Targeted blocking of gene expression for CGRP receptors elevates pulmonary artery pressure in hypoxic rats

Xin Qing and Ingegerd M. Keith

Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706

Submitted 23 October 2002 ; accepted in final form 28 February 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously described the protection by calcitonin gene-related peptide (CGRP) against hypoxic pulmonary hypertension. Here, we examine the roles of its putative receptor RDC-1 and receptor activity-modifying protein (RAMP) 1 in mediating this protection by selectively inhibiting their synthesis. RAMP1 is an accessory protein for another putative CGRP receptor, calcitonin receptor-like receptor. Antisense oligodeoxyribonucleotides (ASODNs, 5 mg·kg-1·day-1 or 5 and 10 mg·kg-1·day-1 for RDC-1) targeting RAMP1 and RDC-1 mRNAs were chronically infused to the pulmonary circulation of male Sprague-Dawley rats during 7 days of normoxia or hypobaric hypoxia (380 mmHg), and {alpha}-CGRP ASODN was used as a technical control. CGRP, RAMP1, and RDC-1 ASODNs significantly elevated pulmonary artery pressure (PPA) in chronic hypoxic rats compared with hypoxic mismatched ASODN (MMODN) and saline vehicle controls. CGRP and RAMP1 ASODNs raised PPA in normoxic rats briefly exposed to 10% O2 above MMODN and saline controls. Moreover, normoxic rats treated with CGRP ASODN had higher basal pulmonary vascular tone compared with controls. These data confirm the protective role of CGRP in the pulmonary circulation and suggest that endogenous RAMP1 and RDC-1 are essential in regulation of PPA in hypoxia. This is the first in vivo evidence supporting RDC-1 and RAMP1 as functional CGRP receptor and receptor component.

antisense oligodeoxyribonucleotide; calcitonin gene-related peptide; RDC-1; receptor activity-modifying protein 1; in vivo gene targeting


PULMONARY HYPERTENSION (PH) is a serious and often fatal disease. Airway hypoxia is a common cause of PH. Hypoxic PH (HPH) occurs in many lung diseases such as persistent PH of the newborn, adult respiratory distress syndrome, chronic obstructive pulmonary disease, high altitude PH and edema, sleep apnea, inflammation, and other causes that interfere with airway oxygenation. An imbalance between pulmonary vasoconstrictors and vasodilators has been suggested as an initiating factor in PH (16). The mRNA and protein levels of the most potent vasoconstrictor peptide, endothelin-1, were elevated in the lungs of patients and hypoxic rats with PH (6, 10, 12, 35). However, absence of vasodilation rather than active vasoconstriction has been proposed as a cause of PH (4, 24, 38).

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are structurally related peptides with potent vasodilatory effects. In fact, CGRP is the most potent peptide vasodilator discovered thus far and ADM's vasodilatory effect is second only to CGRP (39). The two isoforms of CGRP, {alpha}-CGRP and {beta}-CGRP, are both present in lung tissue. {alpha}-CGRP is a 37-amino acid neuropeptide generated from an alternatively spliced transcript of the gene encoding calcitonin (3, 31), whereas {beta}-CGRP is the product of a separate gene. It differs from {alpha}-CGRP by one amino acid in humans and three in rats. Although there is no reported difference in receptor sensitivity or vasodilatory action between the two isoforms, {alpha}-CGRP is the major type of CGRP in the lung. It has been localized with immunocytochemistry and in situ hybridization to airway epithelial neuroendocrine cells and interstitial ganglia (20) and also by immunocytochemistry to epithelial and vascular networks (17, 36). We found early on that CGRP was significantly reduced in left ventricular blood of rats with HPH (18, 34) and persistent PH (21), contributing to an imbalance between vasoconstrictors and vasodilators. We further found that exogenous CGRP prevents the development of HPH and reverses existing HPH via the CGRP type 1 receptor in rats (19, 34). Furthermore, blocking of the CGRP1 receptor with CGRP8–37, immunoprecipitation of endogenous CGRP in the blood stream, or CGRP depletion with capsaicin treatment exacerbated HPH (34, 36); CGRP depletion with capsaicin also significantly increased pulmonary vascular endothelial and medial smooth muscle proliferation under normoxic and hypoxic conditions (36). These results clearly indicate a protective role of endogenous, native CGRP.

CGRP elicits its vasodilatory effect through interaction with G protein-coupled CGRP1 receptors linked to cAMP production. RDC-1 (15) and calcitonin receptor-like receptor (CRLR) (1) are newly cloned putative CGRP1 receptors, both of which belong to the G protein-coupled seven-transmembrane-domain receptor superfamily. Recent information suggests that CRLR can function as either a CGRP1 or an ADM receptor, depending on which of the receptor activity-modifying proteins (RAMP1, RAMP2, or RAMP3) is coexpressed (25). Expression of a functional CGRP1 receptor requires coexpression of CRLR and RAMP1, whereas RAMP2 presents CRLR at the cell surface as an ADM receptor (9, 25, 27). In addition, coexpression of CRLR with RAMP3 may result in ADM (9, 27) or mixed-type ADM/CGRP1 receptors (13). Subsequent reports, including binding and functional assays (cAMP production), support the CRLR/RAMP model (2, 7, 13) but not RDC-1 as a CGRP1 receptor (7). However, all of these studies were done in cultured cells, and there is no in vivo report to date on the physiological function of these agents in intact animals and their lungs. We recently reported upregulation of RDC-1 and RAMP1 mRNAs in hypoxic rat lung and no change in CRLR mRNA, suggesting a functional role for these two agents in regulating the pulmonary circulation (29). Increased RAMP1 mRNA and unchanged CRLR mRNA in hypoxia suggest that CGRP reception at the CRLR is primarily regulated by RAMP1.

In the present study, we investigated a critical role of RAMP1 and RDC-1 in maintenance of normal pulmonary artery pressure (PPA) and their protective effects against HPH by chronic intravenous injection with antisense oligodeoxyribonucleotides (ASODNs) directed at these mRNAs into lungs of rats exposed to normoxia and chronic hypobaric hypoxia. ASODN targeting rat {alpha}-CGRP was used as a technical control. To control for nonspecific effects of treatments with ASODNs, a similar but purposely mismatched ASODN (MMODN) for each antisense sequence was infused similarly.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal treatments. All protocols and surgical procedures employed in this study were reviewed and approved by the Animal Resource Center and the Graduate School of the University of Wisconsin-Madison. Male Sprague-Dawley rats weighing 175–200 g at onset of studies were used for all experimental procedures (n = 108 rats total). The rats were housed in American Association for Accreditation of Laboratory Animal Care-certified facilities and treated humanely according to the approved protocols. Food and water were given ad libitum.

Rats were randomly assigned to treatment and control groups. Phosphorothioate ASODNs and MMODNs for CGRP, RAMP1, or RDC-1 (5 mg·kg-1·day-1 or 5 and 10 mg·kg-1·day-1 for RDC-1) were suspended in sterile saline and infused chronically into the right external jugular vein using a subcutaneous Alzet osmotic minipump (model 2ML1, 10.0 µl/h) over a period of 7 days while rats were exposed to normoxia or hypoxia. Sample sizes varied between four and eight rats for each infusion group (n = 4/group for RAMP1 ASODN, RDC-1 ASODN, and all MMODN infusion groups; n = 8/group for CGRP ASODN infusion groups). Saline controls (n = 6/group) received sterile saline only using similar osmotic minipumps. The loaded pumps were primed by soaking in saline containing bacitracin (0.05 mg/ml) at 37°C for at least 6 h before implantation.

For minipump implantation, each rat was weighed and anesthetized with pentobarbital sodium (42 mg/kg ip). The right jugular vein was cannulated with a PE-10 catheter bridged into a PE-50 catheter. An Alzet osmotic minipump was then connected to the PE-50 catheter and placed subcutaneously at the back of the neck between the scapulas. After overnight recovery, the rats were placed unrestrained in individual cages in normoxia (ambient room air) or in a hypobaric hypoxic chamber (barometric pressure 380 mmHg, equivalent to an inspired O2 level of 10%, ambient room air CO2 level, ambient humidity; Biotron, University of Wisconsin-Madison) for 7 days. The chamber was opened daily for a maximum of 30 min for feeding and cleaning.

ASODNs and MMODNs. Phosphorothioate ASODNs for specific mRNAs were designed, synthesized, and purified by Molecular Research Laboratory (Herndon, VA). Likewise, MMODNs were synthesized to serve as controls. The sequences of oligodeoxyribonucleotides against rat {alpha}-CGRP, rat RAMP1, and rat RDC-1 mRNAs used in this study were: CGRP antisense, 5'-CTG AAG GTC CCT GCG GCG GC-3' (targeting CGRP only, not calcitonin); CGRP mismatch, 5'-GTG ACG TCC GCG GCG GCT AC-3'; RAMP1 antisense, 5'-CCA TGG CAA GCA GAG CCC C-3'; RAMP1 mismatch, 5'-GCA TGC CAG CCA GCC GAA C-3'; RDC-1 antisense, 5'-CTT GGC CTG GAT ATT CAC CC-3'; and RDC-1 mismatch, 5'-CCC ACT TAT AGG TCC GGT TC-3'. The sequence homologies were checked against the reported DNA sequences available in the BLAST program to limit the chances of hybridization to other known targets.

Arterial blood pressure recording and tissue processing. At the end of each experiment, rats from the hypobaric hypoxic chamber were transferred to the laboratory and placed temporarily in a normobaric hypoxic chamber under continuous flow of 10% O2 in N2. Rats were anesthetized with pentobarbital sodium (42 mg/kg ip), and heart rate (beats/min) and mean systemic blood pressure (Psyst) were recorded from the common carotid artery while rats were spontaneously breathing. The trachea was then cannulated for ventilation with room air using a Harvard rodent ventilator set at 1.75 ml tidal volume and a respiratory rate of 70 breaths/min. A midline 2-cm-long thoracotomy was performed, and mean PPA was recorded through a 20-gauge angiocath catheter inserted into the pulmonary artery. After PPA measurements under normoxic conditions (room air), rats from normoxic groups were ventilated with 10% O2 for up to 5 min (acute hypoxia), and PPA was recorded again. All blood pressure measurements utilized a Statham pressure transducer and a Gould recorder, and mean pressures were obtained using the "mean" function on the Gould recorder.

Blood was drawn with a needle inserted into the left ventricle and collected into a 10-ml syringe pretreated with EDTA (1 drop of 2% EDTA/ml blood; Sigma) and aprotinin (200 KIU/ml blood; Sigma). The blood was centrifuged at 2,000 rpm at 4°C for 20 min, and hematocrit (HCT, packed cell volume/blood volume) was determined.

Lungs were then perfused with heparinized saline (0.4% heparin) at ~20 mmHg pressure using a Masterflex pump (Cole Parmer Instrument, Chicago, IL), removed, and weighed. The lung weight was normalized by body weight as a rough indicator of lung edema. One 3-mm-thick transverse slice was sampled from the middle region of the right lower (diaphragmatic) lobe and fixed in Bouins solution for measurement of medial thickness index of small pulmonary arteries and immunocytochemistry. The remaining lung was treated with RNAlater (Ambion, Austin, TX) and stored at -20°C for subsequent mRNA assays.

After lung collection, the heart was excised, fixed in Bouins solution, and later dissected. The ratio of the weight of the right ventricular free wall (RV) to that of the left ventricle with septum (LV+S) was measured and used as an indicator of right ventricular hypertrophy (RVH) when the ratio was significantly elevated.

Morphometric evaluation of pulmonary vascular medial thickness. Five-micrometer-thick paraffin sections of lung samples were stained with Miller elastin stain, and medial thickness index was determined "blindly" on cross-sectioned, circular pulmonary vessels ranging from 20 to 40 µm in outer diameter by applying Image Pro Plus software on digital images imported by the use of a Spot camera and a Nikon Eclipse 600 microscope as previously described (30). In brief, the average vessel diameter (2r) was calculated from the total area inside the circumference of the external elastic lamina of the vessel [2r = 2(area/{pi})1/2]. The medial surface area (located between the inner and outer elastic laminae) was measured and normalized by division by the average outer diameter of each vessel. The normalized values served as the medial thickness index. A minimum of 10 vessels per rat was measured. The individual means for each rat were determined and used for the calculation of the mean and SE of each treated group.

Immunocytochemical technique. For immunocytochemical detection of CGRP in paraffin sections, the labeled-[strept]-avidin-biotin method (HISTOSTAIN-SP; Zymed Laboratories, South San Francisco, CA) was employed. Five-micrometer-thick histological sections of lung samples were mounted on poly-L-lysine-coated slides, deparaffinized in two changes of xylene followed by two changes of 100% alcohol, and immersed in 3% H2O2 in methanol for 10 min followed by rinses in PBS (2 min, 3 times). Sections were then treated with 10% nonimmune goat serum for 10 min and incubated for 1 h with the rabbit anti-rat CGRP primary antiserum (Peninsula Laboratories, San Carlos, CA) in a humid chamber. Optimal working dilution for anti-CGRP was 1:500 using this procedure. After being rinsed in PBS, sections were incubated with biotinylated secondary antibody and then treated with the enzyme conjugate according to the staining protocol provided by the manufacturer. Peroxidase activity was demonstrated using 3-amino-9-ethylcarbazole-H2O2 as substrate. Finally, the sections were counterstained with hematoxylin. All slides were treated in the same manner to ensure precise comparison. Negative controls were subjected to the same procedures, except that they were incubated with a nonimmune rabbit serum instead of the same concentration of primary antiserum. Staining specificity was also assessed by substituting the primary antiserum with the same antiserum (1:500 dilution) after it had been preabsorbed by incubation with the antigen (50 µg synthetic rat {alpha}-CGRP peptide/ml) overnight before use.

To quantitatively analyze the expression of CGRP after CGRP ASODN treatment, immunocytochemical staining was performed as above except that sections were incubated for 2 h at room temperature with primary antiserum at 1:10,000 and that the peroxidase activity was revealed using the nickel enhancement method (diaminobenzidine/nickel-cobalt kit; Zymed). This dilution and procedure were arrived at by performing a dilution series and trying different incubation times to obtain a combination that would allow reliable distinction between various intensities of immunoreactivity and with minimal variation between batches. Counterstaining with hematoxylin was omitted to avoid interference with the immunoreactivity. The immunocytochemistry data were quantified blindly on coded sections using Image Pro Plus software on digital images imported by the use of a Spot camera and a Nikon Eclipse 600 microscope. All measurements were performed on four tissue sections per rat cut from the paraffin blocks 50 µm apart. These sections were cut from the same region of the lung for all rats studied, in the same plane of section, and with approximately equal areas. To quantitatively analyze CGRP-like immunoreactivity (LI) in neuroendocrine cells, we counted the number of positive neuroendocrine cells, with the results expressed as number of positive cells per area of lung. Increased number of CGRP-positive neuroendocrine cells has been reported in the lungs of chronically hypoxic rats using this method (32). Quantitative analysis of immunoreactive nerve fibers was carried out in pulmonary arteries with a diameter of ~1,000 µm, and the number of immunoreactive nerve fibers was normalized to the surface area of the blood vessel wall including intima, media, and adventitia. The measured values per lung section were averaged for each animal, and the mean values were determined for each group based on these averages.

Total RNA extraction and semiquantitative RT-PCR. Total RNA was isolated from rat lungs using an RNeasy Minikit (QIAGEN, Valencia, CA) according to the instructions provided by the manufacturer. Samples were then treated with RQ1 RNase-free DNase (Promega, Madison, WI) and subjected to semiquantitative RT-PCR as previously reported (29). Briefly, 1 µg of DNase-pretreated total RNA was reverse transcribed with random primers in 20-µl reactions using an RT system (Promega). PCR amplification of each gene product was carried out in parallel 50-µl reactions using PCR Core Systems I (Promega) with 1 µM of specific forward and reverse primers and 2 µl of RT product. The 18S rRNA was used as an internal control for sample loading. Amplifications were carried out for 32 cycles for RAMP1, 28 cycles for RDC-1, and 15 cycles for 18S rRNA. The specific forward and reverse primers were designed based on published cDNA sequences of rat RAMP1 and rat RDC-1 (GenBank accession nos. AB028933 [GenBank] and AJ010828 [GenBank] ). Their sequences and the sizes of the amplified cDNAs were: RAMP1 primers, 5'-ACT GGG GAA AGA CCA TAG GGA G-3' and 5'-AGT CAT GAG CAG TGT GAC CGT A-3', 230 bp; RDC-1 primers, 5'-ATG CCC AAC AAG AAT GTG CTG-3' and 5'-ATA GCT GGA GGT GCT GGT GAA-3', 357 bp. The primers for 18S rRNA (5'-CCG CAG CTA GGA ATA ATG GA-3' and 5'-GAG TCA AAT TAA GCC GCA CG-3') yielded a 400-bp product.

After PCR, a 10-µl aliquot from each PCR amplification was loaded onto 1.5% agarose gels with 0.5 µg/ml of ethidium bromide and subjected to electrophoresis. Gels were photographed using Polaroid 667 film and digitized using an Epson 636 scanner. Band density analysis was performed on a PC using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) to determine the quantity of PCR product. To account for differences in the amounts of starting RNA between samples, the density of each RAMP1 or RDC-1 band was normalized by division by the density of the 18S rRNA band 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 saline controls was arbitrarily set at 1.0. Each of the normalized values was divided by the mean value of the saline controls to generate the relative expression levels. To verify their identity, the RT-PCR products were purified and sequenced by the Biotechnology Center of University of Wisconsin-Madison. Results confirmed the specific PCR product identity.

Statistical analysis. Results are expressed as group means ± SE. Data were evaluated using Student's t-test and one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons. Means were considered significantly different at P <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PPA, Psyst, and heart rate. The effects of ASODNs and MMODNs directed at CGRP, RAMP1, and RDC-1 on the mean PPA are shown in Figs. 1, 2, and 3, respectively. Normoxic rats treated with CGRP ASODN had higher basal vascular tone compared with saline controls. Acute hypoxia further elevated PPA in these rats to levels significantly higher than their acute hypoxia controls. In 1-wk hypoxic rats, CGRP ASODN increased PPA even further to levels 37% above hypoxic controls. CGRP MMODN control treatment did not affect PPA. Surprisingly, normoxic rats treated with RAMP1 ASODN had normal basal vascular tone. However, acute hypoxia further elevated PPA in these rats, and RAMP1 ASODN increased PPA in 1-wk hypoxic rats to levels 22% above 1-wk hypoxic controls. We suspected that the weaker response to RAMP1 ASODN, compared with the response to CGRP ASODN, was a dose factor. Therefore, a double-strength dose (10 mg·kg-1·day-1) was used in addition for the RDC-1 study. Similar to RAMP1 ASODN, normoxic rats treated with RDC-1 ASODN at 5 mg·kg-1·day-1 had normal basal vascular tone, and significantly increased PPA was noticed in ASODN-treated 1-wk hypoxic rats (22 and 26% above MMODN and saline controls). Moreover, infusion of RDC-1 ASODN at 10 mg·kg-1·day-1 resulted in an even higher increase of PPA in 1-wk hypoxic rats compared with the low dose. PPA was increased to 57 and 71% above saline and dose-matched, MMODN-treated 1-wk hypoxic controls. However, basal vascular tone was unaffected by high dose of RDC-1 ASODN in normoxic rats, and even more surprisingly the PPA response to acute hypoxia was unaffected by RDC-1 ASODN at either dose.



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Fig. 1. Mean pulmonary artery pressure (PPA) of normoxia- and hypoxia-treated rats infused with calcitonin gene-related peptide (CGRP) antisense oligodeoxyribonucleotides (ASODN), CGRP mismatched oligodeoxyribonucleotides (MMODN), and saline (controls). 1w, 1 Wk. Values are means ± SE. {dagger}Significantly higher than matched normoxic group, {ddagger}significantly higher than matched acute hypoxic group, and *significantly higher than matched saline and MMODN controls by Student-Newman-Keuls test (P <= 0.05).

 


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Fig. 2. PPA of normoxia- and hypoxia-treated rats infused with receptor activity-modifying protein (RAMP1) ASODN, RAMP1 MMODN, and saline (controls). Values are means ± SE. {dagger}Significantly higher than matched normoxic group, {ddagger}significantly higher than matched acute hypoxic group, and *significantly higher than matched saline and MMODN controls by Student-Newman-Keuls test (P <= 0.05).

 


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Fig. 3. PPA of normoxia- and hypoxia-treated rats infused with RDC-1 ASODN, RDC-1 MMODN, and saline (controls). Values are means ± SE. {dagger}Significantly higher than matched normoxic group, {ddagger}significantly higher than matched acute hypoxic group, *significantly higher than matched saline and MMODN controls, and #significantly higher than low dose by Student-Newman-Keuls test (P <= 0.05).

 

Mean Psyst (Fig. 4) and heart rate [average for all rats = 418 ± 3.6 beats/min (mean ± SE; n = 108), data not shown] were unaffected by 1-wk hypoxia and remained unchanged by all ASODN and MMODN at the doses used.



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Fig. 4. Mean systemic blood pressure (Psyst) of normoxia- and hypoxia-treated rats infused with ASODNs and MMODNs against CGRP, RAMP1, and RDC-1 mRNAs and saline. Values are means ± SE. There were no significant differences between any groups in any experiment by Student's t-test and Student-Newman-Keuls test (P <= 0.05).

 

Morphometric changes, HCT, and lung edema. Right ventricular weight index [RV/(LV+S)] (Fig. 5A) and medial thickness index of small pulmonary arteries (Fig. 5B, representative groups only) were measured as indicators of chronic HPH. RV/(LV+S) was significantly increased by 1-wk hypoxia. Although an increasing tendency of RVH index was noticed in some ASODN-treated animals, none of these changes were statistically significant. This is probably due to the limited sensitivity of this method and because 1 wk is insufficient time for ASODN to induce significant RVH. A similar increasing trend of the degree of pulmonary arterial remodeling (pulmonary arterial medial thickness index) was also observed in hypoxic groups and some ASODN-treated groups although without statistical significance.



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Fig. 5. A: right ventricular weight index [RV/(LV+S)] of normoxia- and hypoxia-treated rats infused with ASODNs and MMODNs against CGRP, RAMP1, and RDC-1 mRNAs and saline. RV, right ventricle; LV+S, left ventricle plus septum. Values are means ± SE. {dagger}Significantly higher than matched normoxic group using Student's t-test (P <= 0.05). There was no significant difference between any saline, MMODN-, and ASODN-infused groups in normoxic and in hypoxic rats by Student-Newman-Keuls test (P <= 0.05). B: medial thickness index of small pulmonary arteries (outer diameter ranging from 20 to 40 µm) of normoxia- and hypoxia-treated rats infused with selected ASODNs, MMODNs, and saline. Data from CGRP ASODN-treated normoxic rats and RDC-1 ASODN-treated hypoxic rats (10 mg·kg-1·day-1) were selected to show effects of ASODNs on pulmonary vascular remodeling in normoxic and hypoxic rats because of their greatest effects on PPA elevation compared with other ASODN treatments used. Their matched saline and MMODN control means were no different from one another, and those data were thus pooled within normoxic rats and hypoxic rats separately for further clarity. Values are means ± SE. There was no significant difference between any controls and ASODN-infused groups by Student-Newman-Keuls test (P <= 0.05).

 

HCT and the ratio of lung weight to body weight were significantly increased by 1-wk hypoxic exposure but were unaffected by either ASODN or MMODN treatment (data not shown).

Immunocytochemistry. Immunocytochemical detection of CGRP-LI with the 3-amino-9-ethylcarbazole detection system yielded a reddish/brown product observed in airway epithelial neuroendocrine cells and nerve fibers of the airway mucosa (Fig. 6A). CGRP-LI was also localized in nerve fibers of the tunica intima and tunica adventitia of blood vessels and around vascular smooth muscle of the tunica media (Fig. 7A). By subjective observation, the intensity of immunoreactivity and the relative numbers of positive cells were reduced in the lungs of CGRP ASODN-treated normoxic and hypoxic rats (Fig. 6). CGRP ASODN treatment also decreased the number of visible positive nerve fibers associated with blood vessels (Fig. 7). However, CGRP MMODN treatments had no such effect.



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Fig. 6. Immunohistochemical localization of CGRP neuropeptide expression in the lungs of normoxia (A, B, and C)- and hypoxia (D, E, and F)-treated rats after saline (A and D), CGRP MMODN (B and E), and CGRP ASODN (C and F) infusion. Positive CGRP-like immunoreactivity (LI) was detected in the airway epithelial neuroendocrine cells (large arrows) and nerve fibers (thin arrows) of the airway mucosa. CGRP peptide levels appear to be reduced in the CGRP ASODN groups compared with matched controls, as indicated by diminished intensity and distribution of immunoreactivity. AL, alveolar lumen; BL, bronchiolar lumen.

 


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Fig. 7. CGRP-LI in pulmonary blood vessels of normoxia (A, B, and C)- and hypoxia (D, E, and F)-treated rats after saline (A and D), CGRP MMODN (B and E), and CGRP ASODN (C and F) infusion. Arrows indicate positive nerve fibers in the tunica intima (TI) and tunica adventitia (TA) of blood vessels and around vascular smooth muscle of the tunica media (TM). The numbers of visible positive nerve fibers associated with blood vessels appear to be decreased in the CGRP ASODN groups compared with matched saline and MMODN controls.

 

The quantitative analysis of CGRP-LI in neuroendocrine cells using the nickel enhancement method revealed that CGRP-LI in the airway epithelium was significantly elevated in hypoxic rats and was reduced in CGRP ASODN-treated normoxic and hypoxic rats, although the change in normoxic rats was not significant (Fig. 8, top). The validity of this quantitation method was confirmed by our results that showed significantly higher numbers of neuroendocrine cells displaying CGRP-LI among hypoxic control rats compared with normoxic controls. Furthermore, the overall densities of CGRP-LI in the nerve fibers associated with pulmonary arteries were significantly decreased in CGRP ASODN-treated normoxic and hypoxic rats (Fig. 8, bottom). There were no differences in these parameters between any saline and MMODN-treated groups.



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Fig. 8. CGRP-LI in airway epithelial neuroendocrine cells and clusters (top) and nerve fibers of pulmonary arteries (bottom) in the lungs of normoxia- and hypoxia-treated rats infused with CGRP ASODN, CGRP MMODN, and saline (controls). Immunoreactive cell numbers per lung area (top) and number of immunoreactive nerve profiles normalized to the area of pulmonary artery wall (bottom) are shown. Values are means ± SE. {dagger}Significantly higher than matched normoxic group and *significantly lower than matched saline and MMODN controls by Student-Newman-Keuls test (P <= 0.05).

 

Semiquantitative RT-PCR. Due to the unavailability of antibodies against RAMP1 and RDC-1, semiquantitative RT-PCR was performed to demonstrate the inhibitory effects of ASODNs on expression of targeted gene. Gel electrophoresis of the RT-PCR products with RAMP1 and RDC-1 primers revealed a single band of ~230 bp and a product of ~357 bp, respectively, as previously reported (29). These bands corresponded in size to the RT-PCR products expected from rat RAMP1 and RDC-1 mRNA, respectively. Sequence analysis of these RT-PCR products confirmed their identities. RAMP1 mRNA level was decreased by 27 and 9% with RAMP1 ASODN treatment in normoxic and hypoxic rat lungs, respectively, but unaffected by RAMP1 MMODN (Fig. 9). As shown in Fig. 10, treatment with RDC-1 ASODN at 5 mg·kg-1·day-1 significantly reduced basal RDC-1 mRNA level by ~20% compared with its matched saline and MMODN controls in normoxic rat lungs but did not significantly affect the RDC-1 mRNA expression in hypoxic rat lungs. Moreover, RDC-1 ASODN at 10 mg·kg-1·day-1 reduced basal and hypoxia-stimulated RDC-1 mRNA levels by 25 and 30%, respectively, whereas MMODN had no significant effect. This confirms the specificity of the RAMP1 and RDC-1 ASODNs employed in this study.



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Fig. 9. RAMP1 mRNA levels in the lungs of rats after RAMP1 ASODN treatment and in controls. A: representative RT-PCR products for RAMP1 and 18S rRNA from saline, RAMP1 MMODN-(MM), and RAMP1 ASODN-treated (AS) rats. The experiment shown is representative of at least 3 repetitions. B: quantitative densitometry data of RT-PCR products for RAMP1 mRNAs normalized for 18S rRNA from each group of rats. Semiquantitative RT-PCR shows a ~27% decrease in RAMP1 mRNA by RAMP1 ASODN in normoxic rats and a ~10% reduction in 7-day hypoxic rats compared with their matched saline and RAMP1 MMODN controls. Values are means ± SE. By Student-Newman-Keuls test, #P <= 0.05 vs. saline control and *P <= 0.05 vs. MMODN control. Mean values of the saline control groups were arbitrarily set at 1.0.

 


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Fig. 10. RDC-1 mRNA levels in the lungs of rats after RDC-1 ASODN treatment. A: representative RT-PCR products for RDC-1 and 18S rRNA from saline, RDC-1 MMODN-, and RDC-1 ASODN-treated rats. The experiment shown is representative of at least 3 repetitions. B: quantitative densitometry data of RT-PCR products for RDC-1 mRNAs normalized for 18S rRNA from each group of rats. Semiquantitative RT-PCR shows a ~20% decrease in RDC-1 mRNA by RDC-1 ASODN (5 mg·kg-1·day-1) in normoxic rats. An ~25% and an ~30% reduction was caused by RDC-1 ASODN at 10 mg·kg-1·day-1 in normoxic and hypoxic rat lungs, respectively. RDC-1 mRNA expression was unaffected by RDC-1 MMODN. Values are means ± SE. By Student-Newman-Keuls test, #P <= 0.05 vs. saline control, *P <= 0.05 vs. matched MMODN control, and {dagger}P <= 0.05 vs. low dose. Mean values of the saline control groups were arbitrarily set at 1.0.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previously published results showed that immunoneutralization and receptor blocking of endogenous CGRP augmented PPA and RVH in hypoxia (34). We further found that CGRP depletion with capsaicin exacerbated HPH and significantly increased pulmonary vascular endothelial and medial smooth muscle proliferation under both normoxic and hypoxic conditions (36). These results suggested an essential role of endogenous CGRP and its receptors in pulmonary circulation. The studies described here confirm the same lack of protection against HPH by gene-targeted antisense inhibition of CGRP expression, suggesting that antisense technology provides a precise, fast, and efficient way to knock down specific gene expression in the study of gene function. Also, the increased PPA in CGRP ASODN-treated normoxic rats suggests that CGRP is required in maintenance of normal PPA. Mean Psyst was unaffected by all ASODNs and MMODNs at the doses used, suggesting that endogenous CGRP and its receptors may be more important in the pulmonary circulation than in the systemic circulation.

Although blood pressure is also regulated by change in rate of flow (cardiac output), the unchanged heart rate and Psyst suggest that the increased PPA is more likely caused by factors within the lung, for example, by increased resistance from vasoconstriction and possibly vascular remodeling. Increasing tendency of pulmonary arterial medial thickness index was noticed in some ASODN-treated groups although without statistical significance. Longer duration of hypoxia and ASODN infusion (e.g., 3–4 wk) may provide greater remodeling as well as greater RV/(LV+S) ratios in hypoxic ASODN treatment groups compared with hypoxic control groups. Although the HPH is not yet fully developed by that time and the mechanisms, which participate in the increase of pulmonary vascular resistance, may differ from those in the fully developed HPH, we elected to use 7 days of hypoxia because of the high costs of the phosphorothioate ASODNs and MMODNs. Further studies are necessary to clarify whether CGRP release affects vasoconstriction in early stages of HPH and/or the hypoxic remodeling of pulmonary blood vessels. We previously found that in 16-day hypoxic rats, removal of CGRP by capsaicin treatment resulted in significantly increased medial thickness and number of endothelial cells of pulmonary vessels compared with vehicle-treated hypoxic controls (36). This suggests that CGRP directly or indirectly affects vascular remodeling in cases of prolonged hypoxia. Furthermore, we also observed in our laboratory that infusion of CGRP decreased PPA dose dependently in an in vitro isolated perfused lung preparation under conditions of elevated pulmonary vasomotor tone (34). This suggests that these precontracted vessels are reactive to CGRP under nonhypoxic conditions. It further suggests that CGRP could have a relaxing effect on pulmonary vessels in early stages of HPH. Moreover, the fact that acute hypoxia to normoxic rats in the present study caused further elevation of PPA among CGRP antisense rats compared with saline controls supports a role of CGRP in alleviating early hypoxic vasoconstriction. A direct link between the vasoconstriction and muscularization of pulmonary blood vessels remains questionable. Moreover, cardiovascular remodeling may not be directly linked to CGRP, its receptors, and RAMPs but could occur concomitantly. For example, disruption of natriuretic peptide receptor A, with high affinity for atrial and brain natriuretic peptide, worsens hypoxia-induced cardiac hypertrophy (22) and increases susceptibility to HPH (40).

RV/(LV+S) was significantly increased in response to 1 wk of hypoxia, but no further detectable change was induced by ASODN under hypoxia. The change in PPA in response to hypoxia is close to that observed in response to CGRP inhibition in hypoxic animals, so it is not clear why an effect on RVH was not seen. Besides the limitation of the RVH measurement method, there are other factors that could be responsible for the observation. For example, RV/(LV+S) had already been elevated in rats exposed to hypoxia, and therefore further elevation caused by antisense inhibition may require more aggressive antisense inhibition. Moreover, whereas elevated PPA is a direct result from altered vascular reactivity, tissue remodeling is known to be lagging behind the onset of pressure changes. In our lab, lung vascular remodeling was found to be a slower phenomenon that usually required a minimum of 2 wk for significant changes by 10% oxygen in our Sprague-Dawley rat strain, and RV thickening required a minimum of 1 wk. This suggests that factors other than CGRP and its receptors may contribute to RV and pulmonary vascular medial thickening. Therefore, RV and vascular remodeling must not necessarily occur in concordance with the elevation of PPA. However, the precise mechanisms are not clear yet based on our current knowledge.

It is now widely accepted that CRLR is in fact the CGRP1/ADM receptor and that its receptor specificity and activity are conferred by RAMPs, which are single-transmembrane-domain proteins required to transport CRLR to the cell membrane, define the pharmacology, and determine its glycosylation state (25). Based solely on in vitro cell lines, CRLR coexpressed with RAMP1 functioned as a CGRP1 receptor in Xenopus oocytes, HEK-293T cells, and a rabbit endothelial cell line (9, 25, 27). On the other hand, cotransfection of CRLR with RAMP2 and RAMP3 resulted in ADM receptors and mixed-type ADM/CGRP receptors, respectively (9, 13, 25, 27). In the present study, we examined the role of endogenous RAMP1 in the protective regulation of PPA. We found that RAMP1 is essential in regulation of pulmonary circulation in hypoxia when basal tone is elevated, but not in normoxia. This is the first in vivo report on the physiological function of RAMPs in the whole animal.

The canine orphan receptor RDC-1 was originally cloned from dog thyroid cDNA using a PCR-based method with degenerate primers that correspond to consensus sequences of transmembrane domains 3 and 6 of other G protein-coupled receptors (23) and was later identified as a CGRP1 receptor using COS-7 cells transfected with RDC-1. These cells showed a dose-dependent increase of cAMP in response to rat {alpha}-CGRP in a CGRP8–37-sensitive manner, and high affinity of this receptor for CGRP and CGRP8–37 was confirmed by ligand-binding studies (15). RDC-1 was thus concluded to be a CGRP1 receptor. However, some recent reports questioned this view. For example, the expression of RDC-1 in Xenopus oocytes and HEK-293T cells did not induce binding or the cAMP response to CGRP (25). RDC-1 mRNA levels in eight rat tissues did not correlate with CGRP binding capacity (R = 0.09, P = 0.82) (7). The distribution of RDC-1 mRNA in the brain by in situ hybridization did not match that of CGRP receptor binding sites (14). Moreover, the complete loss of mRNA for RDC-1 induced by ANG II or antisense oligonucleotides did not affect CGRP-evoked increase in cAMP formation (11).

We previously reported hypoxia-induced upregulation of RDC-1 mRNA in rat lung (29), suggesting that RDC-1 may play a role in regulation of the pulmonary circulation. In the present study, treatment of ASODN targeting RDC-1 mRNA resulted in elevated PPA in chronic hypoxic rats, supporting its functional role in regulation of PPA in hypoxia. However, the mechanism of this regulation is unknown, and many questions remain.

It is surprising that CGRP ASODN increased mean PPA in normoxic and in acute hypoxic rats, whereas all other ASODNs only did so in chronic hypoxic rats even though identical or higher reduction levels of specific mRNA were achieved by ASODN in normoxic rats. This could suggest that multiple receptors exist for CGRP ligand. If the functions of these receptors are redundant, blocking either one of them separately may not affect CGRP's vasodilatory action in normoxia when basal tone is normal because the amount of CGRP receptors exceeds the density needed. However, when basal vascular tone is elevated, e.g., during hypoxia, more free CGRP receptors may be needed. Hence, blocking receptor expression could result in higher PPA effects. Also, many infused exogenous pulmonary vasodilators, including CGRP, exert vasodilatory actions under conditions of high vascular tone only. Thus CGRP/CGRP receptor-induced vasodilation could be more effective during hypoxia, when pulmonary vascular tone is elevated. We further speculate that the disparity in functional efficacy between different ASODNs could also be explained by somewhat different properties of these ligands/receptors, or it could be a dose effect due to different efficiency of individual ASODNs. Moreover, the increase of basal vascular tone by CGRP antisense treatment could account for the potentiation of hypoxic pulmonary vasoconstriction in these normoxic rats when acutely exposed to hypoxia. In addition, ~25–30% reduction of RDC-1 mRNA was noticed in both high-dose RDC-1 antisense-treated normoxic and hypoxic groups, but their net changes in amounts could be different due to the documented upregulation of mRNA expression on hypoxia (29).

The major principle of ASODN application is that ASODN-stimulated RNase H can digest the mRNA strand of the mRNA-ASODN hybrid (8, 26, 37). However, ASODN may also act through other mechanisms. For example, ASODN-mRNA hybrid formation causes a steric or conformational obstacle for ribosome from reading through and prevents protein translation. ASODN may also bind to specific sites of genomic DNA and mRNA to prevent transcription, mRNA splicing, and mRNA transport out of the nucleus to the cytoplasm. No matter what mechanism(s) ASODN acts through, it is the change in protein that needs to be measured because the main goal of using ASODN is a reduction in specific protein (28). However, it is difficult to detect CGRP in rat lung by Western blot analysis (data not shown) due to the low expression level of CGRP in the lung. In the present study, we thus used immunocytochemistry to demonstrate the expected CGRP ASODN-induced reduction of CGRP peptide. Unfortunately, there are currently no antibodies against RAMP1 and RDC-1 proteins. Therefore, semiquantitative RT-PCR was used to test the effectiveness of the RAMP1 and RDC-1 ASODNs. Both immunocytochemistry and semiquantitative RT-PCR confirmed the effectiveness of the ASODNs used in the present study. Although the changes in target gene expression, as assessed by RT-PCR, appeared to be quite small, this much inhibition may be sufficient to produce specific physiological effects. In many cases, the reduction in a specific protein in response to antisense treatment in vivo tends to be relatively slight (~20%) due to low uptake efficiency, but significant physiological changes have been detected (28). mRNA levels of RAMP1 and RDC-1 were not significantly reduced by their specific ASODNs in hypoxic rat lung at the dose of 5 mg·kg-1·day-1, but PPA was significantly elevated in these rats, suggesting reduction of protein. The undetected change in specific mRNAs in hypoxic lung may be related to their upregulation after hypoxia, which makes the decrease relatively small. The unchanged mRNA levels may also be in part because tissue samples were assayed that include cells that were not reached or affected by the ASODN, and thus the mRNA inhibitory effects of ASODN administration are likely masked by the unchanged or even increased amount of targeted mRNA in those cells that were not affected, or because the RT-PCR may simply not be sensitive enough to detect small changes in mRNA. It is also possible that ASODN may act through preventing translation without reducing mRNA as mentioned above.

Due to the rapid degradation of the oligodeoxyribonucleotides in their natural form in blood and cells by exo- and endonucleases, we used phosphorothioated oligos to prolong their activity. One potential problem caused by phosphorothioation is that phosphorothioate oligodeoxyribonucleotides, especially those with four repeated guanine residues, tend to bind to proteins nonspecifically (33), and the nonspecific DNA-protein binding may result in some biological effects (5). Therefore, the guanine quartet sequences were avoided in our ASODN and MMODN, and more importantly, for each specific ASODN, we used a MMODN as control. In the present study, only the ASODNs produced an increase in PPA and a reduction in specific protein/mRNA, whereas the MMODNs were ineffective, indicating that the PPA effects were caused by antisense inhibition of the target gene expression, not by nonspecific action of phosphorothioate oligodeoxyribonucleotides.

In conclusion, results of our study demonstrated that in vivo treatments of ASODNs for CGRP, RAMP1, and RDC-1 elevate mean PPA in chronic hypoxic rats and that CGRP ASODN also causes higher mean PPA in normoxic and in acute hypoxic rats. These data confirm CGRP's protective role in the pulmonary circulation and suggest that endogenous RAMP1 and RDC-1 are essential in regulation of PPA in hypoxia.


    ACKNOWLEDGMENTS
 
We thank Dr. Mark Brownfield for technical assistance and the Biotechnology Center, University of Wisconsin-Madison for primer synthesis and sequence analysis. Hypobaric hypoxic exposures utilized the Biotron (University of Wisconsin-Madison).

This work was supported by a grant from the American Lung Association.


    FOOTNOTES
 

Address for reprint requests and other correspondence: I. M. Keith, Dept. of Comparative Biosciences, Univ. of Wisconsin-Madison, School of Veterinary Medicine, AHABS Bldg., 1656 Linden Drive, 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.


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