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Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
PATTERNS OF EPISODIC HYPOXIA:...
LONG-TERM EFFECTS OF...
LONG-TERM EFFECTS OF...
LONG-TERM EFFECTS OF...
SUMMARY AND FUTURE DIRECTIONS
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To the majority of the population, recurrent episodes of hypoxia are more likely encountered in life than sustained hypoxia. Until recently, much of the information on the long-term effects of intermittent hypoxia has come from studies on human subjects experiencing chronic recurrent apneas. Recent development of animal models of intermittent hypoxia and techniques for exposing cell cultures to alternating cycles of hypoxia have led to new information on the effects of episodic hypoxia on oxygen-sensing mechanisms in the carotid body chemoreceptors and regulation of gene expression. The purpose of this review is to highlight some recent studies on the effects of intermittent hypoxia on oxygen sensing at the carotid bodies and regulation of gene expression. In a rodent model, chronic intermittent hypoxia selectively enhances hypoxic sensitivity of the carotid body chemoreceptors. More interestingly, chronic intermittent hypoxia also induces a novel form of plasticity in the carotid body, leading to long-term facilitation in the sensory discharge. Studies on cell cultures reveal that intermittent hypoxia is more potent in activating activator protein-1 and hypoxia-inducible factor-1 transcription factors than sustained hypoxia. Moreover, some evidence suggests that intermittent hypoxia utilizes intracellular signaling pathways distinct from sustained hypoxia. Reactive oxygen species generated during the reoxygenation phase of intermittent hypoxia might play a key role in the effects of intermittent hypoxia on carotid body function and gene expression. Global gene profile analysis in cell cultures suggests that certain genes are selectively affected by intermittent hypoxia, some upregulated and some downregulated. It is suggested that, in intact animals, coordinated gene regulation of gene expression might be critical for eliciting phenotypic changes in the cardiorespiratory systems in response to intermittent hypoxia. It is hoped that future studies will unravel new mechanisms that are unique to intermittent hypoxia that may lead to a better understanding of the changes in the cardiorespiratory systems and new therapies for diseases associated with chronic recurrent episodes of hypoxia.
carotid body; reactive oxygen species; apneas; transcription factors; gene regulation
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
PATTERNS OF EPISODIC HYPOXIA:...
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FOR PEOPLE WHO RESIDE at or near sea level, recurrent episodes of hypoxia are encountered more frequently in life than sustained hypoxia. Transient episodes of hypoxia can occur, for example, with air swallowing during meals that result in elevation of the diaphragm. Moreover, episodic or intermittent hypoxia is associated with many pathophysiological situations, including sleep apneas and apneas in premature infants and in humans who have lung diseases such as chronic obstructive pulmonary disease, asthma, or pulmonary fibrosis. It has been well documented that chronic intermittent hypoxia (CIH) leads to pulmonary as well as systemic hypertension, myocardial and brain infarctions, cognitive dysfunction, and sudden death in elderly. Acute responses to hypoxia that occur within seconds to minutes depend entirely on the O2-sensing ability of the peripheral arterial chemoreceptors, especially the carotid bodies (glomus cells are considered to be the primary O2-sensing cells) (26). On the other hand, almost every mammalian cell, albeit to different degrees, responds to sustained hypoxia (lasting hours to days) by altering gene expression and de novo protein synthesis (7, 32). However, little is known about how intermittent hypoxia affects the O2-sensing mechanisms at the carotid body or on O2-regulated gene expression. The purpose of this minireview is to summarize what is known about the effects of intermittent hypoxia on ventilation and circulation and to highlight some recent studies that suggest that intermittent hypoxia modulates O2 sensing at the carotid body as well as gene regulation via generation of reactive oxygen species (ROS). Because of the constraints of space, the focus of this article will be on the effects of episodic hypoxia in adult life rather than in neonates.
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PATTERNS OF EPISODIC HYPOXIA: HUMAN AND EXPERIMENTAL ANIMAL MODELS |
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Three patterns of intermittent hypoxia are most commonly
encountered, and they are schematically depicted in Fig.
1. The first pattern (Fig. 1A)
is commonly seen in sleep apneas (either obstructive or central
apneas). The duration of each hypoxic episode lasts anywhere between 10 and 40 s, depending on the length of apneas, which are
interspersed with normoxic periods that last several minutes. Patients
with obstructive or central apneas experience repeated bouts of hypoxia
for nearly 7-8 h (i.e., as long as sleep cycle lasts). The number
of hypoxic episodes varies among subjects and depends on the etiology
of the disease [i.e., obstructive or central apneas
(10)]. Fletcher (12) developed a rodent model mimicking the effects of this pattern of intermittent hypoxia. The second pattern (Fig. 1B) of intermittent hypoxia is
encountered, for example, during rapid ascent and descent from high
altitude and also in patients with chronic lung disease wherein
ventilation is depressed during sleep. In this pattern, the hypoxic
episode can last as long as minutes to hours. This pattern of
intermittent hypoxia has been utilized in human subjects (18,
33) and experimental animals (6) to investigate the
effects of episodic hypoxia on the ventilatory control system. The
third pattern (Fig. 1C) is seen with periodic types of
breathing (e.g., Chyne-Stokes, Kusmal, and so forth). In this pattern
of intermittent hypoxia, periods of hyperventilation are followed by
apneas lasting several seconds to minutes. Unlike the first two
patterns, there is very little information on the long-term effects of
the third pattern of intermittent hypoxia. Because the first pattern of
recurrent apneas is the most clinically relevant and there is more
experimental information on this paradigm, this article will focus on
this pattern of hypoxia.
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LONG-TERM EFFECTS OF INTERMITTENT HYPOXIA ON THE VENTILATORY AND CIRCULATORY SYSTEMS |
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Effects of Intermittent Hypoxia Involving Recurrent Apnea Pattern
Much of the information on the long-term effects of intermittent hypoxia on the ventilatory control system has come from studies on patients with recurrent apneas (sleep or central apneas). Many of the patients exhibit an enhanced ventilatory response to hypoxia (HVR) (10). In contrast, respiratory responses to hypercapnia are less affected, unaltered, or even blunted (10). The enhanced HVR can be reversed by treating sleep apneas with continuous positive airway pressure. However, some studies suggest that an attenuated HVR occurs in patients with chronic recurrent episodes of hypoxia, and this decrease in HVR is associated with alcohol consumption (10).We have recently examined the long-term effects of intermittent hypoxia on HVR by mimicking recurrent apneas in a rodent model. In these experiments, awake rats were exposed to 15 s of 5% O2 followed by 5 min of 21% O2 (9 episodes per hour, 8 h/day, for 10 days). HVRs were subsequently examined under anesthetized, paralyzed, and mechanically ventilated conditions using efferent phrenic nerve activity as an index of neural respiration. Baseline minute neural respiration was 38% greater, and the magnitude of ventilatory stimulation caused by acute hypoxia (12% inspired O2 fraction) was 56% greater in animals conditioned with 10 days of intermittent hypoxia. Greenberg et al. (15) reported no obvious increases in HVR in rats exposed to 30 days of intermittent hypoxia with a similar pattern mimicking recurrent apneas. It may be that HVR becomes adapted after 30 days compared with 2 wk of intermittent hypoxia. However, this possibility requires further investigation.
After repeated episodes of hypoxia, breathing remains elevated for nearly 1 h in experimental animals and humans (25). This prolonged activation of breathing in response to episodic hypoxia is often termed long-term facilitation (LTF) (25). LTF is considered to be a hallmark of intermittent hypoxia because a comparable duration of continuous hypoxia does not cause long-lasting activation of breathing. It has been postulated that LTF, by increasing tone in the upper airways, may thereby prevent collapse and possibly help in alleviating airway obstruction that occurs in obstructive sleep apneas (25). We found that rats conditioned with 10 days of intermittent hypoxia exhibit marked potentiation in LTF in respiratory motor output (i.e., efferent phrenic activity). It is likely that this enhanced LTF contributes to increased basal ventilation during the day time in patients with sleep apneas (10), dogs exposed to CIH (8), and in rats exposed to 10 days of intermittent hypoxia.
It has been reported that 30 days of intermittent hypoxia increases blood pressure and sympathetic nerve activity in rats (14, 15), similar to that seen in patients experiencing chronic recurrent episodes of hypoxia (34). However, the experimental paradigm for intermittent hypoxia differs from that seen in patients with recurrent apnea in that transient hypoxic episodes are accompanied by increased CO2 in the latter but not the former. Therefore, to assess whether the long-term effects of apneas are due to hypoxia or hypercapnia or a combination of both, Fletcher et al. (13) exposed rats to brief episodes of hypoxia alone or in combination with CO2. They found that addition of CO2 had no further effect on the increase in blood pressure in response to 30 days of intermittent hypoxia alone. It follows that the long-term effects of recurrent apneas are most likely due to hypoxia rather than to CO2. Whether addition of CO2 influences the long-term effects of intermittent hypoxia on HVR, however, remains to be investigated.
Effects of Intermittent Hypoxia With a Pattern Resembling Ascent and Descent to High Altitude
Several Russian studies reported that 4-h hypoxic exposure per day for 2 wk enhanced the ventilatory response to hypoxia and athletic performance in healthy human subjects (18, 33). Recently, Mohamed and Duffin (21) also reported enhanced HVR in healthy human subjects exposed to 4 h of hypoxia per day for 2 wk. Interestingly, these investigators found that ventilatory sensitivity to hypoxia is diminished immediately after a single episode of 4 h of sustained hypoxia. These observations suggest that repeated conditioning with hypoxia is necessary to elicit enhanced sensitivity to hypoxia. Ling et al. (19) exposed rats to 5 min of 11% O2 followed by room air for 5 min (12 h/night for 7 consecutive nights). They found that HVRs were augmented in animals conditioned with this pattern of intermittent hypoxia. Thus, in both humans and experimental animals, long-term intermittent hypoxia with a pattern resembling ascent and descent to altitude enhances the HVR. However, whether this pattern of intermittent hypoxia also increases blood pressure is not known.| |
LONG-TERM EFFECTS OF INTERMITTENT HYPOXIA ON OXYGEN SENSING AT THE CAROTID BODY |
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It has been suggested that carotid body chemoreceptors constitute the "front-line" defense system for detecting changes in arterial blood gases during apneas, much more so than other respiratory chemoreceptors (e.g., central chemoreceptors). This is because the circulation time from lungs to carotid bodies (~6 s) is relatively shorter than to central areas and therefore the carotid bodies have already responded to the hypoxic blood before the central areas are exposed. Consequently, it is thought that enhanced HVR and increases in blood pressure are the result of increased carotid body activity and/or chemoreceptor gain in the central nervous system. Supporting the idea that changes in carotid body activity are responsible for the adaptations to CIH are the findings that chronic ablation of sinus nerves prevents elevation in blood pressure and sympathetic nerve activity caused by CIH (12) and that glomectomized patients with sleep apneas no longer develop high blood pressures (34). Although these studies suggest that recurrent hypoxic episodes might affect carotid bodies, there have been no studies that directly examined the carotid body activity in animals exposed to CIH.
Recently, Peng and colleagues (23, 24) examined the long-term effects of intermittent hypoxia (pattern similar to recurrent apneas) on carotid body sensory responses to hypoxia in rats. These investigators found that baseline sensory discharge of the carotid bodies and the magnitude of the sensory response to hypoxia were greater in rats exposed to 10 days of intermittent hypoxia (78 and 47%, respectively). Similar results were also obtained using an in vitro carotid body preparation, suggesting that the enhanced carotid body sensitivity to hypoxia is not secondary to alterations in blood pressure and/or circulating vasoactive hormones. On the other hand, the carotid body sensory response to hyperoxic hypercapnia (5% CO2 balance O2) was found to be unaffected by 10 days of intermittent hypoxia. These observations suggest that the carotid body sensory response to hypoxia, but not to CO2, is selectively enhanced in response to long-term intermittent hypoxia. However, whether the carotid body response to hypoxia is also affected by other patterns of intermittent hypoxia remains to be investigated.
Chronic Episodic Hypoxia Induces LTF in Carotid Body Sensory Activity
It has been well documented that afferent inputs from carotid bodies are essential for LTF in respiratory motor output resulting from repetitive hypoxic exposures (25). However, whether episodic hypoxia causes LTF in the carotid body sensory discharge has not been examined. Peng et al. (23, 24) examined the effects of episodic hypoxia on the sensory discharge of the carotid bodies in rats. In these experiments, anesthetized rats were subjected to 10 episodes of hypoxia (12% O2), each episode lasting 15 s, interspersed with 5 min of normoxia. As expected, carotid body activity increased in response to each episode of hypoxia. After the last episode of hypoxia was terminated, the baseline discharge returned promptly back to control levels. However, when this protocol was repeated in animals conditioned with 10 days of intermittent hypoxia (8 h/day; pattern as depicted in Fig. 1A), episodic hypoxia resulted in a prolonged elevation in baseline carotid body sensory activity that lasted nearly 1 h (Fig. 2). This increase in baseline activity after episodic hypoxia occurred despite maintaining arterial blood pressure and blood-gas levels. These observations suggest that LTF is not induced in carotid bodies in response to repetitive hypoxia, whereas conditioning with long-term exposure to intermittent hypoxia induces LTF in carotid body sensory activity. However, whether other patterns of intermittent hypoxia also induce LTF in the carotid body activity remains to be studied.
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Significance of Altered Carotid Body Function Caused by Intermittent Hypoxia
What might be the significance of increased peripheral chemoreceptor sensitivity after CIH? The increased chemoreceptor sensitivity can lead to hyperventilation, thus driving the respiratory controller below the apneic threshold for CO2, thus leading to more apneas. In other words, the enhanced chemosensitivity to hypoxia might act as a "positive feedback," thereby exacerbating the occurrence of apneas. Perhaps this might explain why chronic obstructive apneas eventually lead to central apneas. Moreover, LTF in the carotid body activity might also lead to stimulation of the sympathetic nervous system, resulting in a sustained elevation in blood pressure. Thus changes in the carotid body activity caused by CIH might initiate a cascade of events that lead to pathophysiological changes. Figure 3 illustrates the possible consequences of heightened carotid body sensitivity to hypoxia on the cardiorespiratory systems.
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Mechanisms Associated With Altered Carotid Body Function in Response to CIH
Evidence for the involvement of ROS.
Peng and colleagues (23, 24) exposed rats to 4 h of
continuous hypoxia, which is equivalent to cumulative duration of 10 days of intermittent hypoxia. They found that the sensory response to
hypoxia was blunted in response to 4 h of continuous exposure to
isobaric hypoxia. These observations are in sharp contrast to those
seen with intermittent hypoxia. Furthermore, there was also no
induction of LTF in the carotid body sensory discharge after exposure
to continuous hypoxia. It follows that the patterns of hypoxia, i.e.,
repetitive or continuous, have profoundly different effects on carotid
body function. The major difference between intermittent and continuous
hypoxia is the episodic oxygenation in the former but not the latter.
In this respect, intermittent hypoxia seems to resemble
ischemia-reperfusion. It is well known that during reperfusion,
there is enhanced cellular generation of ROS, especially of
O
1 · day1), a potent
scavenger of superoxide ions, and then subjected to intermittent
hypoxia for 10 days. In these animals, 10 days of intermittent hypoxia
did not result in enhanced hypoxic sensitivity of the carotid body.
Also, the magnitude of LTF in the sensory discharge was markedly
attenuated. Together, these observations support the idea that ROS
might play an essential role in intermittent hypoxia-induced
alterations in the carotid body function.
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LONG-TERM EFFECTS OF INTERMITTENT HYPOXIA AT THE MOLECULAR LEVEL |
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It is being increasingly recognized that activation of specific genes is an important mechanism by which sustained hypoxia triggers long-term adaptive responses. In fact, it may be that these long-term adaptive changes are more crucial to the long-term survival in humans than the immediate ventilatory and cardiovascular changes that systemic hypoxia produces. A variety of redox-sensitive transcription factors have been identified. Much attention has been focused on the effects of hypoxia on activator protein-1 (AP-1; Ref. 28) and hypoxia-inducible factor-1 (HIF-1; Ref. 32). It is more than likely that the molecular mechanisms underlying long-term effects of intermittent hypoxia on the ventilatory and circulatory systems in intact animals involve transcriptional activation of a multitude of genes. Thus it is of considerable importance to examine whether intermittent hypoxia activates transcription factors and gene expression and, if so, by what mechanisms.
Activation of Immediate Early Response Genes and AP-1 Transcription Factor by Intermittent Hypoxia
Immediate early responsive genes (IERGs) can be divided into several superfamilies, and c-fos is one of the most extensively studied member of the IERGs. It has been well established that IERGs form transacting proteins that bind to the DNA sequence, TGATTCA, the consensus-binding site for transcription factor AP-1 (5). The AP-1 complex is formed from heterodimers of one of either the Jun or Fos family of proteins or homodimers of Jun (17). The AP-1 binding motif is a relatively common constituent of transcriptional regulatory elements involved in the activation of several target genes during hypoxia, including that of tyrosine hydroxylase (TH), which encodes the key enzyme in catecholamine synthesis (20, 22).Greenberg et al. (16) reported increased c-Fos protein expression in the central nervous system of rats exposed to 30 days of a recurrent apnea pattern of intermittent hypoxia. Although this an interesting observation, it is not known whether the increased c-Fos protein is due to activation of c-fos gene or posttranslational modification, and it is not certain whether intermittent hypoxia affects AP-1 activity. These questions can be best addressed in cell culture models. To begin to understand the effects of intermittent hypoxia on gene expression, Adhikary and colleagues (1, 2) devised a technique wherein cell cultures could be exposed to alternating cycles of brief hypoxia and reoxygenation mimicking recurrent apneas. Intermittent hypoxia increased c-fos mRNA expression in PC-12 cells. In contrast, an equivalent, cumulative duration of sustained hypoxia had no effect; a longer duration of sustained hypoxia was necessary to activate c-fos mRNA. These observations suggest that intermittent hypoxia is relatively more potent in activating c-fos than sustained hypoxia. Similar results were obtained with human vascular endothelial cells, implying that the effects of intermittent hypoxia are not cell selective. When c-fos was activated by sustained hypoxia, mRNA levels returned to controls within 30 min after termination of the hypoxic stimulus. Interestingly, c-fos mRNA levels remain elevated as long as 5 h after termination of intermittent hypoxia. This long-lasting activation of c-fos mRNA expression by intermittent hypoxia resembles LTF in respiratory motor activity caused by episodic hypoxia (25). These findings seem to suggest that episodic but not sustained hypoxia leads to long-lasting activation of genes. However, future studies are necessary to delineate the mechanisms underlying the prolonged activation of c-fos expression by intermittent hypoxia.
The effect of intermittent hypoxia on AP-1 activity was examined using a reporter gene assay (3). Intermittent hypoxia increased reporter gene activity driven by two copies of AP-1. Once again, a comparable cumulative duration of hypoxia had no effect. Together, these observations suggest that intermittent hypoxia is a potent activator of immediate early genes such as c-fos and AP-1 transcription factor activity. Interestingly, intermittent but not sustained hypoxia results in prolonged activation of c-fos expression resembling LTF in the systemic response.
Activation of HIF Transcription Factor by Intermittent Hypoxia
A number of studies have shown that HIF-1 mediates transcriptional regulation of genes that encode proteins associated with maintaining O2 homeostasis (30-32). HIF-1 is a heterodimer consisting of HIF-1
and HIF-1
(also known as the aryl
hydrocarbon nuclear translocator) subunits. Of the two subunits, the
expression of HIF-1 is tightly regulated by O2, whereas
HIF-1 is constitutively expressed. Recently, two new HIF-related
factors have been identified, HIF-2 (also called EPAS-1) and HIF-3.
HIF-1 and HIF-1 are expressed in most human and rodent tissues,
whereas the expression of HIF-2 and HIF-3 is more cell selective
(32). HIF-1 activity primarily depends on expression of
the HIF-1 subunit (32). Yuan et al. (36)
examined the effect of the recurrent pattern of intermittent hypoxia on
HIF-1 and HIF-2 protein expression in PC-12 cells. They found that
intermittent hypoxia increased both HIF-1 and HIF-2 expression.
Using a reporter gene assay (with reporter gene driven by hypoxic
responsive element, HRE), they also determined whether intermittent
hypoxia increases HIF-1-mediated transcriptional activation.
Intermittent hypoxia increased HRE-luciferase activity progressively
with increasing durations of intermittent hypoxia. Like
c-fos expression, comparable durations of cumulative
sustained hypoxia had no significant effect on HRE-luciferase activity. These observations demonstrate that intermittent hypoxia activates HIF-1 activity.
Signaling Pathways Associated With Transcription Factor Activation by Intermittent Hypoxia
Evidence for the involvement of ROS.
The following lines of evidence support the idea that ROS are also
associated with the activation of transcription factors. Scavengers of
superoxide ions prevented upregulation of c-fos and
attenuated AP-1- and HRE-luciferase activation caused by intermittent hypoxia (3, 36). In addition, it has been reported that
cells exposed to intermittent hypoxia have markedly decreased levels of
aconitase activity, suggesting increased generation of superoxide ions.
However, much remains to be studied to further establish the
involvement of ROS in transcription factor activation by intermittent hypoxia. Multiple enzymes of either mitochondrial or extramitochondrial origin can generate ROS. However, which of these enzymes contribute to
generation of ROS during intermittent hypoxia requires further investigation. Moreover, although the experiments described suggest the
possible involvement of O
cannot be excluded. Clearly, the role of ROS in regulation of gene
expression by intermittent hypoxia requires further detailed investigations. The possible involvement of ROS in regulation of gene
expression by intermittent hypoxia is presented in Fig. 4. In addition,
it is not known whether other patterns of intermittent hypoxia also
activate transcription factors and, if so, whether ROS play a role.
Protein kinases. Several intracellular signaling pathways that contribute to AP-1 activation by sustained hypoxia have been identified (28, 29). For instance, activation of c-fos and AP-1 by hypoxia in PC-12 cells involves Ca2+ signaling pathways. Voltage-gated Ca2+ influx is required for c-fos induction via activation of nonreceptor protein tyrosine kinase, c-Src, and the GTP-binding protein Ras (28). This in turn initiates a sequential kinase cascade that activates mitogen-activated protein kinases (MAPKs), resulting in transactivation of SRE and Ca/CRE cis-elements, leading to stimulation of c-fos transcription (28). The resulting Fos protein in turn increases AP-1 activity. Adhikary et al. (1) reported that 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, a chelator of Ca2+, abolishes intermittent hypoxia-induced activation of c-fos transcription in PC-12 cells. However, nitrendipine, an L-type Ca2+ channel blocker at doses that completely prevented c-fos activation by sustained hypoxia, caused only a modest inhibition of intermittent hypoxia-induced c-fos activation. These observations suggest that Ca2+ plays a role in c-fos activation by intermittent hypoxia. However, unlike sustained hypoxia, intermittent hypoxia might be using Ca2+ channels other than L-type and/or mobilizing intracellular stores.
During hypoxia, HIF-1 is strongly phosphorylated by p42 and
p44 but not by p38 MAPKs or Jun kinase, which in turn contributes to
the transactivation of HIF-1 complex (32). MAPK inhibitors attenuate HRE-reporter gene activation by hypoxia in PC-12 cells (11). Phosphoinositol-3 (PI-3) kinase inhibitors have also
been shown to inhibit the accumulation of HIF-1 protein and
attenuate hypoxia-induced HRE-luciferase activation (9).
Yuan et al. (36) examined the effects of MAPK and PI-3
kinase inhibitors on HRE-reporter gene activation by intermittent
hypoxia. These investigators found that neither MAPK nor PI-3 kinase
inhibitors prevented HRE-reporter gene activation by intermittent
hypoxia. These observations are intriguing in view of the previous
studies showing that MAPK and PI-3 kinase signaling pathways are
essential for HRE-activation by sustained hypoxia (9, 11).
However, Yuan et al. (36) found that BAPTA-AM or
Ca2+/calmodulin kinase inhibitors prevent HRE-luciferase
activation by intermittent hypoxia. Thus these studies, although
preliminary, suggest that signaling pathways associated with
transcription factor activation by intermittent hypoxia are distinct
from those used by sustained hypoxia. Perhaps this may also explain to
some extent why intermittent hypoxia is more potent in activating gene expression than sustained hypoxia.
Evidence for Selective Regulation of Multiple Genes by Intermittent Hypoxia
Although we are beginning to understand the transcription factors involved in adaptive responses to intermittent hypoxia, equally important are the target genes that are activated and that produce proteins responsible for the phenotypic adaptations. Given that AP-1 and HIF-1 regulate a variety of genes, it would not be surprising that intermittent hypoxia affects the expression of several genes, and coordinated regulation of multiple genes is essential for long-term effects of intermittent hypoxia on the cardiorespiratory systems. Conventional Northern blot or RT-PCR assays may not be adequate to analyze the expression of multiple genes in a given sample. However, the recent development of gene chip hybridization technology makes it possible to assess the relative mRNA expression levels of a large number of genes simultaneously in a given sample. We compared the effects of intermittent hypoxia with sustained hypoxia on global gene profiles in PC-12 cells using the Affymatrix gene chip technique. Analysis of the data of over 1,000 genes revealed that there are two prominent clusters of genes that are affected only by intermittent but not by sustained hypoxia. Of the two, surprisingly, one cluster of 46 genes was downregulated by more than twofold in response to intermittent hypoxia. The genes in the other cluster (172 genes) were upregulated by intermittent hypoxia. Examples of genes that are upregulated by intermittent hypoxia include adenylyl cyclase VI, choline kinase, glucose transporter, endothelin B receptors, Shaker-type K+ channels, and TH. Examples of the genes that are downregulated, however, include cyclooxygenases, cytochrome-c oxidases, and the dopamine D1 receptor. The fact that a variety of genes are selectively affected by intermittent hypoxia further supports the idea that the molecular mechanisms that lead to programmed cardiorespiratory alterations by episodic hypoxia are unique to those elicited by sustained hypoxia. Further studies are necessary to investigate the mechanisms associated with upregulation as well as downregulation of gene expression by intermittent hypoxia.| |
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In this review, I have attempted to summarize the long-term effects of different patterns of intermittent hypoxia on ventilation and blood pressure. It should be evident that intermittent hypoxia affects HVR and increases blood pressure, and reflexes from the carotid body play an essential role. The recent development of a rodent model of intermittent hypoxia allows direct examination of carotid body activity in response to CIH. Available evidence indicates that intermittent hypoxia, resembling the pattern of recurrent apneas in humans, enhances the sensory response to hypoxia and induces LTF in the carotid body. Moreover, these effects may play a critical role in causing pathophysiological alterations in cardiorespiratory systems. Also of note is that a comparable duration of sustained hypoxia does not cause such alterations in the carotid body function, indicating that the pattern of hypoxic stimulus (i.e., repetitive vs. sustained) is critical.
The recent development of techniques to expose cell cultures to alternating cycles hypoxia and reoxygenation permit investigations on the effects of intermittent hypoxia on transcription factor activation and gene regulation. Emerging evidence suggests that intermittent hypoxia is more potent in activating AP-1 and HIF-1 transcription factors than sustained hypoxia of a comparable duration. Furthermore, it appears, from the limited information available to date, that the signaling pathways associated with the cellular actions of intermittent hypoxia may be different from that described for sustained hypoxia. Also the data suggest that ROS, generated during the reoxygenation phase of intermittent hypoxia (the pattern resembling recurrent apneas), plays a key role in eliciting changes in carotid body activity and transcription factor activation caused by intermittent hypoxia. However, much remains to be studied about the cellular mechanisms associated with altered carotid body function and gene expression in response to intermittent hypoxia. As a first step, analysis of global profile of gene expression in isolated cells suggests that, for a given duration of hypoxia, intermittent hypoxia selectively activates certain genes, and some of them are downregulated and some upregulated. Therefore, the long-term effects of intermittent hypoxia on the cardiorespiratory systems more than likely involve coordinated regulation of gene expression. Future studies in intact animals using DNA microarray analysis might provide new insights into the molecular basis of long-term effects of intermittent hypoxia on the cardiorespiratory systems. However, virtually nothing is known about the effects of intermittent hypoxia on the posttranslational modification of proteins associated with regulation of the cardiorespiratory systems.
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ACKNOWLEDGEMENTS |
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I am grateful to Dr. J. L. Overholt for critical reading of the manuscript, Dr. David Kline for preparation of figures, and Marianne Sperk for assistance with references.
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FOOTNOTES |
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The research in author's laboratory is supported by National Heart, Lung, and Blood Institute Grants HL-25830 and HL-66448.
Address for reprint requests and other correspondence: N. R. Prabhakar, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106 (E-mail: nrp{at}po.cwru.edu).
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