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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:41-49 (2007)
© 2007 The Gerontological Society of America

Age-Related Changes in Adrenomedullin Expression and Hypoxia-Inducible Factor-1 Activity in the Rat Lung and Their Responses to Hypoxia

Isabel S. S. Hwang, Man Lung Fung, Emily C. Liong, George L. Tipoe and Fai Tang

Departments of 1 Physiology and 2 Anatomy, and 3 the Research Centre of Heart, Brain, Hormone and Healthy Aging, Faculty of Medicine, the University of Hong Kong, Pokfulam, China.

Address correspondence to Fai Tang, PhD, Department of Physiology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR. E-mail: ftang{at}hkucc.hku.hk


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Male rats aged 3 months, 12 months and 20 months were subjected to breathing 8% oxygen for 6 hours. Lung preproadrenomedullin (AM) messenger RNA (mRNA) levels were measured by solution hybridization-RNase protection assay while AM was measured by radioimmunoassay. The binding of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) to DNA was determined by electrophoretic mobility shift. There was an age-related increase in basal levels of preproAM mRNA and AM and of the binding of hypoxia-inducible factor (HIF) to DNA. Upon hypoxic stimulation, HIF binding to DNA increased in the young and middle-aged rats, but not in the old rats. AM gene expression increased in response to hypoxia in rats of all ages, but the increase was much less in the old rats. AM peptide levels in the lung decreased with age in hypoxia. In a separate experiment, male rats aged 3 months and 20 months were subjected to hypoxia as described above. PreproAM, calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) mRNA, HIF-1 and peptidyl-glycine-amidating monooxygenase (PAM) mRNA levels were measured by reverse transcription–polymerase chain reaction. All except PAM showed a decrease in basal levels and a diminished response to hypoxia in the old rats. Polysome profiling demonstrated decreases in the percentages of translatable preproAM mRNA in response to hypoxia, with a greater decrease in the old than the young rats. It is concluded that an age-dependent decrease in the hypoxic response of the AM system in the lung was associated with high basal levels of HIF activity and AM expression in the old rats, and a lower proportion of translatable preproAM mRNA in the old rats in response to hypoxia. Thus, the HIF-AM pathway may be impaired in the aged lung, and other mechanisms may be present to maintain an AM response to hypoxia.

Hypoxia is known to stimulate adrenomedullin (AM) gene expression in a variety of tissues in culture (1–3), and this stimulation is mediated by the binding of hypoxia-inducible factor (HIF) to the promotor region of AM (1). HIF-1 is overexpressed under hypoxic conditions (4). In the rat, acute hypoxia (1% of O2 for 6 hours) has been reported to increase AM and AM receptor gene expression in a number of organs, including the lung (5).

One of the hallmarks of aging is the decrease in the ability to adapt to or cope with adverse conditions. For instance, the tolerance to ischemia and hypoxia is reduced in aged human myocardium (6). Aged persons are more prone to the development of a multitude of chronic diseases such as chronic obstructive pulmonary disease, which may lead to different extents of hypoxia. In chronic obstructive pulmonary disease, plasma AM level was found to be elevated (7). Even in apparently healthy elderly persons, there are decreases in air flow and in the exchange surface area and compliance of the lung compared with young persons (8), and some mechanisms may have been activated to compensate for these decreases. The response in the AM system may be one of these mechanisms. In old rats, the serotonin-dependent augmentation of respiratory motor output is reduced (9).

AM is important for its effects on vasodilation, which increases blood flow to the lung (10), and bronchodilation (11), which increases ventilation. We hypothesize that there may be a compensatory increase in AM gene expression in old rats, and this elevated basal state may compromise their subsequent response to hypoxia. Acute hypoxia has resulted in an increase in AM gene expression and level in the rat lung (5). However, the effect of aging on lung AM gene expression has never been reported, let alone the response to hypoxia. If our hypothesis is correct, there should be an increase in basal AM gene expression in the old rats and a decrease in response to hypoxia. We report here the results of our study on the effect of acute hypoxia in rats of 3 ages: 3 months (young), 12 months (middle-aged), and 20 months (old).


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animal Studies
All procedures were in accordance with the rules and regulation as set down by the Committee for the Use of Animals for Teaching and Research, Faculty of Medicine, the University of Hong Kong. Male Sprague-Dawley rats aged 3 months, 12 months, or 20 months were obtained from the Laboratory Animal Unit, Faculty of Medicine, the University of Hong Kong and were subjected to hypoxia from breathing 8% O2 for 6 hours in a 300-liter acrylic chamber with free access to water and chow. As reported previously (12), the oxygen fraction inside the chamber was established by a mixture of room air and nitrogen, which was regulated by an oxygen analyzer (Vacumetrics Inc., Ventura, CA). Carbon dioxide was absorbed by soda lime granules, and excess humidity was removed by a desiccator. The rats were immediately used in experiments after being taken out of the chamber. For the normoxic control, age-matched rats were kept in room air in the same room. All rats were killed by decapitation from 3:30 to 5:30 PM, and plasma samples were collected into chilled tubes containing aprotinin (500 KIU/mL blood) and EDTA (1 mg/mL blood) as described previously (13). The lungs were dissected out and cut into small blocks before freezing in liquid nitrogen for storage at –70°C. In a second experiment, 5 young (3-month-old) and 4 old (20-month-old) rats were subjected to hypoxia as described above while a similar number of rats served as control. Lung tissues were obtained for reverse transcription–polymerase chain reaction (RT–PCR) and polysome analysis.

Measurement of Plasma AM Level by Radioimmunoassay
Plasma AM was extracted by passage through pre-equilibrated C18 Sep columns, as described previously (13). In brief, after purification by Sep columns, the peptide was eluted with 60% high-performance liquid chromatography (HPLC) grade acetonitrile in 1% trifluoroacetic acid (TFA), dried in a speed-vacuum concentrator (Savant, Farmingdale, NY), reconstituted in 0.5 mL of radioimmunoassay buffer, and further purified with a nanospin column (molecular weight cutoff point, 10 kd; Millipore Corporation, Bedford, MA). Duplicate samples of AM standards (0–500 pg/100 µL) and samples were incubated at 4°C with 50 µL of 125I-AM (10,000 c.p.m.) and 100 µL of AM antiserum (Peninsula Laboratories, Belmont, CA) (13,14). After incubation overnight for 18 hours, 100 µL of normal rabbit gamma globulin (1:100 dilution; Antibodies Inc., Davis, CA), goat anti-rabbit serum (1:10 dilution; Antibodies Inc.), and 10% polyethylene glycol were added. The supernatants were aspirated after centrifugation at 1800 x g for 1 hour, and the pellets were counted in a gamma counter (Cobra II Auto-Gamma Counting System; Packard, Meriden, CT). The sensitivity of the assay was between 1 and 5 pg/tube.

Extraction and Assay of AM in Lung
Frozen lung tissues were extracted in 2 N acetic acid and boiled in a water bath for 10 minutes to inactivate proteases. Aliquots (50 µL) were taken for protein determination. The remaining homogenates were lyophilized and stored at –20°C. The extracts were later reconstituted in radioimmunoassay buffer and subjected to radioimmunoassay as described above.

Protein Determination
A 50 µL aliquot of tissue homogenate or standard (bovine serum albumin [BSA]) was boiled with 1 N sodium hydroxide for 10 minutes. After chilling on ice, 50 µL of the boiled sample was mixed with 2.5 mL of Bio-Rad protein assay reagent (Hercules, CA) for 10 minutes, and measured with a spectrophotometer (LKB Ultraspec II; Biochrom, Cambridge, U.K.) at 595 nm. The concentration of AM was then calculated and expressed as femtomoles per milligram of protein.

Quantification of Preproadrenomedullin Messenger RNA by Solution–Hybridization RNase Protection Assay
Total RNA was extracted from lung samples by TRIZOL. The details of the extraction method and hybridization assay have been described in more detail (14,15). Plasmid preproAM complementary DNA (cDNA) and ß-actin cDNA (gifts from Dr Dominic Autelitano, Baker Medical Research Institute, Prahran, Australia) were transformed into Escherichia coli JM109. Plasmid DNAs were linearized with restriction enzymes (For preproAM: EcoRI for synthesis of probe and BAMH1 for standard; For ß-actin: EcoRI for probe and HindIII for standard). The standard RNA and the riboprobes were synthesized using polymerases (For preproAM: T7 for probe and SP6 for standard; For ß-actin: SP6 for probe and T7 for standard). RNA tissue samples (5 µg) or standards (1–50 pg) were incubated with 100,000 c.p.m. of 32P-labeled AM RNA riboprobe or 32P-labeled ß-actin RNA probe in hybridization buffer. The reaction mixture was denatured at 85°C for 5 minutes and then incubated at 45°C overnight. After digestion by proteinase K, the hybrids were extracted with a phenol/chloroform mixture and precipitated with isopropanol. The pellets were loaded onto a 4% polyacrylamide gel (19:1 Bis), and the gel was run for 1 hour at 4°C, then vacuum dried. The hybrid bands were visualized by exposing to film (RX AIF 2025 cm; FUJIFILM, Japan) in a cassette with intensifying screens at –70°C for 48 hours. With the radiographic film as a template, the hybrid bands on the gel were cut out and their radioactivities counted with a liquid scintillation counter (Tri-carb 2000AC; Packard). The amount of preproAM messenger RNA (mRNA) and ß-actin mRNA in various tissues was determined by a standard curve. The results are presented as picograms of preproAM mRNA per picogram of actin mRNA.

Nuclear Protein Extraction and DNA Electrophoretic Mobility Gel Shift Assay
Nuclear protein extraction was conducted by using the frozen lung samples. As described previously (16), nuclear proteins were extracted from 100 mg of lung tissue by homogenization in 500 µL of buffer 1 (250 mM sucrose, 15 mM NaCl, 5 mM EDTA, 1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 1 mM dithiothreitol, 15 mM Tris-HCl pH 7.9, 60 mM KCl). All buffers contained protease inhibitors (0.5 µL of leupeptin, aprotinin; 2.5 µL of phenylmethylsulfonyl fluoride (PMSF) to prevent protein degradation. The homogenate was centrifuged at 3100 x g for 10 minutes at 4°C. The pellet was resuspended in 500 µL of Buffer 2 (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl). The suspension was centrifuged at 4500 x g for 10 minutes at 4°C, and the pellet was resuspended in 400 µL of Buffer 3 (0.5 mM HEPES pH 7.9, 0.75 mM MgCl2, 0.5 mM EDTA, 0.5 M KCl, 12.5% glycerol, 0.1% Nonidet). The suspension was rocked for 30 minutes and then centrifuged at 21,000 x g for 30 minutes at 4°C. The supernatant was collected and dialyzed against Buffer 4 (10 mM Tris-HCl pH 7.9, 5 mM MgCl2, 1 mM EDTA, 10 mM KCl, 20% glycerol, 0.1% Nonidet) overnight inside a cold room. The dialyzed supernatant containing the nuclear proteins was frozen at –70°C for subsequent use. The protein concentration was determined using the standard bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL). The probe for detection of specific nuclear proteins DNA binding for HIF-1 was 50 nmol desalted nucleotides sequence with HIF-1 binding site 5'-GCCCTACGTGCTGTCTCA-3' (Invitrogen Hong Kong Ltd., Life Technologies, Hong Kong, China). Phosphorylation reaction of this oligonucleotide DNA primer was performed by mixing 2 µL of consensus oligo, 1 µL of T4 polynucleotide kinase 10X buffer, 1 µL of [{gamma}-32p]ATP (3000 Ci/mmol at 10 mCi/mL), 5 µL of nuclease-free water, and T4 polynucleotide kinase (5–10 U/µL) at 37°C for 10 minutes; the reaction was stopped by adding 1 µL of 0.5 M EDTA. The labeled oligonucleotide was separated from the unincorporated nucleotides by chromatography through a G-25 spin column equilibrated in Tris-EDTA(TE) buffer. For nuclear proteins and DNA interaction, 24 µg of nuclear protein extract/20 µL is mixed with 3 µL of 10X reaction buffer, 2 µL of single-strand DNA, and 1 µL of labeled oligomer. The mixture was incubated for 20 minutes at room temperature and then applied to a 7% acrylamide nondenaturing gel. Loading dye (2 µL) was used as a negative control. The DNA–protein complexes were subjected to electrophoresis at room temperature in 0.5X Tris–Borate–EDTA (TBE) buffer at 100V for approximately 3 hours. The gel was applied to a filter paper and dried at 50°C for 30 minutes. Autoradiography was carried out overnight at –70°C.

RT–PCR of mRNAs of PreproAM, Calcitonin Receptor-Like Receptor, Receptor Activity Modifying Proteins, HIF-1, and Peptidyl-Glycine Alpha-Amidating Monooxygenase
The semiquantitative RT–PCR method (15,17) was used to determine the mRNA levels of the preproAM, calcitonin receptor-like receptor (CRLR), receptor activity modifying proteins (RAMPs), HIF-1{alpha}, and peptidyl-glycine alpha-amidating monooxygenase (PAM) in the lung samples. In brief, total RNA (5 µg) was transcribed into cDNA with Superscript II reverse transcriptase (Life Technologies) according to the manufacturer's instructions. After RT, the mRNA levels of PreproAM, CRLR, RAMP1–3, HIF-1{alpha}, and PAM were measured by semiquantitative PCR with the specific forward and reverse primers as shown in Table 1. ß-actin was used as an internal control for normalization of the cDNA level on an MJ Research thermal cycler (MJ Research, La Jolla, CA). The PCR amplification profile for PreproAM and ß-actin was activation for 15 minutes at 94°C followed by 32 and 28 cycles of PCR using denaturation at 94°C for 1 minute, annealing at 56°C and 59°C for 1 minute, and extension at 72°C for 1 minute, whereas that of CRLR and RAMP1–3 consisted of 30 cycles of PCR with annealing temperature at 60°C instead of 56°C. The PCR profile for HIF-1{alpha} and PAM consisted of 35 cycles and 32 cycles of PCR with an annealing temperature both at 60°C. The elongation temperature for both HIF-1{alpha} and PAM was 72°C. The gene-specific PCR products were separated on 1.5% agarose gel together with the ß-actin PCR product, stained with ethidium bromide and were analyzed using a gel dot system (GeneTools; Syngene, Frederick, MD). The mRNA expression levels of the test samples were expressed as ratios of the levels of the gene-specific PCR product relative to levels of ß-actin, as measured by densitometric analysis. The numbers of cycles used for all gene products were within the linear range of PCR amplication. The PCR products were cut from agarose gel, purified by a QIAquick Gel Extraction kit (Qiagen Gmbh, Hilden, Germany), and their identities were confirmed by nucleotide sequencing in the Genome Research Centre (the University of Hong Kong) using an ABI PRISM 3100 Genetic Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA).


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  		Table 1. Primers Used in RT–PCR Experiments.

 
Polysome Analysis
Polysome analysis was performed as described by Lang and colleagues (18). Small blocks of lung tissue were homogenized in a polysome lysis buffer containing 100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.4), cycloheximide at 100 µg/mL, 0.5% Nonidet P40, and RNasin at 20 U/mL (Promega). The homogenates were centrifuged at 17,850 x g for 5 minutes at 4°C, and a 1-mL supernatant was loaded onto a 7-mL 10%–50% linear sucrose gradient. Gradients were centrifuged at 64,000 x g for 2 hours at 4°C in a Beckman SW70Ti rotor (Fullerton, CA). Following centrifugation, 32 fractions of 0.5 mL each were collected from the bottom of the gradients. Optical density of each fraction was measured at a wavelength of 260. Fractions numbered 1–20 (translatable) and 21–32 (nontranslatable) were pooled, and total RNA was extracted with TRIZOL reagent. PreproAM mRNA levels from translatable and nontranslatable regions on the polysome profile were analyzed by RT–PCR.

Statistics
One-way or two-way analysis of variance (ANOVA) was performed as appropriate, followed by post hoc Fisher's Least Significant Differences test. A p value of <.05 is considered significant. Data were presented as mean ± standard error of the mean.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Plasma and Lung AM Levels
There was no increase in plasma AM levels with age, and hypoxia resulted in an increase in plasma AM levels in the young rats only (Figure 1). One-way ANOVA indicates an increase in the basal (normoxic) levels and a decrease in hypoxic levels of lung AM with age (p <.05). There were increases in lung AM levels in response to hypoxia in the young and middle-aged rats but not in the old rats (p <.005, p <.05; Figure 2).


Figure 01
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  		Figure 1. Plasma levels of immunoreactive adrenomedullin (AM) following 6 hours of acute hypoxia conditions in young, middle-aged, and old rats. All values are shown as mean ± standard error of the mean. n = 8 for NY, 7 for NM, 6 for NO, 8 for HY and HM, and 5 for HO (N = normoxic; H = hypoxic; Y = young; M = middle-aged; O = old). * p <.001 compared with normoxic rats

 

Figure 02
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  		Figure 2. Levels of immunoreactive adrenomedullin (AM) in lungs following 6 hours of acute hypoxia conditions in young, middle-aged, and old rats. n = 7 for NY, 5 for NM, 5 for NO, 8 for HY, 8 for HM, and 4 for HO (N = normoxic; H = hypoxic; Y = young; M = middle-aged; O = old). **p <.005, *p <.05 between normoxic and hypoxic rats; +p <.05, ++p <.005 between young, middle-aged, and old hypoxic rats. {ddagger}p <.05 between young and old normoxic rats

 
Lung PreproAM mRNA Levels
For the first study, a two-way ANOVA shows an increase in lung preproAM mRNA levels during aging [F(2,31) = 33.22] and in response to hypoxia at all ages [F(1,35) = 33.613; p <.001] but the magnitude of response diminished in the old rats (from about 700% in the young to <200% in the old; p <.001, p <.01, p <.05, respectively for young, middle-aged, and old, Figure 3). A representative x-ray recording of the preproAM hybrids is shown in Figure 4. This was corroborated by the findings in the second experiment, in which there was a treatment effect [F(1,14) = 55.97] and the hypoxic levels in both the young and the old rats were significantly higher than in the normoxic ones (Figure 6).


Figure 03
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  		Figure 3. Levels of preproadrenomedullin (AM) messenger RNA (mRNA) in lungs following 6 hours of acute hypoxic conditions in young, middle-aged, and old rats. n = 8 for NY, 7 for NM, 5 for NO, 9 for HY, 8 for HM, and 5 for HO (N = normoxic; H = hypoxic; Y = young; M = middle-aged; O = old). *p <.05, **p <.01, ***p <.001 between normoxic and hypoxic rats. {ddagger}p <.01 between young and old rats

 

Figure 04
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  		Figure 4. Polyacrylamide gel electrophoresis (PAGE) of preproadrenomedullin (AM) (top) and ß-actin messenger RNA (mRNA) hybrids in rat lung. Lanes 1–6: Standard of AM and ß-actin (0, 1, 5, 10, 25, 50 µg). Lanes 7–17: Samples: NY (7,10), NM (8,11), NO (9), HY (12,15), HM (13,16), HO (14,17). N = normoxic; H = hypoxic; Y = young; M = middle-aged; O = old

 

Figure 06
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  		Figure 6. Messenger RNA (mRNA) levels of preproadrenomedullin (AM), calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) in lungs following 6 hours of acute hypoxia in young and old rats. n = 5 for NY and HY, and 4 for NO and HO rats. *p <.01, **p <.001 when compared to normoxic rats. {dagger}p <.05 and {dagger}{dagger}p <.005 between young and old normoxic rats

 
HIF-1 Binding to DNA
There was an age-related increase in HIF-1 binding to DNA [F(2,31) = 13.282; p <.001]. In response to hypoxia, this binding increased in both the young and the middle-aged, but not in the old rats (Figure 5). An x-ray recording of electrophoretic mobility gel shift assay (EMSA) is also shown in Figure 5.


Figure 05
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  		Figure 5. Hypoxia-inducible factor-1 (HIF-1) binding to DNA in the rat lungs in normoxia and hypoxia. Levels of HIF-1 DNA binding activity are determined by DNA electromobility gel shift assay. Each group had n = 5–8 samples. *p <.05, **p <.005 compared with normoxic rats, +p <.05 compared with young rats. Top: Lanes 1–6: Samples, respectively, of NY, HY, NM, HM, NO, and HO (N = normoxic; H = hypoxic; Y = young; M = middle-aged; O = old)

 
Lung mRNA Levels of CRLR, RAMPs, HIF-1, and Peptidyl-Glycine-Amidating Monooxygenase
The second study shows an increase in lung CRLR and RAMP1–3 mRNA levels (Figure 6) and HIF-1 mRNA level (Table 2) in response to hypoxia for both young and old rats. There were treatment effects for CRLR, RAMP1–3, and HIF-1{alpha} mRNA levels with F values of 44.2, 43.6, 26.2, 40.1, and 168.8, respectively. The normoxic levels of CRLR, RAMP1, RAMP2, and HIF-1{alpha} mRNAs were all higher in the old rats than in the young ones. However, there was no difference in mRNA level of PAM in lungs of both age groups under either normoxic or hypoxic conditions.


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  		Table 2. Messenger RNA (mRNA) Levels of HIF-1{alpha} and PAM in Young and Old Rats Under Normoxic and Hypoxic Conditions.

 
Polysome Analysis of Lung PreproAM mRNA
Translation of preproAM mRNA was decreased during hypoxia in lung of both young and old rats when compared to the control (Figure 7). There were both treatment and age effects for the change in the percentages of translatable preproAM mRNA [F(1,14) = 25.73 and 23.64) but only a treatment effect for the change in translatable actin mRNA [F(1.14) = 29.44). Hypoxia caused a greater decrease in translatable preproAM mRNA in the old rats (47%) than in the young rats (24%).


Figure 07
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  		Figure 7. a, Percentages of translatable and nontranslatable preproadrenomedullin (AM) and ß-actin messenger RNA (mRNA) in lung tissues of young and old rats in normoxic and hypoxic conditions. *p <.05, **p <.01, ***p <.001 between normoxic and hypoxic rats. b and c, Polymerase chain reaction products of complementary DNAs transcribed from preproAM and actin mRNA as shown on an agarose gel. T = Translatable mRNA; UT = untranslatable mRNA; M = marker (ØX174 RF DNA HaeIII digest); Y = young; O = old; N = normoxic; H = hypoxic

 

   DISCUSSION
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 Materials and Methods
 Results
 Discussion
References
 
In the basal (normoxic) state, there were age-related increases in AM gene expression and peptide levels in the old rats. In addition, the mRNA levels of CRLR and RAMPs were all increased. These findings suggest that there may be an increase in the action of AM in the lung of old rats under normoxic conditions. However, there was no increase in mRNA levels of PAM, which is responsible for the formation of the mature form of AM (19), or the relative amount of translatable to nontranslatable preproAM mRNA, indicating that the increase in AM was mainly due to the increase in preproAM mRNA level. As the mRNA levels of HIF-1{alpha} and the binding of HIF-1{alpha} to DNA of the lung also increased with age, there might be a relationship between HIF-1{alpha} and the AM system.

We report here for the first time an age-related increase in the basal level of preproAM mRNA and AM level in the lung concomitant with an increase of HIF-1{alpha} binding to DNA. This increase in HIF-1 binding to DNA does not agree with previous findings of a decrease in binding in old mice (20,21). Frankel-Denkberg and colleagues actually found an increase in HIF protein, but the binding decreased with age. This augmentation in HIF-1 binding and AM gene expression in the old rats would suggest that AM may play a role in restoring impaired lung function back to normal at old age.

Our finding of an increase in AM gene expression and level in the lung of the young rats in response to hypoxia is similar to the result reported by Holbauer and colleagues (5), and may be related to an increase in HIF-1{alpha} gene expression and level (22) and binding of the protein to the promoter of AM gene (1). We have found a greater than 3-fold increase in HIF-1 gene expression in response to hypoxia. The increase in HIF-1{alpha} binding to DNA could also be due to a decrease in proteasomal degradation of the HIF-1{alpha} protein under hypoxic conditions (23). Thus the increase in HIF-1{alpha} binding may play an important role in stimulating the AM system in the lung. Cytokine released in response to hypoxia (24) may increase HIF-1{alpha} expression (25,26) and binding (27). Hypoxia (28,29) and cytokines (30) can also enhance the production of nitric oxide, which can stabilize HIF-1{alpha} (31) and augment AM gene expression (5). It is likely that both an increase in gene transcription and a decrease in mRNA degradation contributed to the rise in preproAM mRNA here as both an upregulation in gene transcription (17,32) and an increase in half-life of preproAM mRNA (2,32) by hypoxia have been reported.

Upon exposure to acute hypoxia, the old rats did not show any increase in AM peptide, and the increase in preproAM mRNA was decreased. There were concomitant decreases in the response of CRLR and RAMP mRNA levels in the old hypoxic rats, which, like the preproAM mRNA level, might be related to the higher basal (normoxic) levels found in the old rats. These changes may also be related to a decrease in the response of HIF-1 binding to DNA with age such that, in the old rat, there was no longer any increase of HIF-DNA binding over the basal level. The finding that the response to hypoxia was diminished but not abrogated suggests that there are other mechanisms that may regulate the AM system in addition to HIF-1{alpha} binding under hypoxic conditions. Cytokines, which are known to directly increase AM expression (33–35), are likely candidates. Whereas the preproAM levels in hypoxia did not change with age, there was a decrease in the AM level in the middle-aged and old rats compared with the young rats in the hypoxic state. In the hypoxic old rats, the lack of a change in AM peptide level despite an increase in AM mRNA could be explained by a decrease in the translation of preproAM mRNA into protein. This decrease in translatable preproAM mRNA in response to hypoxia was greater in the old than in the young rats. An inhibition in translation of preproAM mRNA of a greater magnitude has been reported in metabolically inhibited vascular smooth muscles (36). The lack of an increase in AM peptide in response to hypoxia in the old rats may suggest a decrease in the AM action, but the increase in gene expression may have at least partially compensated for the decrease in translation. However, this study does not rule out the possibility of an age-related change in the release and/or degradation of the peptide.

Conclusion
The decrease in the responses of HIF-1 activity and preproAM mRNA in the old rats under hypoxic conditions may be associated with high basal levels of the activities in normoxia. The lack of an increase in AM peptide in the hypoxic old rats may suggest an impairment of AM function. The HIF-1{alpha} mechanism may play an active role in regulating the AM system in the maintenance of physiological functions in the lung of old rats under normoxic conditions and in the response to hypoxia in the lung of young ones. Other mechanisms may be present to maintain a hypoxic response in the AM system in old rats.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by a CRCG grant and a grant from the Elaine GCF Tso Memorial Fund Committee of Management of the University of Hong Kong.

We thank Molly P. F. Wong and W. B. Wong for their technical assistance, and Dr. Dominic Autelitano for the gifts of preproAM and ß-actin cDNAs.

Part of the data was presented in the 4th Symposium of Adrenomedullin and Proadenomedullin N-20 peptide in Zurich, Switzerland, 2004.


   Footnotes
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 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received March 7, 2006

Accepted June 29, 2006


   References
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 Materials and Methods
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 Discussion
 References
 

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