- Email: [email protected]
Estrogen increases β-adrenergic binding in the preterm fetal rabbit lung
Thuresson-Klein, Moawad, and Hedqvist
nary alveolar surfactant content in rabbits. Fed Proc 1977;36:541. 14. Olsen DB. Neurohumoral-hormonal secretory stimulation of pulmonary surfactant in the rat. Physiologist 1972; 15:230. 15. Moawad AH, River LP, Lin CoCo Estrogen increases 13adrenergic binding in the preterm fetal rabbit lung. AM J OBSTETGYNECOL 1985;151:514. 16. Liggins Gc. Fetal influences on uterine contractility. In:
Estrogen increases rabbit lung
~-adrenergic
February 15, 1985 Am J Obstet Gynecol
Josimovich JB, ed. Uterine contraction-side effects of steroidal contraceptives. New York: John Wiley & Sons, 1973:205-21. 17. Cheng JB, et al. Glucocorticoids increase pulmonary 13adrenergic receptors in fetal rabbit. Endocrinology 1980; 107: 1646. 18. Le Lemos RA, et al. Acceleration of appearance of pulmonary surfactant in the fetal lamb by administration of corticosteroids. Am Rev Respir Dis 1970; 102:459.
binding in the preterm fetal
Atef H_ Moawad, M.D., L. Philip River, B.A., and Chin-Chu Lin, M.D. Chicago, Illinois Tritium-labeled dihydroalprenolol was used to quantify l3-adrenergic-receptor sites in day 28 fetal rabbit lung tissue. Each of the amniotic sacs of pregnant New Zealand White rabbits on day 26 of gestation was injected in vivo with estrogen (estradiol phosphate, 1.6 f.Lg) in one horn and normal saline solution in the contralateral one. The animals were put to death 48 hours later and the fetal lung tissues were assayed. Estrogen increased the number of l3-adrenergic-receptor sites in the treatment group compared to the control group (216 versus 163 fmollmg of protein, p < 0.02 by paired t test). In the presence of estrogen, l3-adrenergic-receptor activity is enhanced in the preterm fetal rabbit. This effect may be implicated in the l3-adrenergic mediation of phosphplipid synthesis and release in fetal alveolar cells. (AM J OBSTET GYNECOL 1985;151 :514-9.)
Key words: I3-Adrenergic receptor, estrogen, fetal rabbit lung, phospholipid synthesis and release, surfactant Our aim in this study was to assess the effect of estrogen on the lung l3-adrenergic-receptor sites in rabbit fetuses in vivo, as determined by radioligand binding assays. Previously we have reported that exogenous estrogen and endogenous estrogen increase the number of l3-adrenergic-receptor sites in immature and mature rabbit lung tissue.' I3-Adrenergic stimulation has been shown to accelerate surfactant production in the fetallung!·3 Given that estrogen affects the number of l3-adrenergic-receptor sites in mature and immature rabbit lungs, these experiments were designed to investigate whether the same mechanism is functional during fetal development. It is also important to know whether this mechanism is implicated in the process of synthesis and release of the lung surfactant.
Material and methods We used tritium-labeled dihydroalprenolol, a reversible and potent l3-adrenergic agonist with pharFrom the Department of Obstetrics and Gynecology, Pritzker School of Medicine, and the Perinatal (;enter, The University of Chicago. Supported by the Mother's Aid Fund of the Chicago Lying-In Hospital. Presented at the Thirtieth Annual Meeting of the Society for Gynecologic Investigation, Washington, D. c., March 17-20, 1983. Reprint requests: Atef H. Moawad, M.D., Department of Obstetrics and Gynecology, University of Chicago, Chicago Lying-In Hospital, 5841 South Maryland Ave., Chicago, IL 60637 . .::
..
macologic characteristics consistent with binding to l3-adrenergic receptors, to quantify the numbers of 13adrenergic-receptor sites in the fetal New Zealand White rabbit lung following estrogen treatment. Pharmacologic agents. Dihydroalprenolol with a specific activity of 49.1 Ci/mmol was obtained from New England Nuclear. (- )-Norepinephrine hydrochloride, (- i-epinephrine bitartrate, (- )-alprenolol, (- )-isoproterenol hydrochloride, (+ )-isoproterenol hydrochloride, and estradiol phosphate were obtained from Sigma Chemical Company. (+ i-Propranolol, (- )-propranolol, and ( + i-norepinephrine bitartrate were a gift from Dr. Leon Goldberg, Department of Pharmacology, The University of Chicago. Treatment regimen. Six mature female New Zealand White rabbits were bred, with day zero of gestation taken as the day of breeding. On day 26 of gestation the rabbits were anesthetized with 40 mg/kg of ketamine, 16 mg/kg of xylazine, and 2.5% halothane gas. The two uterine horns were exposed and 1.6 fLg of estradiol phosphate in 20 fLl of sterile saline solution was injected via a 30-gauge needle into the amniotic sacs of each of the fetuses on one side. The fetuses in the contralateral uterine horn received 20 fLl of sterile saline solution alone. The maternal abdomen was closed and the rabbit allowed to recover. Each rabbit
I3-Adrenergic binding in preterm fetal lung
Volume 151 Number 4
515
Fig.!. A. Time course to equilibrium for specific dihydroalprenolol binding. The inset shows the pseudo-first-order kinetic plot of dihydronalprenolol binding. SB t represents the concentration of specific bound dihydroalprenolol at time t; SB,q, the concentration of dihydroalprenolol specifically bound at equilibrium. The slope of the line defined by the equation 1n[SB,,/(SBcq - SB t ) ] = Kob , is 7.9 X 10- 3 sec-I. From the relationship KI = (Kob, - K,)/[DHAJ, where K, is the first-order rate constant for reversal (2.652 x 10- 3 sec-I; see Fig. I, B), KI = 5.1361 X 10- 4 nmoJlL -I seC I. Parallel experiments (not shown) demonstrated that the dihydroalprenolol-binding site complex was stable for more than I hour. B, The reversal of dihydroalprenolol binding. After dihydroalprenolol binding reached equilibrium, non labeled alprenolol was added to a concentration of 10 fLmoJlL (time zero). The inset shows the first-order reversal of the dihydroalprenolol-binding site complex; the line is determined by the equation -In(SB/SB,q) = K,t. From these data, K, = 2.65 X 10- 3 sec- I and t '/2 = 377 seconds.
was put to death on day 28 of gestation and the fetuses delivered by hysterotomy. A total of 43 fetuses were used; for each litter, the lungs of the fetuses receiving estrogen were pooled, as were those of the control fetuses. There were two stillbirths in this study series and none of the animals delivered fetuses prematurely. Preparation of lung tissue. The fetal lungs were removed intact, weighed, and placed immediately into ice-cold Tris buffer (50 mmol/L, pH 7.4). The lung tissue was homogenized for three IS-second intervals
at 0° C on a Brinkman Polytron homogenizer at one half speed. Thirty-second cooling periods separated each homogenization. The homogenate was centrifuged at 315 X g for 12 minutes in a Beckman RC-58 refrigerated centrifuge at 0° C to remove cellular debris. The supernatant was centrifuged at 11,400 x g for IS minutes, and the membrane pellet was then resuspended in I ml of Tris buffer, 50 mmollL (pH 7.4). The specimens were flash frozen and stored in liquid nitrogen and assayed within I week. Protein concentration was determined by the
516 Moawad, River, and Lin
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method of Bradford: with bovine serum albumin used as the standard. Dihydroalprenolol binding assay. Dihydroalpreno101 was diluted in Tris buffer, 50 mmollL (pH 7.4). Nonlabeled alprenolol at a concentration of 1000 X the dissociation constant (Ko) was used to define nonspecific binding. The assay and all binding were performed in a total volume of 0.25 ml, consisting of 1.25 to 25 nmol of dihydroalprenolol, 0.25 mg of membrane protein, and 0.1 mmollL of hydrochloric acid by itself or with added adrenergic agents. Incubation was for 15 minutes at 30° C and was terminated by the addition of 5 ml of ice-cold Tris buffer, 50 mmol/L. The samples were then immediately filtered over Whatman GF/C filters under vacuum. The tubes were rinsed with 5 ml of wash buffer, and the filters were then washed with an additional 5 ml of wash buffer. The filters were dried
under increased vacuum and then counted in Biofluor (New England Nuclear) scintillation fluid in a Beckman LS-8000 automatic scintillation counter. Specific binding was determined by subtracting radioactivity bound in the presence of 10 /A-mollL of alprenolol, a concentration of approximately 1000 X the Ko of dihydroalprenolol in this tissue, from that bound in the presence of dihydroalprenolol alone. Specific binding constituted 50% to 60% of total radioactivity bound and was a linear function of added membrane protein across the concentration range used in this study. Ko and the number of dihydroalprenolol-binding sites were calculated by means of Scatchard plotting and linear regression analysis.
Results Dihydroalprenolol binding to fetal rabbit lung membrane fraction. A series of experiments were performed in order to verify that dihydroalprenolol binding was consistent with j3-adtenergic-receptor activity. Dihydroalprenolol binding should be rapid, reversible, and saturable, and adrenergic competitors should show a rank order of potencies parallel to their pharmacologic potencies and compete for binding stereoselectively, with (- )isomers much more potent than (+ )isomers . The kinetics of dihydroalprenolol binding are illustrated in Fig. 1, A and B. Equilibrium was reached by 8 minutes and remained stable in excess of 1 hour. The addition of 10 /A-mol/L of alprenolol (Fig. 1, B) demonstrates the reversibility of dihydroalprenolol binding, with a half-time (tY2) of approximately 240 seconds. The dissociation of specifically bound dihydroalpren0101 followed first-order kinetics, with a rate constant of reversal (K z ) of 2.5120 X 10- 3 sec'. The forward rate constant (K,), determined from the relationship K, = (Kob' - K2/[DHA]), where Kob, = observed rate constant and DHA = dihydroalprenolol, was 5.1361 X 10- 4 nmollL -, sec-', yielding a kinetically derived estimate of the Ko for dihydroalprenolol binding (Ko = K2 /K,) of 4.89 nmollL. Equilibrium studies across a range of dihydroalpren0101 concentrations show that specific binding is saturable (Fig. 2, A), with 163 ± 12 fmol (mean ± SEM) bound per milligram of protein in lung tissue hom fetal control rabbits. Scatchard analysis (Fig. 2, B) indicated that this binding is to a single class of receptors. The mean value of Ko from these experiments was 4.35 ± 0.82 nmol/L, in good agreement with the kinetically derived value. The results of competitive binding experiments are shown in Fig. 3, A to D. For isoproterenol, propranolol, and norepinephrine (Fig. 3, A to C), the ( - )stereoisomers were far more potent than the ( + )stereoisomers. The biologically inactive ( + )isomer of norepinephrine
I3-Adrenergic binding in preterm fetal lung
Volume 151 Number 4
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Comment The present experiments demonstrate an increase in the number of fetal rabbit lung l3-adrenergic-receptor sites under the influence of estrogen when compared to littermate controls. This result is consistent with our previous findings in the lung tissue of rabbits, after parturition.' Given the known rise in endogenous estrogen in the pregnant rabbit near term 5 and the welldocumented impact of l3-adrenergic-receptor activity on fetal lung surfactant production,2.3 it appears likely
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GROUP Fig. 4. Results of the dihydroalprenolol-binding assay in the two treatment groups of preterm fetal rabbits. Comparisons of the differences in the mean values of l3-adrenergic-receptor site numbers between the control and estrogen group were statistically significant (p < 0.02 by paired t test).
518 Moawad, River, and Lin
February 15, 1985 Obstet Gynecol
Am J
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Fig. 5. Diagram, expanded from Liggins,12 showing possible connections between fetal cortisol surge, subsequent increased estrogen and decreased progesterone, and both fetal lung maturation and preparation of the myometrium for labor. that estrogen plays a role in the maturation process of the prenatal rabbit lung. Because we have not studied the absorption route of estrogen from the amniotic fluid, we do not know whether it reached the fetus by swallowing, by respiratory movements, or through the fetal skin. However, since we have proper controls with saline solution injections in littermates the route of absorption is immaterial to the significance of our results-we know that the estrogen injected in the fetal compartments was the cause of the r3-adrenergic lung maturation. Because the assay for r3-adrenergic receptors is performed on whole-lung homogenates the question whether this binding represents receptors in the lung parenchyma, in the tracheobronchial elements, and/or in the lung vasculature must be considered. Concurrent with these experiments we have evaluated the effect of estrogen on type II alveolar cell ultrastructure and lamellar body formation in the fetal rat by means of the electron microscope. 6 These studies clearly demonstrate that estrogen exerts a positive effect on the time of the first appearance of lamellar bodies, their rate of proliferation, and their release into the alveolar space. Other investigators have found similar results with the use of light microscopy in the fetal rabbit. 7 Since direct r3adrenergic stimulation is known to produce a similar acceleration of surfactant production, this argues in favor of a direct role for estrogen in the lung parenchyma. However, other drugs and hormones, including the corticosteroids and aminophylline, stimulate r3-adrenergic-receptor formation in the fetallung. 9• 11 Fur-
ther work with the use of isolated type II alveolar cells in cell culture is needed to resolve these twin issues of which cell type is being stimulated and the chain of events leading to its stimulation. The model proposed in Fig. 5, while not specific to the rabbit, is useful for depicting the effects of estrogen, progesterone, and the corticosteroids on both the pregnant uterus and the fetal lung, effects which may link the onset of labor contractility to the maturation of fetal lung surfactant. REFERENCES I. Moawad AH, River LP, Kilpatrick SJ The effect of es-
2. 3.
4.
5. 6.
7. 8.
trogen and progesterone on l3-adrenergic receptor activity in rabbit lung tissue. AM J OBSTET GYNECOL 1982; 144:608. . Kanjanapone Y, Hartig-Beecken I, Epstein MF. Effect of isoxsuprine on fetal lung surfactant in rabbits. Pediatr Res 1980; 14:278. Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats: Release of phosphatidykholine in response to l3-adrenergic stimulation. J Clin Invest 1979; 63:378. Bradford MM. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248. Challis JRG, Davies IJ, Ryan KJ The concentrations of progesterone, estrone and estradiol-17 in the plasma of pregnant rabbits. Endocrinology 1973;93:971. Thuresson-Klein A, Moawad AH, Hedqvist P. Estrogen stimulates formation of lamellar bodies and release of surfactant in the rat fetal lung. AM J OBSTET GYNECOL 1985;151 :506. Khosla SS, Walker Smith GJ, Parks PA, et al. Effects of estrogen on fetal rabbit lung maturation: morphological and biochemical studies. Pediatr Res 1981;15:1274. Khosla SS, Brehier A, Eisenfeld AJ, et al. Influence of sex
I3-Adrenergic binding in preterm fetal lung
Volume 151 Number 4
hormones on lung maturation in the fetal rabbit. Biochim Biophys Acta 1983;750:112. 9. Cheng JB, Goldfein A, Ballard PL, et al. Glucocorticoids increase pulmonary l3-adrenergic receptors in fetal rabbit. Endocrinology 1980; 107: 1646. 10. DeLemos RA, Shermeta DW, Kuelson JH, et al. Acceleration of appearance of pulmonary surfactant in the fetal lamb by administration of corticosteroids. Am Rev Respir Dis 1970;102:459.
II. Ayromlooi J, Bandyopadhyay S, Neogi A, et al. Effect of aminophylline on fetal lung maturation [Abstract 378]. Washington, DC. Society for Gynecological Investigation, thirtieth annual meeting, March 17-20, 1983. 12. Liggins GC. Fetal influences on uterine contractility. In: Josimovich JB, ed. Uterine contraction-side effects of steroidal contraceptives. New York: John Wiley, 1973: 205-21.
Changes in the synthesis and metabolism of prostaglandins by human fetal membranes and decidua at labor Karolina A. Skinner, M.Sc., and John R. G. Challis, Ph.D., D.Sc. London, Ontario, Canada The production of prostaglandins by dispersed cells from human amnion, chorion, and decidua was examined at term before the onset of labor and at spontaneous vaginal delivery. In order to obtain detailed information about relative prostaglandin production rates, the time course of prostaglandin output was examined by incubating the cells for up to 4 hours and measuring the cumulative output of prostaglandin E2, prostaglandin F2a and 13,14-dihydro-15-keto-prostaglandin F2a in the incubation media. The output of all three prostaglandins was low in tissues obtained before the onset of labor. At labor there was an increased production of prostaglandins E2 and F2a in amnion and a small increase in the output of prostaglandin E2 , prostaglandin F2ao and 13,14-dihydro-15-keto-prostaglandin F2a in decidua. In contrast, chorionic cells obtained at spontaneous vaginal delivery showed high levels of 13,14-dihydro-15-ketoprostaglandin F2a in the media with no net production of prostaglandin E2 or F2a . These data suggest a high rate of specific in vitro prostaglandin synthesis in amnion and decidua at labor, accompanied by a high rate of prostaglandin metabolism in chorion. (AM J OSSTET GVNECOL 1985;151 :519-23.)
Key words: Prostaglandin, amnion, chorion, decidua Several lines of evidence suggest a role for prostaglandins in human parturition. Exogenous prostaglandins have been shown to induce labor, I and in monkeys the onset of parturition can be delayed by prostaglandin synthesis inhibitors." Concentrations of prostaglandins in peripheral plasma,3 amniotic fluid:· 6 and tissues 7 increase at labor. The likely sources of these increased prostaglandins at parturition are the intrauterine tissues. Many researchers have reported that homogenates 8 . 9 and whole cells lo . II from these tissues have the capacity to produce prostaglandins. This production has been reported to increase at labor by some authors, Il. 12 while other From the Medical Research Council Group in Reproductive Biology, the Departments of Obstetrics and Gynecology and Physiology, Research Institute, St. joseph's Hospital, and the University of Western Ontario. This work was supported by the Medical Research Council of Canada (Group Grant in Reproductive Biology, J. R. G. C.) and by an Ontario Graduate Scholarship (K. A. S.). Sponsored by the Society for Gynecologic Investigation. Reprint requests: Ms. K. A. Skinner, Research Institute, St. joseph's Hospital, 268 Grosvenor St., London, Ontario, Canada N6A 4V2.
authors ,o. 13 have found little or no increase. There are also conflicting reports about the relative amounts of the primary prostaglandins produced by these tissues. 10·12.11 Some of this conflict may be due to differences in the lengths of the incubation times used in these studies. Therefore we have performed detailed time-course experiments in order to examine the effects of labor on the type of prostaglandins produced by dispersed cells from human amnion, chorion, decidua, and placenta. Prostaglandin synthesis was studied at term before the onset of labor and at spontaneous delivery. The relative rates of production of prostaglandin E2 , prostaglandin F2a , and l3,14-dihydro-15-keto-prostaglandin F2a from endogenous precursors were examined to determine the pattern of prostaglandin production for each tissue and to measure changes in the output of individual prostaglandins with time of incubation.
Material and methods Subjects. Fetal membranes, with attached decidua and placenta, were obtained from women who were
519
nary alveolar surfactant content in rabbits. Fed Proc 1977;36:541. 14. Olsen DB. Neurohumoral-hormonal secretory stimulation of pulmonary surfactant in the rat. Physiologist 1972; 15:230. 15. Moawad AH, River LP, Lin CoCo Estrogen increases 13adrenergic binding in the preterm fetal rabbit lung. AM J OBSTETGYNECOL 1985;151:514. 16. Liggins Gc. Fetal influences on uterine contractility. In:
Estrogen increases rabbit lung
~-adrenergic
February 15, 1985 Am J Obstet Gynecol
Josimovich JB, ed. Uterine contraction-side effects of steroidal contraceptives. New York: John Wiley & Sons, 1973:205-21. 17. Cheng JB, et al. Glucocorticoids increase pulmonary 13adrenergic receptors in fetal rabbit. Endocrinology 1980; 107: 1646. 18. Le Lemos RA, et al. Acceleration of appearance of pulmonary surfactant in the fetal lamb by administration of corticosteroids. Am Rev Respir Dis 1970; 102:459.
binding in the preterm fetal
Atef H_ Moawad, M.D., L. Philip River, B.A., and Chin-Chu Lin, M.D. Chicago, Illinois Tritium-labeled dihydroalprenolol was used to quantify l3-adrenergic-receptor sites in day 28 fetal rabbit lung tissue. Each of the amniotic sacs of pregnant New Zealand White rabbits on day 26 of gestation was injected in vivo with estrogen (estradiol phosphate, 1.6 f.Lg) in one horn and normal saline solution in the contralateral one. The animals were put to death 48 hours later and the fetal lung tissues were assayed. Estrogen increased the number of l3-adrenergic-receptor sites in the treatment group compared to the control group (216 versus 163 fmollmg of protein, p < 0.02 by paired t test). In the presence of estrogen, l3-adrenergic-receptor activity is enhanced in the preterm fetal rabbit. This effect may be implicated in the l3-adrenergic mediation of phosphplipid synthesis and release in fetal alveolar cells. (AM J OBSTET GYNECOL 1985;151 :514-9.)
Key words: I3-Adrenergic receptor, estrogen, fetal rabbit lung, phospholipid synthesis and release, surfactant Our aim in this study was to assess the effect of estrogen on the lung l3-adrenergic-receptor sites in rabbit fetuses in vivo, as determined by radioligand binding assays. Previously we have reported that exogenous estrogen and endogenous estrogen increase the number of l3-adrenergic-receptor sites in immature and mature rabbit lung tissue.' I3-Adrenergic stimulation has been shown to accelerate surfactant production in the fetallung!·3 Given that estrogen affects the number of l3-adrenergic-receptor sites in mature and immature rabbit lungs, these experiments were designed to investigate whether the same mechanism is functional during fetal development. It is also important to know whether this mechanism is implicated in the process of synthesis and release of the lung surfactant.
Material and methods We used tritium-labeled dihydroalprenolol, a reversible and potent l3-adrenergic agonist with pharFrom the Department of Obstetrics and Gynecology, Pritzker School of Medicine, and the Perinatal (;enter, The University of Chicago. Supported by the Mother's Aid Fund of the Chicago Lying-In Hospital. Presented at the Thirtieth Annual Meeting of the Society for Gynecologic Investigation, Washington, D. c., March 17-20, 1983. Reprint requests: Atef H. Moawad, M.D., Department of Obstetrics and Gynecology, University of Chicago, Chicago Lying-In Hospital, 5841 South Maryland Ave., Chicago, IL 60637 . .::
..
macologic characteristics consistent with binding to l3-adrenergic receptors, to quantify the numbers of 13adrenergic-receptor sites in the fetal New Zealand White rabbit lung following estrogen treatment. Pharmacologic agents. Dihydroalprenolol with a specific activity of 49.1 Ci/mmol was obtained from New England Nuclear. (- )-Norepinephrine hydrochloride, (- i-epinephrine bitartrate, (- )-alprenolol, (- )-isoproterenol hydrochloride, (+ )-isoproterenol hydrochloride, and estradiol phosphate were obtained from Sigma Chemical Company. (+ i-Propranolol, (- )-propranolol, and ( + i-norepinephrine bitartrate were a gift from Dr. Leon Goldberg, Department of Pharmacology, The University of Chicago. Treatment regimen. Six mature female New Zealand White rabbits were bred, with day zero of gestation taken as the day of breeding. On day 26 of gestation the rabbits were anesthetized with 40 mg/kg of ketamine, 16 mg/kg of xylazine, and 2.5% halothane gas. The two uterine horns were exposed and 1.6 fLg of estradiol phosphate in 20 fLl of sterile saline solution was injected via a 30-gauge needle into the amniotic sacs of each of the fetuses on one side. The fetuses in the contralateral uterine horn received 20 fLl of sterile saline solution alone. The maternal abdomen was closed and the rabbit allowed to recover. Each rabbit
I3-Adrenergic binding in preterm fetal lung
Volume 151 Number 4
515
Fig.!. A. Time course to equilibrium for specific dihydroalprenolol binding. The inset shows the pseudo-first-order kinetic plot of dihydronalprenolol binding. SB t represents the concentration of specific bound dihydroalprenolol at time t; SB,q, the concentration of dihydroalprenolol specifically bound at equilibrium. The slope of the line defined by the equation 1n[SB,,/(SBcq - SB t ) ] = Kob , is 7.9 X 10- 3 sec-I. From the relationship KI = (Kob, - K,)/[DHAJ, where K, is the first-order rate constant for reversal (2.652 x 10- 3 sec-I; see Fig. I, B), KI = 5.1361 X 10- 4 nmoJlL -I seC I. Parallel experiments (not shown) demonstrated that the dihydroalprenolol-binding site complex was stable for more than I hour. B, The reversal of dihydroalprenolol binding. After dihydroalprenolol binding reached equilibrium, non labeled alprenolol was added to a concentration of 10 fLmoJlL (time zero). The inset shows the first-order reversal of the dihydroalprenolol-binding site complex; the line is determined by the equation -In(SB/SB,q) = K,t. From these data, K, = 2.65 X 10- 3 sec- I and t '/2 = 377 seconds.
was put to death on day 28 of gestation and the fetuses delivered by hysterotomy. A total of 43 fetuses were used; for each litter, the lungs of the fetuses receiving estrogen were pooled, as were those of the control fetuses. There were two stillbirths in this study series and none of the animals delivered fetuses prematurely. Preparation of lung tissue. The fetal lungs were removed intact, weighed, and placed immediately into ice-cold Tris buffer (50 mmol/L, pH 7.4). The lung tissue was homogenized for three IS-second intervals
at 0° C on a Brinkman Polytron homogenizer at one half speed. Thirty-second cooling periods separated each homogenization. The homogenate was centrifuged at 315 X g for 12 minutes in a Beckman RC-58 refrigerated centrifuge at 0° C to remove cellular debris. The supernatant was centrifuged at 11,400 x g for IS minutes, and the membrane pellet was then resuspended in I ml of Tris buffer, 50 mmollL (pH 7.4). The specimens were flash frozen and stored in liquid nitrogen and assayed within I week. Protein concentration was determined by the
516 Moawad, River, and Lin
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February 15, 1985 Am J Obstet Gynecol
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method of Bradford: with bovine serum albumin used as the standard. Dihydroalprenolol binding assay. Dihydroalpreno101 was diluted in Tris buffer, 50 mmollL (pH 7.4). Nonlabeled alprenolol at a concentration of 1000 X the dissociation constant (Ko) was used to define nonspecific binding. The assay and all binding were performed in a total volume of 0.25 ml, consisting of 1.25 to 25 nmol of dihydroalprenolol, 0.25 mg of membrane protein, and 0.1 mmollL of hydrochloric acid by itself or with added adrenergic agents. Incubation was for 15 minutes at 30° C and was terminated by the addition of 5 ml of ice-cold Tris buffer, 50 mmol/L. The samples were then immediately filtered over Whatman GF/C filters under vacuum. The tubes were rinsed with 5 ml of wash buffer, and the filters were then washed with an additional 5 ml of wash buffer. The filters were dried
under increased vacuum and then counted in Biofluor (New England Nuclear) scintillation fluid in a Beckman LS-8000 automatic scintillation counter. Specific binding was determined by subtracting radioactivity bound in the presence of 10 /A-mollL of alprenolol, a concentration of approximately 1000 X the Ko of dihydroalprenolol in this tissue, from that bound in the presence of dihydroalprenolol alone. Specific binding constituted 50% to 60% of total radioactivity bound and was a linear function of added membrane protein across the concentration range used in this study. Ko and the number of dihydroalprenolol-binding sites were calculated by means of Scatchard plotting and linear regression analysis.
Results Dihydroalprenolol binding to fetal rabbit lung membrane fraction. A series of experiments were performed in order to verify that dihydroalprenolol binding was consistent with j3-adtenergic-receptor activity. Dihydroalprenolol binding should be rapid, reversible, and saturable, and adrenergic competitors should show a rank order of potencies parallel to their pharmacologic potencies and compete for binding stereoselectively, with (- )isomers much more potent than (+ )isomers . The kinetics of dihydroalprenolol binding are illustrated in Fig. 1, A and B. Equilibrium was reached by 8 minutes and remained stable in excess of 1 hour. The addition of 10 /A-mol/L of alprenolol (Fig. 1, B) demonstrates the reversibility of dihydroalprenolol binding, with a half-time (tY2) of approximately 240 seconds. The dissociation of specifically bound dihydroalpren0101 followed first-order kinetics, with a rate constant of reversal (K z ) of 2.5120 X 10- 3 sec'. The forward rate constant (K,), determined from the relationship K, = (Kob' - K2/[DHA]), where Kob, = observed rate constant and DHA = dihydroalprenolol, was 5.1361 X 10- 4 nmollL -, sec-', yielding a kinetically derived estimate of the Ko for dihydroalprenolol binding (Ko = K2 /K,) of 4.89 nmollL. Equilibrium studies across a range of dihydroalpren0101 concentrations show that specific binding is saturable (Fig. 2, A), with 163 ± 12 fmol (mean ± SEM) bound per milligram of protein in lung tissue hom fetal control rabbits. Scatchard analysis (Fig. 2, B) indicated that this binding is to a single class of receptors. The mean value of Ko from these experiments was 4.35 ± 0.82 nmol/L, in good agreement with the kinetically derived value. The results of competitive binding experiments are shown in Fig. 3, A to D. For isoproterenol, propranolol, and norepinephrine (Fig. 3, A to C), the ( - )stereoisomers were far more potent than the ( + )stereoisomers. The biologically inactive ( + )isomer of norepinephrine
I3-Adrenergic binding in preterm fetal lung
Volume 151 Number 4
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Comment The present experiments demonstrate an increase in the number of fetal rabbit lung l3-adrenergic-receptor sites under the influence of estrogen when compared to littermate controls. This result is consistent with our previous findings in the lung tissue of rabbits, after parturition.' Given the known rise in endogenous estrogen in the pregnant rabbit near term 5 and the welldocumented impact of l3-adrenergic-receptor activity on fetal lung surfactant production,2.3 it appears likely
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518 Moawad, River, and Lin
February 15, 1985 Obstet Gynecol
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Fig. 5. Diagram, expanded from Liggins,12 showing possible connections between fetal cortisol surge, subsequent increased estrogen and decreased progesterone, and both fetal lung maturation and preparation of the myometrium for labor. that estrogen plays a role in the maturation process of the prenatal rabbit lung. Because we have not studied the absorption route of estrogen from the amniotic fluid, we do not know whether it reached the fetus by swallowing, by respiratory movements, or through the fetal skin. However, since we have proper controls with saline solution injections in littermates the route of absorption is immaterial to the significance of our results-we know that the estrogen injected in the fetal compartments was the cause of the r3-adrenergic lung maturation. Because the assay for r3-adrenergic receptors is performed on whole-lung homogenates the question whether this binding represents receptors in the lung parenchyma, in the tracheobronchial elements, and/or in the lung vasculature must be considered. Concurrent with these experiments we have evaluated the effect of estrogen on type II alveolar cell ultrastructure and lamellar body formation in the fetal rat by means of the electron microscope. 6 These studies clearly demonstrate that estrogen exerts a positive effect on the time of the first appearance of lamellar bodies, their rate of proliferation, and their release into the alveolar space. Other investigators have found similar results with the use of light microscopy in the fetal rabbit. 7 Since direct r3adrenergic stimulation is known to produce a similar acceleration of surfactant production, this argues in favor of a direct role for estrogen in the lung parenchyma. However, other drugs and hormones, including the corticosteroids and aminophylline, stimulate r3-adrenergic-receptor formation in the fetallung. 9• 11 Fur-
ther work with the use of isolated type II alveolar cells in cell culture is needed to resolve these twin issues of which cell type is being stimulated and the chain of events leading to its stimulation. The model proposed in Fig. 5, while not specific to the rabbit, is useful for depicting the effects of estrogen, progesterone, and the corticosteroids on both the pregnant uterus and the fetal lung, effects which may link the onset of labor contractility to the maturation of fetal lung surfactant. REFERENCES I. Moawad AH, River LP, Kilpatrick SJ The effect of es-
2. 3.
4.
5. 6.
7. 8.
trogen and progesterone on l3-adrenergic receptor activity in rabbit lung tissue. AM J OBSTET GYNECOL 1982; 144:608. . Kanjanapone Y, Hartig-Beecken I, Epstein MF. Effect of isoxsuprine on fetal lung surfactant in rabbits. Pediatr Res 1980; 14:278. Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats: Release of phosphatidykholine in response to l3-adrenergic stimulation. J Clin Invest 1979; 63:378. Bradford MM. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248. Challis JRG, Davies IJ, Ryan KJ The concentrations of progesterone, estrone and estradiol-17 in the plasma of pregnant rabbits. Endocrinology 1973;93:971. Thuresson-Klein A, Moawad AH, Hedqvist P. Estrogen stimulates formation of lamellar bodies and release of surfactant in the rat fetal lung. AM J OBSTET GYNECOL 1985;151 :506. Khosla SS, Walker Smith GJ, Parks PA, et al. Effects of estrogen on fetal rabbit lung maturation: morphological and biochemical studies. Pediatr Res 1981;15:1274. Khosla SS, Brehier A, Eisenfeld AJ, et al. Influence of sex
I3-Adrenergic binding in preterm fetal lung
Volume 151 Number 4
hormones on lung maturation in the fetal rabbit. Biochim Biophys Acta 1983;750:112. 9. Cheng JB, Goldfein A, Ballard PL, et al. Glucocorticoids increase pulmonary l3-adrenergic receptors in fetal rabbit. Endocrinology 1980; 107: 1646. 10. DeLemos RA, Shermeta DW, Kuelson JH, et al. Acceleration of appearance of pulmonary surfactant in the fetal lamb by administration of corticosteroids. Am Rev Respir Dis 1970;102:459.
II. Ayromlooi J, Bandyopadhyay S, Neogi A, et al. Effect of aminophylline on fetal lung maturation [Abstract 378]. Washington, DC. Society for Gynecological Investigation, thirtieth annual meeting, March 17-20, 1983. 12. Liggins GC. Fetal influences on uterine contractility. In: Josimovich JB, ed. Uterine contraction-side effects of steroidal contraceptives. New York: John Wiley, 1973: 205-21.
Changes in the synthesis and metabolism of prostaglandins by human fetal membranes and decidua at labor Karolina A. Skinner, M.Sc., and John R. G. Challis, Ph.D., D.Sc. London, Ontario, Canada The production of prostaglandins by dispersed cells from human amnion, chorion, and decidua was examined at term before the onset of labor and at spontaneous vaginal delivery. In order to obtain detailed information about relative prostaglandin production rates, the time course of prostaglandin output was examined by incubating the cells for up to 4 hours and measuring the cumulative output of prostaglandin E2, prostaglandin F2a and 13,14-dihydro-15-keto-prostaglandin F2a in the incubation media. The output of all three prostaglandins was low in tissues obtained before the onset of labor. At labor there was an increased production of prostaglandins E2 and F2a in amnion and a small increase in the output of prostaglandin E2 , prostaglandin F2ao and 13,14-dihydro-15-keto-prostaglandin F2a in decidua. In contrast, chorionic cells obtained at spontaneous vaginal delivery showed high levels of 13,14-dihydro-15-ketoprostaglandin F2a in the media with no net production of prostaglandin E2 or F2a . These data suggest a high rate of specific in vitro prostaglandin synthesis in amnion and decidua at labor, accompanied by a high rate of prostaglandin metabolism in chorion. (AM J OSSTET GVNECOL 1985;151 :519-23.)
Key words: Prostaglandin, amnion, chorion, decidua Several lines of evidence suggest a role for prostaglandins in human parturition. Exogenous prostaglandins have been shown to induce labor, I and in monkeys the onset of parturition can be delayed by prostaglandin synthesis inhibitors." Concentrations of prostaglandins in peripheral plasma,3 amniotic fluid:· 6 and tissues 7 increase at labor. The likely sources of these increased prostaglandins at parturition are the intrauterine tissues. Many researchers have reported that homogenates 8 . 9 and whole cells lo . II from these tissues have the capacity to produce prostaglandins. This production has been reported to increase at labor by some authors, Il. 12 while other From the Medical Research Council Group in Reproductive Biology, the Departments of Obstetrics and Gynecology and Physiology, Research Institute, St. joseph's Hospital, and the University of Western Ontario. This work was supported by the Medical Research Council of Canada (Group Grant in Reproductive Biology, J. R. G. C.) and by an Ontario Graduate Scholarship (K. A. S.). Sponsored by the Society for Gynecologic Investigation. Reprint requests: Ms. K. A. Skinner, Research Institute, St. joseph's Hospital, 268 Grosvenor St., London, Ontario, Canada N6A 4V2.
authors ,o. 13 have found little or no increase. There are also conflicting reports about the relative amounts of the primary prostaglandins produced by these tissues. 10·12.11 Some of this conflict may be due to differences in the lengths of the incubation times used in these studies. Therefore we have performed detailed time-course experiments in order to examine the effects of labor on the type of prostaglandins produced by dispersed cells from human amnion, chorion, decidua, and placenta. Prostaglandin synthesis was studied at term before the onset of labor and at spontaneous delivery. The relative rates of production of prostaglandin E2 , prostaglandin F2a , and l3,14-dihydro-15-keto-prostaglandin F2a from endogenous precursors were examined to determine the pattern of prostaglandin production for each tissue and to measure changes in the output of individual prostaglandins with time of incubation.
Material and methods Subjects. Fetal membranes, with attached decidua and placenta, were obtained from women who were
519