mRNA expression of novel CGRP1 receptors and
their activity-modifying proteins in hypoxic rat lung
Xin
Qing,
John
Svaren, and
Ingegerd M.
Keith
Department of Comparative Biosciences, School of Veterinary
Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
Calcitonin gene-related peptide
(CGRP) is a potent vasodilator. Our group has reported that exogenous
CGRP may prevent or reverse hypoxic pulmonary hypertension in rats. The
vasodilatory action of CGRP is mediated primarily by CGRP1 receptors.
The calcitonin receptor-like receptor (CRLR) and the orphan receptor
RDC-1 have been proposed as CGRP1 receptors, and recent
evidence suggests that CRLR can function as either a CGRP1 receptor or
an adrenomedullin (ADM) receptor. Receptor activity-modifying proteins
(RAMPs) determine the ligand specificity of CRLR: coexpression of CRLR
and RAMP1 results in a CGRP1 receptor, whereas coexpression of CRLR and RAMP2 or -3 results in an ADM receptor. We used qualitative,
semiquantitative, and real-time quantitative RT-PCR to detect and
quantitate the relative expression of these agents in the lungs of rats
exposed to normoxia (n = 3) and 1 and 2 wk of chronic
hypobaric hypoxia (barometric pressure 380 mmHg, equivalent to an
inspired O2 level of 10%; n = 3/time
period). Our results show upregulation of RDC-1, RAMP1, and RAMP3 mRNAs
in hypoxic rat lung and no change in CRLR and RAMP2 mRNAs. These
findings support a functional role for CGRP and ADM receptors in
regulating the adult pulmonary circulation.
semiquantitative reverse transcription-polymerase chain reaction; real-time quantitative reverse transcription-polymerase chain reaction; calcitonin receptor-like receptor; RDC-1; receptor activity-modifying
protein 1; receptor activity-modifying protein 2; receptor
activity-modifying protein 3
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INTRODUCTION |
CALCITONIN GENE-RELATED
PEPTIDE (CGRP) is a 37-amino acid neuropeptide generated from an
alternatively spliced transcript of the calcitonin gene (2,
28). CGRP belongs to a superfamily of related peptides that
consists of calcitonin, CGRP, islet amyloid polypeptide, and
adrenomedullin (ADM). CGRP is predominantly expressed in the central
and peripheral nervous systems (28, 31) and has diverse
biological effects. In the lung, CGRP is abundant in neuroendocrine
cells of the airway epithelium and is also found in sensory nerve
fibers and intrapulmonary parasympathetic neurons (11,
13). CGRP is the most potent endogenous vasodilatory peptide
discovered so far (38). In addition, our results
showed that exogenous CGRP prevents the development of hypoxic
pulmonary hypertension (HPH) and reverses existing HPH in rats
(12, 32).
CGRP receptors have been characterized in a variety of tissues, mainly
through functional binding assays. Specific CGRP binding sites have
been demonstrated widely in both central and peripheral tissues
including lung (19, 34). Several pharmacological and biochemical studies provide evidence for receptor heterogeneity, and at least three classes of CGRP receptors have been
identified through studies with CGRP8-37 and
[acetamidomethylcysteine2,7]
-CGRP
([Cys(ACM)2,7]
-CGRP) (3,
27). The CGRP1 subtype is highly sensitive to the
antagonistic properties of COOH-terminal fragments of CGRP, whereas CGRP2 possesses high affinity for the linear analog
[Cys(ACM)2,7]
-CGRP. Receptors that respond
to both
-CGRP and salmon (but not human) calcitonin with
high affinity are grouped together as a third type. The
vasodilatory responses to CGRP are mediated primarily by CGRP1
receptors because the action could be potently antagonized by
CGRP8-37 (35).
To date, cloning studies and functional assays have claimed several
receptors to be CGRP1 receptors. First, the canine orphan receptor RDC-1 was originally cloned from dog thyroid cDNA by using the
PCR with degenerate primers that correspond to consensus sequences of
transmembrane domains 3 and 6 of other G protein-linked receptors
(17), and RDC-1 has been identified as a CGRP1 receptor (10). Second, a calcitonin receptor-like sequence
[calcitonin receptor-like receptor (CRLR)] was initially cloned in
rat lung (24), and its human homolog has been reported to
be a CGRP1 receptor (1). Most recently, receptor
activity-modifying proteins (RAMPs) were cloned; their biological
functions are transporting CRLR to the cell membrane, determining
its glycosylation state, and defining its pharmacology
(22). Coexpression of RAMP1 and CRLR was found to
create novel CGRP1 receptors in cell lines, whereas RAMP2 or RAMP3
presents CRLR at the cell surface as an ADM receptor (6,
22).
To better understand the mechanisms of the pulmonary vasodilatory
activity of CGRP in HPH, we examined the mRNA expression of these
CGRP1/ADM receptor-related genes in rat lungs during normoxia and
chronic hypoxia by use of qualitative, semiquantitative, and real-time
quantitative RT-PCR.
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MATERIALS AND METHODS |
Animal treatments.
Adult male Sasco Sprague-Dawley rats weighing 175-200 g were
randomly assigned to groups (3 rats/group) in which they were exposed
to normoxia or 1 or 2 wk of hypoxia. Each rat was placed unrestrained
in a separate cage in ambient room air (normoxia) or in a hypobaric
hypoxic chamber with a barometric pressure of 380 mmHg, equivalent to
an inspired O2 level of 10%, ambient humidity, and ambient
room air CO2 level (Biotron, University of
Wisconsin-Madison). The 1-wk hypoxia-treated rats were housed in room
air (normobaric normoxia) for 1 wk before hypoxic exposure to ensure
that all rats were the same age at the end of the study. The
hypoxic chamber was opened twice a week to clean cages and to replenish
food and water. The maximum duration of time that the hypoxic chamber
was opened each time was 30 min. The normoxic rats were housed under similar but normobaric normoxic conditions. Food and water were given
ad libitum. The rats were housed in American Association for
Accreditation of Laboratory Animal Care (AAALAC)-certified facilities
and treated humanely according to protocols approved by the Animal
Resources Center and the Graduate School of the University of
Wisconsin-Madison.
At the end of the hypoxic exposure, the rats were transferred to the
laboratory and placed in a normobaric chamber under a continuous flow
of 10% O2 in N2 before tissue was collected.
Each rat was removed from the chamber and deeply anesthetized with pentobarbital sodium (80 mg/kg ip). The superior mesenteric artery was
cut to drain the blood, and the lungs were rapidly isolated and
immediately flash frozen in liquid nitrogen and stored at
70°C.
Total RNA extraction and DNase digestion.
Total RNA was isolated from 30 mg of the tissue at the peripheral
region of the lungs with the use of an RNeasy Minikit (QIAGEN, Valencia, CA). Before RT-PCR was performed, samples were pretreated with DNase (RQ1 RNase-free DNase; Promega, Madison, WI) according to
the instructions provided by the manufacturer.
Oligonucleotides (primers) and RT-PCR.
The primers for CRLR, RDC-1, RAMP1, and RAMP3 were designed based
on published cDNA sequences of rat CRLR, rat RDC-1, rat RAMP1, and rat
RAMP3 (GenBank accession nos. L27487, AJ010828, AB028933, and AB030944,
respectively). Primer sequences were as follows: CRLR upstream (5'-AAC
AAC AGC ACG CAT GAG AA-3', corresponding to nucleotide residues
1060-1079); CRLR downstream (5'-ACC CCC AGC CAA GAA AAT AA-3',
corresponding to nucleotide residues 1462-1443); RDC-1 upstream
(5'-GTG CAG CAT AAC CAG TGG CC-3', corresponding to nucleotide residues
396-415); RDC-1 downstream (5'-AGC AAA ACC CAA GAT GAC GGA-3',
corresponding to nucleotide residues 749-729); RAMP1 upstream
(5'-ACT GGG GAA AGA CCA TAG GGA G-3', corresponding to nucleotide
residues 3-24); RAMP1 downstream (5'-AGT CAT GAG CAG TGT GAC CGT
A-3', corresponding to nucleotide residues 232-211); RAMP3
upstream (5'-GTA TGC GGT TGC AAT GAG ACA-3', corresponding to
nucleotide residues 81-101); and RAMP3 downstream (5'-TCT TCT AGC
TTG CCA GGC AC-3', corresponding to nucleotide residues 496-477).
The expected lengths of the RT-PCR products were 403 bp for CRLR, 354 bp for RDC-1, 230 bp for RAMP1, and 416 bp for RAMP3.
Appropriate upstream and downstream primers were designed on the basis
of the cDNA sequence of human RAMP2 (GenBank accession no. AJ001015)
because only the human sequence was available for RAMP2 at the time
this study was undertaken. These were upstream primer 5'-CTG GGC GCT
GTC CTG AAT C-3', corresponding to nucleotides 159-177 and
downstream primer 5'-GAG AAG GTG GGC TGC ACC A-3', corresponding to
nucleotides 484-466. According to the human mRNA sequence of
RAMP2, the expected length of the amplified DNA fragment should be 326 bp.
DNase-pretreated total RNA was reverse transcribed and amplified by PCR
with a one-step RT-PCR kit (Access RT-PCR system; Promega) in a total
volume of 50 µl with 0.2 µg of total RNA and 1 µM each upstream
and downstream specific primer corresponding to the sequence of
interest. Reactions were incubated at 48°C for 45 min, heated to
94°C for 2 min, and cycled according to the following parameters:
94°C for 30 s (denaturation), 55°C for 1 min (annealing), and
68°C for 2 min (extension) for a total of 40 cycles. A 7-min final
extension at 68°C was performed after 40 cycles. Each RT-PCR was
repeated at least once to ascertain reproducibility. Negative control,
positive control, and "no-RT" control reactions were performed.
Negative controls were run in which the RNA templates were replaced by
nuclease-free water in the reactions. For the positive control
reaction, we used 2.5 amol or 1 × 106 copies of the
supplied positive control RNA (1.2-kb kanamycin resistance gene mRNA)
with carrier (Escherichia coli rRNA) and the upstream and
downstream control primers (final concentration 1 µM in 50 µl). The amplification product obtained from the positive control reaction should be 323 bp long and an ~220-bp amplification product may be observed, which arises from the amplification of a
sequence in the carrier RNA. For a no-RT control, nuclease-free water was used in place of the reverse transcriptase. In the RAMP2 study, we used lung tissue samples from normoxic and 2-wk
hypoxia-treated rats only, and the RT-PCRs were performed as above.
Ten microliters from each RT-PCR product were loaded on a 1.5% agarose
gel containing 0.5 µg/ml of ethidium bromide and separated by
electrophoresis. DNA ladders were run in the outside lane to confirm
the molecular sizes of the amplified products. To verify the identity
of the RT-PCR products, the major products were excised from the
agarose gels and purified with the Wizard PCR Preps DNA purification
system (Promega) and then subjected to sequence analysis (Biotechnology
Center, University of Wisconsin-Madison). RT-PCR products were directly
purified and sequenced by the Biotechnology Center if agarose gel
electrophoresis showed a single bright band.
Semiquantitative RT-PCR.
To evaluate these CGRP1 and ADM receptor mRNA expression levels
semiquantitatively, 4 µg of DNase-pretreated total RNA were reverse
transcribed with random primers in 80-µl reactions with a
commercially available kit (Reverse Transcription System, Promega). Reactions were incubated at room temperature for 10 min, at 42°C for
1 h, at 99°C for 5 min, and at 0-5°C for 5 min and then
stored at
20°C until use. PCR amplification of each gene product
was carried out in parallel 25-µl reactions using PCR Core Systems I
(Promega) with 1 µM of the aforementioned specific forward and reverse primers (the primer concentration for RAMP3 amplification was
0.2 µM) and 1 µl of RT product (2 µl of RT product was used for
RAMP3). The mixed samples were placed at 95°C for 2 min and then
cycled as follows: 95°C for 30 s, 55°C for 1 min, and 72°C for 2 min. A final extension step of 5 min at 72°C was carried out.
The 18S rRNA was used as an internal control for sample loading. The
upstream primer (5'-CCG CAG CTA GGA ATA ATG GA-3') and the downstream
primer (5'-GAG TCA AAT TAA GCC GCA GG-3') designed for 18S rRNA yielded
a 400-bp product after gel electrophoresis. Amplifications were carried
out for 28 cycles for CRLR, 29 cycles for RDC-1, 32 cycles for RAMP1,
27 cycles for RAMP2, 35 cycles for RAMP3, and 15 cycles for 18S rRNA.
These cycle numbers were determined empirically by sampling the PCR
amplification every three cycles between cycles 12 and
45 and selecting the approximate midpoint of linear
amplification. Because of the abundance of 18S rRNA, it was not
possible to amplify the cDNA of selected genes and 18S in the same
reaction tube within the linear range. Therefore, they were amplified
in separate reaction tubes. Ten microliters from each PCR amplification
were loaded onto 1.5% agarose gels with 0.5 µg/ml of ethidium
bromide and subjected to electrophoresis.
Real-time quantitative RT-PCR.
Total RNA was digested by RQ1 RNase-free DNase, and first-strand cDNA
was synthesized as described in Semiquantitative
RT-PCR. Real-time quantitative RT-PCR for CRLR, RDC-1,
and RAMP1, -2, and -3 was performed based on SYBR Green I assays and/or
the fluorogenic 5'-nuclease assays by a GeneAmp 5700 sequence-detection
system (PE Biosystems, Foster City, CA), with 18S rRNA as an endogenous control to standardize the amount of sample RNA added to a reaction. Primers and probes were designed by Dr. Kathyrn Becker (PE Biosystems) or with the use of Primer Express software (PE Biosystems). Sequences for all primers and probes used in these analyses, except those for
18S, are listed in Table 1. The primers
and probes for 18S (TaqMan rRNA control reagents) and all reagents for
PCR were purchased from PE Biosystems.
SYBR Green I assays were performed on all selected genes with SYBR
Green PCR Master Mix (PE Biosystems). Each tube contained a total
volume of 25 µl and the following: 1× SYBR Green PCR Master Mix, 150 nM forward and reverse primers (50 nM for 18S rRNA), and first-strand
cDNA synthesized from 1 ng (CRLR and 18S), 2 ng (RAMP2 and RAMP3), 10 ng (RDC-1), or 20 ng (RAMP1) of total RNA. The thermal cycling
parameters for PCR were 10 min at 95°C and 40 cycles for 15 s at
95°C and 1 min at 60°C. All experiments were performed in
triplicate. Dissociation curves were run immediately after the
real-time PCR run, and no nonspecific amplification was detected in
this study.
Data were analyzed with the relative standard curve method. Standard
curves of the genes of interest and 18S rRNA were prepared with three
1:2 dilutions (four points, eightfold range) of first-strand cDNA from
one of the samples that was expected to have the highest amount of mRNA
of the gene of interest. For each reaction tube, the amount of target
or endogenous reference was determined from the standard curves. The
mean amount of each sample was calculated from the triplicate
data and was normalized by division by the mean quantity of 18S rRNA
for the same sample. The mean and SE of each treated group were
calculated from the normalized value for each rat in that group. The
mean value of the normoxic group was arbitrarily set at 1.0. Each of
the normalized values was divided by the mean value of the normoxic
group to generate the relative expression levels. Tests for
significance were performed with one-way ANOVA followed by
Student-Newman-Keuls tests, with P < 0.05 taken as significant.
Because of the small amount of RAMP1 mRNA in lung tissue
(22), real-time quantitative RT-PCR was repeated for RAMP1
with the fluorogenic 5'-nuclease assays, which confer higher
specificity. First-strand cDNA reverse transcribed from 20 ng of total
RNA was used to set up 25-µl real-time quantitative PCRs that
consisted of 1× TaqMan Universal PCR Master Mix, 900 nM forward and
reverse primers for the gene of interest or 50 nM primers for 18S rRNA as the endogenous control, and 200 nM TaqMan probe. PCR amplification was carried out with the following temperature profile: 2 min at
50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Agarose gel electrophoretic analysis was used to verify that the amplified product corresponded to the size predicted for the
amplified fragment. Assays were performed in triplicate, and data were
analyzed in the same way as in the SYBR Green I assays.
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RESULTS |
RT-PCR.
Gel electrophoresis of the RT-PCR products with CRLR primers
revealed a band of ~400 bp in each of the three groups. It
corresponded in size to the RT-PCR product expected from rat CRLR mRNA
(403 bp; Fig. 1). Sequence analysis of
this product showed 100% homology with rat CRLR cDNA, indicating that
these RT-PCR products were amplified from rat CRLR mRNA.

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Fig. 1.
Electrophoresis of RT-PCR products (10 µl of each)
corresponding to mRNA encoding the rat calcitonin receptor-like
receptor (CRLR). Lane M, 100-bp DNA ladder as size marker;
lane 1, positive control; lane 2, negative
control; lanes 3, 5, and 7,
"no-RT" controls; lane 4, normoxic lung; lane
6, 1-wk hypoxic lung; lane 8, 2-wk hypoxic lung.
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By RT-PCR, the primers for RDC-1 amplified a product of the expected
size (354 bp) that corresponded to rat mRNA encoding RDC-1 from each of
the three samples (Fig. 2). Sequence
analysis of this product indicated 100% homology with rat RDC-1 cDNA.
Similarly, the primers for RAMP1 amplified a 230-bp product that
corresponded in size to the RT-PCR product expected from rat RAMP1 mRNA
(Fig. 3). Sequence analysis performed on
the product showed 100% homology with rat RAMP1 cDNA.

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Fig. 2.
Electrophoresis of RT-PCR products (10 µl of each)
corresponding to mRNA encoding the rat RDC-1. Lane M, 100-bp
DNA ladder as size marker; lane 1, negative control;
lanes 2, 4, and 6, no-RT controls; lane
3, normoxic lung; lane 5, 1-wk hypoxic lung; lane
7, 2-wk hypoxic lung.
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Fig. 3.
Electrophoresis of RT-PCR products (10 µl of each)
corresponding to mRNA encoding the rat receptor activity-modifying
protein (RAMP) 1. Lane M, 100-bp DNA ladder as size marker;
lane 1, negative control; lanes 2, 4, and
6, no-RT controls; lane 3, normoxic lung;
lane 5, 1-wk hypoxic lung; lane 7, 2-wk hypoxic
lung.
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The RAMP2 primers amplified five bands from both normoxic and hypoxic
total RNA samples. Their sizes were ~500, 375, 325, 275, and 225 bp
(Fig. 4). Sequence analysis was performed
on the five products and revealed that the
325-bp band had a high
degree of identity with human and mouse RAMP2 cDNA (Fig.
5), suggesting that this is the rat
counterpart of RAMP2. The sequence data reported in Fig. 5 contain only
186 bp to avoid including internal unsequenced spacers. This
sequence was submitted to the GenBank/European Molecular Biology
Laboratory database (accession no. AF162778). A new RAMP2 primer
set was designed based on the sequence discovered (Fig. 5), and RT-PCRs
were repeated with the new primers using methods described in
MATERIALS AND METHODS. A single band of 175 bp was
found in all samples (Fig. 6). Sequence
analysis confirmed its identity. The new primer set was used for the
semiquantitative RT-PCR of RAMP2 described in Semiquantitative
and real-time quantitative RT-PCR to compare the rat lung
RAMP2 expression levels during normoxia or after chronic hypoxia.

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Fig. 4.
Electrophoresis of RT-PCR products (10 µl of each)
corresponding to mRNA encoding the rat RAMP2. Lane M, 100-bp
DNA ladder as size marker; lane 1, negative control;
lanes 2 and 4, no-RT controls; lane 3,
normoxic lung; lane 5, 2-wk hypoxic lung. Short arrows at
right, RT-PCR products; long arrow at right,
RAMP2.
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Fig. 5.
Partial cDNA sequence for rat RAMP2 and the alignment result with
published mouse and human homologs. It shares 91 and 80% homology with
mouse and human RAMP2 cDNA sequences, respectively. Partial cDNA
sequence for rat RAMP2 has been submitted to the GenBank/European
Molecular Biology Laboratory database with accession no. AF162778. A
new primer set was designed based on the sequence and was used to
repeat the RT-PCR of RAMP2. It was also used for semiquantitative
RT-PCR. Positions of the new primer set are in boldface. Positions of
the primer pair for real-time quantitative RT-PCR are underlined.
Arrows, amplification direction of 5' 3'. *Nucleotides that are
identical in all 3 species.
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Fig. 6.
RT-PCR for rat RAMP2 was repeated with the new primer set
designed from the partial cDNA sequence we discovered (shown in Fig.
5). Sequences of the primers are 5'-AGA CTT CCA TGG ACT CTG TCA AG-3'
(forward) and 5'-GAG CAG TTG GCA AAG TGT ATC A-3' (reverse).
Electrophoresis of the RT-PCR products (10 µl of each) showed a
single bright band. Lane M, 100-bp DNA ladder as size
marker; lane 1, negative control; lanes 2,
4, and 6, no-RT controls; lane 3,
normoxic lung; lane 5, 1-wk hypoxic lung; lane 7,
2-wk hypoxic lung.
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A band of 416 bp was amplified from each of the three samples with
RAMP3 forward and reverse primers (Fig.
7). Sequence analysis performed on the
product indicated that it was amplified from rat RAMP3.

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Fig. 7.
Electrophoresis of RT-PCR products (10 µl of each)
corresponding to mRNA encoding the rat RAMP3. Lane M, 100-bp
DNA ladder as size marker; lane 1, negative control;
lanes 2, 4, and 6, no-RT controls; lane
3, normoxic lung; lane 5, 1-wk hypoxic lung; lane
7, 2-wk hypoxic lung.
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No band was detected in negative controls and in no-RT controls,
whereas a strong band of 323 bp and a weaker band of ~220 bp were
observed in the positive control as expected.
Semiquantitative and real-time quantitative RT-PCR.
After completion of the initial qualitative analysis that provided
information on the existence of transcripts, it was necessary to
quantify the levels of CRLR, RDC-1, and RAMP1, -2, and -3 transcripts in normoxic and hypoxic rat lungs to further understand these receptors
and RAMPs during normoxia and hypoxia. Semiquantitative RT-PCR (see
Fig. 8) demonstrated that RAMP1 and RDC-1
mRNA expression were increased in the lungs of 1-wk and 2-wk
hypoxia-treated rats when compared with the results in normoxic rats.
In contrast, no significant change in mRNA expression was found for
CRLR or RAMP2. Moreover, there was a trend of increasing RAMP3 mRNA
with hypoxia, although the change was inconsistent because of the high variance of RAMP3 mRNA expression between individual samples. The 18S
rRNA was used as the internal control for cDNA quantity and quality and
was compared across all normoxic and hypoxic lungs. Preliminary
experiments demonstrated that 18S rRNA did not change with 2 wk of
exposure to the level of hypoxia used in this study, whereas other
housekeeping genes, including glyceraldehyde-3-phosphate dehydrogenase and
-actin, increased (data not shown). In
this study, approximately equivalent amounts of 18S rRNA were shown in
each sample.

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Fig. 8.
Semiquantitative RT-PCR analysis of CRLR, RDC-1, RAMP1, RAMP2, and
RAMP3 mRNA expression after normoxic and 1- and 2-wk hypoxic
treatments. Each lane represents an individual rat. 18S rRNA was
amplified as a control to normalize sample loading. The experiment
shown is representative of at least 3 repetitions.
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Quantitative estimates of the relative abundance of CRLR, RDC-1, and
RAMP1, -2, and -3 mRNAs were also obtained with real-time quantitative
RT-PCR after normoxic or hypoxic treatments (Fig. 9). With the use of
SYBR Green I assays, three- and twofold upregulation of RAMP1 mRNA was
detected after 1- and 2-wk hypoxic treatments, respectively, whereas
CRLR and RAMP2 mRNA expression were unchanged. RAMP3 mRNA was
significantly increased by hypoxia, and there was a highly significant
linear correlation between the amounts of mRNA and time spent in
hypoxia (r = 0.904; P = 0.000) with
individual normalized means in the data set (Fig.
10). Moreover, RDC-1 mRNA expression
was upregulated twofold after hypoxia, although the change was not
significant (P = 0.10 by ANOVA). By using a prelabeled fluorescent probe (the fluorogenic 5'-nuclease assays), similar results
with a very small SE were obtained for RAMP1, which suggests higher
sensitivity. The 18S rRNA, which was used as an internal control, was
unchanged by hypoxia according to both the fluorogenic 5'-nuclease and
SYBR Green I assays.

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Fig. 9.
Real-time quantitative RT-PCR analysis of CRLR, RDC-1,
RAMP1, RAMP2, and RAMP3 mRNA expression in normoxic and 1- (1W) and
2-wk (2W) hypoxic rat lungs. SYBR Green I assays were performed on all
selected genes while fluorogenic 5'-nuclease assays were used to repeat
the real-time quantitation of RAMP1 (RAMP1-Probe) because of the small
amount of RAMP1 mRNA in rat lung. Amount of mRNA is expressed relative
to that in normoxic rat lung (n = 3 rats/group). Values
are means ± SE. There were significant increases for RAMP1 (by
both SYBR Green I and 5'-fluorogenic nuclease assays) and RAMP3 mRNAs
(P < 0.05 by ANOVA). *Significant difference from
normoxic rats, P < 0.05 by Student-Newman-Keuls test.
#Significant difference from 1-wk hypoxic rats,
P < 0.05 by Student-Newman-Keuls test.
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Fig. 10.
Correlation between relative quantity of RAMP3 mRNA in
rat lung and the time that the rat was treated with hypoxia
(r = 0.904; P = 0.000). Points
represent normalized means of triplicate samples for individual rats.
Mean value of the normoxic group was arbitrarily set at 1.0.
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DISCUSSION |
CGRP is well known for its vasodilatory action via CGRP1 receptors
located on vascular endothelium and smooth muscle (18, 36). Reversal of HPH by exogenous CGRP has been previously
reported in rats (32), and the protective role of
endogenous CGRP in HPH has been shown (33). Although
specific CGRP binding sites have been reported in the lung, little is
known about the expression of these novel CGRP1 receptors. Our present
study revealed that CRLR, RDC-1, and RAMP1 mRNAs were expressed in the
rat lungs in normoxia and chronic hypoxia. We were not surprised to
detect the expression of CRLR mRNA in the rat lungs because previous studies (1, 5, 24) have demonstrated a high level of CRLR mRNA expression in both human and rat lungs. In the original study of
RDC-1, Northern blot analysis identified the heart and kidney as the
major expression sites of RDC-1 mRNA, with weaker signals in the brain
and spleen and no signal in the stomach, liver, lung, or salivary
glands (10, 17). To our knowledge, the present study is
the first demonstration of RDC-1 mRNA expression in the lung. This
discovery is probably a result of the higher sensitivity of the RT-PCR
method compared with Northern blot analysis.
RAMP1 mRNA was not detected in human lung by Northern blot analysis
(6, 22). However, RAMP1 mRNA was expected to be expressed in lung at a low level (22). This, therefore, led us to
investigate its expression in rat lung by RT-PCR. Our study clearly
indicates that RAMP1 is expressed in rat lung. According to the RAMP
theory, CRLR can function as either a CGRP1 receptor or an ADM
receptor, with receptor specificity determined by a RAMP family; RAMP1
transports CRLR to the plasma membrane as a CGRP1 receptor, whereas
RAMP2 and RAMP3 present CRLR at the cell surface as an ADM receptor (6, 22). The discovery of RAMP1 expression in lung
supports the RAMP theory and may explain the reversal of HPH by
exogenous CGRP (32).
ADM is structurally and functionally related to CGRP. Human ADM is a
52-amino acid polypeptide (14), and rat ADM has 50 residues (29). ADM shares slight amino acid sequence
similarity with CGRP (24% in humans and 27% in rats). However, both
ADM and CGRP have a six-residue ring structure formed by an
intramolecular disulfide bridge between two cysteine residues, and both
also have the COOH-terminal amide structure. These important structural similarities contribute to overlapping biological effects between CGRP
and ADM. The main physiological effect of ADM reported thus far is its
vasodilatory property, which is second only to that of CGRP
(37). In certain vascular beds, the vasodilator action of
ADM is thought to be mediated at least in part by CGRP1 receptors because CGRP8-37 selectively inhibits the vasodilator responses of
ADM (4, 16, 25). However, it is also possible that ADM acts via its own receptor in some vascular beds (7, 9,
23). These observations seem to be consistent with the RAMP
hypothesis (30). ADM can reduce hypoxia-induced pulmonary
hypertension in the rat lung, and part of the response to ADM is
thought to be mediated by CGRP1 receptors (39). However,
the role of ADM is believed to be most important in the late fetal
period (21).
By Northern blot analysis, ADM mRNA was found to be highly expressed in
several tissues, including adrenal medulla, heart, lung, and kidney, in
both rats and humans (15, 29), and immunoreactive ADM has
also been detected in the plasma (8). Specific ADM binding
sites have been reported in many tissues, including heart and lung
(26). Our present RT-PCR analysis clearly demonstrated that RAMP2 and -3 mRNAs are expressed in rat lungs both in normoxia and
after hypoxic treatment. These data are consistent with the localization of specific ADM binding sites and further support the RAMP
theory. Interestingly, RAMP3 is expressed strongly in the human lung by
Northern blot analysis (22), whereas it is only just
detectable by RT-PCR in rat lung in the present study, which suggests
that RAMP3 may not be very important in rat lung.
Using semiquantitative RT-PCR, we found upregulation of RDC-1, RAMP1,
and RAMP3. To obtain more detailed information, we performed real-time
quantitative RT-PCR assays that showed a higher sensitivity than our
conventional semiquantitative RT-PCR. Results of real-time quantitative
RT-PCR were in agreement with those obtained from semiquantitative
RT-PCR. A two- to threefold increase in RAMP1 mRNA was detected after
chronic hypoxia by both the fluorogenic 5'-nuclease assays and SYBR
Green I assays. However, the fluorogenic 5'-nuclease assays tend to be
somewhat more sensitive for detection of low amounts of target such as
RAMP1 mRNA because the use of fluorogenic probes avoids the
complications caused by detection of nonspecific amplification, which
is more of a problem at low target levels. Although use of the
fluorogenic probe may provide higher specificity and sensitivity, we
decided to use SYBR Green I assays in the present study because of the
high cost of the fluorescently labeled TaqMan probes.
Radioligand studies indicated that CGRP binding capacity in the
vascular endothelium was significantly elevated after 5 days of hypoxia
(means ± SE: control 4.6 ± 0.4 vs. hypoxic 16.6 ± 2.4 amol/mm2) (20). This may reflect less
endogenous binding (and more free receptors) induced by the suppression
of pulmonary CGRP release (32). Alternatively, receptor
upregulation could contribute to the increased radioligand binding. Our
present semiquantitative and real-time quantitative RT-PCRs
revealed the regulation of the expression of these receptors after
hypoxic exposure. Both RAMP1 and RDC-1 mRNA expression were
significantly increased, which supported the CGRP1 receptor
upregulation hypothesis. The increase in RAMP1 mRNA and unchanged CRLR
mRNA in hypoxia suggest that CGRP reception at the CRLR is primarily
upregulated by RAMP1.
To better understand these related receptors and their roles during
normoxia and hypoxia, knowledge of their precise localization in lung
tissue would be useful. However, it is impossible to localize most of
these mRNAs by in situ hybridization only, due to the small amounts of
RDC-1, RAMP1, and RAMP3 mRNAs in the rat lung (10, 17,
22).
In summary, our present study demonstrates that CRLR, RDC-1, RAMP1,
RAMP2, and RAMP3 mRNAs are expressed in normoxic and chronic hypoxic
rat lungs. It supports previous reports showing specific CGRP and ADM
binding sites in the lung. Moreover, upregulation of RAMP1, RAMP3, and
RDC-1 mRNA expression with hypoxia is revealed. These findings, as well
as the partial cDNA sequence of rat RAMP2, should benefit further
studies on these CGRP1 and ADM receptors.
 |
ACKNOWLEDGEMENTS |
The authors thank Dr. Junchao Cai (Dept. of Surgery, Univ. of
Wisconsin-Madison) for technical assistance and the Biotechnology Center (Univ. of Wisconsin-Madison) for primer synthesis and sequence analysis.
 |
FOOTNOTES |
This work was supported by an "Innovative Proposal" grant from the
Univ. of Wisconsin-Madison School of Veterinary Medicine.
Address for reprint requests and other correspondence: I. M. Keith, Dept. of Comparative Biosciences, Univ. of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Dr. West, Madison, WI 53706 (E-mail: keithi{at}svm.vetmed.wisc.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 April 2000; accepted in final form 19 October 2000.
 |
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