Acta Physiologica Sinica, June 25, 2003, 55(3): 260－264
Received 2002-10-08 Accepted 2003-01-14
This work was supported by the National Basic Research Priorities Programme of China (G2000056905) and the National Natural Science Foundation of China (30170347).
Corresponding author. Tel: +86-10-62092183; E-mail: tangchaoshu@263.net.cn
Research Paper
Levels of
adrenomedullin and proadrenomedullin N-terminal 20 peptide in myocardium and
aorta of spontaneously hypertensive rats and Wistar-Kyoto rats
QI Yong-Fen1,2, BU Ding-Fang1, SHI Yan-Rong1, LI Ju-Xiang2, PANG Yong-Zheng1,
TANG Chao-Shu1,2，*
1Institute of Cardiovascular Disease, Peking University First Hospital, Beijing 100034;
2Department of Physiology, Peking University Health Science Center, Beijing 100083
Abstract: In this study, we observed the levels of adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) in myocardium and aorta of spontaneously hypertensive rats (SHRs) in comparison with Wistar-kyoto (WKY) rats. Contents of ADM and PAMP were measured by radioimmunoassay (RIA) in plasma, myocardium and aorta. The amount of Pro-ADM mRNA of myocardium and aorta was determined by competitive quantitative reverse transcription polymerase chain reaction (RT-PCR). In SHRs the amounts of Pro-ADM mRNA of myocardium and aorta were 66.7% (P<0.01) and 73% (P<0.01) higher than those in WKY rat, respectively. In SHRs, the levels of ADM in plasma, myocardium and aorta were 29%, 76.7% and 79% (all P<0.01) higher than those in WKY rats, respectively. The level of PAMP in SHRs was increased by 42.5% in plasma (P<0.01), 47.2% in myocardium (P<0.0.1) and 27.3% in aorta (P<0.05) compared to WKY rats, respectively. In addition, the ratio of ADM content to PAMP content in SHRs group was increased compared with that in WKY group (2.0±0.25 vs 1.64±0.3 and 2.2±0.18 vs 1.56±0.28, in myocardium and aorta, respectively, P<0.01). These results suggest that ProADM gene expression is up-regulated and the increase in ADM and PAMP is different in SHRs. The significance of inconsistency of increase in ADM and PAMP in SHRs needs to be further investigated.
Key words: pathophysiology; adrenomedullin; proadrenomedullin N terminal 20 peptide (PAMP); spontaneously hypertensive rat; mRNA
自发性高血压大鼠和Wistar-Kyoto大鼠心血管组织肾上腺髓质素和肾上腺髓质素原N-末端20肽的水平
齐永芬1,2, 卜定方1, 石彦荣1, 李菊香2, 庞永正1, 唐朝枢1,2,*
1北京大学第一医院心血管病研究所, 北京 100034; 2北京大学医学部生理学系, 北京 100083
摘要: 本工作观察了自发性高血压大鼠(SHRs)和Wistar-kyoto (WKY)大鼠心肌和主动脉肾上腺髓质素(adrenomedullin, ADM)和肾上腺髓质素原N-末端20肽(proadrenomedullin N terminal 20 peptide, PAMP)的水平。以放射免疫分析方法测定血浆、 心肌和主动脉ADM含量。用竞争性定量逆转录多聚酶链式反应(RT-PCR)方法测定心肌和主动脉ProADM mRNA含量。结果发现, SHRs心肌和主动脉ProADM mRNA水平分别比WKY大鼠高66.7% 和73%(均P<0.01)。SHRs血浆、心肌和主动脉ADM-ir含量分别较WKY大鼠高29%、76.7%和79%(均P<0.01)。SHRs血浆、心肌和主动脉PAMP-ir水平分别较WKY大鼠高42.5%(P<0.01)、47.2%(P<0.01)和27.3%(P<0.05)。另外, SHRs 的ADM 和PAMP的比值较WKY大鼠明显增高(心肌和主动脉分别为2.0±0.25 vs 1.64±0.3和 2.2±0.18 vs 1.56±0.28)。结果提示, SHRs心肌和主动脉ProADM基因表达上调, ADM和PAMP水平升高, 但二者升高的比例不一致。SHRs 的ADM和PAMP升高不一致的病理生理意义有待进一步研究。
关键词: 病理生理学; 肾上腺髓质素; 肾上腺髓质素原N末端20肽; 自发性高血压大鼠; mRNA
中图分类号: Q463
Adrenomedullin (ADM), a novel cardiovascular-active peptide, was first isolated from a human pheochromocytoma by Kitamura et al. in 1993[1]. Human adrenomedullin has 52 amino acid residues, with a single disulfide bond between residues 16 and 21 and an amidated tyrosine near the carboxyl terminus. Rat adrenomedullin consists of 50 amino acids, in which only 6 amino acids are different from those of the human peptide. ADM exists not only in adrenal gland but also in vascular endothelial cells, vascular smooth muscle cells (VSMCs), fibroblasts, phagocytes, osteoblasts, and nerve cells[2]. ADM is a potent vasodilator having hypotensive, natriuretic and diuretic actions, and it is also an inhibitor of VSMC proliferation. ADM is derived from a precursor molecule preproadrenomedullin (Prepro-ADM), which is composed of 185 amino acid residues. Proadrenomedullin (Pro-ADM) is formed after removal of the signal peptide of 21 amino acid residues at the N-terminal of Prepro-ADM during the transport of the molecule across cell membrane. ProADM is cleaved into different active fragments at different cleavage sites by proteases. ADM is a PreproADM95-146 fragment; PAMP (proadrenomedullin N-terminal 20 peptide, PreproADM22-41, PAMP) is a weak vasodilator and an inhibitor of the VSMC proliferation, and an inhibitor of catecholamine releasing[3, 4]. Recent studies show that different fragments from the same peptide precursor have different biological effects as relatively independent peptides, and functions in vivo of these peptides are modified and regulated by each other[5]. It was reported that ADM and PAMP were increased in hypertension and heart failure[6,7]. However, their complicated interactions and variations are not yet clearly elucidated under disease condition. Here we report the changes in ADM and PAMP contents in plasma, myocardium and aorta, ProADM mRNA level in myocardium and aorta, using spontaneously hypertensive rats (SHRs) to explore the interactions of different peptides. Our results show that PAMP and ADM contents increase in plasma, myocardium and aorta, and ProADM mRNA expression is upregulated in SHRs, in comparison with those in the control group Wistar-kyoto (WKY) rats. It is interesting that the ratio of ADM content to PAMP content was increased in SHR group. The results indicate that during hypertension there is not only change in ProADM gene expression, but also alteration in interactive relationship among ProADM-derived peptide fragments.
1 MATERIALS AND METHODS
1.1 Materials. SHRs and WKY (n=6, each) rats were obtained from the Experimental Animals Center at Fu Wai Hospital. Animals were treated following the Guidlines of Animal Experiments from the Committee of Medical Ethics Experimental Animal Center of Peking University Health Science Center. Radioimmunoassay (RIA) kits for ADM and PAMP were purchased from Phoenix Pharmaceutical Inc. Trizol was obtained from GIBCO BRL. dNTPs, M-MuLV reverse transcriptase, Oligo (dT) 15 primer and Taq DNA polymerase were provided by Promega Co. Oligonucleotide primers, ADM-S: 5'-CTCGACACTTCCTCGCAGTT, and ADM-A: 5'-GCTGGAGCTGAGTGTGTCTG used for quantitative PCR, ADM-T: 5'-AGTGTGTCTGCCTTGAGGGCTGATCTTGTT used for making internal competitive standard, and β-actin-S: 5'-ATCTGGCACCACACCTTC and β-actin-A: 5'-AGCCAGGTCCAGACGCA used for sample loading calibration were synthesized by SBS Co.
1.2 Experimental protocol. SHRs and WKY rats, weighing 250－300 g, were fed with ordinary food and allowed to drink freely pre-experiment. Blood pressure of rats was measured by tail cuff methods. Rats were anesthetized with urethane (1 g/kg, i.p.), and blood samples were drawn from the abdominal aorta using tubes containing 1 mg/ml of EDTA-2Na and 500 kallikrein inhibitory units (KIU)/ml of aprotinin. Plasma was obtained by centrifugation at 3000 r/min for 10 min at 4℃ and stored at －70℃ until use. Heart was removed and weighed. Aorta was removed. 100 mg of the left ventricle and aorta were acidified with 1 mol/L acetic acid, boiled for 10 min and homogenized, then the supernatant was stored at －70℃ until use for RIA. The left ventricle and aorta from another half of rats were taken for RNA extraction.
1.3 Preparation of internal competitive standard for quantitative PCR[8,9]. PCR was performed using primers ProADM-S and ProADM-A and a cDNA template transcribed from total RNA of rat adrenal gland, which yielded a 446 bp fragment of ProADM cDNA. For making the internal competitive standard, rat adrenal gland cDNA was first amplified using primers ProADM-S and ProADM-T, then the PCR product was reamplified using ProADM-S and ProADM-A as the primers. This competitor had the same sequence as the 446 bp fragment, except a fragment of 70 bp upstream the site of ProADM-A primer was deleted. The 446 bp fragment and the competitor were cloned into pBluescript (SK+) vector. The plasmids were purified, linearized, and quantitated by spectrophotometry.
1.4 Measurement of ProADM mRNA[9,10]. Total RNA from myocardium and aorta was extracted by using the Trizol reagent. 1 μg of total RNA was reverse-transcribed into single strand cDNA using oligo(dT)15 primer and M-MuLV reverse transcriptase. Quantitative PCR was performed according to our previous report[9]. Using this method we obtained two bands (446 bp and 376 bp). PCR product was separated in a 1.5% agarose gel, and stained with ethidine bromide. Ratio of optical density of the two bands (446 bp and 376 bp) was measured by using the Gel Documentation System. Amplification of ProADM cDNA and the internal competitive standard was confirmed by digestion of the PCR products with restriction enzyme Bgl II. A standard curve of the ratio was drawn using the same conditions as described above, except that left ventricle and aorta cDNA were replaced by a series of dilution of the plasmid containing the 446 bp DNA fragment[11]. The amount of ProADM mRNA in the sample was then obtained from the standard curve. To calibrate the sample loaded in quantitative PCR, β-actin cDNA was determined at the same time. Calibrated amount of ADM mRNA was used for further analysis.
1.5 RIA of ADM and PAMP[12]． The extracts of left ventricle and aorta were loaded onto a Sep-Pak C18 cartridge equilibrated with saline. After the cartridge was washed with 2.5 ml of saline and 10% of acetonitrile in 0.1% trifluoroacetic acid (TFA), the absorbed material was eluted with 2 ml of 50% acetonitrile in 0.1% TFA. The elution was lyophilized and subjected to RIA for ADM and PAMP. The RIA incubation buffer for ADM or PAMP was 0.05 mol/L sodium phosphate buffer (pH 7.4), containing 0.5% bovine serum albumin, 1% Triton X-100, 0.08 mol/L NaCl, 0.025 mol/L EDTA-2Na, 0.05% NaN3 and 500 KIU/ml of aprotinin. The RIA was performed according to the protocol for ADM or PAMP radioimmunoassay kits. The IC50 of ADM was 25.6 pg/tube, and the cross-reactivity with rat ADM was 100%. And the cross-reactivity with prodrenomedullin N terminal 20 peptide, amylin and endothelin was 0. The IC50 of PAMP was 20－54 pg/tube, and the cross-reactivity with rat PAMP was 100%. And the cross-reactivity with ADM, amylin was 0.
1.6 Statistical analysis． Data are expressed as mean±SD. Student's t test was used to test the overall statistical significance, and P<0.05 was considered to be significant.
2 RESULTS
2.1 Measurement of blood pressure and heart weight in SHRs and WKY rats
In SHRs the systolic pressure was twice higher than that in WKY rats (28.8±1.6 vs 14.4±1.12 kPa, P<0.01). The ratio of heart weight to body weight (heart coefficient) in SHRs was 41.4% (4.51±0.39 vs 3.19±0.36 mg/g, P< 0.01) higher than that of WKY rats.
2.2 ProADM mRNA levels in left ventricle and aorta
To eliminate the errors from variation of exponential amplification and the plateau effect inherited in PCR, and to obtain relatively accurate and reproducible results from a tiny amount of samples, the competitive quantitative RT-PCR by adding an internal competitive standard into the PCR system was used in this study. The results showed that ProADM mRNA content in SHRs was increased by 66.7% (P<0.01) in myocardium, and 73% (P<0.01) in the aorta, respectively, compared with that in WKY rats (Fig.1).
Fig.1.Changes in relative amount of ProADM cDNA in SHRs. Data are the mean±SD, n=6; **P<0.01, compared with control. SHRs, spontaneously hypertensive rats. ADM, adrenomedullin. ProADM, proadrenomedullin.
2.3 ir-ADM and ir-PAMP assay in plasma, myocardium and aorta
In SHRs, the contents of ADM in plasma, myocardium and aorta were 25%, 50% and 140% (all P<0.01) higher than those of WKY rats, respectively. The levels of PAMP in plasma, myocardium and aorta were 53% (P<0.01), 44% (P<0.01) and 42% (P<0.05) higher than those of WKY rats, respectively. Compared with those of the WKY rats, both plasma ADM and PAMP contents were elevated in SHRs, however, the ratio of plasma ADM to plasma PAMP was not significantly altered (1.19±0.26 vs 1.29±0.15, P>0.05). Both ADM and PAMP contents in SHRs were elevated in myocardium and aorta, but the levels of their elevation were inconsistent. The values of ADM content to PAMP content ratio in myocardium and aorta were significantly increased in comparison with those of WKY rats. In addition, there was a significant correlation between PAMP content and ADM content. The correlation coefficients were 0.71 for plasma, (P<0.05), 0.89 for myocardium, (P<0.01) and 0.82 for aorta (P<0.01, see Table 1).
Table １.Changes in ADM and PAMP of myocardium and aorta of SHRs
|
|
|
ADM |
PAMP |
ADM/PAMP |
|
Plasma (pmol/L) |
WKY |
0.51±0.092 |
0.4±0.03 |
1.28±0.15 |
|
|
SHRs |
0.66±0.07** |
0.57±0.11** |
1.19±0.26 |
|
Myocardium (fmol/mg Pro) |
WKY |
0.60±0.13 |
0.36±0.035 |
1.64±0.30 |
|
|
SHRs |
1.06±0.087** |
0.53±0.038** |
1.99±0.25 |
|
Aorta (fmol/mg Pro) |
WKY |
0.86±0.16 |
0.55±0.069 |
1.56±0.28 |
|
|
SHRs |
1.54±0.13** |
0.70±0.089* |
2.2±0.18** |
P<0.05, **P<0.01, compared with control. ADM, adrenomedullin; PAMP, proadrenomedullin N-terminal 20 peptide.
3 DISCUSSION
Both ADM and PAMP are derived from a common precursor ProADM and they play different roles in physiological processes and exist as an independent factor. ADM is a potent vasodilator having hypotensive, natriuretic and diuretic actions, and it also inhibits the proliferation of VSMCs[13]. PAMP is a weak vasodilator and can inhibit the proliferation of VSMCs[3] and the release of catecholamine. Synergistic effect of ADM and PAMP on vascular dilatation was found that co-administration of ADM and PAMP resulted in a severe hypotension. However, PAMP antagonizes certain biological effects of ADM. Moody et al.[14] observed on the teratocarcinoma cells that ADM elevated cellular cAMP content, stimulat-ed transiently c-fos mRNA expression and [3H]-thymidine incorporation. Whereas PAMP inhibited the above mentioned effects of ADM. A lot of clinical and experimental data showed that plasma ADM and PAMP levels increased obviously during hypertension, myocardial ischemia and heart failure, and this was thought to be an important compensatory mechanism in cardiovascular diseases.
In the present study it was found that SHRs had higher blood pressure, hypertrophic heart, and elevated levels of ADM and PAMP in plasma, myocardium and aorta, which is in accordance with the results reported in literature. In addition, the elevation of ADM and PAMP contents was parallel to the increase in ProADM mRNA levels in SHR myocardium and aorta. The results indicate that during hypertension the increase in cardiovascular ADM and PAMP contents is possibly mediated through the overexpression of ProADM gene.
Several binding sites for activator protein-2 (AP-2) and cAMP-regulated elements were found in the 5' regulatory region of ProADM gene, which is located in chromosome 11 in humans[13]. It was reported that induction of AP-2, secondary to activation of phospholipase C and protein kinase C, might be the mechanism by which growth factors such as fibroblast growth factor and platelet and epidermal-derived growth factor stimulate the transcription of ADM gene in VSMCs[13]. In disease conditions including hypoxia, hypertension and heart failure, ADM gene is upregulated[15－18]. Recently, nuclear factor-κB (NF-κB) sites on the promoter of the ProADM gene were found. Pro-ADM transcription was activated through NF-κB pathway by cytokines, shear stress, hypoxia and ischemia[13]. In hypertension the relationship between ProADM expression and cAMP-regulated element, and the NF-κB pathways needs to be further investigated.
It is interesting that the ratio of ADM to PAMP is variable. In WKY rats the ratio of ADM to PAMP is 1.29±0.15, 1.64±0.3 and 1.56±0.28 in plasma, myocardium and aorta, respectively. The ratio of ADM to PAMP in myocardium and aorta was increased (21.9% P>0.05, and 42.3%, P<0.01, respectively). The present study indicates that hypertension maintains a higher ADM /PAPM ratio which is possibly correlated with endogenous peptidase activities. The increased level of ADM and PAMP in SHRs might play an important role in protecting the cardiovascular system. Administration of exogenous ADM or transfer of ADM gene attenuated experimental hypertension, myocardial infarction and heart failure and vascular remodeling[19]. However, in the mice in which the entire ADM gene was deleted, the homozygous knockout proved lethal at the embryonic stage, which indicates the importance of ADM in the development of cardiovascular system. In addition, binding of receptor with ligand is an important way to clear active peptides in tissues. It needs to be further investigated that whether or not the clearance of PAMP is more effective than that of ADM due to the binding of these two peptides with their respective receptors.
Many researches showed that high ADM and PAMP production occurred in certain diseases[15－18]. Treatment of hypertensive rats with administration of exogenous ADM gene has a new clinical prospect[20]. Our research shows that the level of different enzymatic peptides of ProADM is inconsistent. Proadrenomedullin is further cleaved by endogenous peptidases, resulting in the production of four important peptides. Besides ADM and PAMP, ADT (adrenotensin, PreproADM153-185) causes vasoconstriction, elevation of blood pressure and VSMC proliferation[21]. PreproADM45-92 is a weak vasodilator. Because these different peptides from the same precursor have different biological effects, the significance of the changes in the ratio of these peptides in hypertension needs further investigation.
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