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Am J Physiol Regul Integr Comp Physiol 279: R1625-R1633, 2000;
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Vol. 279, Issue 5, R1625-R1633, November 2000

PDGF-beta receptor expression and ventilatory acclimatization to hypoxia in the rat

Oscar A. Alea1, Marc A. Czapla2, Joseph A. Lasky3, Narong Simakajornboon1, Evelyne Gozal4, and David Gozal4

Constance S. Kaufman Pediatric Pulmonary Research Laboratory, Departments of 1 Pediatrics, 2 Physiology, and 3 Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112; and 4 Kosair Children's Hospital Research Institute, Departments of Pediatrics, Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of platelet-derived growth factor-beta (PDGF-beta ) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-beta receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O2, and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-beta receptor expression. Although no significant changes in PDGF-beta receptor mRNA occurred over time, marked attenuation of PDGF-beta receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH (r: -0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB (n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia (n = 10). We conclude that decreased expression of PDGF-beta receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-beta receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.

growth factors; nucleus of solitary tract; brain stem; chronic hypoxia; platelet-derived growth factor-beta


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ACUTE VENTILATORY RESPONSE to hypoxia in adult mammalian species is biphasic. After an initial ventilatory enhancement, there is a subsequent decrease in ventilation to levels that, although exceeding those during normoxia, are lower than the peak early ventilatory increase (47). However, sustained exposure to environmental hypoxia will lead to a time-dependent increase in ventilation, which has been termed ventilatory acclimatization to hypoxia (VAH) and occurs in humans, goats, ponies, and also in rats (10, 14, 17, 30, 32, 40, 47). Although increased sensitivity of arterial chemoreceptors has been shown to contribute to VAH (5, 6, 16, 41, 54, 59), it has become increasingly clear that central mechanisms are also operative in this process (13, 50, 60) and may contribute to the increased ventilatory output that is readily measurable when normoxic or isocapnic hypoxic mixtures are breathed (1, 9). Indeed, the process of adaptation to hypoxia that results in VAH appears to involve both ends of the primary synaptic pathways underlying the ventilatory response, such that development of increased sensitivity of the peripheral chemoreceptors occurs in parallel with integration of afferent input and amplification of centrally generated efferent output (13).

In a previous study, we found that the early phase of the acute hypoxic ventilatory response is attenuated by platelet-derived growth factor BB (PDGF-BB) but not by PDGF-AA in the rat dorsocaudal brain stem (19), such that this attenuation is mediated by activation of PDGF-beta receptors within the nucleus of the solitary tract (nTS) (19). Furthermore, the second or late phase of the biphasic hypoxic ventilatory response was dependent on the presence and activation of PDGF-beta receptors, such that pharmacological inhibition of PDGF-beta receptors in the rat resulted in significant attenuation of the hypoxic ventilatory depression (19). Similarly, diminished expression of PDGF-beta receptors in transgenic mice was associated with almost complete abolition of the typical ventilatory decline that characteristically occurs with ongoing hypoxia (19). Additional examination revealed that both PDGF-B chain and PDGF-beta receptors, but not PDGF-alpha receptors, were abundantly expressed in a large proportion of nTS neurons undergoing functional recruitment (as evidenced by c-Fos induction) during hypoxia, and that acute hypoxia induced PDGF-B chain mRNA enhancements (19). On the basis of these data, we hypothesized that VAH could be mediated at least in part by temporal changes in PDGF-beta receptor function, such that reciprocal relationships would emerge between the magnitude of VAH and the expression of PDGF-beta receptor within the nTS.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

The experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the National Research Council publication, Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Survival experiments were performed under pentobarbital sodium anesthesia (50 mg/kg ip) on 38 male Sprague-Dawley young adult rats (200-225 g) as previously described (37). In one set of experiments (group I), a small diameter hole was drilled into the occipital skull, and a small cannula (22G; Plastics One, Roanoke, VA) was surgically implanted at, or in close proximity to, the nTS according to standard stereotaxic coordinates (-13.85 mm bregma, 0.2 mm off midline, 8.0 mm depth) (44). The cannula was then connected via PE-50 tubing to preloaded osmotic pumps (model 1002; Alza, Palo Alto, CA) preset to deliver the designated solution at 0.25 µl/h, consisting of either vehicle (Veh) or PDGF-BB (80 nM). After surgery, animals were allowed to recover for 24 h as demonstrated by return to spontaneous feeding and drinking patterns.

In a second set of experiments (group II), a small cannula was placed as in group I and occluded with a guide obturator. Rats were then allowed to recover for 24 h, after which 100 nl of L-glutamate solution (L-Glu; 1 µM) were microinjected with the animal in a barometric chamber to verify that the anticipated ventilatory enhancements indeed occurred and thus confirm adequate position of the cannula. In addition, 100-nl nTS microinjections of Veh and PDGF-BB (80 nM) were carried out 2 h apart and, 15-20 min after each injection, hypoxic challenges (10% O2 balanced in N2) were conducted for 20 min to demonstrate the anticipated attenuation of the hypoxic response (19). Rats that had a positive response, i.e., increased ventilation after L-Glu administration, and that also displayed a PDGF-BB-induced attenuation of their peak hypoxic response compared with vehicle (19), were then placed in an environmental chamber and exposed to chronic hypoxia for 14 days. Microinjections of L-Glu, PDGF-BB, and Veh and corresponding acute hypoxic challenges were repeated at days 7 and 14.

At the conclusion of the experiments in groups I and II, animals were anesthetized with pentobarbital (50 mg/kg ip), microinjected with 100 nl of 20% methylene blue, and transcardially perfused with 100 ml of PBS (0.01 M at pH 7.4), followed by 500 ml of fixative containing 4% paraformaldehyde, 7.85 g lysine, and 1.075 g sodium-metaperiodate in PBS. The brains were removed from the skull and preserved in 4% paraformaldehyde until processing. To verify adequate position of the cannula, serial brain stem sections were obtained, and the position of the tip of the cannula, as well as the overall extent of the diffusion of methylene blue, was verified by light microscopy. If the placement of the cannula tip was outside the boundaries corresponding to the nTS, the experimental data from that animal were not included in data analysis.

Exposures to Hypoxia

In group I, ventilatory measures were recorded in room air conditions and during a 20-min exposure to 10% O2 in N2 by use of a preset gas mixture, 1 day before (-1), on the day of surgery (0), and on days 1, 2, 4, and 7 after implantation of the osmotic pump. To ascertain that no degeneration of PDGF-BB occurred within the osmotic pump over the 7-day duration of these experiments, samples of PDGF-BB (80 nM) were kept in an incubator at 37°C for 7 days and 100-nl microinjections were then carried out on day 7 in those animals receiving chronic administration of vehicle via the osmotic pump, followed by a 20-min hypoxic challenge 20 min after administration.

Group II animals underwent 14 days of chronic hypoxic exposures by sojourning in a custom-designed chamber (volume ~0.2 m3; Oxycycler, Reming Bioinstruments, Redfield, NY) that was operated under a 12:12-h light-dark cycle. O2 concentration was continuously measured by an O2 analyzer and maintained at 10% by a servo-controlled system, such that deviations from the desired concentration were met by addition of N2 or O2 through computer-driven solenoid valves. Ambient CO2 in the chamber was continuously monitored and maintained at <1,500 ppm by appropriate adjustments in the overall chamber ventilation. Humidity was maintained at 40-50%, while ambient temperature was kept at 22-24°C. On days 0, 7, and 14 of hypoxia, 100-nl microinjections of Veh and PDGF-BB (80 nM) were performed 2 h apart into the nTS after collection of baseline ventilatory measures in room air for 15 min. Fifteen to twenty minutes after each microinjection, 20-min hypoxic challenges (10% O2 balanced in N2) were performed, after which animals were returned to the chamber or euthanized with a pentobarbital overdose for assessment of cannula location. In addition, on days 0, 1, 7, and 14 of chronic hypoxia, L-Glu microinjections were also performed, and ventilatory responses were measured as described in the next section.

Ventilatory and Cardiovascular Recordings

Cardiorespiratory measures were continuously acquired in the freely behaving animals by use of the barometric method (Buxco Electronics, Troy, NY) (2, 43). To minimize the long-term effect of signal drift due to temperature and pressure changes outside the chamber, a reference chamber of equal size in which temperature was measured using a T-type thermocouple was used. In addition, a correction factor was incorporated into the software routine to account for inspiratory and expiratory barometric asymmetries (15). Environmental temperature was maintained slightly below the thermoneutral range (24-26°C). At least 60 min before the start of each protocol, animals were allowed to acclimate to the chamber, in which humidified air (70-90% relative humidity) was passed through at a rate of 4 l/min as appropriate, using a precision flow pump-reservoir system. Pressure changes in the chamber due to the inspiratory and expiratory temperature changes were measured using a high gain differential pressure transducer (model MP45-1, Validyne) (11). Analog signals were continuously digitized and analyzed on-line by a microcomputer software program (Buxco Electronics). A rejection algorithm was included in the breath-by-breath analysis routine and allowed for accurate rejection of motion-induced artifacts. Tidal volume, respiratory frequency, and minute ventilation (VE) were computed and stored for subsequent off-line analysis.

Measurement of blood gas values. Arterial blood samples were obtained from the implanted arterial catheter in the rats subjected to experiment I. After withdrawal of 75-100 µl of blood in the dead space of the catheter, another 150 µl were sampled for immediate analysis of PO2, PCO2, and pH with a blood gas analyzer (model 178, Ciba Corning). Measurements were always performed before the hypoxic gas switch and during the last minute of each hypoxic challenge.

Immunoblotting of PDGF-beta receptor. Pooled dorsocaudal brain stem tissues (n = 5-6 animals/sample) primarily corresponding to the nTS were harvested from normoxic or chronically hypoxic rats by punch sampling with a 17-gauge thin-walled hypodermic needle as previously described (21). Six separate experiments corresponding to a total of 30-36 rats/condition were conducted. Samples were homogenized in lysis buffer (PBS pH 7.6, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 20 mM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride). Supernatants obtained from 30-min centrifugation at 15,000 g were assayed for protein content (Bio-Rad, Hercules, CA). Three hundred micrograms of protein were used for the immunoprecipitation of PDGF-beta receptor protein and incubated overnight at 4°C with 12 µg of PDGF-beta receptor antibody (UBI, Lake Placid, NY) in a total volume of 250 µl, after which 20 µl of protein A-Sepharose were added and incubated for 1 h at 4°C. The immunoprecipitates were washed three times with lysis buffer and resuspended in a mixture of 20 µl lysis buffer and 20 µl SDS-sample buffer (0.5 M Tris, pH 6.5, 20% glycerol, 4% SDS, and 100 mM dithiothreitol). Proteins were separated by electrophoresis on a 8% Tris-glycine gel (Novex, San Diego, CA) and transferred to a 0.2-µm nitrocellulose membrane. Nonspecific binding was blocked by 1 h of incubation with 5% BSA in TBS-Tween 20 (TBS-T). The membrane was incubated overnight at 4°C with PDGF-beta receptor antibody (1:300; UBI). Membranes were washed with TBS-T and incubated with secondary antibodies for 1 h. After extensive washing, proteins were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Northern Blots

Pooled dorsocaudal rat brain stem specimens (n = 2-3/sample) primarily corresponding to nTS regions were immediately frozen in liquid nitrogen then stored at -70°C until use. Three separate experiments, corresponding to a total of 6-9 rats/condition, were conducted. Total RNA was isolated from the samples using a guanadinium/cesium chloride isolation method, exactly as previously described (29). Fifteen micrograms of total rat brain or lung RNA (as control) in denaturation buffer were added per well of a 1.2% formaldehyde/agarose gel and separated overnight via electrophoresis. RNA was transferred from the gel to a nylon membrane (Immobilon-N) by capillary action overnight. Prehybridization took place for 3 h at 62°C, and hybridization took place overnight at 62°C. cDNA templates were random-primed with 32P-radiolabeled dCTP by the random primer method using the Ready-To-Go DNA labeling kit (Pharmacia). A plasmid designated b15a, which includes the sequence for the rat PDGF-beta receptor cDNA, was kindly provided by Dr. Michael Pech, Basel, Switzerland. The EcoR I/Hind III restriction enzyme fragment from the b15a plasmid was used as a template to generate the radiolabeled PDGF-beta receptor probe. A plasmid with the murine 18S cDNA construct was obtained from American Type Culture Collection (Rockville, MD) and used to generate labeled probes as a loading control. Labeled probes were separated from unincorporated nucleotides using the TE Midi SELECT-D, G-50 spin columns (5 Prime-3 Prime, Boulder, CO). Hybridized membranes were washed with 2× standard sodium citrate (SSC) with 0.5% SDS, then 0.2× SSC with 0.5% SDS at 37 and 62°C. Images were generated for the hybridized autoradiographic signal by exposing the membrane to Biomax Film (Kodak) for 2 days. The hybridized autoradiographic signal was quantified using a Fuji phosphoimager plate (Fugix BAS 1000) and the McBAS 2.5 software (Fuji USA, Stanford, CT). PDGF-beta receptor results were adjusted using the 18S loading control.

Data Analysis

Values are reported as means ± SD. Baseline ventilation before each hypoxic run was defined as the average of ventilatory measures during the 3-min period immediately preceding the gas switch. For ventilatory challenges, mean VE values in 1-min bins were calculated, and the peak VE value of the hypoxic run was considered as representative of the hypoxic ventilatory response. To normalize across the various experiments, the overall peak VE increase during hypoxia was calculated using the normoxic baseline preceding each challenge and was therefore expressed as %baseline.

For Western and Northern blot procedures, semiquantitative analysis of the bands was performed by scanning densitometry or by using the count values derived from the phosphorimager plates. Comparisons across treatment groups of numerical data were performed using either Student's t-tests or two-way ANOVA (treatment and time or hypoxia) followed by Newman-Keuls post hoc tests, as appropriate. Linear regression between PDGF-beta receptor levels and corresponding normoxic ventilation was also examined in the rats exposed to chronic hypoxia, and the correlation coefficient was calculated from the regression analysis. A P value of <0.05 was considered to achieve statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Group I Experiments

As previously shown with acute nTS microinjections of PDGF-BB (19), continuous administration of the growth factor within nTS locations (Fig. 1) resulted in significant attenuation of the acute ventilatory response to hypoxia (HVR) at days 1 and 2 compared with days -1 and 0 (Fig. 2; Table 1; n = 8; P < 0.001) and compared with Veh-treated rats (n = 8; P < 0.001). However, at day 4, the PDGF-BB effect on HVR was not as pronounced, and by day 7, the HVR reduction had virtually disappeared [Table 1; P < 0.0001 ANOVA for time; P was nonsignificant (NS) day 0 vs. day 7]. To ascertain that PDGF-BB was still active at the later time points, and that the attenuation of the PDGF-BB effect was not the result of diminished PDGF-BB activity over time, on day 7 we performed in Veh-treated rats 100-nl microinjections of PDGF-BB kept for 7 days at 37°C, and we then assessed their HVR. Significant attenuation of HVR occurred after PDGF-BB administration (Fig. 2). Arterial blood gases confirmed the dynamic ventilatory changes associated with prolonged PDGF-BB administration, such that PCO2 was higher in PDGF-BB-treated rats during acute hypoxic challenges than in Veh-treated animals at days 1 and 4 (Table 2). This is in contrast with day 7, at which time point PCO2 during the hypoxic challenge was similar in both treatment groups (Table 2). Of note, the microcannulas were not in place in four rats.


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  		Fig. 1.   A: representative photograph of a site (arrow) in which continuous microinjection was performed for 7 days with an osmotic pump. XII, hypoglossal nucleus; nTS, nucleus of the solitary tract. B: summary diagram for all microinjection locations for 8 rats receiving chronic administration of platelet-derived growth factor BB (PDGF-BB) () and 8 rats receiving vehicle (triangle ). Distance from bregma is indicated at bottom and is based on the atlas by Paxinos and Watson (44).



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  		Fig. 2.   Mean ventilatory responses to a 20-min hypoxic challenge in conscious freely behaving rats receiving chronic nTS administration of PDGF-BB (started at arrow; ; n = 8) or vehicle (Veh; open circle ; n = 8). To normalize across minute ventilation (VE) changes at each time point, the peak hypoxic ventilatory response (HVR) was expressed as the %increase in VE from its corresponding baseline (0%). In the PDGF-BB treatment group, HVR was similar before initiation of PDGF-BB infusion (days pre and 0). However, HVR was significantly attenuated by PDGF-BB at days 1, 2, and 4 (*P < 0.0001), although the effect was reduced at day 4 (day 4 vs. day 1 and day 2: P < 0.01), and at day 7, PDGF-BB and vehicle groups had similar HVR. In Veh-treated rats, a single 100-nl microinjection of PDGF-BB stored at 37°C for 7 days elicited significant attenuation of HVR (triangle , n = 8; P < 0.001 vs. Veh), which was comparable in magnitude to results seen in the PDGF-BB group (P = NS).


                              
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  		Table 1.   Baseline and peak ventilation during a 20-min hypoxic challenge with 10% O2 in conscious freely behaving rats before and during chronic nTS administration of PDGF-BB or vehicle


                              
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  		Table 2.   Mean blood gases during the last minute of a 20-min hypoxic challenge with 10% O2 in conscious freely behaving rats before and during chronic nTS administration of PDGF-BB or vehicle

Group II Experiments

Microinjection of L-Glu within the nTS at days 0, 1, 7, and 14 of chronic hypoxia revealed small but significant increments in the magnitude of VE enhancements elicited by this treatment at days 7 and 14 compared with days 0 and 1 (Fig. 3; n = 10; P < 0.01 ANOVA).


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  		Fig. 3.   Mean VE changes expressed as %change from corresponding baseline (0%) in 10 freely behaving rats exposed to chronic hypoxia (10% O2 balance N2) after 100-nl nTS microinjection of L-glutamate (L-Glu). Significant enhancements of the L-Glu ventilatory effect occurred at 7 and 14 days (*P < 0.01, ANOVA; n = 10).

As previously shown, PDGF-BB at day 0 was associated with attenuation of HVR (19). In contrast, identical treatments at days 7 and 14 of chronic hypoxia failed to elicit any significant effect on HVR (Fig. 4; n = 10). In the other eight animals designated for these experiments, the cannula was either not in the right location (n = 2) or became dislodged during the chronic hypoxic exposure (n = 6), such that data were not available.


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  		Fig. 4.   Mean ventilatory responses to a 20-min acute hypoxic challenge in conscious freely behaving rats exposed to chronic hypoxia (10% O2 balance N2) after 100-nl nTS microinjection of Veh (; n = 10) and PDGF-BB (; n = 10). The significant attenuation of the acute HVR by PDGF-BB was apparent on day 0 (PDGF-BB vs. Veh: P < 0.001) but not at days 7 and 14 of chronic hypoxia (P = NS).

Chronic Exposure to Hypoxia

When rats not undergoing any surgery were exposed to chronic hypoxia, ventilatory measurements performed in normoxia revealed time-dependent VE increases indicative of VAH (Fig. 5; n = 34/time point). These animals were then euthanized for tissue harvesting for PDGF-beta receptor immunoreactivity (see next section).


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  		Fig. 5.   Mean ventilatory changes (Delta VE) during normoxia in 34 conscious rats over the course of a 14-day exposure to chronic hypoxia (10% O2 balance N2). *P < 0.01 vs. normoxic baseline (established 1 day before beginning of hypoxic exposures).

PDGF-beta Immunoblots and Northern Blots

Exposure to chronic hypoxia was associated with decreases in PDGF-beta receptor immunoreactivity that became statistically significant at 7 and 14 days (Fig. 6). Linear regression of normoxic ventilation (as a correlate measure of VAH) plotted against PDGF-beta receptor densitometric readings revealed a significant relationship between these two measurements (r: -0.56; P < 0.005; Fig. 7).


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  		Fig. 6.   Top: representative Western blot of PDGF-beta receptor immunoprecipitates probed with an antibody against the PDGF-beta receptor from dorsocaudal brain stem lysates harvested from rats at 0, 1, 2, 7, and 14 days of hypoxia (10% O2 balance N2). Decreases in PDGF-beta receptor immunoreactivity are apparent at day 2 and continue to decrease thereafter. Bottom: mean scanning densitometry values (expressed in arbitrary units) from 6 sets of dorsocaudal brain stem tissue lysates exposed to room air or hypoxia (10% O2 balance N2) for 1, 2, 7, or 14 days.



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  		Fig. 7.   Scattergram of PDGF-beta receptor immunoreactivity expressed as a ratio with normoxic control in the denominator plotted against corresponding mean ventilatory measurements during normoxia (VE, expressed as a ratio with normoxic control in the denominator) from rats exposed to 1 day (), 2 days (open circle ), 7 days (triangle ), and 14 days (black-down-triangle ) of hypoxia (10% O2 balance N2). A significant linear relationship emerged as shown by the straight line (dotted lines indicate 95% confidence intervals) as follows: y = 1.28-0.23 × x (r: -0.56; P < 0.005).

In contrast, no significant changes occurred over time during prolonged hypoxia in PDGF-beta receptor mRNA (Fig. 8).


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  		Fig. 8.   Top and middle: representative Northern blots for PDGF-beta receptor and S18 (as internal control) in dorsocaudal brain stem harvested from rats at 0, 1, 2, 7, and 14 days of hypoxia (10% O2 balance N2). A control blot from a normoxic rat lung is also shown. No consistent changes in PDGF-beta receptor mRNA were apparent. Bottom: mean phosphoimager counts (adjusted for S18 internal control and expressed as degrees of change from day 0) from 3 sets of dorsocaudal brain stem tissue exposed to room air or hypoxia (10% O2 balance N2) for 1, 2, 7, or 14 days. No significant changes in PDGF-beta receptor mRNA density occurred over time (P = NS).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that long-term administration of PDGF-BB is associated with blunting of its attenuating effect on the acute hypoxic ventilatory response and that such changes are concordant with the temporal characteristics of VAH emergence in the rat. This observation is further reinforced by the decreasing effect of acute PDGF-BB administration on the attenuation of the acute ventilatory response to hypoxia in rats exposed to chronic hypoxia at time points in which VAH is either developing or fully established. Thus the physiological data and the changes in PDGF-beta receptor expression suggest that the latter may underlie components of VAH. Indeed, evidence for decreasing expression of the PDGF-beta receptor, albeit without significant changes in receptor mRNA, occurred over time during exposure to chronic hypoxia within the dorsocaudal brain stem, and such changes in PDGF-beta receptor expression correlated with ventilatory evidence of VAH.

Ventilatory Measurements

The magnitude and time course of ventilatory changes associated with VAH vary across the various mammalian species, ranging from a few hours and ventilatory increases during normoxia by 30-60% above prehypoxic normoxic levels to up to several weeks and doubling of ventilatory output during hypoxia (6, 10, 32). The ventilatory measurements during normoxia in the chronically hypoxic rats (Fig. 3), as well as the overall increases in the acute ventilatory response to L-Glu (Fig. 4) and to acute hypoxia after microinjection of vehicle in group II rats (data not shown) are in close concordance with previous studies on VAH in rats (1, 40, 51) and display some degree of biological variability, particularly during the first day of chronic hypoxic exposure (see Fig. 7). Notwithstanding this interindividual variability in ventilatory adaptation, the abolition of the ventilatory effect of PDGF-BB on the acute ventilatory response, after either long-term administration of this growth factor or after chronic hypoxia, suggests that PDGF-related pathways have undergone functional modifications during the process of acclimatization in this species.

Peripheral and Central Mechanisms of VAH

In an interesting series of experiments using an extracorporeal oxygenator to allow for separate control of blood gas characteristics at the carotid body and brain tissue levels, application of sustained carotid body hypoxia in the absence of brain tissue hypoxia in goats was found to be a prerequisite for the development of VAH in the goat (4, 7). Furthermore, initiation of the VAH process appeared to be critically dependent on the increased afferent neural input originating from peripheral chemoreceptor activation during hypoxia, such that elimination of peripheral chemosensory input virtually abolished VAH (3, 41). Furthermore, chronic exposure of glomus type 1 cells to chronic hypoxia resulted in increased cell excitability and calcium mobilization, resulting in part from recruitment of cAMP-dependent pathways (56) as well as from changes in sodium and potassium channel conductances (22).

In contrast, application of isolated brain hypoxia to awake goats did not induce VAH, suggesting that central mechanisms may not be operative in the initial process of ventilatory acclimatization (61). However, administration of carbon monoxide to awake ponies, such as to induce a reduction of arterial oxygen content without stimulation of the carotid chemoreceptors, resulted in ventilatory increases over time that were compatible with VAH, thereby suggesting that both an early peripherally mediated component and a subsequent central component of VAH occur and are required for the complete expression of VAH (31). To further explore the nature of this central component to VAH, 31P nuclear magnetic resonance spectroscopy of brain was performed in humans to assess whether brain intracellular acidosis provided the supplemental stimulus to ventilatory acclimatization to high altitude (18). However, these experiments clearly ruled out the possibility that changes in intracellular pH contribute to VAH (18). In addition, no definitive role was found for endogenous opioids and serotonin centrally, or for dopamine peripherally, as potential modulators of VAH (23, 27, 28, 38, 39, 42, 45, 46, 60).

More recently, Schmitt et al. (51) reported that tyrosine hydroxylase content and norepinephrine turnover exclusively increased in the caudal part of the nTS in rats, were preceded by ventilatory evidence of VAH, and, more importantly, that the biochemical and physiological parameters were highly correlated. Thus VAH is associated with intrinsic modifications of gene expression within dorsocaudal brain stem neurons that primarily subserve components of the ventilatory reflex arc (12, 55). Such genomic modifications may in turn induce changes in the physiological properties of these neurons, as shown by Nolan and Waldrop (35), who found enhanced discharge frequencies in response to hypoxia in ventrolateral medullary neurons from rats after acclimatization to hypoxia compared with normoxic controls. In addition, Dwinnel and Powell (13) recently demonstrated centrally mediated increases in phrenic nerve output developing over the course of acclimatization to hypoxia in rats. We believe that the modest, albeit significant, increases in ventilation in response to microinjection of L-Glu in the acclimatized rats provide additional evidence for evolving changes in the responsivity of nTS neurons to excitatory stimuli such as increased peripheral chemoreceptor input. Therefore, we support the current conceptual framework for development of VAH to include early dependency on peripheral chemoreceptor integrity, which is followed by substantial alterations in the physiological properties of brain stem neurons. In this context, the present study further adds to this concept and proposes an important role for PDGF-beta receptors in mediating components of such centrally dependent VAH in a rat model.

Role of PDGF-BB and PDGF-beta Receptors in the Hypoxic Ventilatory Response

It is now quite well established that nuclei within the brain stem show high levels of expression for both the PDGF-B chain (49) and the PDGF-beta receptors (19, 53). In contrast, PDGF-alpha receptors are preferentially expressed in glial cells and display different topographic abundance compared with PDGF-beta receptors (19, 48, 58, 62-64).

The abundance of PDGF-related pathways within many of the nuclei underlying the neural substrate for the hypoxic ventilatory response was highly suggestive that PDGF isoforms and their receptors could play a functional role in both the immediate and the long-term mechanisms of adaptation to hypoxia. Several lines of evidence led to this assumption. 1) PDGF-B chain expression is greatly increased in neural regions after focal ischemia (24-26, 36), where it has been assigned a putative role in the prevention of neuronal cell death (8). 2) Activation of PDGF-beta receptors by their specific ligand, PDGF-BB, will inhibit N-methyl-D-aspartate (NMDA) receptor-dependent excitatory postsynaptic currents in CA1 pyramidal neurons of rat hippocampal slices (57), possibly through a recently uncovered pathway that involves activation of GTPase-activating protein of Ras (RasGAP) and binding of RasGAP to the Src kinase to form a complex that prevents the phosphorylation of phospholipase C (50). Phospholipase C is prominently involved in mediating the ventilatory response to hypoxia within an NMDA glutamate receptor-protein kinase C pathway (18, 21, 52). 3) Pharmacological studies from our laboratory have demonstrated that PDGF-BB attenuates the acute phase of the hypoxic ventilatory response, whereas reduction of PDGF-beta receptor function by use of either transgenic or pharmacological approaches prevents development of the hypoxic ventilatory depression that characteristically follows the acute response (19). Therefore, in the setting of acute hypoxia, activation of PDGF-beta receptors within dorsocaudal brain stem nuclei reduces the magnitude of the acute hypoxic ventilatory response and thereby modulates the onset of the late phase of this response. We now show that development of VAH is closely linked to the reduction of PDGF-beta receptor expression in the dorsocaudal brain stem. Because the decreased expression of the receptor as evidenced by the Western blots was not accompanied by a parallel decrease in PDGF-beta receptor mRNA, it is possible that receptor downregulation may have occurred via sustained autophosphorylation, followed by internalization and degradation of the receptor through the ubiquitin proteasome proteolytic pathway (33, 34). However, we cannot exclude with certainty that the absence of PDGF-beta receptor mRNA changes with chronic hypoxia may be due to alterations in posttranscriptional regulation.

In summary, we present novel evidence supporting a role for PDGF-beta receptors within the dorsocaudal brain stem in mediating ventilatory components that characterize the phenomenon of ventilatory acclimatization to hypoxia. These findings further suggest that central mechanisms involving PDGF are indeed operative in VAH and that VAH may in fact represent a complex interplay between peripheral and central neurons. Each of these two intimately linked systems undergoes complex cellular and molecular changes that will ultimately result in optimal organismal adaptation and survival.

Perspectives

Differential activation of glutamate receptors, such as NMDA receptors (37), and growth factor receptors, such as the PDGF-beta receptor (19) within the nTS during the early phases (<1 h) of the hypoxic ventilatory response, suggests that the unique downstream signaling intracellular pathways linked to these receptors are not only involved in moment-to-moment ventilatory output regulation but may also be critical for induction of protective and/or adaptive genes that permit ventilatory acclimatization, as shown in the present study. It is therefore possible that the time domains of complex receptor-receptor and protein-protein interactions induced by hypoxia will in turn induce transcriptional and posttranslational regulation of multiple genes aiming to secure functional adaptation, cell survival, and/or programmed cell death and synaptic plasticity.


    ACKNOWLEDGEMENTS

We thank Dr. Samir El-Dahr for constructive criticisms and suggestions throughout the duration of this research project.


    FOOTNOTES

This study was supported in part by grants from the American Lung Association (CI-002-N), the National Institutes of Health (RO1 HL-65270 and F31 DA-05948), a Maternal and Child Health Training Grant (MCJ-229163), and the American Heart Association (AHA-0050442N).

Address for reprint requests and other correspondence: D. Gozal, Kosair Children's Hospital Research Institute, Univ. of Louisville School of Medicine, 570 S. Preston St., Ste. 321, Louisville, KY 40202 (E-mail: d0goza01{at}gwise.louisville.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 February 2000; accepted in final form 12 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 279(5):R1625-R1633
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