Role of endothelin and endothelin A-type receptor in
adaptation of the carotid body to chronic hypoxia
J.
Chen,
L.
He,
B.
Dinger,
L.
Stensaas, and
S.
Fidone
Department of Physiology, University of Utah School of
Medicine, Salt Lake City, Utah 84108
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ABSTRACT |
Chronic exposure in a
low-PO2 environment (i.e., chronic hypoxia, CH)
elicits an elevated hypoxic ventilatory response and increased hypoxic
chemosensitivity in arterial chemoreceptors in the carotid body. In the
present study, we examine the hypothesis that changes in
chemosensitivity are mediated by endothelin (ET), a 21-amino-acid
peptide, and ETA receptors, both of which are normally
expressed by O2-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ETA
expression in type I cells after 3, 7, and 14 days of CH (380 Torr).
Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked
by acute hypoxia. In normal carotid body, the specific ETA
antagonist BQ-123 (5 µM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the
hypoxia-evoked discharge by 20% (P < 0.01). This
inhibitory effect progressed to 45% at day 9 of CH and to
nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the
presence of BQ-123, the magnitude of the activity evoked by hypoxia did
not differ in normal vs. CH preparations, indicating that the increased
activity was the result of endogenous ET acting on an increasing number of ETA. Collectively, our data suggest that ET and
ETA autoreceptors on O2-sensitive type I cells
play a critical role in CH-induced increased chemosensitivity in the
rat carotid body.
chemoreceptor; chemosensitivity; chemotransduction; hypoxic
ventilatory response; ventalitory acclimatization to hypoxia
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INTRODUCTION |
EXPOSURE TO LOW
AMBIENT O2 elicits a number of molecular, cellular,
and systemic adjustments that collectively mitigate hypoxemia and
promote homeostasis (40). An increase in ventilation is the earliest and most prominent of the adaptive changes elicited by
acute hypoxia. However, chronic exposure to low O2 (i.e.,
chronic hypoxia, CH) evokes an additional time-dependent increase in
minute volume known as ventilatory acclimatization to hypoxia (VAH; see Ref. 5). VAH has been observed in humans during sojourns
to high altitude and in animals exposed in controlled
low-O2 environments. VAH is associated with an increased
hypoxic ventilatory response (HVR), an index of hypoxic ventilatory
drive that is assessed by exposure to an acute hypoxic challenge
(39). Enhanced hypoxic chemosensitivity in the carotid
body, which is manifest as an elevated hypoxia-evoked carotid sinus
nerve (CSN) response, is an important physiological mechanism
underlying changes in ventilatory function during chronic exposure
(3, 5, 38).
Chemotransduction in the carotid body occurs in specialized
O2-sensitive type I cells. Current views suggest that
hypoxia evokes a cascade of events in type I cells, including membrane depolarization, Ca2+ influx, and the release of multiple
biogenic amine and neuropeptide neurotransmitters that excite synaptic
terminals of the CSN (16). Previous efforts to explain the
CH-induced increase in chemosensitivity have been focused primarily on
alterations in neurotransmitter actions (reviewed in Ref.
6). These efforts have identified important changes in the
synthesis, storage, and turnover of the numerous endogenous neuroactive
agents present in type I cells (e.g., dopamine, norepinephrine, ACh,
serotonin, and substance P), but attempts to demonstrate direct
involvement of particular neurotransmitters and/or their receptors in
increased chemosensitivity have produced negative results and/or
conflicting sets of data (e.g., see Refs. 23,
24, and 37).
On the other hand, recent studies in other O2-sensitive
tissues, namely the lung and heart, have shown that the vasoactive peptide endothelin-1 (ET-1) and its receptor (ETA)
are critically involved in physiological and morphological adjustments
in these tissues elicited by sustained exposure to low O2.
ET-1 and ETA are substantially upregulated during CH
(27, 28), and, most importantly, specific ETA
antagonists are able to prevent CH-induced vascular wall thickening,
hypertrophy of the right heart, and pulmonary hypertension induced by
exposure to CH (7, 10, 12, 13, 33).
In a previous communication, we reported that ET- and peptide-like
immunoreactivity is present in rat carotid body type I cells and that
exposure to CH enhances ET immunostaining in these cells
(18). The present study confirms and extends these
immunocytochemical findings and provides a quantitative evaluation of
ET-1 and ETA gene expression. Correlative
electrophysiological and pharmacological experiments demonstrate that
ET is involved in increased carotid body chemosensitivity elicited by
CH. Our data not only show that CH elicits an upregulation of ET-1
peptide and ETA protein in type I cells but that these
changes are also correlated with an enhancement of the hypoxia-evoked
chemoreceptor discharge. Furthermore, between days 3 and
9 of CH, the elevated hypoxia-evoked chemoreceptor nerve
activity becomes increasingly sensitive to inhibition by the specific
ETA antagonist BQ-123.
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MATERIALS AND METHODS |
Animals and exposure to hypobaric hypoxia.
Sixty-two adult male albino rats (180-200 g; Sprague-Dawley
derived; Simonsen, Gilroy, CA) were housed in standard rodent cages
with 24-h access to pellet food and water. Cages containing two to four
rats were placed in a hypobaric chamber where pressures were reduced
incrementally from ambient (~640 Torr at Salt Lake City, 1,400 m)
over a 24- to 36-h period and then were maintained at 380 Torr,
equivalent to 5,500 m. The chamber was continuously flushed with fresh
room air, and the internal temperature was maintained at
20-22°C. The hypobaric chamber was opened every 2 days to
replenish food and water. All animals exposed to the hypobaric
environment survived for up to 16 days without signs of discomfort.
Age-matched control male rats were similarly housed outside the chamber.
Immunocytochemical localization of ET peptides and
ETA protein.
Normal (n = 4) and CH (n = 12; 4 each
at 3, 7, and 14 days) rats were anesthetized with ketamine (10 mg/100 g
im) plus xylazine (0.9 mg/100 g im) and perfused intracardially with
ice-cold 4% paraformaldehyde in 0.1 M PBS. Carotid bodies were
removed, cleaned of surrounding connective tissue, immersed in the same
fixative for 1 h, rinsed in 15% sucrose-PBS for 2 h, and
stored at 4°C in 30% sucrose-PBS overnight. Cryostat sections (4 µm) were thaw-mounted on gelatin-subbed slides. Sections were first
exposed to avidin-biotin preblocking reagents (20 min; Vector) and
incubated at 4°C overnight in primary antibody [anti-ET peptide
(Peninsula); anti-ETA protein (Maine Biotechnology
Services)] diluted 1:16,000 or 1:2,000 in PBS containing 0.3%
Triton X-100. Sections were then rinsed in PBS at room temperature,
incubated for 2 h in biotinylated goat anti-rabbit IgG (Vector),
rinsed in PBS for 20 min, incubated in avidin-biotinylated horseradish
peroxidase complex (2 h; Vector Elite kit), and treated with
3',3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide.
According to supplier specifications, anti-ET cross-reacts equally
(100%) with rat ET-1, human or canine ET-2, and human or porcine
big-ET (a large precursor molecule for ET peptides). The antibody
reacts minimally with ET-3 (0.04%) or closely related peptides (e.g.,
2% sarafotoxin). In all experiments, normal and CH tissue samples and
frozen sections were processed simultaneously, and all incubation and
reaction conditions were identical. In selected sections, the primary
antibody was omitted; no immunostaining was observed in these specimens.
RNA extraction and competitive RT-PCR.
In accord with the instructions in a kit (Totally RNA; Ambion, Austin,
TX) total RNA was extracted from six to eight carotid bodies pooled
from multiple rats (i.e., 3 or 4 normal and 3 or 4 CH animals for each
experiment). The final RNA pellet was resuspended in 75% ethanol,
sedimented, vacuum dried (2 min), dissolved in water, and used
immediately for PCR or stored at
20°C. Protein in the extract was
measured by a modified Lowry method. After removal of contaminating DNA
(MessageClean Kit; Gene Hunter, Nashville, TN), first-strand cDNA was
synthesized using 1 µl of the total RNA incubated at 42°C for 15 min in 20 µl of 10 mM Tris · HCl buffer (pH 8.3) containing
50 mM KCl, 2 mM MgCl2, 20 units RNase inhibitor, 2.5 µM
oligo(dT)16, and 50 units of Moloney murine leukemia virus
RT plus 1 mM of each dNTP. The reaction mixture was denatured at 99°C
for 5 min and chilled to 5°C for 5 min.
The cDNA was amplified with specific primers in a competitive reaction
that included a mimic molecule as internal standard in the reaction
mix. Mimic molecules were constructed in accord with instructions
provided in a kit (Clontech, Palo Alto, CA) and contained upstream and
downstream primer sites identical to the target cDNA but with different
intervening sequence and slightly different base pair size, which
allowed coamplification in the same tube and subsequent gel separation
from the target product. PCR reactions occurred in 20-80 µl of
buffer solution containing MgCl2 (final concentration 1.5 mM), AmpliTaq DNA polymerase (final concentration 5.0 U/100
µl), and 32P-end-labeled primers (final concentration 0.5 µM). PCR was initiated at 95°C for 2 min followed by 35 cycles
consisting of 30 s at 95°C, 30 s at 60°C, and 30 s
at 72°C, with the final cycle extended to 2 min at 72°C, followed
by termination at 4°C. Primer sequences for pre-pro-ET-1 were
"upstream" 5'-TAC AGA GAC CAG AAG TTG ATA C-3' and "downstream"
5'-TAG AAG CCG GAC AGA TGT TC-3' and for the ETA gene
upstream 5'-TTC GTC ATG GTA CCC TTC GA-3 and downstream 5'-GAT ACT CGT
TCC ATT CAT GG-3'. Quantification of the target sequence was obtained
by amplifying a dilution series of the mimic molecule in a constant
amount of target cDNA derived from the tissue. A plot of the
target-to-mimic ratio on the y-axis (quantified from the
radiolabeled products separated on an agarose gel) verses the
reciprocal of the concentration of added mimic on the x-axis is used to estimate the target concentration, which is the point on the
graph where the target and mimic products are equal (i.e., ratio = 1). The corresponding x-intercept is the theoretical
equivalent of the original target mRNA concentration. Values from
normal and CH preparations are expressed per milligram of protein to account for differences in tissue weight.
Electrophysiological recording of CSN activity.
Under ketamine/xylazine anesthesia, and with the aid of a dissecting
microscope, the carotid artery bifurcations containing the carotid
bodies were located and removed from 25 rats after exposure to CH for
0-16 days. The excised tissue was placed in a lucite chamber
containing 100% O2-equilibrated modified Tyrode solution
at 0-4°C (in mM: 112 NaCl, 4.7 KCl, 2.2 CaCl2, 1.1 MgCl2, 42 sodium glutamate, 5 HEPES buffer, and 5.6 glucose; pH = 7.4). Each carotid body along with its attached
nerve was carefully dissected from the artery and cleaned of
surrounding connective tissue. Preparations were then placed in a
conventional superfusion chamber where the carotid body was
continuously superfused (up to 4 h) with modified Tyrode solution
maintained at 37°C and equilibrated with a selected gas mixture. The
CSN was drawn up into the tip (~100 µm ID) of a glass suction
electrode for monopolar recording of chemoreceptor activity. Sufficient
suction was applied to seal the electrode tip against the connective
tissue ring encircling the junction of the carotid body and CSN. The
bath was grounded with a Ag/AgCl2 wire, and neural activity
was led to an AC-coupled preamplifier, filtered, and transferred to a
window discriminator and a frequency-to-voltage converter. Signals were
processed by an analog-to-digital/digital-to-analog converter for
display of frequency histograms on a personal computer monitor.
After neural recording, the CSN was carefully removed, and carotid body
wet weight was determined in a Cahn electrobalance equipped with a humidified weighing chamber. Data were expressed as impulses per second
and were analyzed using ANOVA with the Bonferroni multiple comparison
posttests or paired t-tests.
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RESULTS |
Localization of ET-like immunoreactivity in normal and chronically
hypoxic rat carotid body.
Figure 1 shows the effects of CH on ET
immunostaining in the rat carotid body. In accord with previous studies
that reported that exposure to CH for 14 days elicited a marked
increase in ET immunoreactivity in O2-sensitive type I
cells (18), the present results confirm this increase and
show that the hypoxia-induced elevation of ET peptide expression is
recognizable in most type I chemosensory cells after only 3 days of CH
exposure, during the period of incremental pressure reduction to 380 Torr (see MATERIALS AND METHODS). Although the intensity of
ET immunostaining was similar in carotid bodies after 3 vs. 7 days of
CH exposure, ET immunostaining was markedly enhanced in type I cells
after 14 days of CH, indicating additional peptide production and
storage. By contrast, the levels of reaction product were substantially lower in normoxic animals. In all conditions, staining occurred in
virtually all type I cells as a fine granular precipitate throughout the cytoplasm, whereas the large ovoid nuclei of these cells remained unstained. Importantly, ET immunoreactivity appeared after CH in many
reactive endothelial cells whose somata protruded into the lumen of
dilated sinusoidal blood vessels. However, other tissue components,
including nerve fibers, fibroblasts, type II cells, and other vascular
endothelial cells of arteries and veins, were not stained.

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Fig. 1.
Endothelin immunoreactivity in normal and chronically hypoxic rat
carotid body. A: immunostaining is localized to cell
cytoplasm in type I cells in normal carotid body. Incremental increases
in immunostaining intensity occur after 3 (B), 7 (C), and 14 (D) days of chronic hypoxia (380 Torr). Arrows in D indicate large prominent endothelial
cells protruding into vascular lumen. Scale bar = 20 µm.
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ETA localization in normal and chronically hypoxic rat
carotid body.
In normal carotid bodies, ETA immunoreactivity occurred in
nearly all type I cells as a fine granular reaction product throughout the cytoplasm (Fig. 2A). After
3 and 7 days of CH (Fig. 2, B and C), there were
slight to moderate elevations in ETA immunostaining. However, after 14 days of CH, receptor immunoreactivity in type I cells
was substantially elevated (Fig. 2D). In all preparations, no ETA immunoreactivity was found in type II cells or in
nerve fibers, Schwann cells, fibroblasts, and blood vessels surrounding the lobules.

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Fig. 2.
Immunoreactivity for endothelin receptor (ETA) protein
in normal and chronically hypoxic rat carotid body. A: in
normal tissue, ETA is expressed in chemosensory type I
cells. Immunostaining intensity is noticeably increased after
days 3 (B) and 7 (C) of CH
and is markedly elevated after 14 days of CH (D). Scale
bar = 20 µm.
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Expression of ET-1 peptide and ETA genes in normal and
chronically hypoxic carotid bodies.
The elevated levels of ET peptide and ETA protein
immunostaining observed after CH suggest possible increased expression
of respective peptide- and protein-specific genes. Figures
3 and 4
present analyses of quantitative RT-PCR assays for mRNAs coding for the
ET-1 precursor molecule, pre-pro-ET-1, and ETA protein, respectively. The marked x-intercepts in Fig. 3 indicate
estimated amounts of pre-pro-ET-1 cDNA (corresponding to tissue mRNA)
in normal (Fig. 3A) and CH (Fig. 3B) carotid
bodies. These data, when expressed per milligram of protein in tissue
extracts, indicate a 180-fold increase in the level of pre-pro-ET-1
mRNA in carotid bodies after 14 days of CH. A replicate of this
experiment in a second group of four normal and four CH rats similarly
indicated a 170-fold increase in the expression of the pre-pro-ET-1
transcript. Figure 4 shows a similar evaluation in rat carotid body of
ETA gene expression. In this experiment, 14 days of CH
resulted in a 14-fold increase in ETA transcript levels. In
three replicate experiments, the mean relative increase in
ETA mRNA was 15.1 ± 1.97-fold (mean ± SE,
n = 4; P = 0.0056 vs. hypothetical mean of 1.0).

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Fig. 3.
Quantitative assessment of endothelin (ET)-1 mRNA
transcript level in normal (A) and in 14-day chronic hypoxia
(CH; B) rat carotid body. X-axis: initial
concentration of mimic DNA molecule (internal standard) that contains
primer sequence identical to ET-1 target transcript. Y-axis:
ratio of target-to-mimic concentration in reaction mix after 35 cycles
of PCR. Marked intercepts indicate equal concentrations of target and
mimic molecules. Transcript ratio: concentration of ET-1 transcript in
hypoxic/normoxic carotid bodies expressed per mg protein. Transcript
ratio in replicate experiment indicated 170.7-fold increase in tissue
levels of ET-1 mRNA after 14 days of CH. cpm, Counts/min.
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Fig. 4.
Effect of 14 days of CH on ETA mRNA levels in
rat carotid body. Details as in Fig. 3. In 3 replicate experiments, the
mean CH-induced increase in ETA transcript was 15.1 ± 1.97-fold (mean ± SE, P = 0.0056).
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Effect of CH on resting and stimulus-evoked CSN activity.
CH in the rat induces VAH and an elevated HVR, but adaptive changes in
chemoreceptor nerve activity in this species have not been documented.
We evaluated basal (normoxia) and hypoxia-evoked CSN chemoreceptor
activity in vitro after in vivo exposure to CH for selected periods
lasting up to 16 days. Figure 5
summarizes basal nerve activity recorded in solutions equilibrated at
PO2 = 450 Torr and after reducing bath
PO2 to 120 Torr for 150 s (acute hypoxia),
which elicited submaximal increases in chemoreceptor activity.
Recordings from multiple preparations were highly reproducible (SE
<10%), and the data show that CSN activity at both
PO2 levels progressively increases after
exposure to CH. Significant changes are first observed after 3 days of
CH, and both basal nerve activity and the response to acute hypoxia
continued to increase up to day 9 of CH exposure. No further
increases were observed in preparations from animals exposed to CH for
12, 14, and 16 days. In normal carotid body/CSN preparations, basal
nerve activity was 15.53 ± 1.39 (SE) impulses/s, and this value
was elevated to 95.10 ± 7.54 impulses/s after 9 days of CH
(P < 0.001). The 150-s averaged nerve discharge rate
during superfusion at PO2 = 120 Torr was 186.4 ± 12.8 impulses/s in normal vs. 390.6 ± 30.2 impulses/s in 9-day CH preparations (P < 0.001).

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Fig. 5.
Effect of CH on basal (normoxic) and acute
hypoxia-stimulated carotid sinus nerve (CSN) activity. Basal nerve
activity evaluated in superfusion solutions equilibrated at bath
PO2 = 450 Torr. CSN responses to hypoxia
are expressed as impulses (imp)/s and averaged over a 150-s period of
acute hypoxia at bath PO2 = 120 Torr.
* P < 0.05 and *** P < 0.001 vs. activity in normal (i.e., 0 days CH) preparations (ANOVA).
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Effect of the ETA antagonist BQ-123 on CSN activity.
The participation of endogenous ET peptide in the generation of
chemoreceptor nerve discharge was evaluated using the specific ETA antagonist BQ-123. Figure
6 shows examples of integrated CSN activity in normal (left) and 3-day CH (right)
carotid body/CSN preparations. In each experiment, after establishing
the basal rate of nerve discharge, we lowered the bath
PO2 to ~120 Torr (see
PO2 trace, Fig. 6) for 150 s to evoke the
"control" hypoxic discharge. This was followed by a 2.5-min
superfusion with solution at PO2 = 450 Torr
containing 5 µM BQ-123, a drug concentration sufficient to saturate
ETA (21, 22). A second hypoxic stimulus involved superfusion with 5 µM BQ-123 as the bath
PO2 was again lowered to 120 Torr for 150 s. After a 15- to 20-min wash in the absence of the antagonist
(superfusion solution equilibrated at PO2 = 450 Torr), a third hypoxic stimulus (PO2 = 120 Torr) evaluated "recovery" of the response. The effect of
BQ-123 on CSN discharge is shown in Fig. 6, where after 3 days of CH,
the control and recovery responses to hypoxia were larger than normal
(see also Fig. 5), and in the presence of BQ-123 the response to
hypoxia was reduced by ~20%.

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Fig. 6.
Effect of CH on the sensitivity of chemoreceptor nerve
discharge to BQ-123. Left: 3 superimposed traces of
integrated CSN activity; separate trace indicates changes in bath
PO2. Basal- and hypoxia-stimulated nerve
activity are minimally altered in the presence of 5 µM BQ-123. After
3 days of CH (right), basal nerve activity is marginally
affected by the ETA antagonist. However, nerve activity
evoked by hypoxia is substantially reduced in the presence of the
drug.
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The experimental protocol described above was employed to evaluate the
effects of BQ-123 on CSN activity elicited by superfusion with
low-O2 solution (PO2 = 120 Torr) after animal exposure to varying periods of CH up to 16 days. The
data summarized in Fig. 7A
demonstrate that the antagonist reduced the hypoxia-evoked (i.e.,
stimulus minus basal) CSN activity by 10.9 ± 1.8% (mean ± SE; n = 13) in preparations from normal animals.
However, after 3 days of CH, BQ-123 exposure diminished the
hypoxia-evoked discharge by ~20%, and this effect progressed to 45%
at day 9 of CH and to nearly 50% after 12, 14, and 16 days
of CH.

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Fig. 7.
A: sensitivity of stimulus-evoked CSN activity
to BQ-123 increases progressively as CH is extended up to 16 days. Data
were normalized to the control response to hypoxia (bath
PO2 = 120 Torr). B: effect of
0-16 days of CH on carotid body size. Note that changes in the
sensitivity of the nerve discharge to BQ-123 precede organ enlargement,
which is significant after 7-16 days of hypoxia. Also, weight of
the carotid body increases markedly between 9 and 12 days of exposure,
but the drug effect is unchanged. ** P < 0.01 and
*** P < 0.001 vs. control nerve response or normal
(0-day) weight.
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The effect of CH on carotid body size is a potentially important factor
for interpretation of these data in our in vitro superfused preparations. Increased organ mass may significantly steepen
O2 gradients, which could result in an enhanced hypoxic
stimulus and potentiation of endogenous peptide release. However, the
progressive increase in carotid body wet weight (Fig. 7B)
after exposure to CH suggests that increased organ size does not
directly account for the inhibitory effect of BQ-123. In this regard,
it is important to note that, although significantly enhanced
sensitivity to the drug is evident on days 3 and
5 of CH, the size of the carotid body has not yet increased.
Furthermore, a substantial increase in organ wet weight occurs between
days 9 and 12 of CH, but this change is not
accompanied by a parallel change in the effect of BQ-123. The wet
weight data indicate that organ size is increased three- to fourfold
after 12-16 days of hypoxia, consistent with prior studies
employing quantitative morphological techniques (26).
The analysis of the CH nerve recording data presented in Fig.
8 compares the averaged CSN activity
evoked (stimulus minus basal) by hypoxia
(PO2 = 120 Torr) in the presence of 5 µM
BQ-123 with nerve activity evoked in normal preparations (i.e., 0 days CH) exposed to the drug. In CH preparations, the mean evoked nerve activity in the presence of the ETA antagonist was always
greater than activity evoked in normal preparations. However, a one-way ANOVA of these data, combined with Bonferroni multiple comparison posttests, suggests that significant differences do not exist between
normal and CH groups, with the exception of the 12-day CH group vs.
normal (P < 0.05). In this series of experiments, most
data were obtained from CH groups consisting of only three or four
preparations, a statistical minimum. However, in 15 preparations examined after 5 days of CH (i.e., preparations demonstrating increased
chemosensitivity before CH-induced organ growth), the data demonstrate
levels of evoked nerve activity in the presence of BQ-123 that are
indistinguishable from normal (P > 0.05; at
= 0.05 the statistical test has a power of 40% to detect a 22% difference in the mean).

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Fig. 8.
BQ-123 occludes the usual increase in stimulus-evoked
chemoreceptor activity after CH. Data show averaged nerve activity
evoked (stimulated minus basal activity) by acute hypoxia in the
presence of 5 µM BQ-123. ANOVA analysis indicates that activity
evoked in the presence of the drug after 3, 5, 7, 9, and 16 days of CH
does not differ from normal (0 days; P > 0.05, Bonferroni multiple-comparison test). After 12 days of CH, the increase
is marginally significant (* P < 0.05, see text).
Values in parentheses are no. of experiments.
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The effectiveness of the ETA antagonist was further
explored in normal (n = 10) vs. 5-day CH
(n = 11) preparations exposed to the full range of bath
PO2 levels (40-450 Torr). The data
presented in Fig. 9 show that, in normal
preparations, the percent depression of CSN activity by BQ-123 is
increased with decreasing PO2. Furthermore, in
5-day CH preparations exposed to PO2 180, 120, 100, and 40 Torr, the drug was significantly more effective at reducing
the nerve discharge rate. At higher bath PO2
levels (i.e., 200 and 450 Torr), the drug caused only a marginal
depression of nerve activity that did not differ between normal and
5-day CH preparations.

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Fig. 9.
Relationship between PO2 and the
effects of BQ-123 on CSN discharge recorded in vitro in normal vs. CH
rats. Note that, in both normal and CH preparations, the inhibitory
effect of the ETA antagonist is greater with increasingly
severe hypoxia. In moderate to severe hypoxia (i.e., 180, 120, 100, and
40 Torr), the drug is more effective in CH preparations.
* P < 0.05 and *** P < 0.001 vs. normal. However, at higher PO2 levels (200 and 450 Torr), the discharge is minimally depressed in the presence of
the antagonist. [BQ-123], BQ-123 concentration.
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The failure of BQ-123 to completely block the CH-induced increase in
CSN discharge when bath PO2 levels are
relatively high is further documented in Fig.
10, which presents data from
preparations superfused at PO2 = 450 Torr,
after exposure to CH for 0-16 days. The receptor-saturating dose
of BQ-123 (5 µM) caused a significant reduction in nerve activity in
both normal and CH preparations. However, unlike the complete occlusion
of the CH-induced increase in chemoreceptor activity observed at
PO2 of 120 or lower, BQ-123 only partially
depressed the elevated nerve activity in preparations superfused at 450 Torr, after exposure to CH for 5-16 days. (Nerve activity in the
presence of BQ-123 was not significantly different in 0-day vs. 3-day
preparations.)

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Fig. 10.
Effect of BQ-123 on resting CSN discharge in normal and
CH carotid body. Tissues were removed from normal (0 days) and CH
animals and superfused in vitro at bath
PO2 = 450 Torr. Note that the
ETA antagonist only partially blocks the CH-induced
increase in basal nerve activity. Values in parentheses are no. of
experiments. * P < 0.05 vs. nerve discharge rate
without drug (paired t-test).
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DISCUSSION |
The present study was designed to elucidate the physiological role
of endogenous ET in the chemoreceptor response of the normal carotid
body and to evaluate its involvement in the dynamic physiological adjustments during exposure to CH. McQueen and colleagues
(30) were the first to demonstrate that intravenous
injection of ET peptide elevates respiratory minute volume and elicits
CSN excitation in rat carotid body. Of particular interest was the
observation that these effects were blocked by the specific
ETA antagonist FR-139317. Autoradiographic studies using
125I-labeled ET peptides further demonstrated specific ET
binding sites both in carotid body lobules and in surrounding
microvascular elements (30, 36). Chen et al. (8,
9) reported that ET peptides potentiated hypoxia-evoked nerve
activity when applied to rat and rabbit carotid body/CSN preparations
superfused in vitro, where the potent vascular effects of ET-1 are
eliminated. This effect of ET-1 is blocked by BQ-123 but not by the
ETB antagonist IRL-1038. Additional studies have shown that
incubation of intact rat carotid body in ET-1 increases cAMP levels in
type I cells (9). Moreover, in dissociated type I cells
from rabbit, ET-1 potentiates hypoxia-evoked intracellular
Ca2+ responses and voltage-gated Ca2+ currents
(8). Thus the pharmacological effects of ET appear to be
mediated by the following dual mechanisms: on the one hand they involve
cAMP- and Ca2+-dependent mechanisms in type I cells during
hypoxia (8, 9), and, on the other, they activate
hypotensive and pressor effects, which may occur independently of
arterial PO2 when peptide is administered
intravenously (30).
The present immunocytochemical findings confirm earlier studies that
demonstrated ET peptide in type I cells and increased levels of peptide
expression after 2 wk of CH (18). These data further
demonstrate that peptide content is noticeably elevated after only 3 days of low-O2 exposure and that levels in type I cells
continue to increase, resulting in substantially enhanced immunostaining after 14 days of CH. However, the presence of ET in
certain large prominent endothelial cells at 14 days in the largest of
the dilated sinusoidal capillaries indicates a belated vascular effect
that is restricted to the final stage of remodeling when typical
capillaries are entirely absent in the carotid body. It is noteworthy
that studies in other tissues have revealed that ET peptide levels are
regulated primarily via gene transcription (25). Thus the
presence of high levels of ET peptide in type I cells is corroborated
by studies of pre-pro-ET gene expression using the RT-PCR technique
with an internal standard mimic molecule. These data indicate that
transcript levels for the precursor molecule are elevated >100-fold on
day 14 of CH exposure. Smaller effects have been reported in
rat lung, where conventional mRNA hybridization techniques indicated a
three- to fourfold increase in ET gene expression after 28 days of 10%
O2 breathing (27, 28). Interestingly, analysis
of pre-pro-ET gene structure has shown that the proximal promoter
region contains an active binding site for hypoxia-inducible factor-1
and that mutations in this site prevent hypoxia-induced ET expression
in cultured vascular endothelial cells (20). Moreover, in
transgenic mice expressing a pre-pro-ET-1-luciferase gene construct, exposure to 10% O2 for 24 h elicits a sixfold
increase in promoter activity in lung tissue (2).
Less is known about regulation of the ETA gene. Studies in
the heart and lung have shown that CH induces increased receptor transcript levels (27, 28), and our data for 14-day CH
indicate a 15-fold increase in ETA mRNA in the carotid
body. This elevated transcript level agrees with the marked increase in
immunostaining intensity for ETA protein on CH day
14, with smaller changes observed after 3 and 7 days of CH.
Importantly, in all experimental conditions, ETA
immunoreactivity is localized exclusively in type I cells. The
colocalization of ET peptide and the A-type receptor in type I cells
indicates that this endogenous peptide acts via an autocrine or
paracrine mechanism. The suggestion that it has no direct effects on
afferent chemoreceptor nerve terminals is supported by the finding that
ETA protein immunoreactivity is not present in nerve fibers
in the carotid body. This is also consonant with the reports of McQueen
and colleagues (30), who showed that the specific ETA antagonist FR-139317 does not displace
125I-ET binding sites in nodose ganglion, a structure known
to contain a subpopulation of sensory neurons that innervate arterial
chemoreceptors in the aortic bodies near the heart (30).
Endogenous ET peptide and ETA appear to participate
minimally in the generation of chemoreceptor nerve activity in normal preparations, where receptor saturating concentrations of BQ-123 depress the hypoxia-evoked CSN discharge by <11%. However, after 3 days of CH, ~20% of the evoked nerve activity is sensitive to the
antagonist, and this effect is incrementally increased in preparations
exposed up to 9 days of CH, when 45% of the evoked discharge is
blocked. This gradual emergence of sensitivity to the ETA
blocker is paralleled by an increase in the nerve response to a
standardized hypoxic stimulus. Conversely, these changes in drug
sensitivity and nerve activity are not correlated with the time course
of carotid body enlargement induced by CH. Interestingly, in an early
study of ventilatory acclimatization induced in the rat by exposure at
433 Torr (less severe than the 380 Torr used here), Olson and Dempsey
(32) showed that the progressive increase in minute volume
occurs over the first 4 days, a period corresponding to the steepest
phase of developing BQ-123 sensitivity in our preparations. In
addition, a significant portion of the progressive increase in
hypoxia-evoked CSN activity likewise develops within the first 3 days
of CH.
Our nerve recording data also indicate that basal chemoreceptor
activity is increased after CH. These changes were first observed on
day 3, with subsequent incremental increases up to day
9 of exposure. Increased resting CSN discharge after CH has not
been reported in any species (31), but previous studies
have demonstrated a persistent hyperventilation upon returning to
normoxia after CH in animals and humans (11). However,
this phenomenon appears to be present even after 1 day of hypoxia in
rats exposed at 433 Torr (32), whereas our data indicate
that basal CSN activity is unchanged after 24 h of exposure at 380 Torr. Nonetheless, the elevated nerve activity that develops after 3 days in low O2 is likely to support the continuation of
hyperventilation in normoxia. In any case, an important mechanism for
altered basal nerve activity appears to involve the upregulation of
both endogenous ET levels and the number of ETA because
bath application of 5 µM BQ-123 partially inhibits the increase in
resting CSN discharge in CH preparations. The partial effectiveness of
the receptor antagonist under normoxic/hyperoxic conditions suggests
that the CH-induced increase in resting nerve activity involves both
ET-dependent and -independent mechanisms. Earlier observations
suggested the existence of different mechanisms governing basal vs.
hypoxia-evoked neurotransmitter release in type I cells, where in the
presence of 0 mM Ca2+ and 2.1 mM Mg2+, dopamine
release evoked by hypoxia is almost completely abolished, whereas basal
dopamine release is unaltered (15). Such findings are in
accord with classical studies that showed that, although 0 mM
Ca2+ and high Mg2+ fully inhibit evoked ACh
release from motor nerve terminals, these conditions do not alter the
frequency and amplitude of spontaneous miniature end-plate potentials,
suggesting that transmitter release at the resting synapse is the
result of Ca2+-independent (random) fusion of secretory
vesicles with the plasma membrane (14). It is unknown
whether CH increases the rate of Ca2+-independent vesicle
fusion. However, it is noteworthy that previous ultrastructural studies
of rat type I cells have shown that the volume density of vesicles
decreases after 1 wk of CH but returned to normal values after 2 or 3 wk of hypoxia (19).
The inability of BQ-123 to completely block the elevated resting nerve
activity after CH differs from the effect of this drug during moderate
to severe hypoxia, where the CH-induced increase in chemoreceptor
discharge appears to be quantitatively excluded by the ETA
antagonist. This latter finding suggests that increased levels of
endogenous ET acting at ETA may account for the increased chemoreceptor discharge evoked by acute hypoxia in CH preparations. These data strongly suggest that ET peptides and ETA are
essential for induction of enhanced carotid body chemosensitivity by
CH. However, our data do not exclude the involvement of other
neuroactive agents in the adaptation of the chemoresponse. The basic
functional components of the carotid body, namely type I cells and
chemoafferent nerve terminals, comprise a highly complex neurochemical
apparatus containing multiple competing excitatory and inhibitory
neuroactive agents (1, 17, 35). Thus the dynamic
adjustments induced by CH likely involve a complex interplay between
competing endogenous transmitter systems that act in concert to
regulate the functional output of the carotid body. In such a scheme,
the blockade of ETA by BQ-123 may, in addition to blocking
the purely excitatory effects of endogenous ET peptide, influence the
synthesis, release, and actions of competing agents. Although our data
strongly support important roles for ET peptide and ETA,
the complete reversal of increased chemosensitivity by BQ-123 could,
nonetheless, be the fortuitous consequence of interference in one of
several highly interactive and integrated signaling cascades, resulting
in a change in nerve discharge suggestive of an unrealistically simple underlying mechanism. The possible involvement of other mechanisms may
be indicated in 12-day CH preparations, where BQ-123 failed to
completely block the CH-induced increase in stimulus-evoked activity
(Fig. 8). Indeed, numerous studies have demonstrated significant
CH-induced alterations in the synthesis, storage, and turnover of
multiple candidate neurotransmitters and receptors in type I cells and
chemosensory afferent neurons, suggesting their participation in
altered chemosensitivity (see Ref. 6). Involvement of
these factors in adaptive mechanisms will require further detailed investigations.
In conclusion, CH-induced upregulation of ET peptide and
ETA in O2-sensitive type I cells occurs
concurrently with a time-dependent increase in carotid body
chemosensitivity, an adaptive phenomenon that is blocked by the
specific ETA antagonist BQ-123. Our findings indicate that
endogenous ET mediates enhanced type I cell activity and that elevated
CSN activity may result from effects of ET that modulate the actions of
other neurotransmitter(s) at synapses between type I cells and
chemoafferent nerve terminals. This role of ET in the moment-to-moment
function of the carotid body may occur in addition to long-term actions
of this interesting peptide. Indeed, ET is known to act as a mitogenic
agent involved in tissue remodeling and reshaping in the lung and heart
during CH. Thus high levels of ET peptide and ETA could
also participate in hypertrophy and mitotic activity, which occur in
type I cells during sustained exposure to low ambient O2
(4, 29, 34).
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Neurological and
Communicative Disorders and Stroke Grants NS-12636 and NS-07938.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
B. Dinger, Dept. of Physiology, Univ. of Utah School of
Medicine, 410 Chipeta Way, Rm. 155, Salt Lake City, UT
84108 (E-mail:
bruce.dinger{at}m.cc.utah.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.
10.1152/ajplung.00454.2001
Received 26 November 2001; accepted in final form 3 January 2002.
 |
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