Peptides,Vol. 13, pp. 121-123, 1992

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Human Brain Natriuretic Peptide-Like Immunoreactivity in Human Brain K A Z U H I R O T A K A H A S H I , K A Z U H I T O T O T S U N E , M A S A H I K O SONE, M A K O T O O H N E D A , OSAMU MURAKAMI, KEIICHI ITOI AND TORAICHI MOURI*

The Second Department of Internal Medicine and *The Second Department of Pathology, Tohoku University School of Medicine, 1-1 Seiryo-cho, Aoba-Ku, Sendai, Miyagi 980, Japan Received 2 July 1991 TAKAHASH1, K., K. TOTSUNE, M. SONE, M. OHNEDA, O. MURAKAMI, K. ITOI AND T. MOURI. Human brain natriureticpeptide-likeimmunoreactivityin human brain. PEPTIDES 13(1) 121-123, 1992.--The presence of immunoreactive human brain natriuretic peptide in the human brain was studied with a specific radioimmunoassay for human brain natriuretic peptide-32. This assay showed no significantcross-reactionwith human alpha atrial natriuretic peptide, porcine brain natriuretic peptide or rat brain natriuretic peptide. Immunoreactive human brain natriuretic peptide was found in all 5 regions of human brain examined (cerebral cortex, thalamus, cerebellum, pons and hypothalamus) (0.6-6.7 pmol/g wet weight, n = 3). These values were comparable to the concentrations of immunoreactive alpha atrial natriuretic peptide in human brain (0.5-10.1 pmol/g wet weight). However, Sephadex G-50 column chromatography showed that the immunoreactive human brain natriuretic peptide in the human brain eluted earlier than synthetic human brain natriuretic peptide-32. These findings suggest that human brain natriuretic peptide is present in the human brain mainly as larger molecular weight forms. Brain natriuretic peptide

Atrial natriuretic peptide

Radioimmunoassay

Chromatography

Human brain

Extraction

BRAIN natriuretic peptide (BNP) was originally isolated from the porcine brain (9). Porcine BNP consists of 26 amino acid residues (9). It has a structural similarity to atrial natriuretic peptide (ANP) and similar natriuretic and vasodilator actions. The amino acid sequence of the peptide with a structure similar to porcine BNP was deduced using a human cardiac atrium cDNA library (10). This peptide was designated "human brain natriuretic peptide (human BNP)" because it was presumed to be a brain peptide present in the human brain, due to its structural similarity to porcine BNP. Subsequently, human BNP consisting of 32 amino acid residues was isolated from human atrium (3). High concentrations of human BNP-32 have been shown to be present in human cardiac atrium (3,13). However, its presence in the human brain has not yet been clarified. In the present study, the presence of immunoreactive (IR) human BNP in the human brain was studied with a highly sensitive and specific radioimmunoassay for human BNP-32.

The brain tissues (approximately 500 mg per one region) were boiled in 2 ml 1 M acetic acid for 10 min and homogenized in 10 ml 50% methanol in 1 M acetic acid. The homogenates were centrifuged at 24,000 X g for 30 min. The supernatants were separated and dried by air. The resulting materials were reconstituted in assay buffer [0.1 M phosphate buffer, pH 7.4, containing 0.1% (w/v) human serum albumin, 0.1% (v/v) Triton X- 100 and 0.1% (w/v) sodium azide] and assayed. The recovery, which was determined by adding the known amount of synthetic human BNP to the tissues prior to the extraction, was 88 --- 11% (mean + SD, n = 4).

Radioimmunoassay IR-human alpha ANP and IR-human BNP in the tissue extracts were measured by radioimmunoassay. The radioimmunoassay for human alpha ANP was previously reported (7). This assay showed less than 0.001% cross-reaction with human BNP-32, rat BNP and porcine BNP. The antisera to human BNP-32 were raised in rabbits. Synthetic human BNP-32 (Peptide Institute, Minoh-shi, Osaka, Japan) was conjugated with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) by carbodiimide and injected subcutaneously into three rabbits. Sensitive and specific antisera to human BNP were raised in all three rabbits and the most sensitive antiserum raised in one rabbit was used in the following studies. The antiserum was used at a final dilution of 1:40,000. The synthetic human BNP-32 was used as standard and for iodination

METHOD

Materials Human brain tissues were obtained at postmortem from three patients (one male and two females, 69-78 years old) without history of neurological or psychiatric diseases. Five regions of brain (cerebral cortex, thalamus, cerebellum, pons, hypothalamus) were dissected out within three hours postmortem and stored at - 8 0 ° C .

121

122

TAKAHASHI ET AL.

| 00

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1

dilution 2 4

molecular weight positions (Fig. 2A). Material eluting in the position of human BNP-32 was undetectable. Reverse phase HPLC showed that the IR-human BNP in the cortex was mainly eluted in more hydrophobic positions than human BNP-32 (Fig. 2B). A peak was found near the elution position of human BNP32, but not in the identical position. The Sephadex G-50 column chromatography and HPLC profiles of the human hypothalamus are also similar to these profiles, respectively (data not shown).

8 mg

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DISCUSSION 4O

2O

0

016

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9

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3~6

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1~,5 ' 580

human BNP-32 (fmol/tube)

FIG. l. A standard curve of human brain natriuretic peptide-32 (O) and a dilution curve of the human brain extract (cortex) (O).

Human BNP-32 was isolated from human cardiac atrium (3), and high concentrations of IR-human BNP were found in the human heart (3,13). Elevated plasma IR-human BNP concentrations were reported in the patients with heart failure and chronic renal failure (4,5,8), like human alpha ANP. The IRhuman BNP in the human heart and plasma was immunologically and chromatographically identical to the synthetic human BNP-32. Therefore, human BNP-32 is thought to be a cardiac peptide. However, the presence of human BNP-32 in the human brain has not been clarified.

A

with 125INa. Human BNP-32 was iodinated by the modified chloramine-T method (1). Briefly, ten #1 of 10 mg/ml chloramine-T in water was added to 1.5 nmol peptide and 500 uCi 125INa, and the mixture was agitated for 60 seconds. The reaction was terminated by adding 200 tal 10% (w/v) human serum albumin in 0.5 M acetic acid. Iodinated peptide was purified with a Sephadex G-50 superfine column (1.0 X 20 cm), as described previously (7). The assay could detect changes of 1.2 fmol/tube at 95% confidence using duplicate tubes. The cross-reaction with other peptides such as human alpha ANP, porcine BNP or rat BNP was less than 0.01%. Intra- and interassay coefficients of variation were 5.5% (n = 9) and 11.2% (n = 5), respectively.

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Chromatography

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The brain tissues of cerebellum, hypothalamus and cortex were examined as representative regions of the brain. IR-human BNP in these tissues was characterized by Sephadex G-50 superfine column chromatography (1.0 X 37 cm) and reverse phase high performance liquid chromatography (HPLC) using a u Bondapak C18 column (9 X 300 mm) (Waters, Millford, MA). The tissue extracts were reextracted by Sep-Pak C 18 cartridges (Waters) as previously reported (7,12) and loaded onto the columns. The Sephadex G-50 column was eluted with 1 M acetic acid at a flow rate of 6 ml/hour. One-ml fractions were collected, dried by air, reconstituted in assay buffer and assayed. The recovery from the column was 60-108%. The HPLC column was eluted with a linear gradient of acetonitrile from 10% to 60% in 0.1% trifluoroacetic acid over 50 minutes at a flow rate of 1 ml/minute. One-ml fractions were collected, dried by air, reconstituted in assay buffer and assayed. The recovery from the HPLC column was 66-105%.

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RESULTS A dilution curve of the human brain extract (cortex) was parallel with a standard curve of human BNP-32 (Fig. 1). IRhuman BNP was detected in all the regions of brain examined (Table 1). The levels of IR-human BNP in the human brain were comparable to those of IR-human alpha ANP. Sephadex G-50 column chromatography of the IR-human BNP of human cerebellum showed two peaks eluting in higher

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4'0

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FIG. 2. (A) Sephadex G-50 column chromatography (cerebellum) and (B) reverse phase high performance liquid chromatography (cortex). Vo: void volume. BNP and ANP: the elution positions of human BNP-32 and human alpha ANP, respectively.The dotted line indicates a gradient of acetonitrile.

H U M A N BNP IN H U M A N BRAIN

123

TABLE 1 IMMUNOREACTIVEHUMAN BRAINNATRIURETICPEPTIDE AND IMMUNOREACTIVEHUMAN ALPHAATRIALNATRIURETIC PEPTIDE IN THE HUMAN BRAIN(n = 3) IR-Human BNP (pmol/ g wet weight)

IR-HumanAlpha ANP (pmol/g wet weight)

Case

(A)

(B)

(C)

(A)

(B)

(C)

Cortex Thalamus Cerebellum Pons Hypothalamus

2.6 2.5 6.7 3.3 --

0.6 1.6 2.7 2.8 1.7

1.1 0.6 --0.9

2.8 2.2 10.1 3.7 --

0.8 1.2 1.8 3.7 0.6

0.5 0.6 --1.8

--: not available.

In the present study, the presence of h u m a n BNP-like immunoreactivity in the h u m a n brain has been demonstrated. However, Sephadex G-50 c o l u m n chromatography and HPLC showed that the a m o u n t of material eluting in the position of h u m a n BNP-32 was undetectable or very small. In addition, the findings of Sephadex G-50 column chromatography indicate that at least two larger molecular weight forms of IR-BNP were present in the h u m a n brain. ANP is present in the brain and thought to have important roles in the control of fluid balance and blood pressure (6). A large molecular form of I R - h u m a n alpha ANP, probably rANP,

is a predominant form in the h u m a n brain (2). We also confirmed that most of the I R - h u m a n alpha ANP (>90%) eluted in a larger molecular weight region of the Sephadex G-50 c o l u m n chromatography (our unpublished observations). Therefore, it is likely that BNP is also present in the h u m a n brain as larger molecular weight forms. But it is not clear whether these larger molecular weight forms of IR-BNP have similar pharmacological actions to h u m a n BNP-32 or not, and whether these are precursor forms of hBNP-32, their metabolites, or not. Further studies are required to determine the sequence of these materials. Recently, Prof. Matsuo's group has advocated new n o m e n clature for the natriuretic peptides; A-type natriuretic peptide for atrial natriuretic peptide and B-type natriuretic peptide for brain natriuretic peptide (11). The present study has shown that the a m o u n t of h u m a n BNP-32 is very small in the h u m a n brain; therefore, this new nomenclature would be more suitable and clear. The findings of the present study indicate that the a m o u n t of h u m a n BNP-32 itself in the h u m a n brain is very small, but that h u m a n BNP may be present in the h u m a n brain as larger molecular weight forms. ACKNOWLEDGEMENTS This study was supported in part by a research grant from the Miyagi Prefecture Kidney Association. The authors are very grateful to Prof. N. Sasano, the Second Department of Pathology, Tohoku University School of Medicine and Dr. Y. Tachibana, Takeda Hospital for supplying us the postmortem human brain tissues, and Ms. Moil for her secretarial assistance.

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7. Nozuki, M.; Mouri, T.; Itoi, K.; Takahashi, K.; Totsune, K.; Saito, T.; Yoshinaga, K. Plasma concentrations of atrial natriuretic peptide in various diseases. Tohoku J. Exp. Med. 148:439-447; 1986. 8. Ohneda, M.; Totsune, K.; Sone, M.; Mouri, T.; Takahashi, K.; ltoi, K.; Murakami, O.; Saito, T.; Yoshinaga, K. Importance of the measurement of immunoreactive (IR-) human brain natriuretic peptide (hBNP) in plasma. J. Am. Soc. Nepbrol. 1:423; 1990 (abstract). 9. Sudoh, T.; Kangawa, K.; Minamino, N.; Matsuo, H. A new natriuretic peptide in porcine brain. Nature 332:78-81; 1988. 10. Sudoh, T.; Maekawa, K.; Kojima, M.; Minamino, N.; Kangawa, K.; Matsuo, H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem. Biophys. Res. Commun. 159:1427-1434; 1989. 11. Sudoh, T.; Minamino, N.; Kangawa, K.; Matsuo, H. C-type natriuretic peptide (CNP): A new member of natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun. 168: 863-870; 1990. 12. Takahashi, K.; Mouri, T.; Murakami, O.; Itoi, K.; Sone, M.; Ohneda, M.; Nozuki, M.; Yoshinaga, K. Increases of neuropeptide Y-like immunoreactivity in plasma during insulin-induced hypoglycemia in man. Peptides 9:433-435; 1988. 13. Tateyama, H.; Hino, J.; Minamino, N.; Kangawa, K.; Oglhara, T.; Matsuo, H. Characterization of immunoreactive brain natriuretic peptide in human cardiac atrium. Biochem. Biophys. Res. Commun. 166:1080-1087; 1990.