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Alpha-human atrial natriuretic polypeptide (Alpha-hANP) in normal volunteers and patients with heart failure or hypertension
Peptides, Vol. 7, pp. 33-37, 1986. ©AnkhoInternationalInc. Printedin the U.S.A.
0196-9781/86$3.00 + .DO
Alpha-Human Atrial Natriuretic Polypeptide (Alpha-hANP) in Normal Volunteers and Patients with Heart Failure or Hypertension J. T A N G , D. L. S O N G , M. Z. S U E N , C. W. X I E
Beijing Medical University, Beijing 100083, China
D. C H A N G A N D J. K. C H A N G
Peninsula Laboratories, Inc'., Belmont, CA 94002 R e c e i v e d 22 July 1985 TANG, J., D. L. SONG, M. Z. SUEN, C. W. XIE, D. CHANG AND J. K. CHANG. Alpha-human atrial natriuretic polypeptide (Alpha-hANP) in normal volunteers and patients with heart failure or hypertension. PEPTIDES 7(1) 33-37, 1986.--The presence of alpha-hANP immunoreactive material in human heart and plasma was investigated with a specific and sensitive radioimmunoassay and immunohistochemical method. It was found that alpha-hANP immunoreactive staining of specific atrial granules was located around the nucleus of atrial cardiocytes. No immunoreactive staining was found in the ventricle. The content of immunoreactive hANP was 0.5 pmol/mg protein in the atria and 0.11-+0.01 pmol/ml in the plasma of 26 normal volunteers. In 16 patients with congestive heart failure and 26 patients with essential hypertension, the plasma level of immunoreactive alpha-hANP was significantly lower than that in normal humans. The above evidence indicate that alpha-hANP is a putative hormone secreted by human atrium. A relative shortage of alpha-hANP in the circulatory system may be involved in the mechanism of heart failure and hypertension. Alpha-human atrial natriuretic polypeptide Hypertension
Radioimmunoassay
Immunohistochemistry
Heart failure
chased from DAKO (Copenhagen, Denmark). Thyroglobulin and other chemicals were purchased from Sigma Company (St. Louis).
A L P H A - H U M A N atrial natriuretic polypeptide (alphahANP) was extracted from human atria by Kanagawa and Matsuo. Sequence analysis revealed that it is composed of 28 amino acid [3]. This peptide was found, like atrial peptides in the rat [1,2], to have a potent natriuretic, diuretic and hypotensive activity [8,9]. Recently we have set up a radioimmunoassay for rat atriopeptin to study the characterization and location of atriopeptin in the rat [7]. Here we report the results of measurement and localization of alphahANP immunoreactive material in healthy volunteers and in patients with heart failure or hypertension using radioimmunoassay and immunohistochemical techniques.
Preparation of Alpha-hANP Antiserum Alpha-hANP (5 rag) and thyroglobulin (25 rag) were dissolved in 1 ml pH 7.4 0.2 M phosphate buffer. To this solution, 10/zi of 5% glutaraldehyde was added. The solution was kept at room temperature for 40 min. The peptide-thyroglobulin conjugate was dissolved in saline and then emulsified in complete Freund's adjuvant, 1 ml of emulsion containing 1 mg alpha-hANP. The thyroglobulin conjugate was injected subcutaneously into the back of 3 rabbits. For the booster immunization 500/zg antigen emulsified in incomplete Freund's adjuvant was injected into the same animals. The immunization was repeated every two or four weeks and at three months after the first injection the rabbits were bled.
METHOD
Materials" Alpha-hANP was synthesized by the standard solid phase method at Peninsula Laboratories (Belmont, CA) and purified by column chromatography. The final products were more than 98% pure when assessed by high pressure liquid chromatography. Goat anti-rabbit immunoglobulinsand horseradish peroxidase/anti-peroxidase complex were pur-
Iodination of Alpha-hANP Alpha-hANP was labeled with '25I by the Iodogen method [4]. To a prelyophilized iodogen tube containing 10 /zg of
33
TANG k.7 A 1..
34
Peptide
100
~s0
,
0 alpha-
I0 hAI~P
160
% Cross Reactivity
alpha-hANP ...... rat A N P (ll& = alpha-hANP) . A N P (8-33) (lie ~2 a l p h a - h A N P ~,-281 rat A N P (13-28) rat Atriopeptin 111 ..... rat Atriopeptin U A N P (18-28) rat Atriopeptin I . . . . . Somatostatin Oxytocin Arg ~ V a s o p r e s s i n
~lph~-haNP17 28)
Auriculin B (rat A N P 126-150) Auriculin A (rat A N P 126-149)
(pg)
100 I00 9(/ 50 100 27 57 3 0 0 0
,oo 88 10
FIG. I. Radioimmunoassay specificity for alpha-hANP.
B/Bo(%) 100
TABLE l THE
DISTRIBUTION
OF ALPHA-hANP IMMUNOREACTIVITY HUMAN HEART
Region
IN
Alpha-hANP (pmol/mg protein)
60 4C
Atrium right left septum Ventricle
0.5 0.7 0.4 0.1 undetectable
2(
'1 ~
lb 3b 16o abo
alpha°hANPIJCj
plasma
UI
FIG. 2. Displacement curves of r-'Sl-alpha-hANPbinding to specific antiserum by synthetic alpha-hANP and rat plasma.
iodogen, 0.4 nmol peptide in 10/xl of 0.1 M sodium phosphate buffer (pH 7.4) and 0.5 mCi of carrier free Na12SI was added. After 4 min of mixing by vortex, 100 pJ of 10% KI was added to stop the reaction. The iodinated peptide was purified by column chromatography on Sephadex G-25 (2.0x10 cm) eluted with 50% acetic acid containing 0.1% bovine serum albumin. The specific radioactivity of '2~Ilabeled alpha-hANP was 1000 Ci/mmole.
Tissue Extraction Two human hearts were obtained from accidental death victims and the atria were dissected 2--4 hours after death. The atria were homogenized for 2 min in cold 1 N acetic acid (1:10 w/v). The homogenates were heated at 95°C for 10 rain and then centrifuged at 12000 x g for 20 min. The supernatants were lyophilized, redissolved in HzO and radioimmunoassayed. The extraction efficiency was validated by measuring the recovery of 1zSI-hANP added to the tissue before homogenization, which was 75-90%.
Preparation of Plasma Samples Subjects. Twenty-six normal volunteers, 26 patients with essential hypertension and 16 patients with congestive heart failure were studied. Severity of the diseases was graded as follows; hypertension, slight and fluctuated elevation in
arterial pressure (I°), sustained hypertension (II °) or hypertension complicated by obvious impairment of the function of heart, kidney or brain (III°); heart failure, inability of the heart as a pump appearing on mild exertion (II °) or at rest (III°). All the patients had been on medication, antihypertensive drugs for hypertension, digitalis and diuretics for heart failure. The drug treatment was suspended for 3 days in the patients with hypertension before measurement of the plasma alpha-hANP concentration. Unextracted plasma sample. Blood samples (2 ml each) were taken from peripheral vein of the subjects after an overnight fast. The blood was drawn into heparinized polyethylene tubes containing aprotinin (200 KIU/ml). The plasma was separated from the blood by centrifugation at 1700 x g at 4° C for 30 min and then subjected to a second centrifugation at 48000 x g at 4°C for 30 min to precipitate the protein. The supernatants were then assayed for alphahANP immunoreactivity. A plasma blank solution was prepared from the plasma without adding aprotinin, which was incubated at 37°C for 12 hours, centrifuged as the sample and then examined by radioimmunoassay. Extracted plasma sample. The plasma (20 ml) from the healthy volunteer was extracted in 60 ml of 75% methanol containing 0.01 N HCI (1:3 v/v) and centrifuged at 12000 x g for 20 min. The supernatant was mixed with 10 vol. of 1 N acetic acid, and then went through a Sep Pak C,~ cartridge.
HEART FAILURE, HYPERTENSION AND ALPHA-hANP LEVELS
35
log IO(]
=, a. SO
so ~u o
E ~6 N
0
H
[*
HF Normal
FIG. 3. Plasma level of alpha-hANP in normal volunteers (N) and in the patients with essential hypertension (H) or heart failure (HF). The figures at the bottom of the column indicate the number of subjects in each group.
The cartridge was washed with H20 (10 ml) 3 times, and then eluted with 95% methanol containing 0.25 N HCI. The first 3.6 ml of eluate was collected, lyophilized and radioimmunoassayed. The extraction efficiency was validated by measuring the recovery of ]25I-hANP added to the plasma before extraction, which was 52%.
Radioimmunoassay (RIA) Standard aipha-hANP or the atria or plasma sample (50 p.l) were incubated for 17-24 hours at 4°C with antiserum and ]25I alpha-hANP (8000-10000 cpm) in 450/zl of 0.1 M pH 7.4 sodium phosphate buffer. When the unextracted plasma samples were measured, the plasma blank solution (50 tzl) was added into each standard tube instead of the same volume of buffer to rectify the protein concentration and other interfering materials in the plasma. Separation of bound and free antigen was achieved by the addition of 200 ~l of dextran-coated charcoal solution, which was prepared by suspending 0.15 g Dextran T-70 and 1.5 g activated charcoal Norit A in 100 ml 0.05 M pH 7.4 phosphate buffer containing 0.1% bovine serum albumin and 0.9% NaCI, and then centrifuged at 1700 × g for 10 min. The 125I-labeled alphah A N P bound to the antiserum was measured in a gamma spectrometer. In this RIA, the minimal detectable quantity of alpha-hANP was less than 3 pg/tube; intra- and inter-assay variations for multiple measurements of the same sample were 7% and 11%, respectively; the recovery of " c o l d " alpha-hANP (10 ng/ml) added to the plasma blank solution was 90%.
lmmunohistochemieal Procedure The atria and ventricles of human hearts were removed and immersed in 4% paraformaldehyde for 24 hours at 4°C and then washed in 0.1 M sodium phosphate buffer (pH 7.4) containing 15% sucrose for at least 24 hours. Cryostat sections were cut at a thickness of 10/xm and mounted on gelatin coated glass immediately before use. The peroxidase/antiperoxidase technique was used according to Sternberger et al. [5]. Firstly, 3% H202 was added on the tissue sections for 5-10 min to block endogenous peroxidase. The sections were washed with H20, immersed in 0.1 M phosphate buffer containing 0.3% Triton X-100 for 3 min and then incubated for 48 hours at 4°C with alpha-hANP antiserum. The control sections were prepared with the con-
I1"
m"
Hypertension
II'
I1/"
Heart Failure
FIG. 4. Correlation between the plasma level of alpha-hANP and the degree of hypertension or heart failure. Grades of the diseases were described in the Method section.
secutive sections incubated with the antiserum preabsorbed with 20 /xg of alpha-hANP. Then goat antirabbit immunoglobulin and horseradish peroxidase/antihorseradish peroxidase complex were applied to the sections at room temperature for 40 min, washed with phosphate buffer to remove free antibodies. Finally, the sections were covered with 3,3'diaminobenzidine tetrahydrochloride (60 mg/100 ml Tris-HCl buffer) and 0.003% H202 for 5-10 min, washed and embedded in permount. RESULTS
Characteristics of the Antiserum fi~r Alpha-hANP The antiserum was used at a final dilution of 1:20000 giving approximately 40% binding of the added tracer in the absence of unlabeled alpha-hANP. It has 100% crossreactivity with rat A N P (28 amino acid peptide) and atriopeptin III, has much less cross-reactivity with rat atriopeptin I and II, and does not cross-react with somatostatin, oxytocin and ArgS-vasopressin (Fig. 1).
Distribution and Concentration of Alpha-hANP lmmunoreactivity in the Human Heart The total content of alpha-hANP immunoreactivity detected in normal human atria is 0.5 pmol/mg protein with the highest level in the right atrium and the lowest in the interatrial septum. No immunoreactivity was found in human ventricles (Table 1).
Plasma Level of AIpha-hANP Immunoreactivity Figure 2 shows that unextracted plasma (12.5-100 p.I) and synthetic alpha-hANP caused parallel displacement of 12~Ialpha-hANP binding to the antiserum. The level of alphah A N P immunoreactive material in the unextracted plasma samples was 0.11+0.01 pmol/ml in the normal volunteers, 0.05-+0.01 pmol/ml in hypertensive patients and 0.02-+0.006 pmol/ml in heart failure patients, indicating a significantly decreased plasma level of alpha-hANP in either hypertensive or heart failure cases (Fig. 3). There was a close correlation between the plasma level of alpha-hANP and the degree of the diseases. As compared with the control level (0.11-+0.01 pmol/ml), plasma alpha-hANP concentrations in I °, II ° and III° hypertension decreased by 42-+24%, 64_+11% and 80-+9%; while those in II ° and III° heart failure decreased by
36
T A N G / : ' 7 AL.
B FIG. 5. Lightfield photograph illustrating alpha-hANP immunoreactivity (dark) in atrial cardiocytes of human (10x40). (A): Pattern of alpha-hANP immunoreactive vesicles in right atrium by standard PAP protocol. (B): Absence of staining when primary antiserum was preabsorbed with alpha-hANP.
65+11% and 88_+6%, respectively. It was evident that the severer hypertension or heart failure was, the lower the plasma level was (Fig. 4). In extracted plasma sample, the level of alpha-hANP immunoreactivity in the healthy volunteer was 0.06 pmol/ml of plasma, which would yield a value quite similar to that obtained from the unextracted plasma sample, if it was rectified according to the extraction efficiency (52%). Immunohistoehemical Study in the Human Heart Specific alpha-hANP immunoreactive staining granules were observed in almost every part of the atrium. These granules were located mainly around the nucleus of atrial cardiocytes, especially at either end. When the antiserum was preabsorbed with 20/xg/ml of alpha-hANP, the staining of these atrial granules was reduced or nonexistent (Fig. 5). Incubation of the sections with serum from nonimmunized rabbits failed to reveal any staining, No immunoreactive staining was found in the ventricle. DISCUSSION
The present study provides direct proofs for the existence of alpha-hANP immunoreactive material in human atrium and plasma. Using radioimmunoassay and immunoperoxidase method, we found high level of alpha-hANP immunoreactiv-
ity existed in almost every part of human atrium with the highest level in the right atrium, while no immunoreactivity was found in the ventricle. These results demonstrated a distribution of atrial peptide in the human heart similar to that in the rat heart [7]. Alpha-hANP immunoreactivity was also detected in the human plasma. A wide range of plasma alpha-hANP concentration, from 37.7 to 900 pg/ml, has been reported by different groups [6,10]. The value reported here was in middle range. The considerable variance in plasma level of alphahANP measured by RIA may be due to the difference in methodology, such as, difference in properties of plasma sample, extracted or unextracted, fresh or frozen; difference in peptidase inhibitors added, and in characters of the antiserum. Moreover, difference in salt intake of ordinary diet may be another reason for the variance. Here we have tried a simple and available method for direct measurement of unextracted plasma samples in clinical applications. Using this method, a very small volume of plasma (50 txl) was necessary for the RIA and it gave a same range of alpha-hANP level as was detected in the extracted plasma sample when considering the extraction efficiency. The validity of the direct assay was demonstrated by a good parallelism between the inhibitory effect of unextracted plasma and synthetic alpha-hANP on ~2aI-alpha-hANP binding to specific antiserum. An important consideration is the protein concentration in the unextracted sample which should be minimized
HEART F A I L U R E , H Y P E R T E N S I O N AND ALPHA-hANP LEVELS as much as possible. We chose high speed centrifugation to precipitate the protein in the plasma instead of Sephadex chromatography, because it is more convenient for measuring a large number of samples. In addition, the preincubated plasma blank solution was added to the standard curve tube to compensate for the difference in the protein concentration between sample tubes and standard curve tubes. We have previously demonstrated that atriopeptin in rat atrium could be released by depolarization induced by KC1 in the Ca ++ dependent manner [9], and there was a high level of atriopeptin immunoreactivity in the plasma of rats [11]. These results suggest that atrial peptides produced by the atrium may be released into the circulation and act as plasma
37
hormones. Recently, the potent effects of atrial peptides on cardiovascular functions [8,11] have generated interest.in relation to possible implications in cardiovascular diseases. Our data show significant decreases in alpha-hANP plasma level in patients with heart failure and essential hypertension. The degree of the decrease was well corresponding to the severity of the diseases. This suggested that a relative shortage of alpha-hANP in the circulatory system may be involved in the pathogenesis of heart failure and hypertension. The possibility that diminished release or increased metabolism of alpha-hANP could be related to decreased plasma level is currently under investigation.
REFERENCES 1. Currie, M. G., D. M. Geller, B. R. Cole, N. R. Siegel, K. F. Fok, S. P. Adams, S. R. Eubanks, G. R. GaUupi and P. Needleman. Purification and sequence analysis ofbioactive atrial peptide. Science 233: 67-69, 1984. 2. Flynn, T. G., M. L. de Bold and A. J. de Bold. The amino acid sequence of atrial peptides with potent diuretic and natriuretic properties. Biochern Biophys Res Commun 117: 85%865, 1983. 3. Kanagawa, K. and H. Matsuo. Purification and complete amino acid sequence of alpha-human atrial natriuretic polypeptide (alpha-hANP). Biochem Biophys Res Commun 118: 131-139, 1984. 4. Salacinski, P. R. P., C. Mclean, J. E. C. Sykes, V. V. Clements-Jones and P. J. Lowry. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1, 3, 4, 6-tetra-chloro-3ct, 6a, -diphenyl glycouril (iodogen). Anal Biochem 117: 136-146, 1981. 5. Sternberger, L. A. lmmunocytochemistry, 2nd edition. New York: John Wiley and Sons, 1979. 6. Sugawara, A., K. Nakao, N. Morii, M. Sakamoto, M. Suda, M. Shimokura, Y. Kiso, M. Kihara, Y. Yamori and K. Nishimura. Alpha-human atrial natriuretic polypeptide is released from the heart and circulates in the body. Biochem Biophys Res Commun 129: 43%446, 1985.
7. Tang, J., H. Fei, C. W. Xie, M. Z. Suen and J. S. Han, R. J. Webber, D. Chang and J. K. Chang. Characterization and localization of atriopeptin in rat atrium. Peptides 5:1173-1177, 1984. 8. Tang, J., R. J, Webber, D. Chang, J. K. Chang, J. Kiang and E. T. Wei. Depressor and natriuretic activities of several atrial peptides. Regul Pept 9: 53--59, 1984. 9. Tang, J., H. Fei, C. W. Xie, D. L. Song, L. Ni, Z. S. Ji, M. Z. Suen, R. J. Webber, D. Chang and J. K. Chang. The heart as a new endocrine organ and its clinical significance. Chin J Intern Med 23: 789, 1984. 10. Towle, A. C., S.-E. Ricksten, A. Pettersson, H. Herlitz, M. Aurell, G. Berglund, O. Anderson, J. Hedner and T. Hedner. Measurement of alpha-hANP (human atrial natriuretic factor) in human plasma of normal and hypertensive patients. Second European Meeting on Hypertension, Milan, Italy, 1985, 11. Xie, C. W., D. L. Song, G. F. Ding and J. Tang. The heart as a new endocrine organ: The effects of atriopeptin on the arterial blood pressure in SHR. Fed Proc 44: 499, 1985.
0196-9781/86$3.00 + .DO
Alpha-Human Atrial Natriuretic Polypeptide (Alpha-hANP) in Normal Volunteers and Patients with Heart Failure or Hypertension J. T A N G , D. L. S O N G , M. Z. S U E N , C. W. X I E
Beijing Medical University, Beijing 100083, China
D. C H A N G A N D J. K. C H A N G
Peninsula Laboratories, Inc'., Belmont, CA 94002 R e c e i v e d 22 July 1985 TANG, J., D. L. SONG, M. Z. SUEN, C. W. XIE, D. CHANG AND J. K. CHANG. Alpha-human atrial natriuretic polypeptide (Alpha-hANP) in normal volunteers and patients with heart failure or hypertension. PEPTIDES 7(1) 33-37, 1986.--The presence of alpha-hANP immunoreactive material in human heart and plasma was investigated with a specific and sensitive radioimmunoassay and immunohistochemical method. It was found that alpha-hANP immunoreactive staining of specific atrial granules was located around the nucleus of atrial cardiocytes. No immunoreactive staining was found in the ventricle. The content of immunoreactive hANP was 0.5 pmol/mg protein in the atria and 0.11-+0.01 pmol/ml in the plasma of 26 normal volunteers. In 16 patients with congestive heart failure and 26 patients with essential hypertension, the plasma level of immunoreactive alpha-hANP was significantly lower than that in normal humans. The above evidence indicate that alpha-hANP is a putative hormone secreted by human atrium. A relative shortage of alpha-hANP in the circulatory system may be involved in the mechanism of heart failure and hypertension. Alpha-human atrial natriuretic polypeptide Hypertension
Radioimmunoassay
Immunohistochemistry
Heart failure
chased from DAKO (Copenhagen, Denmark). Thyroglobulin and other chemicals were purchased from Sigma Company (St. Louis).
A L P H A - H U M A N atrial natriuretic polypeptide (alphahANP) was extracted from human atria by Kanagawa and Matsuo. Sequence analysis revealed that it is composed of 28 amino acid [3]. This peptide was found, like atrial peptides in the rat [1,2], to have a potent natriuretic, diuretic and hypotensive activity [8,9]. Recently we have set up a radioimmunoassay for rat atriopeptin to study the characterization and location of atriopeptin in the rat [7]. Here we report the results of measurement and localization of alphahANP immunoreactive material in healthy volunteers and in patients with heart failure or hypertension using radioimmunoassay and immunohistochemical techniques.
Preparation of Alpha-hANP Antiserum Alpha-hANP (5 rag) and thyroglobulin (25 rag) were dissolved in 1 ml pH 7.4 0.2 M phosphate buffer. To this solution, 10/zi of 5% glutaraldehyde was added. The solution was kept at room temperature for 40 min. The peptide-thyroglobulin conjugate was dissolved in saline and then emulsified in complete Freund's adjuvant, 1 ml of emulsion containing 1 mg alpha-hANP. The thyroglobulin conjugate was injected subcutaneously into the back of 3 rabbits. For the booster immunization 500/zg antigen emulsified in incomplete Freund's adjuvant was injected into the same animals. The immunization was repeated every two or four weeks and at three months after the first injection the rabbits were bled.
METHOD
Materials" Alpha-hANP was synthesized by the standard solid phase method at Peninsula Laboratories (Belmont, CA) and purified by column chromatography. The final products were more than 98% pure when assessed by high pressure liquid chromatography. Goat anti-rabbit immunoglobulinsand horseradish peroxidase/anti-peroxidase complex were pur-
Iodination of Alpha-hANP Alpha-hANP was labeled with '25I by the Iodogen method [4]. To a prelyophilized iodogen tube containing 10 /zg of
33
TANG k.7 A 1..
34
Peptide
100
~s0
,
0 alpha-
I0 hAI~P
160
% Cross Reactivity
alpha-hANP ...... rat A N P (ll& = alpha-hANP) . A N P (8-33) (lie ~2 a l p h a - h A N P ~,-281 rat A N P (13-28) rat Atriopeptin 111 ..... rat Atriopeptin U A N P (18-28) rat Atriopeptin I . . . . . Somatostatin Oxytocin Arg ~ V a s o p r e s s i n
~lph~-haNP17 28)
Auriculin B (rat A N P 126-150) Auriculin A (rat A N P 126-149)
(pg)
100 I00 9(/ 50 100 27 57 3 0 0 0
,oo 88 10
FIG. I. Radioimmunoassay specificity for alpha-hANP.
B/Bo(%) 100
TABLE l THE
DISTRIBUTION
OF ALPHA-hANP IMMUNOREACTIVITY HUMAN HEART
Region
IN
Alpha-hANP (pmol/mg protein)
60 4C
Atrium right left septum Ventricle
0.5 0.7 0.4 0.1 undetectable
2(
'1 ~
lb 3b 16o abo
alpha°hANPIJCj
plasma
UI
FIG. 2. Displacement curves of r-'Sl-alpha-hANPbinding to specific antiserum by synthetic alpha-hANP and rat plasma.
iodogen, 0.4 nmol peptide in 10/xl of 0.1 M sodium phosphate buffer (pH 7.4) and 0.5 mCi of carrier free Na12SI was added. After 4 min of mixing by vortex, 100 pJ of 10% KI was added to stop the reaction. The iodinated peptide was purified by column chromatography on Sephadex G-25 (2.0x10 cm) eluted with 50% acetic acid containing 0.1% bovine serum albumin. The specific radioactivity of '2~Ilabeled alpha-hANP was 1000 Ci/mmole.
Tissue Extraction Two human hearts were obtained from accidental death victims and the atria were dissected 2--4 hours after death. The atria were homogenized for 2 min in cold 1 N acetic acid (1:10 w/v). The homogenates were heated at 95°C for 10 rain and then centrifuged at 12000 x g for 20 min. The supernatants were lyophilized, redissolved in HzO and radioimmunoassayed. The extraction efficiency was validated by measuring the recovery of 1zSI-hANP added to the tissue before homogenization, which was 75-90%.
Preparation of Plasma Samples Subjects. Twenty-six normal volunteers, 26 patients with essential hypertension and 16 patients with congestive heart failure were studied. Severity of the diseases was graded as follows; hypertension, slight and fluctuated elevation in
arterial pressure (I°), sustained hypertension (II °) or hypertension complicated by obvious impairment of the function of heart, kidney or brain (III°); heart failure, inability of the heart as a pump appearing on mild exertion (II °) or at rest (III°). All the patients had been on medication, antihypertensive drugs for hypertension, digitalis and diuretics for heart failure. The drug treatment was suspended for 3 days in the patients with hypertension before measurement of the plasma alpha-hANP concentration. Unextracted plasma sample. Blood samples (2 ml each) were taken from peripheral vein of the subjects after an overnight fast. The blood was drawn into heparinized polyethylene tubes containing aprotinin (200 KIU/ml). The plasma was separated from the blood by centrifugation at 1700 x g at 4° C for 30 min and then subjected to a second centrifugation at 48000 x g at 4°C for 30 min to precipitate the protein. The supernatants were then assayed for alphahANP immunoreactivity. A plasma blank solution was prepared from the plasma without adding aprotinin, which was incubated at 37°C for 12 hours, centrifuged as the sample and then examined by radioimmunoassay. Extracted plasma sample. The plasma (20 ml) from the healthy volunteer was extracted in 60 ml of 75% methanol containing 0.01 N HCI (1:3 v/v) and centrifuged at 12000 x g for 20 min. The supernatant was mixed with 10 vol. of 1 N acetic acid, and then went through a Sep Pak C,~ cartridge.
HEART FAILURE, HYPERTENSION AND ALPHA-hANP LEVELS
35
log IO(]
=, a. SO
so ~u o
E ~6 N
0
H
[*
HF Normal
FIG. 3. Plasma level of alpha-hANP in normal volunteers (N) and in the patients with essential hypertension (H) or heart failure (HF). The figures at the bottom of the column indicate the number of subjects in each group.
The cartridge was washed with H20 (10 ml) 3 times, and then eluted with 95% methanol containing 0.25 N HCI. The first 3.6 ml of eluate was collected, lyophilized and radioimmunoassayed. The extraction efficiency was validated by measuring the recovery of ]25I-hANP added to the plasma before extraction, which was 52%.
Radioimmunoassay (RIA) Standard aipha-hANP or the atria or plasma sample (50 p.l) were incubated for 17-24 hours at 4°C with antiserum and ]25I alpha-hANP (8000-10000 cpm) in 450/zl of 0.1 M pH 7.4 sodium phosphate buffer. When the unextracted plasma samples were measured, the plasma blank solution (50 tzl) was added into each standard tube instead of the same volume of buffer to rectify the protein concentration and other interfering materials in the plasma. Separation of bound and free antigen was achieved by the addition of 200 ~l of dextran-coated charcoal solution, which was prepared by suspending 0.15 g Dextran T-70 and 1.5 g activated charcoal Norit A in 100 ml 0.05 M pH 7.4 phosphate buffer containing 0.1% bovine serum albumin and 0.9% NaCI, and then centrifuged at 1700 × g for 10 min. The 125I-labeled alphah A N P bound to the antiserum was measured in a gamma spectrometer. In this RIA, the minimal detectable quantity of alpha-hANP was less than 3 pg/tube; intra- and inter-assay variations for multiple measurements of the same sample were 7% and 11%, respectively; the recovery of " c o l d " alpha-hANP (10 ng/ml) added to the plasma blank solution was 90%.
lmmunohistochemieal Procedure The atria and ventricles of human hearts were removed and immersed in 4% paraformaldehyde for 24 hours at 4°C and then washed in 0.1 M sodium phosphate buffer (pH 7.4) containing 15% sucrose for at least 24 hours. Cryostat sections were cut at a thickness of 10/xm and mounted on gelatin coated glass immediately before use. The peroxidase/antiperoxidase technique was used according to Sternberger et al. [5]. Firstly, 3% H202 was added on the tissue sections for 5-10 min to block endogenous peroxidase. The sections were washed with H20, immersed in 0.1 M phosphate buffer containing 0.3% Triton X-100 for 3 min and then incubated for 48 hours at 4°C with alpha-hANP antiserum. The control sections were prepared with the con-
I1"
m"
Hypertension
II'
I1/"
Heart Failure
FIG. 4. Correlation between the plasma level of alpha-hANP and the degree of hypertension or heart failure. Grades of the diseases were described in the Method section.
secutive sections incubated with the antiserum preabsorbed with 20 /xg of alpha-hANP. Then goat antirabbit immunoglobulin and horseradish peroxidase/antihorseradish peroxidase complex were applied to the sections at room temperature for 40 min, washed with phosphate buffer to remove free antibodies. Finally, the sections were covered with 3,3'diaminobenzidine tetrahydrochloride (60 mg/100 ml Tris-HCl buffer) and 0.003% H202 for 5-10 min, washed and embedded in permount. RESULTS
Characteristics of the Antiserum fi~r Alpha-hANP The antiserum was used at a final dilution of 1:20000 giving approximately 40% binding of the added tracer in the absence of unlabeled alpha-hANP. It has 100% crossreactivity with rat A N P (28 amino acid peptide) and atriopeptin III, has much less cross-reactivity with rat atriopeptin I and II, and does not cross-react with somatostatin, oxytocin and ArgS-vasopressin (Fig. 1).
Distribution and Concentration of Alpha-hANP lmmunoreactivity in the Human Heart The total content of alpha-hANP immunoreactivity detected in normal human atria is 0.5 pmol/mg protein with the highest level in the right atrium and the lowest in the interatrial septum. No immunoreactivity was found in human ventricles (Table 1).
Plasma Level of AIpha-hANP Immunoreactivity Figure 2 shows that unextracted plasma (12.5-100 p.I) and synthetic alpha-hANP caused parallel displacement of 12~Ialpha-hANP binding to the antiserum. The level of alphah A N P immunoreactive material in the unextracted plasma samples was 0.11+0.01 pmol/ml in the normal volunteers, 0.05-+0.01 pmol/ml in hypertensive patients and 0.02-+0.006 pmol/ml in heart failure patients, indicating a significantly decreased plasma level of alpha-hANP in either hypertensive or heart failure cases (Fig. 3). There was a close correlation between the plasma level of alpha-hANP and the degree of the diseases. As compared with the control level (0.11-+0.01 pmol/ml), plasma alpha-hANP concentrations in I °, II ° and III° hypertension decreased by 42-+24%, 64_+11% and 80-+9%; while those in II ° and III° heart failure decreased by
36
T A N G / : ' 7 AL.
B FIG. 5. Lightfield photograph illustrating alpha-hANP immunoreactivity (dark) in atrial cardiocytes of human (10x40). (A): Pattern of alpha-hANP immunoreactive vesicles in right atrium by standard PAP protocol. (B): Absence of staining when primary antiserum was preabsorbed with alpha-hANP.
65+11% and 88_+6%, respectively. It was evident that the severer hypertension or heart failure was, the lower the plasma level was (Fig. 4). In extracted plasma sample, the level of alpha-hANP immunoreactivity in the healthy volunteer was 0.06 pmol/ml of plasma, which would yield a value quite similar to that obtained from the unextracted plasma sample, if it was rectified according to the extraction efficiency (52%). Immunohistoehemical Study in the Human Heart Specific alpha-hANP immunoreactive staining granules were observed in almost every part of the atrium. These granules were located mainly around the nucleus of atrial cardiocytes, especially at either end. When the antiserum was preabsorbed with 20/xg/ml of alpha-hANP, the staining of these atrial granules was reduced or nonexistent (Fig. 5). Incubation of the sections with serum from nonimmunized rabbits failed to reveal any staining, No immunoreactive staining was found in the ventricle. DISCUSSION
The present study provides direct proofs for the existence of alpha-hANP immunoreactive material in human atrium and plasma. Using radioimmunoassay and immunoperoxidase method, we found high level of alpha-hANP immunoreactiv-
ity existed in almost every part of human atrium with the highest level in the right atrium, while no immunoreactivity was found in the ventricle. These results demonstrated a distribution of atrial peptide in the human heart similar to that in the rat heart [7]. Alpha-hANP immunoreactivity was also detected in the human plasma. A wide range of plasma alpha-hANP concentration, from 37.7 to 900 pg/ml, has been reported by different groups [6,10]. The value reported here was in middle range. The considerable variance in plasma level of alphahANP measured by RIA may be due to the difference in methodology, such as, difference in properties of plasma sample, extracted or unextracted, fresh or frozen; difference in peptidase inhibitors added, and in characters of the antiserum. Moreover, difference in salt intake of ordinary diet may be another reason for the variance. Here we have tried a simple and available method for direct measurement of unextracted plasma samples in clinical applications. Using this method, a very small volume of plasma (50 txl) was necessary for the RIA and it gave a same range of alpha-hANP level as was detected in the extracted plasma sample when considering the extraction efficiency. The validity of the direct assay was demonstrated by a good parallelism between the inhibitory effect of unextracted plasma and synthetic alpha-hANP on ~2aI-alpha-hANP binding to specific antiserum. An important consideration is the protein concentration in the unextracted sample which should be minimized
HEART F A I L U R E , H Y P E R T E N S I O N AND ALPHA-hANP LEVELS as much as possible. We chose high speed centrifugation to precipitate the protein in the plasma instead of Sephadex chromatography, because it is more convenient for measuring a large number of samples. In addition, the preincubated plasma blank solution was added to the standard curve tube to compensate for the difference in the protein concentration between sample tubes and standard curve tubes. We have previously demonstrated that atriopeptin in rat atrium could be released by depolarization induced by KC1 in the Ca ++ dependent manner [9], and there was a high level of atriopeptin immunoreactivity in the plasma of rats [11]. These results suggest that atrial peptides produced by the atrium may be released into the circulation and act as plasma
37
hormones. Recently, the potent effects of atrial peptides on cardiovascular functions [8,11] have generated interest.in relation to possible implications in cardiovascular diseases. Our data show significant decreases in alpha-hANP plasma level in patients with heart failure and essential hypertension. The degree of the decrease was well corresponding to the severity of the diseases. This suggested that a relative shortage of alpha-hANP in the circulatory system may be involved in the pathogenesis of heart failure and hypertension. The possibility that diminished release or increased metabolism of alpha-hANP could be related to decreased plasma level is currently under investigation.
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7. Tang, J., H. Fei, C. W. Xie, M. Z. Suen and J. S. Han, R. J. Webber, D. Chang and J. K. Chang. Characterization and localization of atriopeptin in rat atrium. Peptides 5:1173-1177, 1984. 8. Tang, J., R. J, Webber, D. Chang, J. K. Chang, J. Kiang and E. T. Wei. Depressor and natriuretic activities of several atrial peptides. Regul Pept 9: 53--59, 1984. 9. Tang, J., H. Fei, C. W. Xie, D. L. Song, L. Ni, Z. S. Ji, M. Z. Suen, R. J. Webber, D. Chang and J. K. Chang. The heart as a new endocrine organ and its clinical significance. Chin J Intern Med 23: 789, 1984. 10. Towle, A. C., S.-E. Ricksten, A. Pettersson, H. Herlitz, M. Aurell, G. Berglund, O. Anderson, J. Hedner and T. Hedner. Measurement of alpha-hANP (human atrial natriuretic factor) in human plasma of normal and hypertensive patients. Second European Meeting on Hypertension, Milan, Italy, 1985, 11. Xie, C. W., D. L. Song, G. F. Ding and J. Tang. The heart as a new endocrine organ: The effects of atriopeptin on the arterial blood pressure in SHR. Fed Proc 44: 499, 1985.