is dependent on membrane depolarization, but the cause of the depolarization is unclear. At least three types of potassium currents inhibited by hypoxia have been described: 1) a fast-activating, rapidly inactivating current in rabbit glomus cells (42), 2) a BK-type current (large conductance calcium-dependent potassium current) in rat cells (Fig. 1,line 1) (46), and 3) a non-voltage-dependent leak potassium conductance in rat cells (Fig. 1, line 2) (8). The importance of each of these currents in mediating hypoxia transduction is unresolved, but it is perhaps important that two have been shown to change developmentally. The BK-type current is smaller in glomus cells of 4-day-old rats compared with 11-day-adult rats (Fig. 1, line 1) (28), and exposure of rats to postnatal hypoxia (which appears to delay the maturation process) suppresses the magnitude of the hypoxia-sensitive BK current (Fig. 2,point 1) (52). Like the BK current, the leak potassium current also matures during the postnatal period and this maturation is inhibited by postnatal hypoxia (Fig. 2, point 2) (13, 50). In addition to a reduction in the oxygen-sensitivity potassium currents, chronic hypoxia results in glomus cell hypertrophy and an increase in calcium current density (29). Despite the larger calcium currents, hypoxia-induced changes in nerve activity and catecholamine release are reduced in these carotid bodies (18). However, paradoxically, basal secretion rates of catecholamine during normoxia are high (18, 32). Thus a maturation in the membrane currents that mediate glomus cell depolarization during hypoxia may account for all the downstream changes: an increased response of intracellular calcium to hypoxia, an increase in the magnitude of secretion with hypoxia, and an increase in the magnitude of the nerve response.
TROPHIC AND DEPOLARIZING INFLUENCES
In addition to cellular and subcellular changes within the chemoreceptor complex (e.g., ion channel current or regulation of calcium levels), the newborn period is critical in determining neuronal survival and neuronal phenotype. There is significant loss of chemoafferent neurons if the carotid body is removed from the nerve terminals at a critical time in the newborn period, and the loss may be prevented by application of brain-derived neurotrophic factor (BDNF) (31). In “knockout” mice lacking BDNF, there is a severe underdevelopment of carotid body innervation and altered respiratory drive (23). BDNF is transiently expressed in the carotid body during a period corresponding to the onset of innervation and at a time when petrosal neurons are dependent on BDNF for survival (7, 31). Therefore, it appears that, during the fetal and neonatal period, release of trophic factors by the carotid body is critical for establishment and maintenance of the afferent neural innervation of the carotid body by petrosal ganglion neurons. This is not true in later life, when surgical resection or destruction of the carotid body does not result in degeneration of sinus nerve fibers (48).
Postnatal Po 2 also plays a major role in determining neuronal survival. Rats reared in 60% oxygen for 4 wk suffered a 41% loss of unmyelinated, sinus nerve axons (Fig. 2,point 3) and a reduction in carotid body volume compared with control rats (Fig. 2, point 4) (24). This correlated with a loss of neurons in the petrosal ganglia but no loss of neurons in a control (nodose) ganglia, and no change in neuronal number was observed after a similar hyperoxia exposure in mature rats. With obvious parallels to the neuronal cell loss experienced after carotid body (i.e., BDNF) removal, a reasonable speculation is that oxygen tension regulates the expression or release of neurotrophic factors from the carotid body, and this is critical for maintaining petrosal chemoreceptor neurons in early life. If neurons are lost during this time, then they are lost forever.
In addition to maintaining neuronal survival, the postnatal level of neuronal activity may be critical in determining transmitter phenotype of chemoreceptor afferent neurons. With normal development, ∼40% of neurons with projections to the carotid body are positive for tyrosine hydroxylase expression (25). This is a much higher proportion than for the whole petrosal ganglia (10–20%). Because all petrosal neurons may demonstrate the tyrosine hydroxylase phenotype if depolarized at critical periods of early life (30), it is likely that the level of chemoreceptor activity in early life determines the number of carotid body afferents that are tyrosine hydroxylase positive. In contrast, the level of depolarization has no effect on tyrosine hydroxylase expression in later life (30). Although the physiological consequences of changes in the proportion of tyrosine hydroxylase phenotypes are not presently clear, it is clear that the period around the time of birth is critical in determining the survival of chemoafferent neurons and establishing their gene expression.
IMPLICATIONS FOR HUMANS
Chemoreceptors play an essential role in detection of hypoxia and initiating an appropriate reflex response. In addition to increasing ventilation, chemostimulation results in arousal from sleep and switching from nasal to oral breathing. Although the consequences of removal of the peripheral chemoreceptors may be relatively minor in the adult, the loss of peripheral chemoreceptor input may be especially critical in the newborn period. Denervation around the time of birth has resulted in severe respiratory disturbances in rats (34, 35), pigs (14, 19), and lambs (10, 11). This leads to the speculation that a reduction in peripheral chemoreceptor function may expose the newborn to respiratory instability and possible unexpected death. Indeed, abnormalities in carotid body size or transmitter content have been reported in victims of sudden infant death syndrome (47). Although it is impossible to definitively know the importance of these findings, it suggests that failure of a peripheral chemoreceptor may have contributed to the syndrome.
The maturational change in peripheral chemosensitivity, which appears to be triggered by the rise in Po 2 around the time of birth, occurs at many levels, from a determination of the afferent neuronal survival to glomus cell calcium responsiveness and transmitter release. Interference with the normal maturational cues may have prolonged and severe consequences. Postnatal hyperoxia causes a severe loss of carotid body volume and degeneration of sinus nerve afferent fibers. This suggests that additional care be exercised in monitoring or regulating postnatal Po 2 levels, since higher levels may result in neuronal loss and lower levels may result in a prolongation of immaturity and poor sensitivity to hypoxia.
FUTURE DIRECTIONS AND UNANSWERED QUESTIONS
Our ultimate goal is to understand the mechanism of hypoxia transduction well enough to allow a pharmacological manipulation of chemosensitivity. This would be useful to assure enough chemosensitivity to protect against prolonged apnea and desaturation but low enough to control dyspnea. Two major unanswered questions are in the way of achieving this goal: What is the actual sensor for hypoxia and how does this sensor ultimately generate increased spiking activity? Dr. Prabhakar considers the first issue elsewhere in this series (47a). The second issue may be ripe for addressing using molecular biological tools.
As pointed out above, the role of candidate neurotransmitters in mediating nerve excitation is unresolved. Perhaps dopamine is directly involved in this process, but a pharmacological approach has not resolved this issue very well. Although we know that the magnitude of catecholamine secretion is generally correlated with the level of nerve activity, use of dopamine antagonists generally failed to suppress the nerve response to hypoxia. This may imply that dopamine serves as an inhibitory modulator but may also suggest that it is difficult to pharmacologically inhibit the dopamine receptors on the afferent nerve endings. Rather than seeking to resolve this question through ever-increasing doses of antagonists, the question may be better resolved with the use of genetically altered mice with targeted deletions of dopamine receptors. Individual mice lacking D1through D4 isoforms have all been produced but have not been examined for their chemoresponsiveness. Similarly, mice with targeted disruptions of NK1 (neurokinin) receptors (the receptor for substance P, a purported excitatory transmitter), endothelial nitric oxide synthase (NOS) and neuronal NOS enzymes, and NADPH oxidase (oxygen-sensitive enzymes with a purported roles in carotid body oxygen sensing) have been produced, and physiological studies regarding the consequences of these disruptions on carotid body function have just started (21, 38).
We seem to have a good start in understanding the developmental changes occurring in the postnatal period, that is, changes in nerve branching, survival, calcium responsiveness, ion channel mechanisms, and transmitter release. In the near future, we hope that a coherent and consistent picture of how the carotid body functions can be synthesized, which we can use to better appreciate the critical developmental changes that occur during the newborn period.
Address for reprint requests and other correspondence: D. F. Donnelly, Dept. of Pediatrics, Division of Respiratory Medicine, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520 (E-mail:).
Second in a series of invited mini-reviews on “Hypoxia Influence on Gene Expression.”
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