0021-972x/92/7502-0547$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 2 Printed in U.S.A.
Atria1 Natriuretic Peptide Receptors Endometrial Stromal Cells* JOHN L. GILILLAND, SABITA LAHIRI, AND Reproductive Washington,
YUEH-CHU LEONARD
L. TSENG, WARTOFSKY
Endocrine and Endocrine-Metabolic D. C. 20307-5001
VLADIMIR
in Human
TROCHE,
Services, Walter Reed Army Medical
Center,
ABSTRACT Atria1 natriuretic peptide (ANP) has been shown to affect water and ion transport and specific ANP binding has been identified in several secretory tissues. ANP commonly acts via stimulation of membranebound particulate guanylate cyclase with the production of cyclic guanosine monophosphate (cGMP). We questioned whether ANP played a role in the complex cyclic transformation of the endometrium into a secretory tissue, and whether its action was cGMP mediated. Endometrium was obtained by biopsy in regularly menstruating women and stromal cells were isolated and cultured for use in this study. ANP competitive binding assays were performed using 12SI-labeled ANP (0.1 nmol/L) and increasing concentrations of unlabeled ANP (O-1000 nmol/L). Optimal binding was obtained after 3-h incubation at 4 C and binding characteristics, including dissociation constant and bind-
ing site quantity, were estimated by Scatchard analysis. Specific, high affinity (dissociation constant, 0.078 + 0.004 nmol/L) and low capacity (4,877 + 1,951 binding sites/cell) ANP binding was identified, with nonspecific binding representing less than or equal to 16% of total binding. Evaluation of ANP-stimulated cyclic nucleotide production revealed an increase in cGMP production, with a 7-fold increase at 1000 nmol/L ANP, and no effect on CAMP production. In conclusion, we have identified specific high affinity receptors for ANP in human endometrial cells, suggesting a role for ANP in endometrial cell function and/or development mediated via cGMP production. We propose that ANP may affect local salt and water metabolism, may be involved in the secretory evolution of glandular and stromal cells, and may further facilitate endometrial development via modulation of local vascular tone and endothelial permeability. (J Clin Endocrinol Metub 75: 547-551, 1992)
A
men (39.8 f 3.4 pmol/L) though no fluctuation was seen during the menstrual cycle. ANP levels in postmenopausal women were not significantly different from those in agematched men but were significantly higher (78.3 + 19.4 pmol/L) than those from young women, suggesting effects mediated by the sex steroids. ANP levels have also been evaluated during pregnancy, with most significant increases noted at 48 h postpartum (22). This increase coincides with the phenomenon of postpartum diuresis and may result from atria1stretch causedby a shift in body fluids into the vascular space. Specific binding sites for ANP have been discovered in the placenta (23) though no physiological role for ANP in placental hemodynamics has yet been elucidated. Potent inhibition of uterine smooth muscle contraction has been demonstrated in the nongravid rat uterus (24). This effect, however, was not demonstrable in the gravid rat uterus and a similar refractoriness to the tocolytic effects of ANP was obtained after treatment of virgin rats with progesterone (25). The endometrium has not yet been evaluated for the presence of ANP or its receptors, nor has ANP’s impact on endometrial development been studied.
TRIAL cardiocyte granules were first described in 1956 (l), though their significance at that time was unknown. It wasn’t until 1981 that de Bold et al. (2) suggested that these granules were the source of a factor affecting the volume regulatory function of the kidney, after demonstrating a rapid and potent natriuretic and diuretic response in rats injected with atria1 extracts. Since that time, multiple additional biological properties of atria1 natriuretic peptide (ANP) have been defined (3-12). Several mechanismsof action of ANP have been demonstrated, the most common of which is via stimulation of membrane-bound or particulate guanylate cyclase with accumulation of cyclic guanosinemonophosphate (cGMP) (13). In addition, inhibition of adenylate cyclase and subsequent CAMP production has been found in some tissues such as vascular tissue (14), adrenal cortex (8, 15), pituitary gland (16), and thyroid gland (17) in associationwith ANP binding. ANP’s effects on the reproductive tract have not yet been clarified, though evidence exists to support its involvement in hypothalamic, pituitary, and gonadal function (18-20). A recent report by Clark et al. (21) studied the influence of gender, age, and the menstrual cycle on plasma ANP. ANP levels in young, menstruating women (mean + SE, 68.1 & 5.5 pmol/L) averaged approximately twice those in young
Materials Primary
Received June 21, 1991. Address requests for reprints to: Dr. Leonard Wartofsky, Chief, Department of Medicine, 7D, Walter Reed Army Medical Center, Washington, D. C. 20307-5001. * The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
and Methods
cell cultures
Endometrium was obtained from regularly menstruating, healthy, adult females undergoing laparoscopy (with general anesthesia) for various indications. Exclusion criteria included pregnancy, abnormal bleeding, amenorrhea, infection, malignancy, and use of oral contraceptive pills or other estrogenor progestin-containing medication. Tissue was obtained in accord with Institutional Review Board guidelines of
547
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548
GILILLAND
the Walter Reed Army Medical Center. Freshly obtained endometrium was washed three times in phenol red-free Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Chemical Co., St. Louis, MO), and then digested with 2.5 g/L Type I collagenase (Worthington Biochemical Corp., Freehold, NJ) in phosphate buffered saline (PBS) for 1 h in a 37 C water bath with frequent, intermittent shaking. The resultant cell suspension was centrifuged at 400 X g for 10 min and the pellet was resuspended in Basal Medium [BM; DMEM supplemented with an antibiotic-antimycotic mixture (100 U/mL penicillin, 100 &mL streptomycin, and 0.25 pg/mL amphotericin-B; GIBCO Laboratories, Grand Island, NY), 2 mmol/L L-glutamine (GIBCO), and 10% fetal bovine serum (Hyclone Laboratories, Logan, UT)] and then placed in 162-cm* culture flasks (Costar, Cambridge, MA) for 30 min at 37 C to allow for stromal cell attachment as described by Schatz et al. (26). After 30 min, the medium with nonadherent cells was aspirated, discarded, and replaced with fresh BM. After 72 h of incubation in a humidified 5% CO2 atmosphere at 37 C, the stromal cell population was further purified by differential trypsinization, as described by Cherny and Findlay (27), with a calciumand magnesium-free PBS solution containing 0.5 g/L trypsin (Sigma) and 0.2 g/L EDTA (Sigma). The cell suspension was then centrifuged at 400 x g for 10 min and the pellet resuspended in BM. Total cell count was determined using trypan blue exclusion on an aliquot of the suspension and cells were plated at a density of 100,000/we11 in 24.well culture plates (Costar). These cells were incubated for an additional 8 days before receptor assay with one fresh medium change 4 days after trypsinization. At the completion of this time, cells were consistently in a monolayer covering the entire surface of the culture well base.
Immunocytochemical
identification
Epithelial cells, unlike cells of mesenchymal origin (e.g. stromal cells), produce keratin (28), which has been used as a discriminator for these two cell types (29). Identification of epithelial and stromal elements in monolayer culture was accomplished using a three-layered glucoseoxidase stain (Vector Laboratories, Burlingame, CA) by sequential treatment with rabbit antihuman keratin (Dako Corporation, Carpinteria, CA) and goat antirabbit antiserum (Vector). After purification by the methods previously described, stromal cells were cultured on standard coverslips. In addition, mixed populations of epithelial and stromal cells cultured on coverslips were used as controls. When complete monolayers were achieved, cells were fixed with cold, absolute methanol. They were then flooded with rabbit antihuman keratin, incubated at 25 C for 1 h, and washed three times with PBS. Visible localization of keratin was then accomplished using an ABCglucose oxidase kit (Vector).
A NP receptor assay ANP cell surface receptors were assayed using the method devised by Leitman and Murad (30). Optimal equilibrium binding conditions for stromal cells were determined before these experiments with a binding study in which specific ANP binding was compared at different incubation temperatures and times (see Results). Cells in 24-well plates were washed twice with DMEM and then incubated for 3 h at 4 C with 200 PL buffer [DMEM, 2 g/L BSA, 20 mmol/L HEPES (Sigma)] containing 0.1 nmol/L [‘251]cu-rat ANPieZs (DuPont, Wilmington, DE) and increasing concentrations (O-100 nmol/L) of unlabeled a-human ANP1-28 (Peninsula Laboratories, Belmont, CA). After washing twice with cold PBS containing 2 g/L BSA, cells were solubilized with 0.1 mol/L NaOH and transferred to glass counting tubes, and the radioactivity was determined in a y-counter. Data were evaluated using Scatchard analysis (31) with LlGAND (32), a computer program that provided unbiased best estimates of the equilibrium dissociation constants (Kd) and of binding site concentration. Nonspecific binding was calculated from wells in which labeled ANP (0.1 nmol/L) and an overwhelming concentration of unlabeled ANP (1000 nmol/L) were coincubated. This value, expressed as a percentage of total added labeled ANP, was then subtracted from total binding to obtain specific binding. Nonspecific binding was calculated for each specimen and for each lot of labeled ANP to control for potential interassay variation. Calculation of the number of binding sites per cell required an accurate estimate of the number of cells per well. On the
ET AL.
JCE & M .1992 Voll5.No2
day of the receptor assay, cell counts were performed in two representative wells from each plate assayed. This was accomplished using trypsin for cell detachment and trypan blue exclusion to identify viable cells. Cell count variability between wells was less than 10%.
Cyclic nucleotide Cyclic nucleotide was evaluated in by Kasagi et al. monolayer culture Hank’s solution, isobutyl-methylxanthine, 1000 nmol/L) of well was collected determination of Wilmington, DE), respectively.
assay production in response to stimulation with ANP endometrial stromal cells using a method developed (33). After washing with DMEM, stromal cells in were incubated for 2 h at 37 C in 250 PL low-salt pH 7.5, with 20 mmol/L HEPES, 0.5 mmol/L 34 g/L BSA, and increasing concentrations (Oa-human ANP. After incubation, buffer from each immediately and stored at -20 C for subsequent cGMP and CAMP concentrations by RIA (DuPont, with assay sensitivities of 0.01 and 0.1 pmol/lOO pL,
Results
We evaluated stromal cells obtained from the endometrium of seven patients at various times during the menstrual cycle. In addition to taking a detailed patient history, a portion of the tissue obtained was sent to pathology for histologic dating and representative stromal cell preparations were evaluated for epithelial contamination as described.No demonstrable epithelial cell contamination was noted in monolayers cultured from purified stromal cell preparations, whereas cultures of mixed cell populations demonstrated keratin staining in epithelial elements. Specific ANP binding variability as a function of incubation temperature and time is depicted graphically in Fig. 1. Receptor binding at 4 C steadily increased with time, reaching a plateau by 3 h, with no demonstrable decrease in binding noted within the time frame studied. In contrast, binding at 25 C and 37 C peaked and then decreasedwith further incubation, a result likely attributable to receptor internalization and degradation under these conditions. Competition binding data, obtained at 4 C, were analyzed and revealed a single set of specific ANP receptors with high affinity (Kd mean of 0.078 + 0.004 nmol/L) and low capacity (mean of 4877 & 1951 binding sites per cell) binding. Nonspecific binding was lessthan or equal to 16%. Patient data
0
1
2 TIME
3
4
(hr)
FIG. 1. Evaluation of time and temperature as variables in ANP binding in cultured human endometrial stromal cells. Specific binding was calculated by subtracting nonspecific binding from total binding. Nonspecific binding was obtained by coincubation of labeled hormone (0.1 nmol/L) and unlabeled hormone (1000 nmol/L).
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ATRIAL
NATRIURETIC
RECEPTORS
are displayed in Table 1 and Fig. 2 is a representative Scatchard Plot (data from patient 2). Although a-rat ANP was used in these binding studies (differing from a-human ANP by only one amino acid), its performance in competition binding assays, in terms of binding affinity similarity, has been previously demonstrated (9). Though no absolute conclusions regarding receptor number variation can be made because the number of patients was small, a trend toward an increase in receptor number in the proliferative phase was noted. Incubation of endometrial stromal cells with ANP resulted in an increase in production of cGMP as measured in the incubation buffer. This increase behaved in a dose-response fashion, with a 3-fold increase at 100 nmol/L and a greater than 7-fold increase at 1000 nmol/L ANP (see Fig. 3). No change in CAMP production was noted after the addition of ANP. Discussion The physiological effects of ANP are wide ranging and involve a large number of target tissues. These effects are TABLE 1. Scatchard from competitive ANP 3h Patient no.
analysis of receptor binding data obtained binding to endometrial stromal cells at 4 C for
Endometrial histopathology
1 2 3 4 5 6 7
Proliferative Proliferative Secretory,POD Secretory,POD Secretory,POD Secretory,POD Secretory,POD
Kd (nmol/L)
Binding sites (no. per cell)b
0.074 0.079 0.081 0.094 0.083 0.078 0.055
15,024 7,642 1,178 5,800 2,020 1,289 1,188
1-2 2-3 4-5 11-12 12-13
Nonspecific binding 66) 9.7 4.9 13.9 3.8 11.7 15.0 16.0
‘Kd (dissociation constant) was estimated by Scatchard analysis using specific-bound data obtained by subtracting nonspecific binding from total binding. ‘Binding site number per cell was also estimated by Scatchard analysis, using specific-bound data and correcting for total cell number. Cell number per well was obtained by averaging cell counts from two representative wells, obtained on the day of competitive binding assay.
0.03 Y 8 B
I
K d = 0.079 nmol/L Binding Sites/Cell
i
0.02 .
z
m
. me
0.01 -
.
\
.
0.00 ! 0
0.25
0.5 BOUND
FIG. 2. Scatchard unlabeled cells.
= 7,642
ANP
0.75
1 1.0
1.25
(nmol/L)
plot of competition binding data with labeled and in monolayers of cultured human endometrial stromal
IN ENDOMETRIAL
STROMA
.32 1
T
.28 ” 5 L 2
.24 .20 -
,P E
.16 -
!i
.12 -
8
.08 .04 I1
0’”
0
0.1
I
1
I
10
I
I
100
I,
1000
ANP (nmol/L) FIG. 3. cGMP stromal
production cells in response
in monolayer to the addition
cultures of human of ANP.
endometrial
mediated by specific cell surface receptors via the production of cGMP as a second messenger.ANP has been shown to affect water and ion transport and specific ANP binding has been identified in several tissues with secretory functions (34-36), leading one to speculate that ANP may be involved in the secretory processesof some tissues, including the endometrium. A detailed understanding of the developmental processes responsible for the complex, cyclical transformation of the endometrium in the ovarian/menstrual cycle is critical to comprehending the many stepsthat must occur for successful establishment and maintenance of a pregnancy. The unique capacity of pregnancy sustenance requires an endometrium that is of adequate depth, vascularity, and nutritional richness, a state that is achieved through a well orchestrated seriesof events, involving multiple factors. Early in the cycle, after menses,the endometrium is very thin and consists of epithelial and compacted stromal elements. Under the influence of estrogen produced by the developing ovarian follicle, these cellular elements proliferate, and by midcycle the endometrium achieves a height some 10 times its initial height. This expansion results not only from an increase in cell number and size but more significantly from a “reinflation” of the stroma with the acquisition of water, ions, and amino acids (37). Another interesting observation is the occurrence throughout the proliferative phase of a small amount of apocrine-like secretion into gland lumina (38), the significance of which is as yet unclear. During the secretory phase, heralded by ovulation and a significant increase in progesterone secretion, proliferation of glandular and stromal elements is inhibited as their energies are directed more toward secretion. Thereafter, endometrial height remains relatively stable. The most significant alterations in the stroma during the secretory phase include stromal edema and a characteristic transfiguration of stromal cells known as predecidualization. Stromal edema, which also occurs to some degree in the proliferative phase, peaks on postovulatory day (POD) 8. Predecidualization, the result of the production and acquisition of cytoplasmic glycogen, is first seen on POD 9 and progresses to involve nearly all stromal cells by the end of the cycle.
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550
GILILLAND
Participation by ANP in endometrial development and function is certainly plausible and, since plasma levels of ANP do not appear to change significantly during the menstrual cycle, one conceivable explanation for regulation of ANP effects in the endometrium is cyclical variation in receptor quantity. Although our data suggesta trend toward increased receptor number in the proliferative phase, additional studies will be necessary to establish a relationship between receptor number and phase of the menstrual cycle. As progesterone has been shown to modulate ANP effects in the myometrium, possibly by affecting receptor number, it may have similar effects in the endometrium. Physiologically, an increase in the number of ANP receptors and a subsequent increase in ANP binding in the proliferative phase would support the process of stromal expansion, which requires significant water and ion acquisition by the tissue. Local production of cGMP may alsoresult in increased blood flow to the endometrium, which would be advantageous in terms of the provision of other hormones and growth factors to a rapidly growing cell population. In conclusion, we have identified specific, high affinity receptors for ANP in human endometrial stromal cells and have demonstrated a responseto ANP binding in these cells, with a significant increase in cGMP production in a doseresponsefashion. We propose that ANP may affect local salt and water metabolism, may be involved in the evolution of glandular and stromal cells, and may further facilitate endometrial development via modulation of local vascular tone and endothelial permeability. Acknowledgments This project was completed with the approval and support Department of Clinical Investigation, Walter Reed Army Medical We are indebted to Dr. Dennis O’Connor for his evaluation histopathology from each specimen and to Dr. Thomas Klein advice and editorial assistance.
of the Center. of the for his
References 1. Kisch B. 1956 Electron microscopy of the atrium of the heart I. Guinea pig. Exp Med Surg. 114:99-112. 2. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. 1981 A rapid and potent natriuretic response to intravenous injection of atria1 myocardial extract in rats. Life Sci. 28:89-94. ’ 3. Currie MG. Geller DM, Cole BR. et al. 1983 Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science. 221:71-3. 4. Abman SH, Accurso FJ. 1991 Sustained fetal pulmonary vasodilation with prolonged atria1 natriuretic factor and cGMP infusions. Am J Physiol. 260:H183-92. 5. Lofton CE, Newman WH, Currie MG. 1990 Atria1 natriuretic peptide regulation of endothelial permeability is mediated by cGMP. Biochem Biophys Res Commun. 172:793-9. 6. Kurtz A, Della Bruna R, Pfeilschifter J, Taugner R, Bauer C. 1986 Atria1 natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process. Proc Nat1 Acad Sci USA. 83:4769-73. 7. Goodfriend TL, Elliott ME, Atlas SA. 1984 Actions of synthetic atria1 natriuretic factor on bovine adrenal glomerulosa. Life Sci. 35:1675-82. 8. MacFarland RT, Zelus BD, Beavo JA. 1991 High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in CAMP and steroidogenesis in adrenal glomerulosa cells. J
ET AL.
JCE & M. 1992 Vol75.No2
Biol Chem. 2661136-42. 9. Sellitti DF, Tseng YL, Wartofsky L. 1989 Receptors for atria1 natriuretic peptide?ANP) and regulation of thyroglobulin secretion bv ANP in human thvroid cells. Life Sci. 45:793-801. 10. Antunes-Rodrigues J, McCann SM, Samson WK. 1986 Central administration of atria1 natriuretic factor inhibits saline preference in the rat. Endocrinology. 118:1726-S. 11. Samson WK, Aguila MC, Martinovic J, Antunes-Rodrigues J, Norris M. 1987 Hypothalamic action of atria1 natriuretic peptide to inhibit vasopressin secretion. Peptides. 8:449-54. K, Nishida Y, Hosomi H, Tanaka S. 1991 Effects of 12. Matsushita atria1 natriuretic peptide on water and NaCl absorption across the intestine. Am J Physiol. 260:R6-12. 13. Waldman SA, Rapoport RM, Murad F. 1984 Atria1 natriuretic factor selectively activates particulate guanylate cyclase and elevated cyclic GMP in rat tissues. J Biol Chem. 259:14332-4. 14. Anand-Srivastava MB, Franks DJ, Cantin M, Genest J. 1984 Atria1 natriuretic factor inhibits adenylate cyclase activity. Biochem Biophys Res Commun. 121:855-62. 15. Anand-Srivastava MB, Genest J, Cantin M. 1985 Inhibitory effect of atria1 natriuretic factor on adenylate cyclase activity in adrenal cortical membranes. FEBS Lett. 181:199-202. MB, Genest J, Cantin M. 1985 Inhibition of 16. Anand-Srivastava pituitary adenylate cyclase by atria1 natriuretic factor. Life Sci. 36:1873-9. 17. Tseng YL, Lahiri S, Sellitti DF, Burman KD, D/Avis JC, Wartofsky L. 1990 Characterization by affinity cross-linking of a receptor for atria1 natriuretic peptide in cultured human thyroid cells associated with reductions in both adenosine 3’,5’-monophosphate production and thyroglobulin secretion. J Clin Endocrinol Metab. 70:528-33. 18. Mukhopadhyay AK, Bohnet HG, Leidenberger FA. 1986 Testosterone production by mouse Leydig cells is stimulated in vitro by atria1 natriuretic factor. FEBS Lett. 202: 11 l-6. 19. Pandey KN, Osteen KG, Inagami T. 1987 Specific receptor-mediated stimulation of progesterone secretion and cGMP accumulation by rat atria1 natriuretic factor in cultured human granulosalutein (G-L) cells, Endocrinology. 121:1195-7. 20. Samson WK, Aguila MC, Bianchi R. 1988 Atria1 natriuretic factor inhibits luteinizing hormone secretion in the rat: evidence for a hypothalamic site of action. Endocrinology. 122:1573-82. 21. Clark BA, Elahi D, Epstein FH. 1990 The influence of gender, age, and the menstrual cycle on plasma atria1 natriuretic peptide. J Clin Endocrinol Metab. 70:349-52. 22. Merkouris RW, Miller FC, Catanzarite V, Rigg LA, Quirk JG, Vesely DL. 1990 Increase in the plasma levels of the N-terminal and C-terminal portions of the prohormone of atria1 natriuretic factor during normal pregnancy. Am J Obstet Gynecol. 162:859-64. 23. Hatiis CG, Grogan DM. 1988 Atria1 natriuretic peptide receptors in normal human placentas. Am J Obstet Gynecol.& 159:587-91. 24. Bek T, Ottesen 8, Fahrenkrug I. 1988 The effect of Galanin, CGRP and ANP on spontaneous smooth muscle activity of rat uterus. Peptides. 9:497-500. W, Varma DR. 1990 Refractoriness of the gravid rat uterus 25. Potvin to tocolytic and biochemical effects of atria1 natriuretic peptide. Br J Pharmacol. 100:341-7. 26. Schatz F, Gordon RE, Laufer N, Gurpide E. 1990 Culture of human endometrial cells under polarizing conditions. Differentiation. 42:184-90. RA, Findlay JK. 1990 Separation and culture of ovine 27. Cherny endometrial epithelial and stromal cells: evidence of morphological and functional polarity. Biol Reprod. 43:241-50. R, Banks-Schlegel S, Pinkus GS. 1980 Immunohisto28. Schlegel chemical localization of keratin in normal human tissues. Lab Invest. 42~91-6. 29. Centola GM, Cisar M, Knab DR. 1984 Establishment and morphologic characterization of normal human endometrium in vitro. In Vitro Cell Dev Biol. 20:451-62. 30. Leitman DC, Murad F. 1986 Comparison of binding and cyclic GMP accumulation by atria1 natriuretic peptides in endothelial cells. Biochim Biophys Acta. 885:74-9. 31. Scatchard G. 1949 The attractions of proteins for small molecules “I
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ATRIAL
NATRIURETIC
RECEPTORS
and ions, Ann NY Acad Sci. 51:660-72. 32. Munson PJ, Rodbard D. 1980 LIGAND: a versatile computerized approach for characterization of l&and-binding systems. Ann Biochem. 107:220-39. 33. Kasagi K, Konishi J, Iida Y, et al. 1982 A new in vitro assay for thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3’,5’-mono-phosphate increase. J Clin Endocrinol Metab. 54:108-14. 34. Pelletier G. 1988 Localization of binding sites for ANF in the rat mammary gland. Peptides. 9:673-5.
IN ENDOMETRIAL
STROMA
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35. Jeandel L, Morrier E, Heisler S. 1989 Atria1 natriuretic peptide stimulates submandibular gland synthesis and secretion of cGMP. Am J I’hysiol. 257:E675-80. 36. Bianchi C, Anand-Srivastava MB, De Lean A, et al. 1986 Localization and characterization of specific receptors for atria1 natriuretic factor in the ciliary processes of the eye. Curr Eye Res. 5:283-93. 37. Speroff L, Glass RH, Kase NL. 1989 Clinical gynecologic endocrinology and infertility. Baltimore: Williams and Wilkins; 266-71. 38. Wynn RM, Harris JA. 1967 Ultrastructural cyclic changes in the human endometrium I. normal preovulatory phase. Fertil Steril. 18:632-48.
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Vol. 75, No. 2 Printed in U.S.A.
Atria1 Natriuretic Peptide Receptors Endometrial Stromal Cells* JOHN L. GILILLAND, SABITA LAHIRI, AND Reproductive Washington,
YUEH-CHU LEONARD
L. TSENG, WARTOFSKY
Endocrine and Endocrine-Metabolic D. C. 20307-5001
VLADIMIR
in Human
TROCHE,
Services, Walter Reed Army Medical
Center,
ABSTRACT Atria1 natriuretic peptide (ANP) has been shown to affect water and ion transport and specific ANP binding has been identified in several secretory tissues. ANP commonly acts via stimulation of membranebound particulate guanylate cyclase with the production of cyclic guanosine monophosphate (cGMP). We questioned whether ANP played a role in the complex cyclic transformation of the endometrium into a secretory tissue, and whether its action was cGMP mediated. Endometrium was obtained by biopsy in regularly menstruating women and stromal cells were isolated and cultured for use in this study. ANP competitive binding assays were performed using 12SI-labeled ANP (0.1 nmol/L) and increasing concentrations of unlabeled ANP (O-1000 nmol/L). Optimal binding was obtained after 3-h incubation at 4 C and binding characteristics, including dissociation constant and bind-
ing site quantity, were estimated by Scatchard analysis. Specific, high affinity (dissociation constant, 0.078 + 0.004 nmol/L) and low capacity (4,877 + 1,951 binding sites/cell) ANP binding was identified, with nonspecific binding representing less than or equal to 16% of total binding. Evaluation of ANP-stimulated cyclic nucleotide production revealed an increase in cGMP production, with a 7-fold increase at 1000 nmol/L ANP, and no effect on CAMP production. In conclusion, we have identified specific high affinity receptors for ANP in human endometrial cells, suggesting a role for ANP in endometrial cell function and/or development mediated via cGMP production. We propose that ANP may affect local salt and water metabolism, may be involved in the secretory evolution of glandular and stromal cells, and may further facilitate endometrial development via modulation of local vascular tone and endothelial permeability. (J Clin Endocrinol Metub 75: 547-551, 1992)
A
men (39.8 f 3.4 pmol/L) though no fluctuation was seen during the menstrual cycle. ANP levels in postmenopausal women were not significantly different from those in agematched men but were significantly higher (78.3 + 19.4 pmol/L) than those from young women, suggesting effects mediated by the sex steroids. ANP levels have also been evaluated during pregnancy, with most significant increases noted at 48 h postpartum (22). This increase coincides with the phenomenon of postpartum diuresis and may result from atria1stretch causedby a shift in body fluids into the vascular space. Specific binding sites for ANP have been discovered in the placenta (23) though no physiological role for ANP in placental hemodynamics has yet been elucidated. Potent inhibition of uterine smooth muscle contraction has been demonstrated in the nongravid rat uterus (24). This effect, however, was not demonstrable in the gravid rat uterus and a similar refractoriness to the tocolytic effects of ANP was obtained after treatment of virgin rats with progesterone (25). The endometrium has not yet been evaluated for the presence of ANP or its receptors, nor has ANP’s impact on endometrial development been studied.
TRIAL cardiocyte granules were first described in 1956 (l), though their significance at that time was unknown. It wasn’t until 1981 that de Bold et al. (2) suggested that these granules were the source of a factor affecting the volume regulatory function of the kidney, after demonstrating a rapid and potent natriuretic and diuretic response in rats injected with atria1 extracts. Since that time, multiple additional biological properties of atria1 natriuretic peptide (ANP) have been defined (3-12). Several mechanismsof action of ANP have been demonstrated, the most common of which is via stimulation of membrane-bound or particulate guanylate cyclase with accumulation of cyclic guanosinemonophosphate (cGMP) (13). In addition, inhibition of adenylate cyclase and subsequent CAMP production has been found in some tissues such as vascular tissue (14), adrenal cortex (8, 15), pituitary gland (16), and thyroid gland (17) in associationwith ANP binding. ANP’s effects on the reproductive tract have not yet been clarified, though evidence exists to support its involvement in hypothalamic, pituitary, and gonadal function (18-20). A recent report by Clark et al. (21) studied the influence of gender, age, and the menstrual cycle on plasma ANP. ANP levels in young, menstruating women (mean + SE, 68.1 & 5.5 pmol/L) averaged approximately twice those in young
Materials Primary
Received June 21, 1991. Address requests for reprints to: Dr. Leonard Wartofsky, Chief, Department of Medicine, 7D, Walter Reed Army Medical Center, Washington, D. C. 20307-5001. * The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
and Methods
cell cultures
Endometrium was obtained from regularly menstruating, healthy, adult females undergoing laparoscopy (with general anesthesia) for various indications. Exclusion criteria included pregnancy, abnormal bleeding, amenorrhea, infection, malignancy, and use of oral contraceptive pills or other estrogenor progestin-containing medication. Tissue was obtained in accord with Institutional Review Board guidelines of
547
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548
GILILLAND
the Walter Reed Army Medical Center. Freshly obtained endometrium was washed three times in phenol red-free Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Chemical Co., St. Louis, MO), and then digested with 2.5 g/L Type I collagenase (Worthington Biochemical Corp., Freehold, NJ) in phosphate buffered saline (PBS) for 1 h in a 37 C water bath with frequent, intermittent shaking. The resultant cell suspension was centrifuged at 400 X g for 10 min and the pellet was resuspended in Basal Medium [BM; DMEM supplemented with an antibiotic-antimycotic mixture (100 U/mL penicillin, 100 &mL streptomycin, and 0.25 pg/mL amphotericin-B; GIBCO Laboratories, Grand Island, NY), 2 mmol/L L-glutamine (GIBCO), and 10% fetal bovine serum (Hyclone Laboratories, Logan, UT)] and then placed in 162-cm* culture flasks (Costar, Cambridge, MA) for 30 min at 37 C to allow for stromal cell attachment as described by Schatz et al. (26). After 30 min, the medium with nonadherent cells was aspirated, discarded, and replaced with fresh BM. After 72 h of incubation in a humidified 5% CO2 atmosphere at 37 C, the stromal cell population was further purified by differential trypsinization, as described by Cherny and Findlay (27), with a calciumand magnesium-free PBS solution containing 0.5 g/L trypsin (Sigma) and 0.2 g/L EDTA (Sigma). The cell suspension was then centrifuged at 400 x g for 10 min and the pellet resuspended in BM. Total cell count was determined using trypan blue exclusion on an aliquot of the suspension and cells were plated at a density of 100,000/we11 in 24.well culture plates (Costar). These cells were incubated for an additional 8 days before receptor assay with one fresh medium change 4 days after trypsinization. At the completion of this time, cells were consistently in a monolayer covering the entire surface of the culture well base.
Immunocytochemical
identification
Epithelial cells, unlike cells of mesenchymal origin (e.g. stromal cells), produce keratin (28), which has been used as a discriminator for these two cell types (29). Identification of epithelial and stromal elements in monolayer culture was accomplished using a three-layered glucoseoxidase stain (Vector Laboratories, Burlingame, CA) by sequential treatment with rabbit antihuman keratin (Dako Corporation, Carpinteria, CA) and goat antirabbit antiserum (Vector). After purification by the methods previously described, stromal cells were cultured on standard coverslips. In addition, mixed populations of epithelial and stromal cells cultured on coverslips were used as controls. When complete monolayers were achieved, cells were fixed with cold, absolute methanol. They were then flooded with rabbit antihuman keratin, incubated at 25 C for 1 h, and washed three times with PBS. Visible localization of keratin was then accomplished using an ABCglucose oxidase kit (Vector).
A NP receptor assay ANP cell surface receptors were assayed using the method devised by Leitman and Murad (30). Optimal equilibrium binding conditions for stromal cells were determined before these experiments with a binding study in which specific ANP binding was compared at different incubation temperatures and times (see Results). Cells in 24-well plates were washed twice with DMEM and then incubated for 3 h at 4 C with 200 PL buffer [DMEM, 2 g/L BSA, 20 mmol/L HEPES (Sigma)] containing 0.1 nmol/L [‘251]cu-rat ANPieZs (DuPont, Wilmington, DE) and increasing concentrations (O-100 nmol/L) of unlabeled a-human ANP1-28 (Peninsula Laboratories, Belmont, CA). After washing twice with cold PBS containing 2 g/L BSA, cells were solubilized with 0.1 mol/L NaOH and transferred to glass counting tubes, and the radioactivity was determined in a y-counter. Data were evaluated using Scatchard analysis (31) with LlGAND (32), a computer program that provided unbiased best estimates of the equilibrium dissociation constants (Kd) and of binding site concentration. Nonspecific binding was calculated from wells in which labeled ANP (0.1 nmol/L) and an overwhelming concentration of unlabeled ANP (1000 nmol/L) were coincubated. This value, expressed as a percentage of total added labeled ANP, was then subtracted from total binding to obtain specific binding. Nonspecific binding was calculated for each specimen and for each lot of labeled ANP to control for potential interassay variation. Calculation of the number of binding sites per cell required an accurate estimate of the number of cells per well. On the
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JCE & M .1992 Voll5.No2
day of the receptor assay, cell counts were performed in two representative wells from each plate assayed. This was accomplished using trypsin for cell detachment and trypan blue exclusion to identify viable cells. Cell count variability between wells was less than 10%.
Cyclic nucleotide Cyclic nucleotide was evaluated in by Kasagi et al. monolayer culture Hank’s solution, isobutyl-methylxanthine, 1000 nmol/L) of well was collected determination of Wilmington, DE), respectively.
assay production in response to stimulation with ANP endometrial stromal cells using a method developed (33). After washing with DMEM, stromal cells in were incubated for 2 h at 37 C in 250 PL low-salt pH 7.5, with 20 mmol/L HEPES, 0.5 mmol/L 34 g/L BSA, and increasing concentrations (Oa-human ANP. After incubation, buffer from each immediately and stored at -20 C for subsequent cGMP and CAMP concentrations by RIA (DuPont, with assay sensitivities of 0.01 and 0.1 pmol/lOO pL,
Results
We evaluated stromal cells obtained from the endometrium of seven patients at various times during the menstrual cycle. In addition to taking a detailed patient history, a portion of the tissue obtained was sent to pathology for histologic dating and representative stromal cell preparations were evaluated for epithelial contamination as described.No demonstrable epithelial cell contamination was noted in monolayers cultured from purified stromal cell preparations, whereas cultures of mixed cell populations demonstrated keratin staining in epithelial elements. Specific ANP binding variability as a function of incubation temperature and time is depicted graphically in Fig. 1. Receptor binding at 4 C steadily increased with time, reaching a plateau by 3 h, with no demonstrable decrease in binding noted within the time frame studied. In contrast, binding at 25 C and 37 C peaked and then decreasedwith further incubation, a result likely attributable to receptor internalization and degradation under these conditions. Competition binding data, obtained at 4 C, were analyzed and revealed a single set of specific ANP receptors with high affinity (Kd mean of 0.078 + 0.004 nmol/L) and low capacity (mean of 4877 & 1951 binding sites per cell) binding. Nonspecific binding was lessthan or equal to 16%. Patient data
0
1
2 TIME
3
4
(hr)
FIG. 1. Evaluation of time and temperature as variables in ANP binding in cultured human endometrial stromal cells. Specific binding was calculated by subtracting nonspecific binding from total binding. Nonspecific binding was obtained by coincubation of labeled hormone (0.1 nmol/L) and unlabeled hormone (1000 nmol/L).
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ATRIAL
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RECEPTORS
are displayed in Table 1 and Fig. 2 is a representative Scatchard Plot (data from patient 2). Although a-rat ANP was used in these binding studies (differing from a-human ANP by only one amino acid), its performance in competition binding assays, in terms of binding affinity similarity, has been previously demonstrated (9). Though no absolute conclusions regarding receptor number variation can be made because the number of patients was small, a trend toward an increase in receptor number in the proliferative phase was noted. Incubation of endometrial stromal cells with ANP resulted in an increase in production of cGMP as measured in the incubation buffer. This increase behaved in a dose-response fashion, with a 3-fold increase at 100 nmol/L and a greater than 7-fold increase at 1000 nmol/L ANP (see Fig. 3). No change in CAMP production was noted after the addition of ANP. Discussion The physiological effects of ANP are wide ranging and involve a large number of target tissues. These effects are TABLE 1. Scatchard from competitive ANP 3h Patient no.
analysis of receptor binding data obtained binding to endometrial stromal cells at 4 C for
Endometrial histopathology
1 2 3 4 5 6 7
Proliferative Proliferative Secretory,POD Secretory,POD Secretory,POD Secretory,POD Secretory,POD
Kd (nmol/L)
Binding sites (no. per cell)b
0.074 0.079 0.081 0.094 0.083 0.078 0.055
15,024 7,642 1,178 5,800 2,020 1,289 1,188
1-2 2-3 4-5 11-12 12-13
Nonspecific binding 66) 9.7 4.9 13.9 3.8 11.7 15.0 16.0
‘Kd (dissociation constant) was estimated by Scatchard analysis using specific-bound data obtained by subtracting nonspecific binding from total binding. ‘Binding site number per cell was also estimated by Scatchard analysis, using specific-bound data and correcting for total cell number. Cell number per well was obtained by averaging cell counts from two representative wells, obtained on the day of competitive binding assay.
0.03 Y 8 B
I
K d = 0.079 nmol/L Binding Sites/Cell
i
0.02 .
z
m
. me
0.01 -
.
\
.
0.00 ! 0
0.25
0.5 BOUND
FIG. 2. Scatchard unlabeled cells.
= 7,642
ANP
0.75
1 1.0
1.25
(nmol/L)
plot of competition binding data with labeled and in monolayers of cultured human endometrial stromal
IN ENDOMETRIAL
STROMA
.32 1
T
.28 ” 5 L 2
.24 .20 -
,P E
.16 -
!i
.12 -
8
.08 .04 I1
0’”
0
0.1
I
1
I
10
I
I
100
I,
1000
ANP (nmol/L) FIG. 3. cGMP stromal
production cells in response
in monolayer to the addition
cultures of human of ANP.
endometrial
mediated by specific cell surface receptors via the production of cGMP as a second messenger.ANP has been shown to affect water and ion transport and specific ANP binding has been identified in several tissues with secretory functions (34-36), leading one to speculate that ANP may be involved in the secretory processesof some tissues, including the endometrium. A detailed understanding of the developmental processes responsible for the complex, cyclical transformation of the endometrium in the ovarian/menstrual cycle is critical to comprehending the many stepsthat must occur for successful establishment and maintenance of a pregnancy. The unique capacity of pregnancy sustenance requires an endometrium that is of adequate depth, vascularity, and nutritional richness, a state that is achieved through a well orchestrated seriesof events, involving multiple factors. Early in the cycle, after menses,the endometrium is very thin and consists of epithelial and compacted stromal elements. Under the influence of estrogen produced by the developing ovarian follicle, these cellular elements proliferate, and by midcycle the endometrium achieves a height some 10 times its initial height. This expansion results not only from an increase in cell number and size but more significantly from a “reinflation” of the stroma with the acquisition of water, ions, and amino acids (37). Another interesting observation is the occurrence throughout the proliferative phase of a small amount of apocrine-like secretion into gland lumina (38), the significance of which is as yet unclear. During the secretory phase, heralded by ovulation and a significant increase in progesterone secretion, proliferation of glandular and stromal elements is inhibited as their energies are directed more toward secretion. Thereafter, endometrial height remains relatively stable. The most significant alterations in the stroma during the secretory phase include stromal edema and a characteristic transfiguration of stromal cells known as predecidualization. Stromal edema, which also occurs to some degree in the proliferative phase, peaks on postovulatory day (POD) 8. Predecidualization, the result of the production and acquisition of cytoplasmic glycogen, is first seen on POD 9 and progresses to involve nearly all stromal cells by the end of the cycle.
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Participation by ANP in endometrial development and function is certainly plausible and, since plasma levels of ANP do not appear to change significantly during the menstrual cycle, one conceivable explanation for regulation of ANP effects in the endometrium is cyclical variation in receptor quantity. Although our data suggesta trend toward increased receptor number in the proliferative phase, additional studies will be necessary to establish a relationship between receptor number and phase of the menstrual cycle. As progesterone has been shown to modulate ANP effects in the myometrium, possibly by affecting receptor number, it may have similar effects in the endometrium. Physiologically, an increase in the number of ANP receptors and a subsequent increase in ANP binding in the proliferative phase would support the process of stromal expansion, which requires significant water and ion acquisition by the tissue. Local production of cGMP may alsoresult in increased blood flow to the endometrium, which would be advantageous in terms of the provision of other hormones and growth factors to a rapidly growing cell population. In conclusion, we have identified specific, high affinity receptors for ANP in human endometrial stromal cells and have demonstrated a responseto ANP binding in these cells, with a significant increase in cGMP production in a doseresponsefashion. We propose that ANP may affect local salt and water metabolism, may be involved in the evolution of glandular and stromal cells, and may further facilitate endometrial development via modulation of local vascular tone and endothelial permeability. Acknowledgments This project was completed with the approval and support Department of Clinical Investigation, Walter Reed Army Medical We are indebted to Dr. Dennis O’Connor for his evaluation histopathology from each specimen and to Dr. Thomas Klein advice and editorial assistance.
of the Center. of the for his
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35. Jeandel L, Morrier E, Heisler S. 1989 Atria1 natriuretic peptide stimulates submandibular gland synthesis and secretion of cGMP. Am J I’hysiol. 257:E675-80. 36. Bianchi C, Anand-Srivastava MB, De Lean A, et al. 1986 Localization and characterization of specific receptors for atria1 natriuretic factor in the ciliary processes of the eye. Curr Eye Res. 5:283-93. 37. Speroff L, Glass RH, Kase NL. 1989 Clinical gynecologic endocrinology and infertility. Baltimore: Williams and Wilkins; 266-71. 38. Wynn RM, Harris JA. 1967 Ultrastructural cyclic changes in the human endometrium I. normal preovulatory phase. Fertil Steril. 18:632-48.
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