BIOCHEMICAL
Vol. 169, No. 2, 1990 June 15, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 462-468
PRODUCTION OF FXDOTRELIN-1 BY RAT CIJLTURED MESANGIAL CELLS Hisato Sakamoto, Sei Sasaki, Yukio Hirata, Taihei Imai*, Kenji Ando**, Takashi Ida, Takeshi Sakurai*, Masashi Yanagisawa", TomohMasaki*, and Fumiaki Marumol Second Department of Internal Medicine, Tokyo Medical Dental University, Yushima, Tokyo 113, Japan *Institute
of
**Kitasato
Received April
Basic Medical Science, University Tsukuba, Ibaraki 305, Japan
Biochemical Laboratories, SMI-Bristol Kanagagawa229, Japan
and
of
Tsukuba,
Ltd, Sagamihara,
26, 1990
Summary: We investigated whether ET-l is synthesized by and released from mesangial cells. ET-l-like immunoreactivity (LI) released into medium increased time-dependently under a serum-free condition. The amounts of ET-l-L1 released into the mediumwas augmented in the presence of fetal calf serum. Reverse-phase HPLCprofile of the conditioned media revealed a major component coeluting with standard ET-l. Northern blot analysis of poly(A)+RNA extracted from mesangial cells showed a single major band corresponding to the size of preproET-1 mRNA (2.3 kb). These findings demonstrate that ET-1 is synthesized by and released from rat mesangial cells and suggest a possibility that it acts on their own cells as an autocrine factor. 0 1990 Academic Press, Inc.
Endothelin-1 isolated
is a novel vasoconstrictive
and sequenced from the supernatant of
endothelial cells
(ET-l)
cells
(1).
receptors
for ET-1 (Z),
breakdown of phosphoinositides,
Ca2+ concentration,
and subsequent contraction
However, it
porcine
and that
aortic
(3,4).
cells
synthesize and secrete endothelin
ET-1
induces
increase in intracellular
and replication
has not yet been determined
cells
the
cultured
originally
Recent studies have demonstrated that rat mesangial
possess specific
receptor-mediated
peptide
and related
present study was designed to test this possibility
of mesangial
whether
peptides.
mesangial Therefore,
by measurement of
lcorrespondence should be addressed to Fumiaki Marumo, The Second Departmment of Internal Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. 0006-291X/90 $1.50 Copyright 6 1990 by Academic Press, fnc. All rights of reproduction in any form reserved.
462
Vol.
169, No. 2, 1990
ET-l-like
BIOCHEMICAL
immunoreactivity
blot analysis
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(LI) released into the medium, and by
of rat preproET-1 mRNAin cultured
Northern
rat mesangial cells.
MATFIRIALSAAD METDODS Cells Culture. Kidneys were removed from IOO-15Og pentobarbital anesthetized male Sprague-Dawley rats. Isolated glomeruli were obt$ined by a sieving method (5,6). The cells were subcultured in 25 cm flask (CORNING,Corning, NY), and grown in RPM11640 supplemented with 15% fetal calf serum (FCS) (GIBCO Laboratories, Grand Island, NY) and 0.006% kanamycin. The cells thus obtained had characteristics of mesangial cells by the morphological features, such as large stellate or spindle shape with many irregular cytoplasmic projections on light microscopy, abundant bundles of contractile filaments on electron microscopy, and the presence of desmine by immunofluorescent staining. The contamination of endothelial cells was excluded by the absence of factor VIIIrelated antigen, morphological features and the fact that endothelial cells do not survive under the conventional culture conditions as employed (7). Fibroblast contamination was also excluded by the cells growth in D-valine-containigg media. Furthermore, t e cells contracted in resp nse to ionomycin (lo- M), angiotensin-II (IO- b M) and endothelin-1 (IO-’ M). Subcultured mesangial cells (3 - 13th passage) were used in the present experiments. Messngial
prewashed RIA of El!-1. Confluent mesangial cells (-1 x 106 cells/flask) with phosphate buffered-saline (PBS) were incubated for 8 or 24 h with 5 ml RPM11640 in the presence and absence of FCS. After incubation, media The supernatant was extracted with Spe-C were collected and centrifuged. cartridge (J.T.Baker Chemical Co., Phillipsburg, NJ) as previously reporte 2 The recovery of ET-1 during the extraction procedure was 64.1+3.7X (8). (mean+SE, n=4). RIA for ET-l was performed essentially in the same manner as recently described (8). In brief, 0.1 ml sample or standard ET-l (Peptide Institute, Inc., Osaka, Japan), 0.1 ml assay buffer and 0.1 ml rabbit antiET-1 serum (final dilution; 1:300,000) IjJyj e incubated for 24 h at 4 -C, followed by the addition of 0.05 ml I-ET-l (83 pmol/ml; specific activity of 74 TBq/mmol, AmershamInternational Plc, UK) and further incubation for 48 h. The separation of the bound from free ligands was performed by the double antibody/polyethylene glycol method (8).
Reverse-phase EPLC. Reverse-phase HPLC was performed using octadecylsilica column (0.45x25cm, 5 urn, Nucleosil, Machery-Nagel, FRG) with a linear gradient of-acetonitrile from 15 to- 60i in 0.09x acid at a flow rate of 1 ml/min. One-ml fractions were trifluoroacetic collected and assayed for ET-I-LI. The recovery of standard ET-1 during the chromatographic procedure was 95%.
Total RNA from blot aw4sis of preproendothelin-1 HA. method (11, confluent rat mesangial cells was extracted by the LiCl-urea and subjected to pol&A)+RNA selection. Both total (10 ug) and poly(A)'RNA gel, (IO ug) were electrophoresed through a 1.1X agarose/formaldehyde transferred to GeneScreen Plus membrane(New England Nuclear, Boston, MA) and hybridized with an 1.2 kb rat preproendothelin-1 cDNA as a probe (9). The autoradiography was performed by exposing the membraneto Kodak KAR-I film at -80% for 8 h. ltortbern
463
Vol.
169,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS Serial
dilution
curve
parallel
cells
was
l-L1
was released
dependently
to that
generated
of standard
in the presence
The amounts of ET-l-L1
released
ET-l
ET-l-L1
from the cells.
increased,
by the conditioned
into
media of
in RIA (Fig.l), released
into
and the absence
mesangial
suggesting the medium
of 10% FCS
medium in the presence
ETtime-
(Fig.2).
of 10% FCS were
+ 3.4 pg/ml (8 h) and 132 + 20 pg/ml (24 h); they were significantly
38.8
(p
Vol. 169, No. 2, 1990 June 15, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 462-468
PRODUCTION OF FXDOTRELIN-1 BY RAT CIJLTURED MESANGIAL CELLS Hisato Sakamoto, Sei Sasaki, Yukio Hirata, Taihei Imai*, Kenji Ando**, Takashi Ida, Takeshi Sakurai*, Masashi Yanagisawa", TomohMasaki*, and Fumiaki Marumol Second Department of Internal Medicine, Tokyo Medical Dental University, Yushima, Tokyo 113, Japan *Institute
of
**Kitasato
Received April
Basic Medical Science, University Tsukuba, Ibaraki 305, Japan
Biochemical Laboratories, SMI-Bristol Kanagagawa229, Japan
and
of
Tsukuba,
Ltd, Sagamihara,
26, 1990
Summary: We investigated whether ET-l is synthesized by and released from mesangial cells. ET-l-like immunoreactivity (LI) released into medium increased time-dependently under a serum-free condition. The amounts of ET-l-L1 released into the mediumwas augmented in the presence of fetal calf serum. Reverse-phase HPLCprofile of the conditioned media revealed a major component coeluting with standard ET-l. Northern blot analysis of poly(A)+RNA extracted from mesangial cells showed a single major band corresponding to the size of preproET-1 mRNA (2.3 kb). These findings demonstrate that ET-1 is synthesized by and released from rat mesangial cells and suggest a possibility that it acts on their own cells as an autocrine factor. 0 1990 Academic Press, Inc.
Endothelin-1 isolated
is a novel vasoconstrictive
and sequenced from the supernatant of
endothelial cells
(ET-l)
cells
(1).
receptors
for ET-1 (Z),
breakdown of phosphoinositides,
Ca2+ concentration,
and subsequent contraction
However, it
porcine
and that
aortic
(3,4).
cells
synthesize and secrete endothelin
ET-1
induces
increase in intracellular
and replication
has not yet been determined
cells
the
cultured
originally
Recent studies have demonstrated that rat mesangial
possess specific
receptor-mediated
peptide
and related
present study was designed to test this possibility
of mesangial
whether
peptides.
mesangial Therefore,
by measurement of
lcorrespondence should be addressed to Fumiaki Marumo, The Second Departmment of Internal Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. 0006-291X/90 $1.50 Copyright 6 1990 by Academic Press, fnc. All rights of reproduction in any form reserved.
462
Vol.
169, No. 2, 1990
ET-l-like
BIOCHEMICAL
immunoreactivity
blot analysis
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(LI) released into the medium, and by
of rat preproET-1 mRNAin cultured
Northern
rat mesangial cells.
MATFIRIALSAAD METDODS Cells Culture. Kidneys were removed from IOO-15Og pentobarbital anesthetized male Sprague-Dawley rats. Isolated glomeruli were obt$ined by a sieving method (5,6). The cells were subcultured in 25 cm flask (CORNING,Corning, NY), and grown in RPM11640 supplemented with 15% fetal calf serum (FCS) (GIBCO Laboratories, Grand Island, NY) and 0.006% kanamycin. The cells thus obtained had characteristics of mesangial cells by the morphological features, such as large stellate or spindle shape with many irregular cytoplasmic projections on light microscopy, abundant bundles of contractile filaments on electron microscopy, and the presence of desmine by immunofluorescent staining. The contamination of endothelial cells was excluded by the absence of factor VIIIrelated antigen, morphological features and the fact that endothelial cells do not survive under the conventional culture conditions as employed (7). Fibroblast contamination was also excluded by the cells growth in D-valine-containigg media. Furthermore, t e cells contracted in resp nse to ionomycin (lo- M), angiotensin-II (IO- b M) and endothelin-1 (IO-’ M). Subcultured mesangial cells (3 - 13th passage) were used in the present experiments. Messngial
prewashed RIA of El!-1. Confluent mesangial cells (-1 x 106 cells/flask) with phosphate buffered-saline (PBS) were incubated for 8 or 24 h with 5 ml RPM11640 in the presence and absence of FCS. After incubation, media The supernatant was extracted with Spe-C were collected and centrifuged. cartridge (J.T.Baker Chemical Co., Phillipsburg, NJ) as previously reporte 2 The recovery of ET-1 during the extraction procedure was 64.1+3.7X (8). (mean+SE, n=4). RIA for ET-l was performed essentially in the same manner as recently described (8). In brief, 0.1 ml sample or standard ET-l (Peptide Institute, Inc., Osaka, Japan), 0.1 ml assay buffer and 0.1 ml rabbit antiET-1 serum (final dilution; 1:300,000) IjJyj e incubated for 24 h at 4 -C, followed by the addition of 0.05 ml I-ET-l (83 pmol/ml; specific activity of 74 TBq/mmol, AmershamInternational Plc, UK) and further incubation for 48 h. The separation of the bound from free ligands was performed by the double antibody/polyethylene glycol method (8).
Reverse-phase EPLC. Reverse-phase HPLC was performed using octadecylsilica column (0.45x25cm, 5 urn, Nucleosil, Machery-Nagel, FRG) with a linear gradient of-acetonitrile from 15 to- 60i in 0.09x acid at a flow rate of 1 ml/min. One-ml fractions were trifluoroacetic collected and assayed for ET-I-LI. The recovery of standard ET-1 during the chromatographic procedure was 95%.
Total RNA from blot aw4sis of preproendothelin-1 HA. method (11, confluent rat mesangial cells was extracted by the LiCl-urea and subjected to pol&A)+RNA selection. Both total (10 ug) and poly(A)'RNA gel, (IO ug) were electrophoresed through a 1.1X agarose/formaldehyde transferred to GeneScreen Plus membrane(New England Nuclear, Boston, MA) and hybridized with an 1.2 kb rat preproendothelin-1 cDNA as a probe (9). The autoradiography was performed by exposing the membraneto Kodak KAR-I film at -80% for 8 h. ltortbern
463
Vol.
169,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS Serial
dilution
curve
parallel
cells
was
l-L1
was released
dependently
to that
generated
of standard
in the presence
The amounts of ET-l-L1
released
ET-l
ET-l-L1
from the cells.
increased,
by the conditioned
into
media of
in RIA (Fig.l), released
into
and the absence
mesangial
suggesting the medium
of 10% FCS
medium in the presence
ETtime-
(Fig.2).
of 10% FCS were
+ 3.4 pg/ml (8 h) and 132 + 20 pg/ml (24 h); they were significantly
38.8
(p
