AJP - Lung Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 280: L547-L554, 2001;
1040-0605/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qing, X.
Right arrow Articles by Keith, I. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qing, X.
Right arrow Articles by Keith, I. M.
Vol. 280, Issue 3, L547-L554, March 2001

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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]alpha -CGRP ([Cys(ACM)2,7]alpha -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]alpha -CGRP. Receptors that respond to both alpha -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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Primers and probes for real-time quantitative RT-PCR

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (30K):
[in this window]
[in a new window]
  		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.

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.


View larger version (17K):
[in this window]
[in a new window]
  		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.



View larger version (25K):
[in this window]
[in a new window]
  		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.

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 approx 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.


View larger version (55K):
[in this window]
[in a new window]
  		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.



View larger version (36K):
[in this window]
[in a new window]
  		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'right-arrow3'. *Nucleotides that are identical in all 3 species.



View larger version (32K):
[in this window]
[in a new window]
  		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.

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.


View larger version (18K):
[in this window]
[in a new window]
  		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.

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 beta -actin, increased (data not shown). In this study, approximately equivalent amounts of 18S rRNA were shown in each sample.


View larger version (67K):
[in this window]
[in a new window]
  		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.

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.


View larger version (33K):
[in this window]
[in a new window]
  		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.



View larger version (12K):
[in this window]
[in a new window]
  		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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aiyar, N, Rand K, Elshourbagy NA, Zeng Z, Adamou JE, Bergsma DJ, and Li Y. A cDNA encoding the calcitonin gene-related peptide type 1 receptor. J Biol Chem 271: 11325-11329, 1996[Abstract/Free Full Text].

2.   Amara, SG, Jonas V, Rosenfeld MG, Ong ES, and Evans RM. Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 298: 240-244, 1982[Medline].

3.   Dennis, T, Fournier A, Cadieux A, Pomerleau F, Jolicoeur FB, St. Pierre S, and Quirion R. hCGRP8-37, a calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery. J Pharmacol Exp Ther 254: 123-128, 1990[Abstract/Free Full Text].

4.   Entzeroth, M, Doods HN, Wieland HA, and Wienen W. Adrenomedullin mediates vasodilation via CGRP1 receptors. Life Sci 56: L19-L25, 1995.

5.   Fluhmann, B, Muff R, Hunziker W, Fischer JA, and Born W. A human orphan calcitonin receptor-like structure. Biochem Biophys Res Commun 206: 341-347, 1995[ISI][Medline].

6.   Fraser, NJ, Wise A, Brown J, McLatchie LM, Main MJ, and Foord SM. The amino terminus of receptor activity-modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor. Mol Pharmacol 55: 1054-1059, 1999[Abstract/Free Full Text].

7.   Hjelmqvist, H, Keil R, Mathai M, Hubschle T, and Gerstberger R. Vasodilation and glomerular binding of adrenomedullin in rabbit kidney are not CGRP receptor mediated. Am J Physiol Regulatory Integrative Comp Physiol 273: R716-R724, 1997[Abstract/Free Full Text].

8.   Ichiki, Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, and Eto T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 338: 6-10, 1994[ISI][Medline].

9.   Kapas, S, Catt KJ, and Clark AJ. Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J Biol Chem 270: 25344-25347, 1995[Abstract/Free Full Text].

10.   Kapas, S, and Clark AJ. Identification of an orphan receptor gene as a type 1 calcitonin gene-related peptide receptor. Biochem Biophys Res Commun 217: 832-838, 1995[ISI][Medline].

11.   Keith, IM, and Ekman R. Calcitonin gene-related peptide in hamster lung and its coexistence with serotonin: a chemical and immunocytochemical study. Regul Pept 22: 315-323, 1988[ISI][Medline].

12.   Keith, IM, Ekman R, Kraiczi H, and Sandler R. CGRP treatment of chronic hypoxia-induced pulmonary hypertension in rats (Abstract). FASEB J 9: A601, 1995.

13.   Keith, IM, Pelto-Huikko M, Schalling M, and Hökfelt T. Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia. Histochemistry 96: 311-315, 1991[ISI][Medline].

14.   Kitamura, K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, and Eto T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192: 553-560, 1993[ISI][Medline].

15.   Kitamura, K, Sakata J, Kangawa K, Kojima M, Matsuo H, and Eto T. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem Biophys Res Commun 194: 720-725, 1993[ISI][Medline]. [Corrigenda. Biochem Biophys Res Commun 202: July 1994, p. 643.]

16.   Lang, MG, Paterno R, Faraci FM, and Heistad DD. Mechanisms of adrenomedullin-induced dilatation of cerebral arterioles. Stroke 28: 181-185, 1997[Abstract/Free Full Text].

17.   Libert, F, Parmentier M, Lefort A, Dinsart C, Van Sande J, Maenhaut C, Simons MJ, Dumont JE, and Vassart G. Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 244: 569-572, 1989[Abstract/Free Full Text].

18.   Luu, TN, Dashwood MR, Chester AH, Muddle JR, and Yacoub MH. Calcitonin gene-related peptide in healthy and atheromatous human epicardial coronary arteries. Function and receptor characterization. J Vasc Res 32: 93-99, 1995[ISI][Medline].

19.   Mak, JC, and Barnes PJ. Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung. Peptides 9: 957-963, 1988[ISI][Medline].

20.   Mannan, MM, Springall DR, Enard C, Moradoghli-Haftvani A, Eddahibi S, Adnot S, and Polak JM. Decreased endothelium-dependent pulmonary vasodilator effect of calcitonin gene-related peptide in hypoxic rats contrasts with increased binding sites. Eur Respir J 8: 2029-2037, 1995[Abstract].

21.   Marinoni, E, Di Iorio R, Alo P, Villaccio B, Alberini A, and Cosmi EV. Immunohistochemical localization of adrenomedullin in fetal and neonatal lung. Pediatr Res 45: 282-285, 1999[ISI][Medline].

22.   McLatchie, LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, and Foord SM. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393: 333-339, 1998[Medline].

23.   Nandha, KA, Taylor GM, Smith DM, Owji AA, Byfield PG, Ghatei MA, and Bloom SR. Specific adrenomedullin binding sites and hypotension in the rat systemic vascular bed. Regul Pept 62: 145-151, 1996[ISI][Medline].

24.   Njuki, F, Nicholl CG, Howard A, Mak JC, Barnes PJ, Girgis SI, and Legon S. A new calcitonin-receptor-like sequence in rat pulmonary blood vessels. Clin Sci (Colch) 85: 385-388, 1993[Medline].

25.   Nuki, C, Kawasaki H, Kitamura K, Takenaga M, Kangawa K, Eto T, and Wada A. Vasodilator effect of adrenomedullin and calcitonin gene-related peptide receptors in rat mesenteric vascular beds. Biochem Biophys Res Commun 196: 245-251, 1993[ISI][Medline].

26.   Owji, AA, Smith DM, Coppock HA, Morgan DG, Bhogal R, Ghatei MA, and Bloom SR. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology 136: 2127-2134, 1995[Abstract].

27.   Quirion, R, Van Rossum D, Dumont Y, St. Pierre S, and Fournier A. Characterization of CGRP1 and CGRP2 receptor subtypes. Ann NY Acad Sci 657: 88-105, 1992[ISI][Medline].

28.   Rosenfeld, MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WW, and Evans RM. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304: 129-135, 1983[Medline].

29.   Sakata, J, Shimokubo T, Kitamura K, Nakamura S, Kangawa K, Matsuo H, and Eto T. Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun 195: 921-927, 1993[ISI][Medline].

30.   Sams, A, and Jansen-Olesen I. Expression of calcitonin receptor-like receptor and receptor-activity-modifying proteins in human cranial arteries. Neurosci Lett 258: 41-44, 1998[ISI][Medline].

31.   Terenghi, G, Polak JM, Ghatei MA, Mulderry PK, Butler JM, Unger WG, and Bloom SR. Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye. J Comp Neurol 233: 506-516, 1985[ISI][Medline].

32.   Tjen-A-Looi, S, Ekman R, Lippton H, Cary J, and Keith I. CGRP and somatostatin modulate chronic hypoxic pulmonary hypertension. Am J Physiol Heart Circ Physiol 263: H681-H690, 1992[Abstract/Free Full Text].

33.   Tjen-A-Looi, S, Kraiczi H, Ekman R, and Keith IM. Sensory CGRP depletion by capsaicin exacerbates hypoxia-induced pulmonary hypertension in rats. Regul Pept 74: 1-10, 1998[ISI][Medline].

34.   Umeda, Y, and Arisawa M. Characterization of calcitonin gene-related peptide (CGRP) receptors in guinea pig lung. Jpn J Pharmacol 51: 377-384, 1989[Medline].

35.   Van Rossum, D, Hanisch UK, and Quirion R. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci Biobehav Rev 21: 649-678, 1997[ISI][Medline].

36.   Watkins, DJ, and Burcher E. Autoradiographic localization of calcitonin gene-related peptide receptors in guinea pig respiratory tract: effect of capsaicin pretreatment. Peptides 16: 683-692, 1995[ISI][Medline].

37.   Wimalawansa, SJ. Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials. Endocr Rev 17: 533-585, 1996[ISI][Medline].

38.   Wimalawansa, SJ. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. Crit Rev Neurobiol 11: 167-239, 1997[ISI][Medline].

39.   Zhao, L, Brown LA, Owji AA, Nunez DJ, Smith DM, Ghatei MA, Bloom SR, and Wilkins MR. Adrenomedullin activity in chronically hypoxic rat lungs. Am J Physiol Heart Circ Physiol 271: H622-H629, 1996[Abstract/Free Full Text].

Am J Physiol Lung Cell Mol Physiol 280(3):L547-L554
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. N. Chattergoon, F. M. D'Souza, W. Deng, H. Chen, A. L. Hyman, P. J. Kadowitz, and J. R. Jeter Jr.
Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells
Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L202 - L211.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
S. D. Brain and A. D. Grant
Vascular Actions of Calcitonin Gene-Related Peptide and Adrenomedullin
Physiol Rev, July 1, 2004; 84(3): 903 - 934.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
X. Qing and I. M. Keith
Targeted blocking of gene expression for CGRP receptors elevates pulmonary artery pressure in hypoxic rats
Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L86 - L96.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
I. Brouns, J. Van Genechten, H. Hayashi, M. Gajda, T. Gomi, G. Burnstock, J.-P. Timmermans, and D. Adriaensen
Dual Sensory Innervation of Pulmonary Neuroepithelial Bodies
Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 275 - 285.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
W. Nowak, N. Parameswaran, C. S. Hall, N. Aiyar, H. V. Sparks, and W. S. Spielman
Novel regulation of adrenomedullin receptor by PDGF: role of receptor activity modifying protein-3
Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1322 - C1331.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qing, X.
Right arrow Articles by Keith, I. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qing, X.
Right arrow Articles by Keith, I. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online