Presence of Adrenomedullin-Like Immunoreactivity in the Human Cerebrospinal Fluid

Peptides, Vol. 18, No. 3, pp. 459–461, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00

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BRIEF COMMUNICATION

Presence of Adrenomedullin-Like Immunoreactivity in the Human Cerebrospinal Fluid KAZUHIRO TAKAHASHI,* 1 MASAHIKO SONE,† FUMITOSHI SATOH,‡ OSAMU MURAKAMI,‡ KAZUHITO TOTSUNE,‡ HIROAKI TANJI,† NOBUYUKI SATO,† HISAO ITO† AND TORAICHI MOURI§ *Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, Sendai, Miyagi 980-77, Japan †Department of Internal Medicine, the National Iwate Hospital, Ichinoseki, Iwate 021, Japan ‡Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi 980-77, Japan §Division of Neuroendocrinology, Mouri Clinic, Natori 981-12, Japan Received 27 September 1996; Accepted 6 November 1996 TAKAHASHI, K., M. SONE, F. SATOH, O. MURAKAMI, K. TOTSUNE, H. TANJI, N. SATO, H. ITO AND T. MOURI. Presence of adrenomedullin-like immunoreactivity in the human cerebrospinal fluid. PEPTIDES 18(3) 459–461, 1997.—The presence of adrenomedullin-like immunoreactivity in the cerebrospinal fluid was studied by radioimmunoassay in 13 subjects with various neurological diseases. The concentrations of adrenomedullin-like immunoreactivity in the cerebrospinal fluid were 9.4 { 3.1 pmol/l (mean { SD, n Å 13). Reverse-phase high performance liquid chromatography of the extract of the pooled cerebrospinal fluid showed that approximately 40% of the adrenomedullin-like immunoreactivity was chromatographically identical to human adrenomedullin(1–52). This is the first report that demonstrates the presence of adrenomedullin-like immunoreactivity in the human cerebrospinal fluid. q 1997 Elsevier Science Inc. Adrenomedullin

Cerebrospinal fluid

Radioimmunoassay

ADRENOMEDULLIN (AM) is a 52-amino acid peptide that was isolated from the human pheochromocytoma (2). AM is a potent vasodilator peptide with stimulating action on platelet cAMP production (2). AM shares about 27% homology at its C-terminal portion (16–52) with calcitonin gene-related peptide (CGRP). cDNA encoding human AM precursor was cloned from human pheochromocytoma cDNA library (3). Immunoreactive AM and AM mRNA were found to be present in various tissues (1,3) and in some tumors, including pheochromocytomas (1,3,10) and pulmonary tumors (5). High expression of AM and AM mRNA was found in the tumor tissue of pheochromocytoma, adrenal medulla, cardiac ventricle, kidney, and lung (2). Initially AM was reported to be absent in the brain (2). However, we and other authors have shown that AM-like immunoreactivity (LI) is present in the brain (10,11,18). Intracerebroventricular injection of AM increased blood pressure (14) and inhibited water drinking in the rat (6). AM inhibited basal and

Chromatography

Choroid plexus

corticotropin-releasing hormone-stimulated ACTH secretion from dispersed anterior pituitary cells in culture (8). In addition, AM binding sites were observed in the rat brain (7). These findings suggest that AM functions as a neuromodulator. AM-LI is present in human plasma and urine (1,4,9). But there has been no report on AM-LI in the cerebrospinal fluid. We therefore studied the presence of AM-LI in the cerebrospinal fluid obtained from patients with various neurological diseases by radioimmunoassay (RIA). METHOD

Samples of cerebrospinal fluid were obtained from 13 patients with various neurological diseases (Table 1), and aliquots of these samples were used in the present study. Informed consent was obtained from each subject. One milliliter of cerebrospinal fluid was acidified with 1 ml of 0.75 mol/l acetic acid containing

1 Requests for reprints should be addressed to Kazuhiro Takahashi, M.D., The Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, 2-1 Seiryo-Machi, Aoba-ku, Sendai, Miyagi, 980-77, Japan.

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TAKAHASHI ET AL. TABLE 1

THE AGE, SEX, AND CLINICAL DIAGNOSIS OF 13 SUBJECTS AND ADRENOMEDULLIN-LIKE IMMUNOREACTIVITY (AM-LI) CONCENTRATIONS IN THE CEREBROSPINAL FLUID

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Age

Sex

Clinical Diagnosis

AM-LI (pmol/l)

52 70 27 52 60 71 79 72 44 77 57 44 32

M M M M M F M F M M F M F

Amyotrophic lateral sclerosis Cerebral aneurysm, postoperative Multiple sclerosis Olivopontocerebellar atrophy, suspected Olivopontocerebellar atrophy Benign postural vertigo, headache Diabetic polyneuropathy Guillain–Barre´ syndrome, suspected Neuro-Behc¸et’s disease Cerebral infarction Headache Mononeuritis multiplex Guillain–Barre´ syndrome

8.4 13.2 8.4 5.9 8.3 5.5 7.9 11.3 9.9 5.4 8.1 14.9 14.4

0.5% (w/v) casein (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and loaded onto a Sep-Pak C-18 cartridge (Waters, Milford, MA). The peptides were eluted with 2 ml of 60% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA). The eluate was dried by air and reconstituted in 0.5 ml of 0.1 mol/l phosphate buffer, pH 7.5, containing 0.5% (w/v) casein, 0.1% (w/v) bovine serum albumin, 0.2% (v/v) Triton X-100, and 0.1% (w/v) sodium azide. Aliquots of 100 and 10 ml were assayed in duplicale in one assay. The recovery of the extraction, which was determined by using pooled cerebrospinal fluid containing a known amount of adrenomedullin, was 101.2%. The RIA was performed as previously reported (10), except for the antiserum against human AM(1–52). The antiserum (No. 102) raised against human AM(1–52) in a rabbit in our laboratory (11) was used at a final dilution of 40,000. The assay could detect changes of 0.5 fmol/tube from zero in duplicate tubes with 95% confidence. The amount of [ 125I]AM added was approximately 4000 cpm/tube. The zero binding was approximately 30% of the total added [ 125I]AM and nonspecific binding was approximately 4% of the total added [ 125I]AM. Values of 20%, 50%, and 80% of AM standard were approximately 50, 8, and 2 fmol/tube, respectively. Cross-reaction with other peptides such as CGRP, amylin, neuropeptide Y, and somatostatin was less than 0.001%. Intra- and interassay coefficients of variation were 9.3% (n Å 10) and 12.7% (n Å 8), respectively. Chromatographic characterization of AM-LI in the pooled cerebrospinal fluid was performed by Sephadex G50 superfine column chromatography (10 1 560 mm) and reverse-phase high performance liquid chromatography (HPLC) using a mBondapak C18 column (3.9 1 300 mm, Waters). Pooled cerebrospinal fluid was extracted with a Sep-Pak C18 cartridge and loaded onto each column. The Sephadex G50 column was eluted with 1 mol/l acetic acid containing 0.5% (w/v) bovine serum albumin at a flow rate of 6 ml/h. The HPLC column was eluted with a linear gradient of acetonitrile containing 0.1% (v/v) TFA from 10% to 60% at a flow rate of 1 ml/min/fraction over 50 min. Each fraction (0.8 ml/fraction in Sephadex G-5- column chromatography and 1 ml/fraction in HPLC) was collected, dried by air, and assayed. RESULTS

A twofold dilution curve of the extract of the pooled cerebrospinal fluid was parallel to the standard curve of AM (data not

shown). The concentrations of AM-LI in the cerebrospinal fluid were 9.4 { 3.1 pmol/l (mean { SD, n Å 13) in 13 patients with various neurological diseases (Table 1). There was no noticable relationship between the levels of AM-LI in the cerebrospinal fluid and the neurological diseases. Sephadex G-50 column chromatography of the extract of the pooled cerebrospinal fluid showed a peak eluting in the position of AM(1–52) and materials eluting in higher molecular weight region [Fig. 1(A)]. Reverse-phase HPLC showed two immunoreactive peaks, one of which was eluting in an identical position to human AM(1–52) [Fig. 1(B)]. The oxidized form of AM was eluted in the same position as AM(1–52). DISCUSSION

The present study has, for the first time, shown the presence of AM-LI in the cerebrospinal fluid. The levels of AM-LI in the cerebrospinal fluid were comparable to those in plasma (1,4,9). No relationship was found between the levels of AM-LI in the

FIG. 1. Chromatographic analyses of the extract of the pooled cerebrospinal fluid. (A) Sephadex G50 column chromatography. (B) Reversephase high performance liquid chromatography. The arrow indicates the elution position of human adrenomedullin(1–52) (AM). The dotted line indicates a gradient of acetonitrile (ACN). Vo , void volume. AM-LI, adrenomedullin-like immunoreactivity.

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ADRENOMEDULLIN IN CEREBROSPINAL FLUID

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cerebrospinal fluid and neurological diseases. A limited number of samples were examined in the present study. We therefore could not exclude the possibility that studies in a larger number of samples will reveal such a relationship in future. Positive immunostaining was shown in the epithelial cells of the choroid plexus (19). We have recently reported the production and the secretion of AM from the cultured cells of choroid plexus carcinoma, a malignant tumor derived from the choroid plexus (16). The choroid plexus may therefore be a major source of AM in the cerebrospinal fluid. AM has a structural similarity to CGRP, which is abundant in the brain (15,17). We have reported the presence of AM in the human brain using the antisera against human AM(1–52) without cross-reaction with CGRP (10,11). AM-LI was localized in the paraventricular, supraoptic, and infundibular nuclei of human hypothalamus (11). In addi-

tion, a variety of cell types can produce AM, such as vascular endothelial cells and smooth muscle cells (12,13) and anterior pituitary cells (19). AM derived from these cells may partly contribute to the AM-LI in the cerebrospinal fluid. The chromatographic analyses showed that approximately 40% of the total AM-LI in the cerebrospinal fluid was eluted in the position of AM ( 1 – 52 ) . The nature of the material eluting in the first peak on the HPLC remains unclear. But the findings by Sephadex G50 column chromatography suggest that larger molecular weight substances, possibly a precursor of AM ( 1 – 52 ) or its processed forms, are present in the human cerebrospinal fluid. The findings in the present study support the idea that AM is a neuromodulator. AM may affect brain function through the cerebrospinal fluid as well as by neurosecretion.

REFERENCES 1. Ichiki, Y.; Kitamura, K.; Kangawa, K.; Kawamoto, M.; Matsuo, H.; Eto, T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 338:6–10; 1994. 2. Kitamura, K.; Kangawa, K.; Kawamoto, M.; et al. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192:553–560; 1993. 3. Kitamura. K.; Sakata, J.; Kangawa, K.; Kojima, M.; Matsuo, H.; Eto, T. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem. Biophys. Res. Commun. 194: 720–725; 1993. 4. Kitamura, K.; Ichiki, Y.; Tanaka, M.; et al. Immunoreactive adrenomedullin in human plasma. FEBS Lett. 341:288–290; 1994. 5. Martinez, A.; Miller, M. J.; Unsworth, E. J.; Siegfried, J. M.; Cuttitta, F. Expression of adrenomedullin in normal human lung and in pulmonary tumors. Endocrinology 136:4099–4105; 1995. 6. Murphy, T. C.; Samson, W. K. The novel vasoactive hormone, adrenomedullin, inhibits water drinking in the rat. Endocrinology 136:2459–2463; 1995. 7. Owji, A. A.; Smith, D. M.; Coppock, H. A.; et al. An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology 136:2127–2134; 1995. 8. Samson, W. K.; Murphy. T.; Schell, D. A. A novel vasoactive peptide, adrenomedullin, inhibits pituitary adrenocorticotropin release. Endocrinology 136:2349–2352; 1995. 9. Sato, K.; Hirata, Y.; Imai, T.; Iwashina, M.; Marumo, F. Characterization of immunoreactive adrenomedullin in human plasma and urine. Life Sci. 57:189–194; 1995. 10. Satoh, F.; Takahashi, K.; Murakami, O.; et al. Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. J. Clin. Endocrinol. Metab. 80:1750–1752; 1995.

11. Satoh, F.; Takahashi, K.; Murakami, O.; et al. Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland. Neurosci. Lett. 203:207–210; 1996. 12. Sugo, S.; Minamino, N.; Kangawa, K.; et al. Endothelial cells actively synthesize and secrete adrenomedullin. Biochem. Biophys. Res. Commun. 201:1160–1166; 1994. 13. Sugo, S.; Minamino, N.; Shoji, H.; et al. Production and secretion of adrenomedullin from vascular smooth muscle cells: Augumented production by tumor necrosis factor-a. Biochem. Biophys. Res. Commun. 203:719–726; 1994. 14. Takahashi, H.; Watanabe, T. X.; Nishimura, M.; et al. Centrally induced vasopressor and sympathetic responses to a novel endogenous peptide, adrenomedullin, in anesthetized rats. Am. J. Hypertens. 7:478–482; 1994. 15. Takahashi, K.; Mouri, T.; Sone, M.; et al. Calcitonin gene-related peptide in the human hypothalamus. Endocrinol. Jpn. 36:409–415; 1989. 16. Takahashi, K.; Satoh, F.; Hara, E.; et al. Production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells. J. Neurochem. 68:726–731; 1997. 17. Tschopp, F. A.; Henke, H.; Petermann, J. B.; et al. Calcitonin generelated peptide and its binding sites in the human central nervous system and pituitary. Proc. Natl. Acad. Sci. USA 82:248–252; 1985. 18. Wang, X.; Yue, T.-L.; Barone, F. C.; et al. Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage. Proc. Natl. Acad. Sci. USA 92:11480–11484; 1995. 19. Washimine, H.; Asada, Y.; Kitamura, K.; et al. Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue. Histochemistry 103:251–254; 1995.

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