Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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We tested the hypotheses that
hypoxic exposure is associated with exacerbated pulmonary hypertension
and right ventricular (RV) enlargement, reduced atrial natriuretic
peptide (ANP) clearance receptor (NPR-C) expression, and enhanced
B-type natriuretic peptide (BNP) expression in the absence of
ANP. Male wild-type [ANP(+/+)], heterozygous [ANP(+/)], and
homozygous [ANP(
/
)] mice were studied after a 5-wk hypoxic
exposure (10% O2). Hypoxia increased RV ANP mRNA and
plasma ANP levels only in ANP(+/+) and ANP(+/
) mice. Hypoxia-induced
increases in RV pressure were significantly greater in ANP(
/
) than
in ANP(+/+) or ANP(+/
) mice (104 ± 17 vs. 45 ± 10 and
63 ± 7%, respectively) as were increases in RV mass (38 ± 4 vs. 26 ± 5 and 29 ± 4%, respectively). NPR-C mRNA levels
were greatly reduced in the kidney, lung, and brain by hypoxia in all three genotypes. RV BNP mRNA and lung and kidney cGMP levels were increased in hypoxic mice. These findings indicate that disrupted ANP
expression worsens hypoxic pulmonary hypertension and RV enlargement but does not alter hypoxia-induced decreases in NPR-C and suggest that
compensatory increases in BNP expression occur in the absence of ANP.
pulmonary hypertension
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INTRODUCTION |
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ATRIAL NATRIURETIC PEPTIDE (ANP) and B-type natriuretic peptide (BNP) play important roles in regulating pulmonary vascular tone and in the pathogenesis of hypoxia-induced pulmonary hypertension (8-11, 20, 22, 27, 31). ANP and BNP are produced in both the cardiac atria and ventricles (3). Under stressful conditions, e.g., pulmonary hypertension, myocardial infarction, cardiac hypertrophy, congestive heart failure, and hypoxia, synthesis and release of both natriuretic peptides are increased (3, 22, 30). ANP and BNP bind to receptors in the pulmonary circulation (4, 25), relaxing preconstricted pulmonary arteries in vitro (32) and inducing pulmonary vasodilation in isolated lungs from chronically hypoxic rats compared with those in air-exposed control rats (6, 8-11, 20). Experiments performed in our laboratory have demonstrated that rats exposed to chronic hypoxia exhibit increased pulmonary arterial pressure and right ventricular hypertrophy in association with an elevation in endogenous plasma ANP levels (9, 10). Intravenous infusion of ANP blunts acute hypoxia-induced pulmonary vasoconstriction in conscious rats (9), and continuous infusion of ANP during hypoxia attenuates the development of pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling while increasing plasma ANP and lung cGMP levels (10). Furthermore, neutralization of endogenous ANP with monoclonal antibodies potentiates hypoxia-induced pulmonary hypertension (11). A recent study by Klinger et al. (20) demonstrated that infusion of BNP attenuates hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in rats. Thus ANP and BNP appear to be endogenous modulators of the pulmonary hypertensive response to hypoxia.
Three receptor subtypes for natriuretic peptides have been identified (4, 25). The ANP-A receptor (NPR-A) and the ANP-B receptor (NPR-B) are membrane-bound guanylate cyclases that mediate most of the biological actions of ANP and BNP through the regulation of intracellular cGMP levels (4). The ANP-C receptor (NPR-C) lacks the intracellular guanylate cyclase domain and is, therefore, uncoupled from cGMP (25). The NPR-C mediates the cellular internalization and subsequent lysosomal degradation of ANP and BNP and is thus thought to function as a clearance receptor (1, 14, 25, 33). Several reports (29-33) have suggested roles in addition to this clearance function.
The NPR-C accounts for the overwhelming majority of ANP and BNP binding sites in most rat tissues, including lung, kidney, and brain, as assessed by radioligand binding techniques and mRNA quantitation (25, 27). NPR-C blockade delays the metabolic clearance of ANP and BNP in vivo and potentiates the biological effects of both peptides (8, 25), and the NPR-C is thus termed the high-capacity natriuretic peptide clearance receptor (25, 33). Klinger et al. and Li et al. have previously demonstrated that both acute and chronic hypoxia cause significant downregulation of NPR-C binding (17) and gene expression without affecting NPR-A or NPR-B gene expression in rat lung (27). It has been reported that ANP itself can downregulate NPR-C expression in cultured smooth muscle cells or endothelial cells through a NPR-A-8-bromo-cGMP-mediated pathway (14, 35). Furthermore, Li et al. (26) have previously demonstrated that hypoxia can act directly on pulmonary smooth muscle cells to cause rapid and significant downregulation of NPR-C. It is unclear whether the decreased expression of the NPR-C gene in the lung of hypoxia-adapted rats is a function of hypoxia per se or of hypoxia-induced increases in ANP and/or BNP levels.
In the present study, we used ANP knockout mice with insertional inactivation of the ANP gene to further test the hypotheses that increased cardiac ANP synthesis and circulating ANP levels during hypoxia protect against the development of hypoxic pulmonary hypertension and that hypoxic exposure can inhibit NPR-C gene expression without enhancing ANP production.
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METHODS |
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ANP knockout mice.
The generation of ANP knockout mice has previously been described in
detail (12). Briefly, a targeting construct was designed to insert a disruptive neomycin resistance gene (neo) into
exon 2 of the mouse pro-ANP gene (Nppa) in embryonic stem
cells of mouse strain 129. Chimeras harboring the mutation were then
mated to mice of strain C57BL/6J (B6) to produce 129 × B6
F1 offspring. Matings between 129 × B6 F1
heterozygotes produced homozygous mutant [ANP(/
)], heterozygous
[ANP(+/
)], and wild-type [ANP(+/+)] F2 offspring in
Mendelian proportions. Male 10- to 14-wk-old ANP(+/+), ANP(+/
), and
ANP(
/
) mice weighing 30-35 g were used in this study. The
animals were obtained from our resident colony that was founded with
pathogen-free breeding pairs. The genotypes were identified by
polymerase chain reaction (PCR) assay of genomic DNA from tail snips
soon after the infants were weaned and were confirmed after the
experiment. The two primer pairs for PCR were 1)
5'-GGG-CAT-CTT-CTG-CTG-GCT-CCT-CAC-TCC-ATC-3' and
5'-TAA-AGC-GCA-TGC-TCC-AGA-CT-3' for the mutant allele and
2) 5'-GGC-TCC-GAG-GGC-CAG-CGA-GCA-GAG-CCC-TCA-3' and
5'-CGT-TCC-CCG-ACC-CAC-GCC-AGC-ATG-GGC-TCC-3' for the normal allele.
All mice were housed in groups of three to four per cage; maintained at
constant humidity (60 ± 5%), temperature (24 ± 1°C), and
light cycle (6 AM to 6 PM); and fed a standard mouse pellet diet
(Ralston Purina Diet) ad libitum. All protocols were approved by the
Institutional Animal Care and Use Committee at the University of
Alabama at Birmingham and were consistent with the Guide for the
Care and Use of Laboratory Animals published by the National Institutes of Health [DHEW Publication No. (NIH) 85-23, revised 1985].
Hypoxic exposure. Mice of all three genotypes were exposed to hypoxia in an 800-liter model 818GBB Plexiglas glove box (Plas Labs, Lansing, MI) beginning at age 10-14 wk for a total of 5 wk as previously described (8-11, 27). Hypoxic exposures (range 10.0 ± 0.5% O2) were accomplished by intermittently adding N2 to the chamber from a liquid N2 reservoir, the gas outflow of which was regulated by a solenoid valve (Peter Paul Electronics, New Britain, CT) controlled by the recorder output of a model 1630 O2 controller (Engineered Systems & Designs). A baralyme CO2 scrubber (Allied Health Care Products, St. Louis, MO) kept the CO2 concentration at <0.2%. Relative humidity within the chamber was kept at <60% with anhydrous CaSO4 and a constant temperature circulator (Polysciences, Niles, IL). Boric acid was used to keep NH3 levels within the chamber at a minimum. Animals were permitted to have standard laboratory chow and tap water ad libitum. Daily animal maintenance was carried out without interruption of the exposures through double ports in the chamber. Normoxic control animals were caged similarly and were exposed to filtered room air for identical periods.
Right ventricular pressure measurement and blood and tissue
collection.
After 5 wk of hypoxic or normoxic exposure, the mice were weighed and
anesthetized with a mixture of ketamine (10 mg/100 g intramuscularly)
and xylazine (1.5 mg/100 g intramuscularly). A silicone catheter was
introduced into the right jugular vein via a venotomy and passed across
the tricuspid valve into the right ventricle (RV) to measure the RV
pressure. The chest remained intact during these measurements. Blood
for ANP assay was obtained from the RV after the hemodynamic
measurements were completed and was placed in iced tubes containing
2.25 mg of EDTA and 1.5 trypsin inhibitor units of aprotinin. Plasma
was separated by centrifugation and stored at 80°C until used for a
radioimmunoassay (RIA) for ANP.
RNA isolation and Northern blot analysis. Total RNA was extracted with the guanidine thiocyanate method, and Northern analysis was performed as previously described (24) with 32P-labeled selective cDNA probes for ANP and BNP (courtesy of Drs. R. Wiegand and H. E. Tolunay, respectively, Monsanto, St. Louis, MO) and cDNA probes for NPR-A, NPR-C, endothelial nitric oxide (NO) synthase (eNOS), and inducible NOS (iNOS) that had been generated in our laboratory by RT followed by the DNA PCR with lung RNA as the template as previously described (27). Between each reprobing, 32P-labeled cDNA was stripped off the membrane by pouring boiling 0.1× saline-sodium citrate-0.1% SDS onto the membrane and shaking for 20 min at room temperature. To quantitate the amount of RNA loaded, the blots were stripped as above and rehybridized with the control probe, the 32P-labeled 18S rRNA-specific oligonucleotide 5'-ACGGTATCTGATCGTCTTCGAACC-3'.
ANP and cGMP measurements. Plasma ANP content was measured by RIA with ANP RIA kits (Peninsula Laboratories, Belmont, CA) after extraction with Sep-Pak C18 cartridges (Waters Associates, Milford, MA) as previously described (8, 10, 11). Tissue cGMP content was measured by RIA with cGMP RIA kits (DuPont NEN Research Products, Boston, MA) as previously described (9). The sensitivities of the ANP and cGMP RIAs are 3 pg and 50 fmol per assay tube, respectively.
Statistical analysis. Results are expressed as means ± SE. Statistical analyses were carried out with the CRUNCH statistical package (CRUNCH Software, Oakland, CA) on an IBM 486-compatible computer. The data were analyzed by two-way analysis of variance (ANOVA) to test for separate and combined effects of genotypes and hypoxia on tissue weights, plasma ANP, RV pressure, tissue mRNA, and cGMP levels. One-way ANOVA followed by the Newman-Keuls test was used to test the effects of genotype on the above variables within the normoxic and hypoxic groups. The unpaired t-test was used to test the effects of hypoxia on the above variables within each genotype. Differences were reported as significant if the P value was < 0.05.
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RESULTS |
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Table 1 reports the BWs and tissue
weights at the end of a 5-wk exposure to hypoxia. Final BWs of the
normoxic control mice were the same among the three genotypes.
ANP(+/+) and ANP(+/) but not ANP(
/
) mice exposed to chronic
hypoxia had slightly but significantly lower BWs than their normoxic
controls. Under normoxic conditions, ANP(
/
) mice had heavier LVs
(as indicated by LV-to-BW ratio) than ANP(+/+) or ANP(+/
) mice.
Chronic hypoxic exposure was not associated with significant LV
enlargement in ANP(+/+) or ANP(+/
) mice but significantly increased
the LV-to-BW ratio in ANP(
/
) mice. The increase in the LV-to-BW
ratio was significantly greater in ANP(
/
) mice than in the other
two groups exposed to chronic hypoxia (significant interaction between
hypoxia and ANP genotype on LV-to-BW ratio, P = 0.03 by
two-way ANOVA). The RV+LV-to-BW ratio was greater in ANP(
/
) mice
than in ANP(+/+) or ANP(+/
) mice under normoxic conditions,
indicating that overall cardiac mass was increased in the absence of
ANP even without the stimulus of hypoxic stress. The RV+LV-to-BW ratios
were significantly greater in all three genotypes exposed to chronic
hypoxia than in their normoxic controls. The increase in RV+LV-to-BW
ratios was significantly greater in ANP(
/
) mice than in the other
two groups exposed to chronic hypoxia (P = 0.03 by
two-way ANOVA). Lung, kidney, and brain weights did not differ among
genotypes under normoxic conditions. Chronic hypoxic exposure increased lung and brain but not kidney weights equally in all three groups.
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No differences in mean RV pressure (MRVP), which was used as an index
of pulmonary arterial pressure, were seen among the three genotypes
under normoxic conditions (Fig.
1A). As anticipated, MRVP
increased in all three genotypes with hypoxic exposure. The MRVP of the
chronically hypoxic ANP(/
) mice was significantly greater than that
of chronically hypoxic ANP(+/+) and ANP(+/
) mice (by one-way ANOVA).
The increase in MRVP was significantly greater in ANP(
/
) mice [a
10.1 ± 1.4 mmHg or 104 ± 17% increase compared with that
in normoxic ANP(
/
) mice] than in ANP(+/+) mice [a 4.0 ± 0.9 mmHg or 45 ± 10% increase compared with that in normoxic
ANP(+/+) mice] or ANP(+/
) mice [a 5.6 ± 2.0 mmHg or 63 ± 7% increase compared with that in normoxic ANP(+/
) mice] exposed
to chronic hypoxia (P = 0.02 by two-way ANOVA). Under normoxic conditions, ANP(
/
) mice had heavier RVs (as indicated by
RV-to-BW ratio; Fig. 1B) than ANP(+/+) or ANP(+/
) mice. RV enlargement developed in all three genotypes of mice exposed to chronic
hypoxia, but the hypoxia-induced increase in the RV-to-BW ratio was
significantly greater in ANP(
/
) mice [a 0.75 ± 0.10 mg/g or
66 ± 12% increase compared with that in normoxic ANP(
/
) mice] than in ANP(+/+) mice [a 0.37 ± 0.07 mg/g or 43 ± 8% increase compared with that in normoxic ANP(+/+) mice] or in
ANP(+/
) mice [a 0.43 ± 0.08 mg/g or 48 ± 10% increase
compared with that in normoxic ANP(+/
) mice] (P = 0.03 by two-way ANOVA). As anticipated, plasma ANP and steady-state RV
ANP mRNA levels were barely detectable in either normoxic or hypoxic
ANP(
/
) mice (Fig. 1C). Plasma ANP levels in ANP(+/+) and
ANP(+/
) mice were not significantly different under either normoxic
or hypoxic conditions. Chronic hypoxic exposure significantly increased
plasma ANP to the same levels in ANP(+/+) and ANP(+/
) mice (Fig.
1C).
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Under normoxic conditions, ANP(+/+) and ANP(+/) mice had the same
steady-state levels of ANP mRNA in the RV (Fig.
2A). Chronic hypoxic exposure
significantly increased RV ANP mRNA levels in both ANP(+/+) and
ANP(+/
) mice, parallel to the changes in plasma ANP levels. ANP mRNA
levels in the LV were barely detectable in ANP(
/
) mice under either
normoxic or hypoxic conditions (Fig. 2B). LV ANP mRNA levels
in ANP(+/+) mice were significantly higher than in ANP(+/
) mice under
normoxic conditions. Chronic hypoxic exposure did not alter LV ANP mRNA
levels in ANP(+/+) and ANP(+/
) mice.
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Steady-state NPR-A mRNA levels in lung did not differ among the three
genotypes under normoxic conditions and remained unchanged after 5 wk
of hypoxic exposure (Fig. 3A).
Under normoxic conditions, steady-state NPR-C mRNA expression in the
lung was positively related to ANP gene copy number but was easily
detectable even in the ANP(/
) group (Fig. 3B). Unlike
NPR-A mRNA levels, NPR-C mRNA levels in the lungs were significantly
reduced in all three groups of mice [85 ± 11, 41 ± 13, and
40 ± 7% decreases in ANP(+/+), ANP(+/
), and ANP(
/
) mice,
respectively] by 5 wk of hypoxic exposure. The "floor" levels of
NPR-C mRNA seen after 5 wk of hypoxia were nearly identical in all
groups despite very different plasma ANP levels. Similar to the lung,
the steady-state NPR-A mRNA levels in the kidney (Fig. 3C)
and brain (Fig. 3E) did not differ among the three genotypes
under normoxic conditions and remained unchanged after hypoxic
exposure. ANP(
/
) mice had significantly lower steady-state NPR-C
mRNA levels in the kidney than ANP(+/+) and ANP(+/
) mice under
normoxic conditions (Fig. 3D). Chronic hypoxic exposure
significantly reduced kidney [73 ± 6, 82 ± 12, and 78 ± 5% decreases in ANP(+/+), ANP(+/
), and ANP(
/
) mice, respectively; Fig. 3D] and brain [85 ± 7, 90 ± 10, and 94 ± 7% decreases in ANP(+/+), ANP(+/
), and ANP(
/
)
mice, respectively; Fig. 3F] NPR-C mRNA levels in all three
groups to similar floor levels as previously shown for the
lung.
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To begin to study the possible mechanism(s) that mediates the
downregulation of NPR-C, we measured steady-state tissue RV and LV BNP
mRNA (Fig. 2); lung, kidney, and brain eNOS (Fig.
4) and iNOS mRNA; and lung and kidney
cGMP (Fig. 5) levels in hypoxia-adapted and control mice. BNP was not measured because of the unavailability of
a selective antibody against mouse BNP. Because the phenotypes of ANP(+/+) and ANP(+/) mice were identical in above studies, we
examined only ANP(+/+) and ANP(
/
) mice in these experiments. Under
normoxic conditions, steady-state BNP mRNA levels in the RV were
slightly but significantly greater in ANP(
/
) mice than in ANP(+/+)
mice (Fig. 2C). Chronic hypoxic exposure significantly increased RV BNP mRNA levels in both groups. RV BNP mRNA levels of
chronically hypoxic ANP(
/
) mice were slightly but significantly greater than those of chronically hypoxic ANP(+/+) mice. LV BNP mRNA
concentrations were two to three times lower than those in the RV.
Under normoxic conditions, BNP mRNA levels were not different among the
three genotypes (Fig. 2D). LV BNP mRNA levels were decreased in ANP(+/+) and ANP(+/
) mice but not in ANP(
/
) mice with 5 wk of
hypoxic exposure. BNP mRNA levels in lung, kidney, and brain were not
detectable by Northern blot analysis in these experiments.
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Steady-state eNOS mRNA levels in the lung did not differ between
ANP(+/+) and ANP(/
) mice under normoxic conditions and increased
significantly to the same level in both groups of mice with 5 wk of
hypoxic exposure (Fig. 4A). eNOS mRNA levels in the kidney
(Fig. 4B) and brain (Fig. 4C) did not differ
between groups under normoxic conditions and remained unchanged in both
groups with 5 wk of hypoxic exposure. Tissue (lung, kidney, and brain) iNOS mRNA levels were undetectable by Northern blot analysis in these experiments.
Under normoxic conditions, cGMP levels in the lung (Fig. 5A)
and kidney (Fig. 5B) were significantly lower in ANP(/
)
mice than in ANP(+/+) mice. Chronic hypoxic exposure significantly increased lung and kidney cGMP content in both groups, but cGMP levels
in chronically hypoxic ANP(
/
) mice were still significantly lower than those in chronically hypoxic ANP(+/+) mice.
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DISCUSSION |
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The present study demonstrated that ANP(/
) mice develop more
severe pulmonary hypertension and RV enlargement than ANP(+/+) or
ANP(+/
) mice during hypoxic exposure, supporting the hypothesis that
ANP has a modulatory role in the development of hypoxia-induced pulmonary hypertension. NPR-C mRNA expression was downregulated in
tissues of all three genotypes despite major differences among the
genotypes in circulating ANP levels, steady-state ANP mRNA expression,
and RV pressures, indicating that hypoxia-induced downregulation of
NPR-C expression is independent of circulating ANP levels in hypoxic
animals. RV BNP and lung eNOS mRNAs as well as lung and kidney cGMP
levels were elevated in hypoxia-adapted animals in the absence of ANP,
suggesting the possibility that these mediators (circulating BNP, local
NO, and cGMP) may have participated in the downregulation of brain and
kidney NPR-C expression.
ANP(/
) mice provide a unique opportunity to examine the pulmonary
hemodynamic and cardiac remodeling responses to chronic hypoxia in an
animal model that is incapable of producing ANP. Our findings that
homozygous ANP(
/
) mice exhibited higher RV pressures and greater RV
enlargement than ANP(+/+) and ANP(+/
) mice in association with barely
detectable cardiac ANP mRNA expression and plasma ANP levels during
chronic hypoxic exposure are in agreement with the recent report by
Klinger et al. (21) in ANP(
/
) mice exposed to
hypobaric (0.5 atm) hypoxia for 3 wk. These authors demonstrated that
after 3 wk of hypobaric hypoxia, ANP(
/
) mice had greater RV peak
pressure (RVPP), RV-to-BW ratio, LV-to-BW ratio, and muscularization of
peripheral pulmonary vessels than ANP(+/+) or ANP(+/
) mice. They also
reported that under normoxic conditions, the RVPP in ANP(
/
) mice
was slightly but significantly greater than that in ANP(+/+) and
ANP(+/
) mice. In the present study, we found that the MRVP was the
same among the three genotypes under normoxic conditions. This minor
disparity between the two reports could be attributed to methodological
differences, including measurement of RVPP in Klinger et al.'s study
vs. MRVP in the present study.
Together, these findings provide further evidence that ANP modulates pulmonary vascular remodeling and protects against the development of pulmonary hypertension in response to hypoxia. These findings support previous observations in other animal models and in humans. Studies from our own laboratory (8, 10) demonstrated that hypoxia-induced ANP secretion provides a compensatory mechanism through the vasodilator and antiproliferative effects of ANP to modulate the development of hypoxia-induced pulmonary hypertension and vascular remodeling in rats exposed to hypoxia for 4 wk. Furthermore, Klinger et al. (18) demonstrated that transgenic mice that overexpress ANP develop less RV hypertension and RV and pulmonary vascular remodeling than nontransgenic control mice after 3 wk of hypobaric hypoxic exposure. A significant positive correlation between plasma ANP level and pulmonary arterial pressure has also been observed in normal human subjects and patients with heart disease (28).
BNP has cardiovascular effects similar to those of ANP, although ANP
and BNP have markedly different affinities for NPR-A and NPR-B
(3). Like ANP, BNP acts through the NPR-A and NPR-B to
enhance intracellular cGMP, relax preconstricted isolated pulmonary arteries, and blunt hypoxic pressor responses in isolated perfused intact rat lungs and rats exposed to hypoxia (6,
20). In contrast to ANP, BNP is mainly produced in cardiac
ventricles and appears to be released over a long time scale in
response to a sustained increase in ventricular afterload
(3, 6, 30, 31). BNP
levels are increased in diseases that affect the ventricle, e.g.,
pulmonary hypertension, myocardial infarction, ventricular dysfunction,
or congestive heart failure (6, 30). It has
been postulated that ANP and BNP participate in a complementary "dual-peptide system" to modulate vascular responses and
intravascular fluid homeostasis and that BNP may act as an emergency
molecule against ventricular overload (3,
31). If this were the case, it might be expected that when
ANP is deficient or lacking, BNP synthesis would be increased and
compensate for the lack of ANP. Our findings that cardiac BNP mRNA
levels were higher in the normoxic ANP(/
) mice than in the normoxic
ANP(+/+) mice and that RV BNP mRNA levels increased in both genotypes
of mice during hypoxic exposure support this hypothesis. A limitation
of the present study is the inability to measure BNP. Mouse BNP antigen
could not be measured because of the unavailability of a selective
antibody for mouse BNP. Future studies in our laboratory are needed to address this deficiency.
A major finding of the present study is that expression of NPR-C is
reduced in the lung, kidney, and brain of all three genotypes of mice
during hypoxia adaptation. A previous study in our laboratory (27) demonstrated that steady-state NPR-C mRNA levels are
selectively downregulated in the lung of hypoxia-adapted rats in
association with increases in circulating levels of ANP. In contrast,
mRNA levels of the guanylate cyclase-coupled NPR-A and NPR-B were
unchanged or increased. Nuclear runoff analysis showed greatly
decreased transcription of the NPR-C gene in the lung of
hypoxia-exposed rats (27). Similarly, Klinger et al.
(17) demonstrated that 125I-ANP binding is
significantly decreased in the lung of hypoxia-adapted rats. The
reduction in tissue NPR-C gene expression may have an important
physiological significance in hypoxia-adapted animals because the
downregulation of NPR-C may contribute to the increase in plasma ANP or
BNP levels observed during hypoxic exposure. Reductions in NPR-C
density on the surface of the ANP and BNP target cells, such as
pulmonary vascular smooth muscle cells, significantly increase ANP and
BNP availability to the NPR-A and NPR-B, neither of which is
downregulated during hypoxic exposure (24), leading to
augmented pulmonary vasodilation and an antiproliferative effect. This
interpretation is supported by the exaggerated vasodilator response of
pulmonary arteries to ANP and BNP under hypoxic conditions demonstrated
in vivo and in vitro by our laboratory (8-11) and other investigators (6, 20, 32).
Jin et al. (8) have shown that administration of
ANP-(423), a selective NPR-C ligand, delays the clearance of ANP from
the circulation and attenuates hypoxia-induced pulmonary hypertension
in the rat. Similarly, Klinger et al. (18) demonstrated
that the hypoxia-associated reduction in tissue concentration of NPR-C
binding sites was associated with a delay in the clearance of ANP from
the circulation.
It has been postulated that the downregulation of NPR-C during hypoxic
exposure is secondary to elevated ANP levels in the circulation and is
mediated by an increase in cGMP production through the activation of
NPR-A or NPR-B and particulate guanylate cyclase (25).
Other mediators that have been shown to regulate ANP receptors in other
systems include growth factors, such as platelet-derived growth factor
and basic fibroblast growth factor, and protein kinase C and protein
kinase A activators (7, 13, 14,
25). Pharmacological treatment of vascular smooth muscle cells with 8-bromo-cGMP (15) or -adrenergic
stimulation (16) has also been reported to downregulate
NPR-C expression. NPR-C in vascular smooth muscle cells is
downregulated by ANP, BNP, and C-type natriuretic peptide (CNP), and
the rank order of potency for this effect is CNP > ANP > BNP, the same as that of the potency of the natriuretic peptides for
cGMP production in vascular smooth muscle cells (15). The
present finding that the cGMP content of lungs and kidneys of both
hypoxic ANP(+/+) and ANP(
/
) mice was increased is consistent with
previous observations by Jin et al. (9) that cGMP levels
are increased in the lung and plasma of hypoxia-adapted rats. However,
the hypothesis that ANP-induced cGMP production is responsible for the
downregulation of NPR-C gene expression during hypoxic exposure is
not supported by our present finding of diminished tissue NPR-C mRNA
and barely detectable plasma ANP levels and cardiac ANP mRNA levels in
hypoxia-adapted ANP(
/
) mice. Our findings that NPR-C mRNA
expression was downregulated in the tissues of hypoxia-adapted
ANP(
/
) mice indicate that hypoxia-induced downregulation of NPR-C
expression is independent of tissue or circulating ANP levels in
hypoxic animals.
Possible mechanisms to explain downregulation of NPR-C in tissues of
hypoxic ANP(/
) mice include compensatory effects of BNP, which is
increased in hypoxia-adapted ANP(
/
) mice, and overexpression of the
NOS-NO-cGMP pathway. NO produced by the pulmonary vascular endothelium
is thought to be generated mainly by eNOS and to modulate pulmonary
vascular responses to a variety of vasoconstrictor stimuli, including
hypoxia (2). The functional significance of eNOS-dependent
NO production in the lung is illustrated by the finding that mice with
targeted disruption of the eNOS gene develop more severe pulmonary
vascular remodeling and hypertension in response to chronic hypoxic
exposure (2). Although constitutively expressed by
vascular endothelial cells, eNOS gene expression is regulated by
various stimuli, including hypoxia (24). Decreased eNOS
expression and NO production have been described in human pulmonary
hypertension (5). However, a more recent study
(24) has found increased NO production and eNOS expression
in the lungs of rats with chronic hypoxia-induced pulmonary
hypertension (24). Our findings of increased eNOS
expression in both hypoxic ANP(+/+) and ANP(
/
) mice are consistent
with the later findings.
Heme oxygenase-1 (HO-1) and CNP could also be involved in the
downregulation of NPR-C observed in hypoxia-adapted ANP(/
) mice.
HO-1 catalyzes the oxidation of heme to generate carbon monoxide and
bilirubin, and carbon monoxide, like NO, stimulates soluble guanylyl
cyclase and increases cellular levels of cGMP (34). CNP
activates NPR-B linked to particulate guanylyl cyclase and generates
cGMP (23). Furthermore, it has been shown that tissue HO-1
(34) and circulating CNP (19) are increased
during chronic hypoxic exposure. The relationships among enhanced
BNP, eNOS, CNP, HO-1, and cGMP production and downregulation of tissue NPR-C in hypoxia-adapted animals in both the presence and absence of
ANP deserve further investigation.
Interestingly, the hearts of ANP(/
) mice under both normoxic and
hypoxic conditions weighed 20-30% more than those of the ANP(+/+)
and ANP(+/
) mice. This inverse relationship of cardiac ANP mRNA and
plasma ANP levels with heart weight is consistent with the previous
demonstration by Klinger et al. (18) that transgenic mice
that overexpress ANP had smaller hearts (30-40% lower heart
weight) than nontransgenic control mice under normoxic conditions.
Taken together, these findings support the interpretation that cardiac
ANP gene expression is involved in the development and/or growth of the
heart. A deficiency of ANP may amplify the humoral or paracrine effects
of various cytokines and/or growth factors on cardiac growth. These
mechanisms merit further investigation.
In summary, this study provides evidence that hypoxic exposure can downregulate tissue NPR-C gene expression independent of changes in ANP levels and expression of NPR-A. Downregulation of NPR-C likely represents an adaptation aimed at reducing ANP clearance from the circulation, thus enhancing the biological effects of ANP and mitigating the severity of hypoxia-induced pulmonary hypertension. The increase in cardiac BNP and pulmonary eNOS expression may provide a compensatory mechanism to modulate the pulmonary vascular response to chronic hypoxia.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-44195, HL-50147, HL-45990, and HL-07457.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y.-F. Chen, 1008 Zeigler Research Bldg., Univ. of Alabama at Birmingham, UAB Station, Birmingham, AL 35294 (E-mail: YFChen{at}uab.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. §1734 solely to indicate this fact.
Received 30 November 1999; accepted in final form 3 March 2000.
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