Secretion of Endothelin-1 and Adrenomedullin by SW-13 Human Adrenocortical Carcinoma Cells
*Kazuhiro Takahashi, *Ayako Yoshinoya, *Osamu Murakami, +Kazuhito Totsune, and *Shigeki Shibahara
*Department of Molecular Biology & Applied Physiology and +Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi, Japan
Summary: The adrenal medulla and pheochromocytomas are known to secrete various neuropeptides and vasoactive peptides. On the other hand, the production and secretion of peptides by adrenocortical tumors have not been studied in detail. The study reported here therefore set out to examine these two functions for two vasoactive peptides, endothelin-1 (ET-1) and adrenomedullin (ADM) in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreac- tive ET (irET) and irADM were detected in the culture medium of
SW-13 cells. Northern blot analysis showed the expression of ET-1 and ADM mRNAs in SW-13 cells. On the other hand, no signifi- cant amounts of calcitonin-gene-related peptide, corticotropin- releasing-hormone, neuropeptide Y or urocortin were secreted by SW-13 cells. This study has shown that ET-1 and ADM are the two unique vasoactive peptides that are produced and secreted by adrenocortical carcinoma cells. Key Words: Endothelin (ET)- Adrenomedullin (ADM)-Adrenal cortex-SW-13-Adrenal car- cinoma.
The adrenal medulla and pheochromocytomas are known to secrete various neuropeptides and vasoactive peptides. On the other hand, the production and secre- tion of peptides by adrenocortical tumors have not been studied in detail. We have recently reported expression of adrenomedullin (ADM), a potent vasodilator peptide, in the tissues of various adrenocortical tumors and SW-13 human adrenocortical carcinoma cells (1). Endothelin-1 (ET-1) is produced and secreted by vari- ous tumors (2,3). However, the secretion of ET-i by SW-13 human adrenocortical carcinoma cells has not been studied. We therefore studied the production and secretion of ET-1 by SW-13 human adrenocortical car- cinoma cells.
METHODS
A human adrenocortical carcinoma cell line, SW-13 (4) was obtained from Health Science Research Resources Bank of Japan Health Sciences Foundation (Osaka, Japan) and cultured as previously reported (1).
The culture medium of SW-13 cells was extracted using a Sep-Pak C18 cartridge (Waters, Milford, MA, U.S.A.) and assayed as previously reported (5,6). Reverse-phase high performance liquid chromatogra- phy (HPLC) was performed as previously reported (1).
Northern blot analysis was performed as previously reported (1). The probe for ET-1 was the BamHI/EcoRI fragment of pBT-hET-1 (7).
RESULTS
Immunoreactive ET (irET) accumulated in the cul- ture medium of SW-13 cells time dependently (Fig. 1). Reverse-phase HPLC showed a single immunoreactive peak eluting in the position of synthetic ET-1 (data not shown). The irET levels in the culture medium were about 44% of immunoreactive adrenomedullin (irADM) levels in SW-13 cells (1). On the other hand, no signif- icant amounts of calcitonin-gene-related peptide, corti- cotropin-releasing hormone, neuropeptide Y or urocortin were secreted by SW-13 cells. Northern blot
15
IR-ET (fmol/105 cells)
10
5
0
4 h
8 h
24 h
32 h
Pheo
SW-13
ADM mRNA
ET-1 mRNA
ß-actin mRNA
analysis showed expression of ET-1 and ADM mRNAs in SW-13 cells (Fig. 2).
DISCUSSION
The present study has shown the production and sec- retion of ET-1 by SW-13 adrenocortical carcinoma cells, as well as ADM (1). Other neuropeptides, including cal- citonin-gene-related peptide and neuropeptide Y, were not secreted by these cells. Thus, ET-1 and ADM are the two unique peptides that are produced by the adrenocor- tical carcinoma. Both peptides have effects on the growth and differentiation of cells. ET-1 is known to have a mito- genic action on various cells including tumor cells (3,8). ADM has an inhibitory effect on the cell growth of vas- cular tissues (9), while it has a stimulatory effect on the tumor cell growth (10). Furthermore, it is known that ET-1 and ADM have modulatory effects on the secretion of adrenocortical steroid hormones (9,11).
Acknowledgments: This study has been supported partly by Grants-in-aid for Scientific Research for Exploratory Research from the Ministry of Education, Science, Sports and Culture of Japan. We thank Professor S.R. Bloom, Dr M.A. Ghatei and Dr P.M. Jones (Hammersmith Hospital, London) for their gift of the endothelin anti- serum (BP6) and the plasmid containing the endothelin-1 cDNA (pBT- hET-1), and Prof T. Yamamoto (Tohoku University) for ß-actin cDNA.
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