0021-972x/92/7505-1230$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright CC)1992 by The Endocrme Society
Synthesis Decidual
Vol. 15, No. 5 Printed m U S.A.
and Release Cells*
of Endothelin-1
TOSHIRO KUBOTA, SHUSAKU KAMADA, TAIHEI IMAI, FUMIAKI MARUMO, AND
YUKIO TAKESHI
HIRATA, AS0
by Human SATORU
Department of Obstetrics and Gynecology (T.K., S.K., T.A.), and Second Department (Y.H., SE., T.I., F.M.), Tokyo Medical and Dental University, Faculty of Medicine,
EGUCHI,
of Internal Medicine Tokyo 113, Japan
ABSTRACT We have studied whether a novel vasoconstrictor, endothelin-1 (ETl), is synthesized by and released from human decidual cells in early pregnancy, and whether ET-l acts directly on their own cells. It was observed that ET-l-like immunoreactivity (ET-l-LI) was released from cultured decidual, but not villous, cells, as a function of time. Reverse-phase high-pressure liquid chromatography of the conditioned media from the decidual cells revealed a major peak of ET-lLI coeluting with standard ET-l. Phorbol myristate acetate, a protein kinase C activator, dose-dependently increased the release of ET-l-L1 from the decidual cells, while a protein kinase C inhibitor, H7, significantly attenuated the stimulatory effect of phorbol myristate acetate on ET-l-L1 release. Northern blot analysis demonstrated the expres-
sion of messenger RNA for prepro-ET-l in the decidual tissue, but no such messenger RNA was observed in the villous tissue. The human decidual tissue contained a noninteracting, single class of binding sites demonstrating higher affinity for ET-1 and ET-2 than ET-3. This would be most consistent with the ETA receptor subtype. An ET-linduced, dose-dependent accumulation of total inositol phosphates was also observed in human decidual cells prelabeled with myo-[“Hlinositol. The present results demonstrate for the first time that human decidual cells in early pregnancy can synthesize and release ET-l. These cells also possess specific functional receptors for ET-1 which are coupled to phosphoinositide hydrolysis. Thus our data suggest a possible role for ET-1 in autocrine and/or paracrine function in human decidual cells. (J Clin Endocrinol Metab 75: 1230-1234, 1992)
E
peptin were purchased from Sigma Chemical (St. Louis, MO); ET-l, ET2, and ET-3 from Peptide Institute (Osaka, Japan); lZ51-ET-1 (specific activity: 74 TBq/mmol), [32P]deoxycystidine triphosphate (111 TBq/ mmol), and myo-[3H]inositol (3.03 TBq/mmol) from Amersham International (Amersham, UK); collagenase (125-200 IU/mg), deoxyribonuclease, RPMI-1640 medium, and fetal calf serum (FCS) from GIBCO (Grand Island, NY); collagen (type-l)-coated plates from Koken Co. (Tokyo, Japan); Spe Cs cartridge from J. T. Baker Chemical Co. (Phillipsburg, NJ); a MagnaGraph nylon membrane from Micron Separations Inc. (Westborough, MA); and Dowex AG l-X8 anion exchange formate resin from Bio-Rad Laboratories (Richmond, CA).
NDOTHELIN
(ET), originally isolated from the supernatant of cultured porcine endothelial cells, is a potent and long-lasting vasoconstrictive peptide with 21 amino acid residues (1). Subsequently, three ET isopeptides, named ET1, ET-2, and ET-3 with different biological activities, have been identified by screening of the human genomic DNA library (2). It has been reported that ET receptors are widely distributed (3-5). The expression of messenger RNA (mRNA) for prepro-ET-l has also been observed in a variety of human tissues (6-4, suggesting diverse actions in various organs. It has been shown that human placental membranes have high-affinity receptors for ET-1 (5), and that ET-l-like immunoreactivity (LI) is present in the human amniotic fluid (9). This suggests its potential role in the human fetoplacental environment. It has also been reported that ET-l-L1 is released from rabbit endometrial cells (10). This study was undertaken to determine whether human decidual cells produce and release ET-l-LI, and whether the cells express receptors for ET-l to mediate response to exogenous ET-l. Subjects
Cell culture The decidual tissue of normal early pregnancies (6-8 weeks of gestation) was quickly removed by dilatation and curettage, thoroughly washed with physiological saline, and cut into multiple blocks from which capillaries and blood clots were eliminated. These specimens were enzymatically dispersed with collagenase (2 mg/mL) and deoxyribonuclease (50 Fg/mL), and large cells were filtered and separated by nylon mesh (500 and 75 pm), as described previously (11). The cells were seeded onto a collagen-coated plate at a density of approximate lo6 viable cells/well in RPMI-1640 medium containing 10% FCS and antibiotics (12). They then were maintained for 24 h in a humidified 37 C atmosphere of 5% C02-95% air until they attached to the floor of the dish. The villous tissue was obtained simultaneously and similarly dispersed and cultured. On the day of the experiment, the cells were incubated with and without compounds tested in RPMI-1640 containing 10% FCS for 2448 h. At the end of incubation, the medium was removed, centrifuged, and stored until assayed. The number of cells was counted by the citric acid-crystal violet method (13).
and Methods
Compounds Compounds acetate (PMA),
were obtained from H7, phenylmethylsulfonyl
several
sources: phorbol fluoride, pepstatin,
myristate and leu-
Received December 9, 199 1. Address all correspondence and requests for reprints to: Toshiro Kubota, M.D., Department of Obstetrics and Gynecology, Tokyo Medical and Dental University, Faculty of Medicine, l-5-45, Yushima, Bunkyoku, Tokyo 113, Japan. *This work was supported in part by Grants-in-Aid (03670773, 2304055, 03268102, 03454512, 03454218) from the Ministry of Education, Science and Culture of Japan and a fund from the Uehara Memorial Foundation.
Reverse-phase
high-pressure
liquid chromatography
The pooled conditioned media (10 mL) were trifluoroacetic acid (TFA), and the supernatant cartridge and eluted with 2 mL 60% acetonitrile/O.l% previously (14,15). The eluates were evaporated to of synthetic ET-1 during the extraction procedure
(HPLC)
acidified with 0.1% applied to a Spe C, TFA as reported dryness; the recovery was 77%. The extract
1230
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ENDOTHELIN of conditioned medium phase HPLC using an Macherey-Nagel, Duren, 60%) of acetonitrile in min. A I-mL fraction recovery of ET-l during
AND
HUMAN
DECIDUAL
CELLS
1231
from the decidual cells was subjected to reverseoctadecylsilica column (0.45 X 25 cm, 5 pm: Germany) eluted with a linear gradient (150.1% TFA for 70 min (14); flow rate was 1 mL/ was collected and assayed for ET-l-LI. The the chromatographic procedure was 95%.
RIA of ET-l The ET-1 RIA was performed essentially as reported previously (15, 16), using ‘2’I-ET-l as a tracer and synthetic ET-I as a standard; the antibody employed in the RIA demonstrated 100% cross-reactivity with ET-l, ET-2, and ET-3, but less than 1% with big ET-l. The final dilution of the antibody was 1:12,000; the sensitivity was 1 fmol/tube and the 50% intercept was 14 fmol/tube. The coefficients of intra- and interassay variability were 3.2% and 8.6%, respectively.
0’
1
0 Northern
I
2.5
5
blot analysis
Total RNA from human decidual and villous tissue in early pregnancy was extracted with the LiCl-urea method and subjected to poly(A)’ RNA selection as previously described (17). Poly(A)’ RNA (10 Kg) was fractionated in a formaldehyde/l. 1% agarose gel electrophoresis and transferred to a MagnaGraph nylon membrane. The probe 3’.noncoding exon from cloned human prepro-ET-1 gene (1) was labeled with [“‘I’] deoxycystidine triphosphate by the random-primed method, and incubated at 42 C for 16 h with the membranes in a hybridization buffer containing 1 mol NaC1/50% (vol/vol) formamide/l% sodium dodecyl sulfate/250 pg/mL of salmon sperm DNA. The membranes were washed with 0.3 mol NaC1/30 mmol sodium citrate/l% sodium dodecyl sulfate at 60 C, and autoradiographed on a Kodak XAR-1 film with an intensifying screen at -80 C for 8-16 h (18).
Binding
1
I
10 ET-l
I
I
I
25 50 100 250 500 1000 (fmol/tube)
FIG. 1. Serial dilution curve of conditioned medium cidual cells in ET-1 RIA. Standard curve of ET-1 curve of conditioned medium (0) are compared.
from human de(0) and dilution
experiments
Binding studies were performed in the same manner as described previously (19). In brief, human decidual tissue in early pregnancy was homogenized in 20 mmol phosphate buffer, pH 7.5, containing 130 mmol NaCl, 1 mmol ethylenediaminetetraacetate, 0.2 mmol phenylmethylsulfonyl fluoride, 10 pg/mL pepstatin, and 10 pg/mL leupeptin. The homogenate was then washed twice, resuspended in the same buffer, and used as membrane preparations. ‘?-ET-l (50 pL) was added to 100 FL of the membrane suspension in the presence and absence of unlabeled ET-l, ET-2, and ET-3. The mixtures were incubated for 30 min at 30 C and then free radioactive ligands were removed by centrifugation. Radioactivity was measured with an Aloca y-counter (ARC300). Specific binding was defined as the total binding minus the nonspecific binding in the presence of an excess (lo-’ mol) of unlabeled ET-l. Binding parameters were calculated by Scatchard analysis of the binding data obtained from equilibrium displacement binding curves.
Measurement
of
inositol phosphates
Human decidual cells (-lo6 cells/well) were prelabeled with 185 KBq myo-[3H]inositol in RPMI-1640 medium for 48 h, washed twice with the same medium containing 10 mmol LiCI, then exposed to ET-1 and ET-3 for 15 min. Incubation was terminated by a rapid withdrawal of the medium, and plates were then flash-frozen using liquid nitrogen and stored at -20 C. The frozen monolayer was scraped free in 0.5 mL of 0.2 mol KC1/5 mmol ethyleneglycol-bis-(@-aminoethyl ether)N,N,N’,N’-tetraacetic acid, pH 7.4, and total inositol phosphates (II’,) were extracted in an acidified aqueous-organic solvent system (chloroform/methanol/water/hydrochloric acid, 2:2:1:0.02). They were separated by chromatographic analysis on Dowex AG l-X8 anion exchange formate resin as previously described (20-22). In the aqueous phase, free [3H]inositol was initially removed with 0.1 mol HCOOH, and the total labeled II’, fraction was then discharged with 0.1 mol HCOOH/ 1.0 mol NH,COOH. Radioactivity in each effluent was determined by a liquid scintillation spectrometer.
4b Retention
50 Time
(min)
FIG. 2. Reverse-phase HPLC profile of medium from cultured decidual cells. A solid (15-60%) of acetonitrile in 0.1% TFA for the retention time of authentic ET-l. Solid
Statistical
extract of the conditioned line shows a linear gradient 70 min. An arrow indicates columns show ET-l-LI.
methods
Data were analyzed by a one-way Bonferroni test for multiple comparisons as mean + SD.
analysis of variance (23). All data were
and the expressed
Results The dilution curve of the conditioned medium from cultured human decidual cells was parallel to a standard curve of ET-l in RIA (Fig. 1). An elution profile of the medium extract from cultured decidual cells on reverse-phase HPLC is shown in Fig. 2; a major component of ET-l-L1 was eluted in the position corresponding to authentic ET-l. The ET-l-L1 was released from the decidual cells into medium as a function of time (Fig. 3); the ET-l-L1 concentration increased linearly during the 12-h incubation, reaching a plateau after 24 h. However, no ET-l-L1 was detected in the medium from cultured villous cells. The protein kinase C (PKC) activator, PMA, dose-dependently (10-7-10-6 mol) increased the release of ET-l-L1 from the decidual cells after 48-h incubation (Fig. 4). The PKC inhibitor, H7, significantly
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KUBOTA
1232
JCE & M. 1992 Vol75.No5
ET AL.
3 loo0 2z 800 2 s
8 2 Hours
in Culture
600 400
7 T
200
L
0
FIG. 3. Release of ET-l-L1 from human decidual cells (0) and villous cells (A) as a function of time. The cells were incubated at 37 C in RPMI-1640 medium with 10% FCS during the 24-h incubation. Each point is the mean of six wells; bar shows SD.
**
lr- *ir** -r 11
(P < 0.01) attenuated the stimulatory effect of PMA on ET-
l-L1 release(Fig. 4). Northern blot analysis of poly(A)+ RNA from human decidual and villous tissuewas performed using complementary DNA for human prepro-ET-l as a probe (Fig. 5). A single hybridization band corresponding to the size (2.3 kb) of mRNA coding for human prepro-ET-l was demonstrable in the decidual tissue, but not in the villous tissue. Competitive binding study of ‘251-ET-1 to the decidual membranes is shown in Fig. 6. Unlabeled ET-1 and ET-2 competitively inhibited the binding of ‘251-ET-1to the membranes with I&s of 10-i’ mol and 5 X lo-” mol, respectively, while unlabeled ET-3 showed a less steep inhibition curve with an IC,, of 8 X 10-l’ mol. Scatchard analysis of the binding data suggested the presence of high-affinity binding sites of ET-l in the decidua (Fig. 6, inset) with an apparent dissociation constant of 4.3 X lo-” mol and a maximal binding capacity of 0.90 pmol/mg protein, ET-l induced dose-dependent (1O-s-1O-6 mol) accumulations of II’, in myo-[3H]inositol prelabeled decidual cells during a 15-min incubation (Fig. 7). The dose-dependent effect of ET-3 was lessthan ET-l. Discussion This is the first report demonstrating that human dccidual cells in early pregnancy synthesize and secrete ET-l. The ET-l-like materials releasedfrom the decidua in vitro appear to be immunologically and physiochemically similar to authentic ET-l, based on the parallelism to ET-1 RIA, and a retention time identical with that of standard ET-l by reverse-phaseHPLC. Recently, it has been reported that ET-l-L1 was detectable in the amniotic fluid of term pregnancy (9) and that preproET mRNA was expressedin the avascular human amnion at term (8). Taken together, these observations suggestthat ETl-L1 present in human amniotic fluid may be derived pri-
,
g
lOOO-
1 2 .5
800-
i! 8 5 IY 7 'i-
600400200-
kl PMA (lo-‘M) H-7 (lo-‘M)
(-) (-)
(-) (+)
(+) C-J
(+) (+I
FIG. 4. Effects of PMA and H7 on the release of ET-l-L1 from human decidual cells. The cells were incubated at 37 C for 48 h in the absence and presence of PMA (1O-8-1O-6 mol) (upper panel), and PMA (10e6 mol) and H7 (W6 mol) (lowerpanel). Each column shows the mean of six wells; bar shows SD. (*), P c 0.001; (**), P < 0.01; (***), P < 0.05 us. control.
marily from decidual cells. The present study clearly demonstrates that human decidual, but not villous, cells secrete ET-l-LI. Furthermore, Northern blot analysis of poly(A)+ RNA from the decidual tissue,but not from the villous tissue, in early pregnancy, reveals the expression of a prepro-ET-l mRNA identical to that expressed in vascular endothelium (18). These findings verify de ~OVOsynthesis of ET-l by and its releasefrom the human decidual cells. The present study further shows that PMA, a PKC activator, significantly increasesthe release of ET-l-L1 from the decidual cells. The PKC inhibitor, H7, significantly attenuates ET-l-L1 release.Yanagisawa et al. (18) have originally demonstrated that a potent PKC activator, tetradecanoylphorbol acetate, caused a marked induction of prepro-ET-l mRNA
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ENDOTHELIN
AND
HUMAN
DECIDUAL
1233
CELLS
4
28s rRNA
6
IOUNO
A---A
8
10
(fmol)
ET-3 l.
I
\
1,
‘0,
L\
0 lo-‘*
lo-”
1o-g
10-10 Endothelins
c
..%....__ ~-. t --------“p 10-e
(M)
FIG. 6. Competitive binding of “9-ET-l to human decidual membranes by unlabeled ET isopeptides. The decidual membranes (100 gg) were incubated at 30 C for 30 min in the absence and the presence of various doses of ET-l (O), ET-2 (O), and ET-3 (A). Specific binding was 73.1% of total binding. Each point is the mean of triplicate samples. Inset, Scatchard plot of binding data.
PPIET-1 2.3 kb
10r
18s rRNA-
B FIG. 5. Northern blot analysis of mRNA in the human decidua (A) and villi (B) of early human pregnancy. Poly(A)+ RNA (10 pg) was hybridized with human prepro-ET-l complementary DNA as a probe. A single band with a size of 2.3 kb was observed in the decidua, but not in the villi. Size markers in kb are indicated on the left.
in human endothelial cells, and stimulates the releaseof ETl-L1 from cultured bovine endothelial cells (16). These findings suggestthat activation of PKC plays an important role in the mechanism of ET-1 synthesis and/or release from decidual cells in a similar fashion as endothelial cells. Three isopeptides of the ET family, ET-l, ET-2, and ET-3, possessa diverse set of pharmacological activities with different potencies (2). Recent studies have suggestedthe existence of two subtypes of ET receptor: the ET-l/ET-2-selective ET subtype (ETA) and the nonisopeptide-selective subtype (ETB) (24, 25). The present binding study clearly shows the presence of a noninteracting, single class of binding sites demonstrating higher-affinity for ET-1 and ET-2 than ET-3 in human decidual tissue. This would suggest a predominance of ET,+receptor subtype in human decidual cells. It has been reported that ET-l stimulates polyphosphoinositide breakdown via a phospholipase C-mediated mech-
I
I 0-
I
10 --O Endothelin
I
lo+
lo-'
1o-6
(M)
FIG. 7. Effect of ET-l on production of total IP, in human decidual cells. After being prelabeled with myo-[3H]inositol for 48 h, cells were exposed to ET-l (0) and ET-3 (0) for 15 min in the presence of 10 mmol LiCl. IP, was extracted by Dowex AG l-X8 anion exchange formate resin. Each point is the mean of six wells from one of three similar experiments. (*), P < 0.001; (**), P < 0.05 us. control.
anism in various cells, such as vascular smooth muscle cells (3), renal mesangial cells (26), and fibroblasts (27). In fact, both ET, and ET* receptors containing seven transmembrane domains belong to the G-proteins-coupled receptor superfamily. In this study, we observed that ET-1 stimulates hydrolysis of phosphoinositide more potently than ET-3 in cultured human decidual cells. These results are consistent with those of binding studies. Since ET-l induces a transient increasein intracellular Ca2+in fura- -loaded human decidual cells (our unpublished observation), the significant increasesin II’, production by ET-l strongly suggestthat ET-l induces intracellular Ca*+ mobilization via phosphoinositide breakdown. The physiological functions of ET-1 in the human decidual
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1234
KUBOTA
cell remain unknown. Recently, Stojilkovic et al. (28) have demonstrated that ET-1 stimulates the release of gonadotropin from rat anterior pituitary cells through the mobilization of an intracellular Ca2+ influx. However, ET-l has no effect on PRL release from cultured human decidual cells (our unpublished observation). It also has been reported that ET1 stimulates the proliferation of many cells, including rat vascular smooth muscle cells (3), fibroblasts (27), glomerular mesangial cells (26), and certain human tumor cells (17). Therefore, it is possible that ET-l may function as a mitogen for human decidual cells. The autocrine and/or paracrine role of ET-1 in human decidual cells needs to be investigated. In conclusion, the present study demonstrates that human decidual cells can synthesize and release ET-l, and possess specific receptors for ET-l functionally coupled to phosphoinositide breakdown. This suggests that ET-1 may have its potential autocrine and/or paracrine function in human decidual cells. Acknowledgments We gratefully acknowledge excellent technical assistance.
Ms. M. Ohba
and M. Kurosawa
for their
References 1. Yanagisawa M, Kunihara H, Kimura S, et al. 1988 A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 332:411-415. 2. Inoue A, Yanagisawa M, Kimura S, et al. 1989 The human endothelin family: three structurally and pharmacologically distinct isopeptides by three separate genes. Proc Nat1 Acad Sci USA. 86:2863-2867. 3. Hirata Y, Yoshimi H, Takata S, et al. 1988 Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commum. 154:868-875. 4. Koseki C, Imai M, Hirata Y, Yanagisawa M, Masaki T. 1989 Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide? Am J Physiol. 256:858-866. 5. Fischli W, Clozel M, Guilly C. 1989 Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay. Life Sci. 44:1429-1436. 6. MacCunber MW, Ross CA, Glaser BM, Snyder SH. 1989 Endothelin: visualization of mRNA by in situ hybridization provides evidence for local action. Proc Nat1 Acad Sci USA. 86:7285-7289. Shichiri M, Hirata Y, Emori T, et al. 1989 Secretion of endothelin and related peptides from renal epithelial cell lines. FEBS Lett. 253:203-206. Sunnergren KP, Word RA, Sambrook JF, MacDonald PC, Casey ML. 1990 Expression and regulation of endothelin precursor mRNA in avascular human amnion. Mol Cel Endocrinol. 68:R7-R14. Usuki S, Saitoh T, Sawamura T, et al. 1990 Increased maternal plasma concentration of endothelin-1 during labor pain or on delivery and the existence of a large amount of endothelin-1 in amniotic fluid. Gynecol Endocrinol. 4:85-97.
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JCE & M. 1992 Vol75.No5
10. Orlando C, Brandi ML, Peri A, et al. 1990 Neurohypophyseal hormone regulation of endothelin secretion from rabbit endometrial cells in primary culture. Endocrinology. 126:1780-1783. 11. Yamamoto T, Nishiyama M, Yanoh K, Naka Y, Naka A, Sugiyama Y. 1989 Regulation df human endometrial and decidual cell f&tion -Role of epidermal growth factor in the decidualization. Proa Clin Biol Res. i94:413-4T4. 12. Kubota T, Kamada S, Taguchi M, Aso T. 1991 The effects of insulin-like growth factor-1 (IGF-I) and IGF-11 on prolactin (PRL) release from human decidua and amniotic fluid PRL. Acta Obstet Gynaecol Jpn. 43:1515-1520. 13. Patterson Jr MK. 1979 Measurement of growth and viability of cell in culture. In: Jacoby WB, Pastan IH. eds Methods Enzymol, Academic Press, New York, 58:141-150. 14. Hirata Y, Matsunaga T, Ando K, Furukawa T, Tsukagoshi H, Marumo F. 1990 Presence of endothelin-l-like immunoreactivity in human cerebrospinal fluid. Biochem Biophys Res Commun. 166:1274-1278. 15. Ando K, Hirata Y, Shichiri M, Emori T, Marumo F. 1990 Presence of immunoreactive endothelin in human plasma. FEBS Lett. 245:164-166. 16. Emori T, Hirata Y, Ohta K, Shichiri M, Marumo F. 1989 Secretory mechanism of immunoreactive endothelin in cultured bovine endothelial cells. Biochem Biophys Res Commun. 160:93-100. 17. Shichiri M, Hirata Y, Nakajima T, et al. 1991 Endothelin-1 is an autocrine/paracrine growth factor for human cancer cell lines. J Clin Invest. 87:1867-1871. 18. Yanagisawa M, Inoue A, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T. 1989 The human preproendothelin-1 gene: possible regulation by endothelial phosphoinositide turnover signaling. J Cardiovasc Pharmacol. 13:513-S17. 19. Hagiwara H, Kozuka M, Ito T, Eguchi S, Hirose S. 1990 Properties of the rat uterus endothelin receptor sites. Biochem Res. 11:93-98. 20. Judd AM, Jarvis WD, MacLeod RM. 1987 Attenuation of pituitary polyphosphoinositide metabolism by protein kinase C activation. Mol Cell Endocrinol. 54:107-114. 21. Javis WD, Judd AM, MacLeod RM. 1989 Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the D2 dopamine receutor. Endocrinoloev. 123:2793-2799. 22. Kubota T; Kuan SI, MacLeYdd RM. 1989 Effect of 17 P-estradiol on phosphoinositide metabolism and prolactin secretion in anterior pituitary cells. Neuroendocrinology. 50:400-405. 23. Grove WM, Andreason NC. 1982 Stimulation tests for many hypotheses in exploratory research. J Nerv Ment Dis. 170:3-8. . 24. Sakurai T, Yanagisawa M, Takuwa Y, et al. 1990 Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 348:732-735. 25. Arai H. Hori S. Aramori I. Ohkubo H. Nakanishi S. 1990 Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 348:730-732. 26. Simonsen MS, Wann S, Mene P, et al. 1989 Endothelin stimulates phospholipase C, Na’/H’ exchange, c-fos expression, and mitogenesis in rat mesangial cells. J Clin Invest. 83:708-712. 27. Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki T. 1989 A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Biol Chem. 238:249-252. 28. Stojilkovic SS, Merelli F, Iida T, Krsmanovic LZ, Catt KJ. 1990 Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior pituitary cells. Science. 248:1663-1666.
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Synthesis Decidual
Vol. 15, No. 5 Printed m U S.A.
and Release Cells*
of Endothelin-1
TOSHIRO KUBOTA, SHUSAKU KAMADA, TAIHEI IMAI, FUMIAKI MARUMO, AND
YUKIO TAKESHI
HIRATA, AS0
by Human SATORU
Department of Obstetrics and Gynecology (T.K., S.K., T.A.), and Second Department (Y.H., SE., T.I., F.M.), Tokyo Medical and Dental University, Faculty of Medicine,
EGUCHI,
of Internal Medicine Tokyo 113, Japan
ABSTRACT We have studied whether a novel vasoconstrictor, endothelin-1 (ETl), is synthesized by and released from human decidual cells in early pregnancy, and whether ET-l acts directly on their own cells. It was observed that ET-l-like immunoreactivity (ET-l-LI) was released from cultured decidual, but not villous, cells, as a function of time. Reverse-phase high-pressure liquid chromatography of the conditioned media from the decidual cells revealed a major peak of ET-lLI coeluting with standard ET-l. Phorbol myristate acetate, a protein kinase C activator, dose-dependently increased the release of ET-l-L1 from the decidual cells, while a protein kinase C inhibitor, H7, significantly attenuated the stimulatory effect of phorbol myristate acetate on ET-l-L1 release. Northern blot analysis demonstrated the expres-
sion of messenger RNA for prepro-ET-l in the decidual tissue, but no such messenger RNA was observed in the villous tissue. The human decidual tissue contained a noninteracting, single class of binding sites demonstrating higher affinity for ET-1 and ET-2 than ET-3. This would be most consistent with the ETA receptor subtype. An ET-linduced, dose-dependent accumulation of total inositol phosphates was also observed in human decidual cells prelabeled with myo-[“Hlinositol. The present results demonstrate for the first time that human decidual cells in early pregnancy can synthesize and release ET-l. These cells also possess specific functional receptors for ET-1 which are coupled to phosphoinositide hydrolysis. Thus our data suggest a possible role for ET-1 in autocrine and/or paracrine function in human decidual cells. (J Clin Endocrinol Metab 75: 1230-1234, 1992)
E
peptin were purchased from Sigma Chemical (St. Louis, MO); ET-l, ET2, and ET-3 from Peptide Institute (Osaka, Japan); lZ51-ET-1 (specific activity: 74 TBq/mmol), [32P]deoxycystidine triphosphate (111 TBq/ mmol), and myo-[3H]inositol (3.03 TBq/mmol) from Amersham International (Amersham, UK); collagenase (125-200 IU/mg), deoxyribonuclease, RPMI-1640 medium, and fetal calf serum (FCS) from GIBCO (Grand Island, NY); collagen (type-l)-coated plates from Koken Co. (Tokyo, Japan); Spe Cs cartridge from J. T. Baker Chemical Co. (Phillipsburg, NJ); a MagnaGraph nylon membrane from Micron Separations Inc. (Westborough, MA); and Dowex AG l-X8 anion exchange formate resin from Bio-Rad Laboratories (Richmond, CA).
NDOTHELIN
(ET), originally isolated from the supernatant of cultured porcine endothelial cells, is a potent and long-lasting vasoconstrictive peptide with 21 amino acid residues (1). Subsequently, three ET isopeptides, named ET1, ET-2, and ET-3 with different biological activities, have been identified by screening of the human genomic DNA library (2). It has been reported that ET receptors are widely distributed (3-5). The expression of messenger RNA (mRNA) for prepro-ET-l has also been observed in a variety of human tissues (6-4, suggesting diverse actions in various organs. It has been shown that human placental membranes have high-affinity receptors for ET-1 (5), and that ET-l-like immunoreactivity (LI) is present in the human amniotic fluid (9). This suggests its potential role in the human fetoplacental environment. It has also been reported that ET-l-L1 is released from rabbit endometrial cells (10). This study was undertaken to determine whether human decidual cells produce and release ET-l-LI, and whether the cells express receptors for ET-l to mediate response to exogenous ET-l. Subjects
Cell culture The decidual tissue of normal early pregnancies (6-8 weeks of gestation) was quickly removed by dilatation and curettage, thoroughly washed with physiological saline, and cut into multiple blocks from which capillaries and blood clots were eliminated. These specimens were enzymatically dispersed with collagenase (2 mg/mL) and deoxyribonuclease (50 Fg/mL), and large cells were filtered and separated by nylon mesh (500 and 75 pm), as described previously (11). The cells were seeded onto a collagen-coated plate at a density of approximate lo6 viable cells/well in RPMI-1640 medium containing 10% FCS and antibiotics (12). They then were maintained for 24 h in a humidified 37 C atmosphere of 5% C02-95% air until they attached to the floor of the dish. The villous tissue was obtained simultaneously and similarly dispersed and cultured. On the day of the experiment, the cells were incubated with and without compounds tested in RPMI-1640 containing 10% FCS for 2448 h. At the end of incubation, the medium was removed, centrifuged, and stored until assayed. The number of cells was counted by the citric acid-crystal violet method (13).
and Methods
Compounds Compounds acetate (PMA),
were obtained from H7, phenylmethylsulfonyl
several
sources: phorbol fluoride, pepstatin,
myristate and leu-
Received December 9, 199 1. Address all correspondence and requests for reprints to: Toshiro Kubota, M.D., Department of Obstetrics and Gynecology, Tokyo Medical and Dental University, Faculty of Medicine, l-5-45, Yushima, Bunkyoku, Tokyo 113, Japan. *This work was supported in part by Grants-in-Aid (03670773, 2304055, 03268102, 03454512, 03454218) from the Ministry of Education, Science and Culture of Japan and a fund from the Uehara Memorial Foundation.
Reverse-phase
high-pressure
liquid chromatography
The pooled conditioned media (10 mL) were trifluoroacetic acid (TFA), and the supernatant cartridge and eluted with 2 mL 60% acetonitrile/O.l% previously (14,15). The eluates were evaporated to of synthetic ET-1 during the extraction procedure
(HPLC)
acidified with 0.1% applied to a Spe C, TFA as reported dryness; the recovery was 77%. The extract
1230
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ENDOTHELIN of conditioned medium phase HPLC using an Macherey-Nagel, Duren, 60%) of acetonitrile in min. A I-mL fraction recovery of ET-l during
AND
HUMAN
DECIDUAL
CELLS
1231
from the decidual cells was subjected to reverseoctadecylsilica column (0.45 X 25 cm, 5 pm: Germany) eluted with a linear gradient (150.1% TFA for 70 min (14); flow rate was 1 mL/ was collected and assayed for ET-l-LI. The the chromatographic procedure was 95%.
RIA of ET-l The ET-1 RIA was performed essentially as reported previously (15, 16), using ‘2’I-ET-l as a tracer and synthetic ET-I as a standard; the antibody employed in the RIA demonstrated 100% cross-reactivity with ET-l, ET-2, and ET-3, but less than 1% with big ET-l. The final dilution of the antibody was 1:12,000; the sensitivity was 1 fmol/tube and the 50% intercept was 14 fmol/tube. The coefficients of intra- and interassay variability were 3.2% and 8.6%, respectively.
0’
1
0 Northern
I
2.5
5
blot analysis
Total RNA from human decidual and villous tissue in early pregnancy was extracted with the LiCl-urea method and subjected to poly(A)’ RNA selection as previously described (17). Poly(A)’ RNA (10 Kg) was fractionated in a formaldehyde/l. 1% agarose gel electrophoresis and transferred to a MagnaGraph nylon membrane. The probe 3’.noncoding exon from cloned human prepro-ET-1 gene (1) was labeled with [“‘I’] deoxycystidine triphosphate by the random-primed method, and incubated at 42 C for 16 h with the membranes in a hybridization buffer containing 1 mol NaC1/50% (vol/vol) formamide/l% sodium dodecyl sulfate/250 pg/mL of salmon sperm DNA. The membranes were washed with 0.3 mol NaC1/30 mmol sodium citrate/l% sodium dodecyl sulfate at 60 C, and autoradiographed on a Kodak XAR-1 film with an intensifying screen at -80 C for 8-16 h (18).
Binding
1
I
10 ET-l
I
I
I
25 50 100 250 500 1000 (fmol/tube)
FIG. 1. Serial dilution curve of conditioned medium cidual cells in ET-1 RIA. Standard curve of ET-1 curve of conditioned medium (0) are compared.
from human de(0) and dilution
experiments
Binding studies were performed in the same manner as described previously (19). In brief, human decidual tissue in early pregnancy was homogenized in 20 mmol phosphate buffer, pH 7.5, containing 130 mmol NaCl, 1 mmol ethylenediaminetetraacetate, 0.2 mmol phenylmethylsulfonyl fluoride, 10 pg/mL pepstatin, and 10 pg/mL leupeptin. The homogenate was then washed twice, resuspended in the same buffer, and used as membrane preparations. ‘?-ET-l (50 pL) was added to 100 FL of the membrane suspension in the presence and absence of unlabeled ET-l, ET-2, and ET-3. The mixtures were incubated for 30 min at 30 C and then free radioactive ligands were removed by centrifugation. Radioactivity was measured with an Aloca y-counter (ARC300). Specific binding was defined as the total binding minus the nonspecific binding in the presence of an excess (lo-’ mol) of unlabeled ET-l. Binding parameters were calculated by Scatchard analysis of the binding data obtained from equilibrium displacement binding curves.
Measurement
of
inositol phosphates
Human decidual cells (-lo6 cells/well) were prelabeled with 185 KBq myo-[3H]inositol in RPMI-1640 medium for 48 h, washed twice with the same medium containing 10 mmol LiCI, then exposed to ET-1 and ET-3 for 15 min. Incubation was terminated by a rapid withdrawal of the medium, and plates were then flash-frozen using liquid nitrogen and stored at -20 C. The frozen monolayer was scraped free in 0.5 mL of 0.2 mol KC1/5 mmol ethyleneglycol-bis-(@-aminoethyl ether)N,N,N’,N’-tetraacetic acid, pH 7.4, and total inositol phosphates (II’,) were extracted in an acidified aqueous-organic solvent system (chloroform/methanol/water/hydrochloric acid, 2:2:1:0.02). They were separated by chromatographic analysis on Dowex AG l-X8 anion exchange formate resin as previously described (20-22). In the aqueous phase, free [3H]inositol was initially removed with 0.1 mol HCOOH, and the total labeled II’, fraction was then discharged with 0.1 mol HCOOH/ 1.0 mol NH,COOH. Radioactivity in each effluent was determined by a liquid scintillation spectrometer.
4b Retention
50 Time
(min)
FIG. 2. Reverse-phase HPLC profile of medium from cultured decidual cells. A solid (15-60%) of acetonitrile in 0.1% TFA for the retention time of authentic ET-l. Solid
Statistical
extract of the conditioned line shows a linear gradient 70 min. An arrow indicates columns show ET-l-LI.
methods
Data were analyzed by a one-way Bonferroni test for multiple comparisons as mean + SD.
analysis of variance (23). All data were
and the expressed
Results The dilution curve of the conditioned medium from cultured human decidual cells was parallel to a standard curve of ET-l in RIA (Fig. 1). An elution profile of the medium extract from cultured decidual cells on reverse-phase HPLC is shown in Fig. 2; a major component of ET-l-L1 was eluted in the position corresponding to authentic ET-l. The ET-l-L1 was released from the decidual cells into medium as a function of time (Fig. 3); the ET-l-L1 concentration increased linearly during the 12-h incubation, reaching a plateau after 24 h. However, no ET-l-L1 was detected in the medium from cultured villous cells. The protein kinase C (PKC) activator, PMA, dose-dependently (10-7-10-6 mol) increased the release of ET-l-L1 from the decidual cells after 48-h incubation (Fig. 4). The PKC inhibitor, H7, significantly
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KUBOTA
1232
JCE & M. 1992 Vol75.No5
ET AL.
3 loo0 2z 800 2 s
8 2 Hours
in Culture
600 400
7 T
200
L
0
FIG. 3. Release of ET-l-L1 from human decidual cells (0) and villous cells (A) as a function of time. The cells were incubated at 37 C in RPMI-1640 medium with 10% FCS during the 24-h incubation. Each point is the mean of six wells; bar shows SD.
**
lr- *ir** -r 11
(P < 0.01) attenuated the stimulatory effect of PMA on ET-
l-L1 release(Fig. 4). Northern blot analysis of poly(A)+ RNA from human decidual and villous tissuewas performed using complementary DNA for human prepro-ET-l as a probe (Fig. 5). A single hybridization band corresponding to the size (2.3 kb) of mRNA coding for human prepro-ET-l was demonstrable in the decidual tissue, but not in the villous tissue. Competitive binding study of ‘251-ET-1 to the decidual membranes is shown in Fig. 6. Unlabeled ET-1 and ET-2 competitively inhibited the binding of ‘251-ET-1to the membranes with I&s of 10-i’ mol and 5 X lo-” mol, respectively, while unlabeled ET-3 showed a less steep inhibition curve with an IC,, of 8 X 10-l’ mol. Scatchard analysis of the binding data suggested the presence of high-affinity binding sites of ET-l in the decidua (Fig. 6, inset) with an apparent dissociation constant of 4.3 X lo-” mol and a maximal binding capacity of 0.90 pmol/mg protein, ET-l induced dose-dependent (1O-s-1O-6 mol) accumulations of II’, in myo-[3H]inositol prelabeled decidual cells during a 15-min incubation (Fig. 7). The dose-dependent effect of ET-3 was lessthan ET-l. Discussion This is the first report demonstrating that human dccidual cells in early pregnancy synthesize and secrete ET-l. The ET-l-like materials releasedfrom the decidua in vitro appear to be immunologically and physiochemically similar to authentic ET-l, based on the parallelism to ET-1 RIA, and a retention time identical with that of standard ET-l by reverse-phaseHPLC. Recently, it has been reported that ET-l-L1 was detectable in the amniotic fluid of term pregnancy (9) and that preproET mRNA was expressedin the avascular human amnion at term (8). Taken together, these observations suggestthat ETl-L1 present in human amniotic fluid may be derived pri-
,
g
lOOO-
1 2 .5
800-
i! 8 5 IY 7 'i-
600400200-
kl PMA (lo-‘M) H-7 (lo-‘M)
(-) (-)
(-) (+)
(+) C-J
(+) (+I
FIG. 4. Effects of PMA and H7 on the release of ET-l-L1 from human decidual cells. The cells were incubated at 37 C for 48 h in the absence and presence of PMA (1O-8-1O-6 mol) (upper panel), and PMA (10e6 mol) and H7 (W6 mol) (lowerpanel). Each column shows the mean of six wells; bar shows SD. (*), P c 0.001; (**), P < 0.01; (***), P < 0.05 us. control.
marily from decidual cells. The present study clearly demonstrates that human decidual, but not villous, cells secrete ET-l-LI. Furthermore, Northern blot analysis of poly(A)+ RNA from the decidual tissue,but not from the villous tissue, in early pregnancy, reveals the expression of a prepro-ET-l mRNA identical to that expressed in vascular endothelium (18). These findings verify de ~OVOsynthesis of ET-l by and its releasefrom the human decidual cells. The present study further shows that PMA, a PKC activator, significantly increasesthe release of ET-l-L1 from the decidual cells. The PKC inhibitor, H7, significantly attenuates ET-l-L1 release.Yanagisawa et al. (18) have originally demonstrated that a potent PKC activator, tetradecanoylphorbol acetate, caused a marked induction of prepro-ET-l mRNA
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ENDOTHELIN
AND
HUMAN
DECIDUAL
1233
CELLS
4
28s rRNA
6
IOUNO
A---A
8
10
(fmol)
ET-3 l.
I
\
1,
‘0,
L\
0 lo-‘*
lo-”
1o-g
10-10 Endothelins
c
..%....__ ~-. t --------“p 10-e
(M)
FIG. 6. Competitive binding of “9-ET-l to human decidual membranes by unlabeled ET isopeptides. The decidual membranes (100 gg) were incubated at 30 C for 30 min in the absence and the presence of various doses of ET-l (O), ET-2 (O), and ET-3 (A). Specific binding was 73.1% of total binding. Each point is the mean of triplicate samples. Inset, Scatchard plot of binding data.
PPIET-1 2.3 kb
10r
18s rRNA-
B FIG. 5. Northern blot analysis of mRNA in the human decidua (A) and villi (B) of early human pregnancy. Poly(A)+ RNA (10 pg) was hybridized with human prepro-ET-l complementary DNA as a probe. A single band with a size of 2.3 kb was observed in the decidua, but not in the villi. Size markers in kb are indicated on the left.
in human endothelial cells, and stimulates the releaseof ETl-L1 from cultured bovine endothelial cells (16). These findings suggestthat activation of PKC plays an important role in the mechanism of ET-1 synthesis and/or release from decidual cells in a similar fashion as endothelial cells. Three isopeptides of the ET family, ET-l, ET-2, and ET-3, possessa diverse set of pharmacological activities with different potencies (2). Recent studies have suggestedthe existence of two subtypes of ET receptor: the ET-l/ET-2-selective ET subtype (ETA) and the nonisopeptide-selective subtype (ETB) (24, 25). The present binding study clearly shows the presence of a noninteracting, single class of binding sites demonstrating higher-affinity for ET-1 and ET-2 than ET-3 in human decidual tissue. This would suggest a predominance of ET,+receptor subtype in human decidual cells. It has been reported that ET-l stimulates polyphosphoinositide breakdown via a phospholipase C-mediated mech-
I
I 0-
I
10 --O Endothelin
I
lo+
lo-'
1o-6
(M)
FIG. 7. Effect of ET-l on production of total IP, in human decidual cells. After being prelabeled with myo-[3H]inositol for 48 h, cells were exposed to ET-l (0) and ET-3 (0) for 15 min in the presence of 10 mmol LiCl. IP, was extracted by Dowex AG l-X8 anion exchange formate resin. Each point is the mean of six wells from one of three similar experiments. (*), P < 0.001; (**), P < 0.05 us. control.
anism in various cells, such as vascular smooth muscle cells (3), renal mesangial cells (26), and fibroblasts (27). In fact, both ET, and ET* receptors containing seven transmembrane domains belong to the G-proteins-coupled receptor superfamily. In this study, we observed that ET-1 stimulates hydrolysis of phosphoinositide more potently than ET-3 in cultured human decidual cells. These results are consistent with those of binding studies. Since ET-l induces a transient increasein intracellular Ca2+in fura- -loaded human decidual cells (our unpublished observation), the significant increasesin II’, production by ET-l strongly suggestthat ET-l induces intracellular Ca*+ mobilization via phosphoinositide breakdown. The physiological functions of ET-1 in the human decidual
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KUBOTA
cell remain unknown. Recently, Stojilkovic et al. (28) have demonstrated that ET-1 stimulates the release of gonadotropin from rat anterior pituitary cells through the mobilization of an intracellular Ca2+ influx. However, ET-l has no effect on PRL release from cultured human decidual cells (our unpublished observation). It also has been reported that ET1 stimulates the proliferation of many cells, including rat vascular smooth muscle cells (3), fibroblasts (27), glomerular mesangial cells (26), and certain human tumor cells (17). Therefore, it is possible that ET-l may function as a mitogen for human decidual cells. The autocrine and/or paracrine role of ET-1 in human decidual cells needs to be investigated. In conclusion, the present study demonstrates that human decidual cells can synthesize and release ET-l, and possess specific receptors for ET-l functionally coupled to phosphoinositide breakdown. This suggests that ET-1 may have its potential autocrine and/or paracrine function in human decidual cells. Acknowledgments We gratefully acknowledge excellent technical assistance.
Ms. M. Ohba
and M. Kurosawa
for their
References 1. Yanagisawa M, Kunihara H, Kimura S, et al. 1988 A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 332:411-415. 2. Inoue A, Yanagisawa M, Kimura S, et al. 1989 The human endothelin family: three structurally and pharmacologically distinct isopeptides by three separate genes. Proc Nat1 Acad Sci USA. 86:2863-2867. 3. Hirata Y, Yoshimi H, Takata S, et al. 1988 Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commum. 154:868-875. 4. Koseki C, Imai M, Hirata Y, Yanagisawa M, Masaki T. 1989 Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide? Am J Physiol. 256:858-866. 5. Fischli W, Clozel M, Guilly C. 1989 Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay. Life Sci. 44:1429-1436. 6. MacCunber MW, Ross CA, Glaser BM, Snyder SH. 1989 Endothelin: visualization of mRNA by in situ hybridization provides evidence for local action. Proc Nat1 Acad Sci USA. 86:7285-7289. Shichiri M, Hirata Y, Emori T, et al. 1989 Secretion of endothelin and related peptides from renal epithelial cell lines. FEBS Lett. 253:203-206. Sunnergren KP, Word RA, Sambrook JF, MacDonald PC, Casey ML. 1990 Expression and regulation of endothelin precursor mRNA in avascular human amnion. Mol Cel Endocrinol. 68:R7-R14. Usuki S, Saitoh T, Sawamura T, et al. 1990 Increased maternal plasma concentration of endothelin-1 during labor pain or on delivery and the existence of a large amount of endothelin-1 in amniotic fluid. Gynecol Endocrinol. 4:85-97.
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10. Orlando C, Brandi ML, Peri A, et al. 1990 Neurohypophyseal hormone regulation of endothelin secretion from rabbit endometrial cells in primary culture. Endocrinology. 126:1780-1783. 11. Yamamoto T, Nishiyama M, Yanoh K, Naka Y, Naka A, Sugiyama Y. 1989 Regulation df human endometrial and decidual cell f&tion -Role of epidermal growth factor in the decidualization. Proa Clin Biol Res. i94:413-4T4. 12. Kubota T, Kamada S, Taguchi M, Aso T. 1991 The effects of insulin-like growth factor-1 (IGF-I) and IGF-11 on prolactin (PRL) release from human decidua and amniotic fluid PRL. Acta Obstet Gynaecol Jpn. 43:1515-1520. 13. Patterson Jr MK. 1979 Measurement of growth and viability of cell in culture. In: Jacoby WB, Pastan IH. eds Methods Enzymol, Academic Press, New York, 58:141-150. 14. Hirata Y, Matsunaga T, Ando K, Furukawa T, Tsukagoshi H, Marumo F. 1990 Presence of endothelin-l-like immunoreactivity in human cerebrospinal fluid. Biochem Biophys Res Commun. 166:1274-1278. 15. Ando K, Hirata Y, Shichiri M, Emori T, Marumo F. 1990 Presence of immunoreactive endothelin in human plasma. FEBS Lett. 245:164-166. 16. Emori T, Hirata Y, Ohta K, Shichiri M, Marumo F. 1989 Secretory mechanism of immunoreactive endothelin in cultured bovine endothelial cells. Biochem Biophys Res Commun. 160:93-100. 17. Shichiri M, Hirata Y, Nakajima T, et al. 1991 Endothelin-1 is an autocrine/paracrine growth factor for human cancer cell lines. J Clin Invest. 87:1867-1871. 18. Yanagisawa M, Inoue A, Takuwa Y, Mitsui Y, Kobayashi M, Masaki T. 1989 The human preproendothelin-1 gene: possible regulation by endothelial phosphoinositide turnover signaling. J Cardiovasc Pharmacol. 13:513-S17. 19. Hagiwara H, Kozuka M, Ito T, Eguchi S, Hirose S. 1990 Properties of the rat uterus endothelin receptor sites. Biochem Res. 11:93-98. 20. Judd AM, Jarvis WD, MacLeod RM. 1987 Attenuation of pituitary polyphosphoinositide metabolism by protein kinase C activation. Mol Cell Endocrinol. 54:107-114. 21. Javis WD, Judd AM, MacLeod RM. 1989 Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the D2 dopamine receutor. Endocrinoloev. 123:2793-2799. 22. Kubota T; Kuan SI, MacLeYdd RM. 1989 Effect of 17 P-estradiol on phosphoinositide metabolism and prolactin secretion in anterior pituitary cells. Neuroendocrinology. 50:400-405. 23. Grove WM, Andreason NC. 1982 Stimulation tests for many hypotheses in exploratory research. J Nerv Ment Dis. 170:3-8. . 24. Sakurai T, Yanagisawa M, Takuwa Y, et al. 1990 Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 348:732-735. 25. Arai H. Hori S. Aramori I. Ohkubo H. Nakanishi S. 1990 Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 348:730-732. 26. Simonsen MS, Wann S, Mene P, et al. 1989 Endothelin stimulates phospholipase C, Na’/H’ exchange, c-fos expression, and mitogenesis in rat mesangial cells. J Clin Invest. 83:708-712. 27. Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki T. 1989 A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Biol Chem. 238:249-252. 28. Stojilkovic SS, Merelli F, Iida T, Krsmanovic LZ, Catt KJ. 1990 Endothelin stimulation of cytosolic calcium and gonadotropin secretion in anterior pituitary cells. Science. 248:1663-1666.
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