Differential responses of circulating and tissue adrenomedullin and gene expression to volume overload

Journal

of Cardiac

Failure Vol. 6 No. 2 2000

Experimental Studies

Differential Responses of Circulating and Tissue Adrenomedullin and Gene Expression to Volume Overload SHUJI HIRANO, MD, TAKUROH IMAMURA, MD, TAKESHI MATSUO, MD, YUICHIRO ISHIYAMA, MD, JOHJI KATO, MD. KAZUO KITAMURA, MD, YASUSHI KOIWAYA, MD, TANENAO ETO, MD Miyizaki,

Japari

ABSTRACT Background: Adrenomedullin (AM), which is produced by various tissues and organs, also circulates in the blood. Circulating AM levels increase during disease states such as essential hypertension, heart failure, and renal failure. However, little is known about how circulating AM or AM production responds to volume overload (VOL). Methods and Results: Progressive VOL was induced in rats by an aortocaval shunt (AC) or by an aortocaval shunt with banding of the abdominal aorta distal to the shunt (AC + B), which created a larger shunt volume. Plasma and tissue AM concentrations. as well as AM gene expression levels, were measured at I, 5. and I4 days after operation. Plasma concentrations of atrial natriuretic peptide (ANP), aldosterone, and renin activity (PRA) were also examined. Pulmonary congestion, pleural effusion, and ascites rapidly progressed in the AC + B group, suggesting that VOL’caused more rapid heart failure under these conditions. Plasma AM concentrations in the AC + B and AC groups at day I compared with those in sham-operated rats were increased by 300% and l40%, respectively, and then gradually declined. The time course of plasma AM over I4 days was similar to that of plasma aldosterone and PRA, but not of plasma ANP or intracardiac filling pressure. The increase in plasma AM was accompanied by upregulated AM gene expression in the lung and aorta and by decreased AM concentrations in the atrium, ventricle, and adrenal gland. Cardiac AM gene expression levels were increased in the hypertrophied ventricles of AC and AC + B rats. Conclusions: The major findings of the present study were I) a rapid increase in plasma AM after the imposition of VOL in association with increased plasma aldosterone and PRA, 2) the contribution of several organs to this increase, and 3) a late increase in the AM messenger RNA (mRNA) level in the ventricles as VOL-induced ventricular hypertrophy developed. Key words: atrial natriuretic peptide, heart failure, aortocaval shunt, ventricular hypertrophy, renin-angiotensin system.

Adrenomedullin that has structural From College,

the First Miyazaki.

(AM) is a potent vasodilator peptide homology with calcitonin gene-related

Dcparmenr Japan.

of hrcrmal

Medicine,

Miyazuki

peptide and amylin (I). Originally isolated from a human pheochromocytoma, the AM gene is also expressed in several normal tissues, including the adrenal medulla, heart, aorta, lung, and kidney (2). Adrenomedullin also circulates in human and rat plasma (3,4). Among the many neurohumoral changes that occur in

Medical

Manuscript received May 25. 1999; revised manuscript received February 21, 2000; revised manuscript accepted February 23, 2000. Reprint requests: Tanenao Eta, MD, First Department of Internal Medicine, Miyazaki Medical College, Kihara 5200. Kiyotake, Miyazaki 889-I 692, Japan. Copyright

0 2000

6s Churchill

response to the inadequate blood volume that is characteristic of heart failure, the activation of the adrenergic nerve and renin-angiotensin system (RAS) maintains blood supply to vital organs. In contrast, atria1 natriuretic peptide (ANP) is thought to act as a counter-regulatory

Lhdrtgsrorre@

1071-9164/00/0602-0003$10.00/0

doi: 10.10.54/jcaf.2000.7277

120

Adrenomedullin in Volume Overload hormone that opposes these effects by causing vasodilation and by increasing salt and water excretion (5). Plasma AM concentrations are elevated in patients with congestive heart failure compared with controls, and these levels are closely correlated with the plasma concentrations of neurohormonal factors such as norepinephrine, ANP, and plasma renin activity (PRA), as well as with hemodynamic parameters such as pulmonary capillary wedge pressure and pulmonary artery pressure (6,7). On the other hand, intravenous injections of AM exert a hypotensive effect that is accompanied by a significant decrease in systemic vascular resistance and increased cardiac output (8,9). The intrarenal administration of AM increases urinary output and sodium excretion (IO). These findings imply that AM, like ANP, acts as a circulatory hormone that counters excessive vasoconstriction and volume retention during heart failure. However, little is known about the response of circulating AM to volume overload (VOL), which organs synthesize AM, or the interactions between AM and other humoral factors. To address these issues, we produced a rat model of acute VOL with or without progressive heart failure and measured plasma and tissue AM concentrations and AM messenger RNA (mRNA) expression at 3 time points. In addition, we compared the plasma concentrations of AM, ANP, aldosterone, PRA, and hemodynamic parameters.

Methods Animals

and Surgery

Male Wistar rats that weighed 220 to 250 g and were obtained from Charles River, Inc (Atsugi, Japan) were housed in a temperature- and light-controlled environment and maintained on standard rat chow (CE-2; CLEA Japan, Tokyo, Japan) with free access to tap water. After an acclimatization period of at least 3 days, the animals were randomly assigned to 3 groups as follows: aortocaval shunt alone (AC) (n = 25), aortocaval shunt plus banding of the abdominal aorta distal to the shunt and immediately proximal to the bifurcation (AC + B) (n = 35) to create a larger shunt volume, and sham-operation (sham) (n = 22). An aortocaval fistula was produced at the union of the segment two-thirds caudal to the renal artery and one-third cephalic to the aortic bifurcation with only 1 puncture by using an 18-G (outer diameter/ 1.25 mm) disposable needle as described by Garcia and Diebold (11). An aortic constriction was induced with a 23-G (outer diameter/O.64 mm) disposable needle. The needle was placed on the abdominal aorta distal to the aortocaval shunt and immediately proximal to the bifurcation. The aorta and the adjacent needle were then tightly ligated. The needle was removed, leaving the vessel constricted to the diameter of the needle. Sham

l

Hirano et al

121

rats (controls) underwent identical surgical procedures with the exception of puncturing and aortic banding. To minimize operative stress, all procedures were completed within I5 minutes, and the aorta of all animals was clamped for I minute. Levels of serum creatinine did not differ in the 3 groups. To confirm shunt volume, we withdrew blood samples (100 FL) from the inferior vena cava proximal to the puncture and determined the partial pressure of oxygen (PoZ) in the samples by using a blood gas analyzer. Values for PO? in the AC + B, AC, and sham groups were 80.9 t 1.4, 65.6 2 1.5, and 48.7 & 1.1 mm Hg (mean + SE), respectively, suggesting that a larger degree of shunt was produced by the AC + B rather than the AC group. Rats were examined and killed at days I, 5, and I4 after the surgical procedure. To evaluate the presence of heart failure, we weighed the lungs and examined them for the prevalence of pleural effusion and ascites. We also observed cumulative death rates over 14 days, which were calculated separately for the AC + B (n = l4), AC (n = 8), and sham (n = 8) groups. Measurement

of Hemodynamics

Rats were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium. One PE-50 catheter (Becton Dickinson and Company, Sparks, MD) was inserted into the ascending aorta and left ventricle (LV) through the right carotid artery, and another was placed in the right atrium (RA) through the right jugular vein. Heart rate (HR), aortic blood pressure (BP), LV enddiastolic pressure (LVEDP), and mean right atria1 pressure (mRAP) were measured with a Statham pressure transducer (model P231D; Gould, Saddle Brook, NJ) connected to a polygraph (model 141-6; San-Ei, Tokyo, Japan). We compared the effect of the banding position of the abdominal aorta on BP and LV weight/body weight (BW) to evaluate the component of pressure overload (POL) in this model. Systolic BP was increased by suprarenal aortic banding (184 + 7 mm Hg and 170 + 4 mm Hg at 7 and 14 days after operation, respectively) compared with the sham rats (125 t 3 mm Hg and 122 2 4 mm Hg at 7 and 14 days after operation, respectively), whereas it was not affected by aortic banding infrarenal and just proximal to the bifurcation, which is in the same position as the AC + B model (131 + 2 mm Hg and 121 t 3 mm Hg at 7 and 14 days after operation, respectively). The LV/BW values were also increased by suprarenal aortic banding (2.84 + 0.07 mg/g and 3.05 2 0.10 mg/g at 7 and 14 days after operation, respectively) compared with sham rats (2.04 + 0.02 mg/g and 2.08 t 0.04 mg/g at 7 and 14 days after operation, respectively), whereas this value was not affected by aortic banding infrarenal and just proximal to the bifurcation (2.07 + 0.04 mg/g and

122

Journal of Cardiac Failure Vol. 6 No. 2 June 2000

2.07 + 0.07 mg/g at 7 and 14 days after operation, respectively). These preliminary results suggest that a POL component is negligible in this AC + B model. Blood Samples Measurement

for AM and ANP

Blood was collected into ice-cooled tubes with 70 pg/mL of aprotinin and 1.5 mg/mL of ethylenediaminetetraacetic acid (EDTA) * 2Na from the arterial catheter that was placed in the ascending aorta. Plasma samples were loaded onto a Sep-Pak C-l 8 cartridge (Waters, Milford, MA) that had been equilibrated with saline. After washing with saline, adsorbed materials were eluted with 60% acetonitrile containing 0.1% trifluoroacetic acid. The eluate was lyophilized and stored until AM and ANP were measured. Tissue Samples for Measuring Weights and AM

Organ

After collecting arterial blood samples, the chest and abdominal cavities were opened to examine the presence of pleural effusion or ascites that would confirm heart failure. The auricular portion of the atria, ventricles, lungs, adrenal glands, and the thoracic to abdominal portion of the aorta were then resected. The right ventricle (RV) was dissected along its septal insertion. The ventricular septum was considered to be part of the LV. These tissues were weighed, boiled in 5 volumes of 1 mol/L acetic acid for 10 minutes to inactivate intrinsic proteases, and homogenized with a Polytron for 2 minutes. The supernatant of the extract obtained by centrifugation at 18,000g for 30 minutes was diluted with an equal volume of distilled water and applied to a Sep-Pak C-18 cartridge. After washing with 0.5 mol/L acetic acid, adsorbed materials were eluted with 60% acetonitrile containing 0.1% trifluoroacetic acid. The eluate was lyophilized and stored until AM was measured. Radioimmunoassay

of AM and ANP

AM levels were measured by a radioimmunoassay (RIA), as described, with an antiserum against human AM[40-52]CONH, that cross-reacts 100% with rat AM. Tracer binding was half maximally inhibited by rat AM at 10 fmol/tube, and AM was detectable within the range of 1 to 256 fmol/tube. Levels of ANP were measured by an RIA with an antiserum against cu-rat ANP at a final dilution of 1:250,000 (12,13). Inhibition was half maximal at 20 fmol/tube, and ANP was measurable over the range of 2 to 512 fmol/tube. The respective intra- and interassay coefficients of variance were 6% and 9% for the assay of AM and 5% and 8% for ANP.

Measurement of PRA and Plasma Aldosterone To measure PRA and plasma aldosterone levels, a separate series of groups of AC (n = 18). AC + B (n = 18). and sham (n = 18) rats were prepared as previously described. At 1.5, and 14 days after operation, blood was collected by decapitation into chilled tubes containing 1.5 mg/mL of EDTA * 2Na and immediately centrifuged at 3,000g for 10 minutes at 4°C. PRA levels were determined by using the Renin RIA Beads kit (Dynabot Co, Osaka, Japan) ( 14) and plasma aldosterone was determined by using the SPAC-S Aldosterone Kit (Daiichi Radioisotope Inc, Tokyo, Japan) (15). RNA Extraction

and Northern

Blots

Thirty micrograms of total RNA that were extracted from the tissues with acid guanidinium thiocyanate-phenol-chloroform (16) was denatured in 1 mol/L glyoxal, 50% dimethylsulfoxide, and 0.01 mol/L NaHzPO, (pH 7.0) for 1 hour at 50°C. Denatured RNA was resolved by electrophoresis on a 1% agarose gel containing 0.01 mol/L NaHzPO, (pH 7.0) transferred to a Zeta-probe membrane (Bio-Rad, Hercules, CA), and fixed by ultraviolet irradiation. The membrane was hybridized at 37°C in 0.5% sodium dodecyl sulfate (SDS), 6 X saline sodium citrate (SSC), 50% formamide, 5 X Denhardt’s solution, and 100 p/mL salmon sperm DNA with a “P-labeled probe. An EwRIIBgI I DNA fragment of rat AM corresponding to nucleotides - 153 - 422 was radiolabeled with [a-“‘P]dCTP by random primed-labeling (17) and used as a hybridization probe. After hybridization for 20 hours, the membrane was washed in 0.1 X SSC and 0.5% SDS at 55°C and band intensity was estimated by using a Bioimage analyzer (BAS 2000; Fuji, Tokyo, Japan). Expression of glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard. Statistical

Analysis

All results are expressed as means t SEM. Differences among 3 groups at each postoperative period were evaluated with the l-way analysis of variance followed by Scheffe’s test. Relationships between variables were studied with linear regression analysis. Differences were considered significant at P < .05.

Results Hemodynamics, Mortality Rate

Organ Weights,

and

Table 1 shows that systolic and diastolic BP values in the AC + B and AC groups at day 1 were significantly decreased compared with those in the sham group. At all

Adrenomedullin in Volume Overload Table 1.

Hctnodynamic

Data

After Day

a~ I, 5. and

14 Days

Operation I

Day 5

Day

14

HR (beats/min) 359 2 I6 42x 2 5* 377 2 17’

Sl1am

AC AC + B Systolic BP (mm Hg) Shan1 AC AC + B Diastolic BP (mm Hg) Sham AC AC + B mRAP (mm Hg) Sham .AC AC + B LVEDP (mm Hg) Sham

AC AC

+ B

I!I4 120 -c 3* 115 + 3’

I23 + 5 I20 -t 6 III 2 IO

I29 z 5 I30 k 3 I25 I+_ 2

+ 3 -+ 5’ 58 + 3;:

87 -t 9 762 II 63 2 IO

I02 z -I 89 z 4”

0.5 2 0.2 I.5 + 0.3 2.7 2 0.4’$

0.5 + 0.3 2.9 f 0.3* 5.9 + o.9+'

3.9 5 0.4 7.2 3- 0.7" 9.9 t- o.9*"

2.5 t 0.3 9.6 + I.J* 21.1 + 7.5’j

135

99 74

82 + 2’ 0.4 2 0.2 2.7 + 0.4” 3.9 + 0.5: 5.1 + 0.5 9.9 + 1.1% 16.2 k 1.7:’

Values are means +- SEM. HR. heart rate: BP. blood pressure; mRAP, mean right atrial pressure: LVEDP. left ventricular end-diastolic pressure: AC + B. aortocaval shunt plus banding group; AC, aortocaval shunt group; and Sham. sham-operation group. * P i .05 I’ sham group on corresponding day. ’ P < .Ol \’ AC group on corresponding day. G P < .Ol 18sham group on corresponding day. ’ P < .05 1’ AC group on corresponding day.

time points, mRAPs and LVEDPs in the AC + B group were significantly higher (P < .Ol) than those in the sham group, and mRAPs and LVEDPs were moderately increased in the AC group compared with those in the sham group. Table 2 shows organ weights relative to BW and the prevalence of ascites or pleural effusion in each group. Both RV to BW and LV to BW ratios were significantly increased at days 5 and 14 in the AC + B group compared with those in the sham group (P < .OI). The RV to BW ratio at days 5 and 14 and LV to BW ratio at day 14 in the AC group significantly increased. The lung to BW ratio in AC + B group was significantly greater than that in sham group at days 5 and 14 (P < .Ol). Pleura1 effusion or ascites at days 1, 5, and 14 appeared in 19%, lOO%, and 73% of the rats in the AC + B group, respectively (Table 2). None of the animals died within 14 days in the AC or sham groups. However, 4 of 14 rats (29%) in the AC + B group that had increased wet lung weight because of severe pulmonary congestion, as well as massive pleural effusion and ascites, died of heart failure within this period. Plasma Levels of AM and ANP, Aldosterone and PRA Fig. 1 shows that plasma AM concentrations in the AC + B and AC groups that were increased by 300%

Hirano et al

l

123

(P < .Ol) and 140% (P < .05), respectively, at day 1 compared with those in the sham group declined to nearly control levels at day 14. Plasma concentrations of ANP in the AC + B and AC groups were increased by 640% and 280%, respectively, at day I, but the level was highest at day 5 and significantly elevated at day 14. Similar to AM, plasma aldosterone and PRA levels were increased at day 1 in relation to VOL severity, and these gradually declined to almost basal values. We considered the possibility that hypoxia in the lower extremities in the AC + B model causes an increase in AM production and in the plasma AM concentration ( 18,l9). We therefore evaluated the plasma AM concentration in rats with aortic banding infrarenal and immediately proximal to the bifurcation, which is the same position as in the AC + B model. However, plasma AM concentrations were not increased in these animals (3.28 + 0.34 fmoI/mL, 3.15 2 0.38 fmol/mL, and 2.59 ? 0.15 fmol/mL at days 1, 5, and 14, respectively) compared with levels in sham rats (3.29 + 0.27 fmol/ mL, 3.19 2 0.30 fmol/mL, and 2.54 + 0.13 fmol/mL at days 1, 5, and 14, respectively). These results suggested that imposing aortic banding immediately proximal to the bifurcation in addition to an aortocaval shunt does not induce additional AM production.

Table 2.

Body

and Organ After Day

Weights Operation

at 1, 5, and

I

Day 5

243 + 2 227 +J* 233 + 3

268 2 2 235 t 9* 229 t -I*

BW (g) Sham AC AC + B RV/BW (mg/g) Sham AC AC + B LVlBW (mg/g) Sham AC AC + B LunglBW (mg/g) Sham AC AC + B Prevalence of ascites or pleural effusion Sham AC AC + B

14 Days

Day

I4

295 2 7 300 t 5 285 t 4

0.67 + 0.03 0.65 + 0.02 0.70 + 0.22

0.68 + 0.02 0.79 t 0.04+ 0.86 t 0.03*

0.59 t 0.02 0.90 2 0.04* 1.01 t 0.04**

2.27 + 0.04 2.19 t- 0.04 2.30 +- 0.04

2. I4 2 0.03 2.32 t 0.12 2.54 t 0.06*

2.11 -+ 0.04 2.80 k 0.05* 3.00 2 0.1 I*

4.50 + 0.06 4.38 2 0.10 4.68 -t 0.33

4.19 t 0.01 4.83 -r- 0.15 8.23 ” 1.20*'

3.95 5.22 6.03

0% (O/8) 0% (0110) 19%(3/16)

0% (O/6) 0% (O/7, 100%(8/8)

2 0.05 t 0.14+ t 0.50*

0%(0/8) 50% (4/8) 73%(8/l I)

Values are means rt SEM. BW. body weight: RVIBW. right ventricle to body weight ratio; LVIBW. left ventricle to body weight ratio; Lung/BW, lung to body weight ratio: AC + B, aortocaval shunt plus banding group: AC, aortocaval shunt group; Sham, sham-operation group. * P < .Ol B sham group on corresponding day. ’ P < .05 1’ sham group on corresponding day. * P < .05 tp AC group on corresponding day. s P < .Ol \’ AC group on corresponding day.

124

Journal

of Cardiac

Failure Vol. 6 No. 2 June 2000

15

600

Fig. 1. Plasmaconcentrationsof adrenomedullin (AM), atrial natriuretic peptide (ANP), aldosterone, and plasma renin activity (PRA) in 3 groups at I,$ and 14 days after operation. Values are means 2 SEM. ** P < .Ol. * P < .05 v sham group. n P < .Ol. p P < .05 v AC group. AC + B, aortocaval shunt plus aortic banding; AC, aortocaval shunt; and sham, sham-operated.

600 Q5

5

400 200

0

0

11 25,

1000

$ a

0.

PRA

*;

600

1 AC+B

600

@ AC q sham

400

” ’ day 1 ’ day 5 ’ day 14 ’

LA

RV

0.6,

I

I

I 0.3

MO.4 :E 2 - 0.2

1 4

Adrenal gland.

13.6~

KidneY

I

2 2 0.2 J3 0.1

0

0.3

1

LV

0.4,

0

Aorta

15

1

Lung

Fig. .I& Tissue concentrations of adrenomedullin (AM) in the right atrium (RA), left atrium (LA): right ventricle (RV), left ventricle f&V), .&renal @tnd, l&ey, aorta, and lung at 1,5, and 14 days after operation. Values are means + SEM. ** P < .Ol. * P < .05 ti sham group. ‘fI F < ;Ql..q P < .05 v AC group. AC -I- B, aortocaval shunt plus aortic banding; AC, aortocaval shunt; and sham, sham-opetited.

Adrenomedullin

in Volume

Overload

l

Hirano et al

125

AMr, GAPDH m . .

.I

AM GAPDH

Fig. 3. Representative Northern blots showing adrenomedullin (AM) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels at I, 5, and 14 days after operation. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; AC + B, aortocaval shunt plus aortic bandin g; AC, aortocaval shunt; and Sham, sham-operated.

Tissue AM Concentrations Fig. 2 shows AM concentrations in various tissues from the 3 groups. Levels of AM were significantly reduced in the right atrium (RA) of the AC + B group compared with those from AC and sham groups. Concentrations of AM in the RA of AC + B and AC groups were obviously reduced at day 5, and these recovered slightly at day 14. Changes in AM concentrations were similar for the left atrium (LA) in AC + B and AC groups. The AM concentrations in the RV of AC + B and AC groups were significantly lower than those of the sham group at day 1 (P < .Ol), and the AM level remained lower at days 5 and 14 in the AC group. Changes in AM levels were comparable in the LV. In the adrenal gland, AM concentrations were decreased in relation to VOL severity at day 1, but recovered to the control level at days 5 and 14. In contrast, AM concentrations of the lungs in the AC + B and AC groups were initially higher than those of sham group and then declined to control levels. The AM levels remained unchanged in the aorta and kidneys of all groups throughout the study. Tissue AM Gene Expression Fig. 3 shows representative RNA blots for AM and GAPDH mRNAs from the various tissues. For quantita-

tive analysis, the band intensity of AM was standardized against that of GAPDH. Percent changes in AM mRNA expression, relative to the sham group, are shown in Fig. 4. The AM mRNA levels of the aorta in the AC + B and AC groups at day 1 were increased by 104% (P C .Ol) and 52% (P < .05), respectively, compared with those in the sham group. They then declined to the control level at day 14. The changes were similar in the lung. In contrast, AM mRNA levels of the RV and LV in AC + B and AC groups remained unchanged at day 1 but were increased at day 14 compared with the sham group. Expression levels in the RA, LA, adrenal glands, and kidneys in the AC + B and AC groups did not differ from those in the sham group at any day examined. Relationships Between AM in Plasma Tissue and Other Parameters

or

Figs. 5A and B show that plasma ANP levels of aI groups were significantly correlated with both mRAP and LVEDP (P < .Ol). However, this relationship was not evident for plasma AM (Figs. 5C and D). The correlation between AM concentrations in the adrenal gland and plasma was significantly negative (P < .Ol), whereas that between AM concentrations in the lung and plasma was positive (P < -01) (Figs. 6A and B).

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Journal of Cardiac Failure Vol. 6 No. 2 June 2000 m 250

@) Adrenal 1 gland

250

Kidney

EtXL

Aorta

w 250,

1

LV

Lung

Fig. 4. Levels of adrenomedullin (AM) gene expressionin AC + B and AC groups relative to those in the sham group. Values are means 31 SEM. ** P < .Ol. *P < .05 v sham group. q P < .05 17AC group. FU, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; AC + B, aortocaval shunt plus aortic banding; AC, aortocaval shunt; sham, sham-operated.

Discussion Systolic BP values between the AC + B and the AC group did not significantly differ, and LV hypertrophy and systolic hypertension did not develop as a result of aortic banding infrarenal and just proximal to. the bifurcation. Therefore, additional POL on the LV did not occur in the AC + B group compared with the AC group, suggesting that AC + B rats can be basically regarded as a model of VOL on the LV. However, components of both VOL and POL may be present on the RV in these shunt models. This is because Liu et al (20) confirmed that sy.stolic and diastolic RV pressure is elevated, and the degree of myocyte hypertrophy in rats with a fistula is greater in the RV than in the LV. High mortality rate, pulmonary congestion, and pleural effision or ascites were also associated with the AC + B group, suggesting a rapid development of heart failure in this group. Thus, ah AC shunt in assdciation with aortic banding distal to the shunt produced a larger degree of VOL and more rapid progressive heart failure than an AC shunt alone. -The present study examined the time course of plasma AM )in .a rat model of acute VOL with or without heart failure. We summarize ,he characteristics of the circulating AM ih thkmodel as follows. Plasma AM levels

are immediately increased during the early stage in relation to VOL severity, and they peak before ANP. Changes in plasma AM levels over the 14 postoperative days are quite similar to those in the plasma aldosterone concentration and PRA. We reported that plasma levels of AM in patients with heart failure are closely correlated with those of ANP, PRA, pulmonary artery pressure, and capillary wedge pressure (6,7). These findings suggested that AM plays a role in preventing inadequate fluid retention and vasoconstriction. We also described that chronically infused AM has a hypotensive effect accompanied by a significant reduction of plasma aldosterone and PRA in the 2-kidney, l-clip hypertensive rat (21). Together with the results of the present study, these results suggest that increased plasma AM levels counterregulate against RAS augmentation during the early stage of VOL. We found that elevated plasma AM concentrations during progressive VOL were accompanied by immediate upregulation and similar changes over 14 days of AM gene expression in the lung and aorta. Lung AM concentrations ipcreased at day 1 in the AC + B group with statistical significance and in the AC group without significance. In addition, the combined data from all stages of each of the groups showed that the AM concentration

Adrenomedullin

in Volume

1q A

Fig. 5. Relationships between plasma concenlralion of ANP (A and B) or AM (C and D) and mRAP or LVEDP. Data of’ 3 groups at all stages are presented. ANP, atrial natriuretic peptide; AM, adrenomedullin; nlRAP, mean right atrial pressure; LVEDP, left ventricular end-diastolic pressure; N.S., not significant.

Overload

Hirano et al

l

127

401 B

-2bplasmaANP1000 1500 (pg/mL) 121 c 9

0

JO-D

r=0.19 n=82 N.S.

.

500 1000 1500 plasmaANP (pg/mL) r=O. 12 n=82

S8 .w 3 4 E 2 0 -2 I Q 10

0

I

5

10

15

20

25

0

plasmaAM (fmol/mL)

of the lung was significantly and positively correlated with that of plasma AM. Although we did not conduct arterio-venous cross blood sampling, these findings suggest that AM synthesized in the lung may partially contribute to the increased plasma AM level in acute VOL. On the other hand, AM is actively secreted from cultured vas&ar endothelial and smooth muscle cells of the rat aorta (22,23). The plasma AM level in the rat model of septic shock induced by lipopolysaccharide is signifi-

I

I

10 15 20 25 plasmaAh4 (fmol/mL) 5

cantly increased, and AM gene expression in the aorta is augmented (24). However, because the AM concentration of the aorta did not change over 14 days (unlike that of the lung), we cannot conclude that AM was secreted from the aorta in the present study. Nonetheless, AM concentrations of the atrium, ventricle, and adrenal gland were immediately decreased without a concurrent reduction of AM mRNA levels. It is also reported that the ventricles of patients with congestive heart failure se-

B 25 I= -0.63

r=0.45 n=82 P
0

n=82 PcO.01

0

-i! 20

Fig. 6. Relationships between plasma and tissueAM concentrations in the adrenalgland (A) and lung (B). Data of 3 groups at all stages are presented. AM, adrenomedullin.

2

0 0

5

10

15

I 20

adrenalgland AM (fmol/mg)

0

0

I 5

I 10

1 15

lung Ah4 (fmo@g)

I 20

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Journal of Cardiac Failure Vol. 6 No. 2 June 2000

Crete AM (25). The AM concentration in the adrenal gland was significantly and negatively correlated with plasma AM. We reported that AM is secreted by cultured neonatal rat cardiomyocytes and by bovine adrenal medullary cells (26,27). Thus, the decrease in AM concentrations in the organs in I)& may result from a rapid release of AM into the circulation from intracellular stores. AM is probably secreted from several organs into the blood when receiving stimuli that may differ among tissues in this VOL model. Inadequate fluid retention and vasoconstriction in heart failure may be counteracted by ANP via its natriuretic and vasodilator actions (5), and its biological properties are similar to those of AM. In addition, ANP is secreted mainly from the cardiac atria in response to atria1 stretch caused by VOL (28,29). Consistent with this, plasma ANP was significantly correlated with mRAP and LVEDP in the present study, but AM was not. Indeed, the time courses of plasma AM and ANP were quite different. The gene expression of AM in vascular endothelial cells is stimulated by shear stress (30), and both angiotensin Ll and aldosterone increase AM secretion from vascular smooth muscle cells (31,32). The present results suggest that circulating AM levels in progressive VOL with or without heart failure are regulated by RAS rather than VOL per se, whereas those of ANP are regulated by the mechanical stretch of VOL rather than by RAS. Further studies are required to define the mechanism(s) regulating AM production and secretion in heart failure. Unrelated to the changes in plasma AM level, AM mRNA expression in the AC + B and AC groups was increased in the hypertrophied ventricle at day 14. However, AM concentrations in hypertrophied ventricles at day 14 were lower than those in sham rats. We reported that AM concentrations in hypertrophied ventricles are increased in Dahl-salt sensitive rats at 3 weeks after salt loading (33) and in renovascular hypertensive rats at 4 weeks after left renal arterial clipping (34). The discrepancies among these and the present study could be because of the models studied and the duration of overload. The ventricular AM mRNA level and ventricular AM concentration were not parallel in the progressive phase of the VOL model, suggesting that the ventricular AM concentration is affected by the summation of changes in the synthesis and secretion of ventricular AM. Furthermore, we recently reported that AM secreted from cardiomyocytes inhibits de novo protein synthesis by these cells, suggesting that AM helps to inhibit cardiomyocyte hypertrophy as a locally acting hormone (26). Thus, ventricular AM may act locally to modulate the development of ventricular hypertrophy. We conclude that a rapid increase in plasma AM after the imposition of VOL is associated with increases in PRA and plasma aldosterone levels, that several organs may contribute to this increase, and that a late increase m

the ventricular AM mRNA level is associated with the development of VOL-induced ventricular hypertrophy.

Acknowledgment The authors would like to thank Dr. Kenji Kangawa (National Cardiovascular Center Research Institute, Osaka, Japan) for providing the anti-a-rANP antiserum used in this study. They would also like to thank Mari Kawamoto for excellent technical assistance and N. Foster for critical reading of the manuscript.

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