Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine

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Enhanced proliferation of fetal rat hepatocytes In pnmary culture induced by ritodrine Hisao Ando, MD, Masahide Kasugai, MD, Yutaka Ishihara, MD, Osamu Kurauchi, MD, Nobuhiko Suganuma, MD, Shigehiko Mizutani, MD, and Yutaka Tomoda, MD Nagoya, Japan OBJECTIVE: Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that ~2-adrenergic receptor stimulation could promote fetal liver growth. STUDY DESIGN: Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. RESULTS: Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. CONCLUSION: These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of ~2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis. (AM J OBSTET GVNECOL 1993;168:693-7.)

Key words: Ritodrine, cell growth, fetal rat hepatocyte

Ritodrine is a ~2-sympathomimetic agent. Its primary use in obstetrics is to inhibit premature labor. I Furthermore, it has recently been used to improve fetal growth retardation." Although ~2-sympathomimetics are known to cross the placental barrier and reach the fetus," 4 there have been no precise reports on the direct effect of ~2-adrenergic receptor agonists on fetal cell proliferation. Liver growth is especially important for the fetus because the liver is believed to be the organ affected first in fetal growth retardation." In this study we used primary cultures of fetal rat hepatocytes, which maintain various functions of the liver in vivo." It is known that ~2-adrenergic receptors are rich in fetal and neonatal rat hepatoeytes, whereas adult rat hepatocytes in vivo have very few ~2-adrenergic receptors." On the other hand, hepatoeytes isolated from regenerating

From the Department of Obstetrics and Gynecology, School of Medicine, Nagoya University. Supported in part fry Ogyaa Donation from theJapan Associationfor Maternal Welfare. Presented in part at the First International Congress of Perinatal Medicine, Tokyo, Japan, November 5-8, 1991. Received for publication April 6, 1992; revised August 12, 1992; accepted September 22, 1992. Reprint requests: Hisao Ando, MD, Department of Obstetrics and Gynecology, School of Medicine, Nagoya University, 65, Tsurumaicho, Showa-ku, Nagoya 466, Japan. Copyright © 1993 Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 611142877

livers exhibit increased sensitivity to ~2-adrenergic receptor stimulation." We hypothesized that ~2-adrener­ gic stimulation could promote fetal liver growth. In the current study we measured the effects of ritodrine on deoxyribonucleic acid (DNA) synthesis and on nucleus number of fetal rat hepatocytes in primary culture. Moreover, the effect of ritodrine on cell cycle was analyzed with flow cytometry. We suggest that ritodrine enhances DNA synthesis in primary culture of fetal rat hepatoeytes.

Material and methods Animals. Wistar strain rats maintained on a regimen of laboratory chow were used. Hepatoeytes for each culture were prepared from 40 to 55 fetal rats (on day 17 of gestation). In some cultures hepatoeytes were prepared from 30 to 40 neonatal rats (2 days old) or from an adult rat (60 days old). Adult rats were anesthetized by intraperitoneal injection of pentobarbital sodium. Neonatal rats were anesthetized with inhalation of diethyl ether. Isolation and monolayer culture of parenchymal hepatocytes. The materials used for cell isolation and culture were as described elsewhere." Fetal and neonatal hepatocytes were isolated by collagenase digestion of liver fragments as previously described by GuguenGuillouzo et al." The in situ collagenase perfusion method of Seglen 10 was used to isolate hepatocytes from adult rat livers. The isolated cells were washed three

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times with cold Eagle's minimal essential medium. In each culture inocula of 4.0 x 104 cells per square centimeter were seeded into about 50 plastic culture dishes (35 mm diameter) coated with collagen type I (Sumitomo Bakelite, Tokyo) in Williams' medium E supplemented with 5% fetal calf serum and gentamicin (40 ug/ml). After 3 hours the medium was replaced by serum-free Williams' medium E containing 10- 9 mol/L dexamethasone (Sigma, St. Louis), which is necessary to obtain viable and functional cells," and 5 units per milliliter aprotinin (Mochida, Tokyo). After 24 hours of culture the medium was replaced by serum- and hormone-free Williams' medium E, and each experiment was started. Hepatocytes were used when viability, assessed by trypan blue, was > 80%. Only the dishes in which viable cells were recognized homogeneously were used in each experiment. The cultures were maintained at 37° C in humidified air containing 5% carbon dioxide. Hepatocytes from fetal, neonatal, and adult rats were cultured more than 10, four, and two times, respectively, in this investigation. Assay of DNA synthesis. After culture for 24 hours 10-" to 10- 4 mol/L ritodrine (Kissei, Matsumoto, Japan) or 10- 6 mol/L epidermal growth factor (EGF) (Sigma) was added to the fresh basal medium as described above. In some cultures 3 x 10- 5 mol/L propranolol (Sigma) was added with 10- 5 mol/L ritodrine. Then tritiated thymidine (2.5 fL Ci/ml, 52.4 Ci/mmol, New England Nuclear, Boston) was added, and culture was continued for 24 hours. DNA synthesis was assayed as incorporation of tritiated thymidine into DNA in 24 hours with or without aphidicolin (10 fLg/ml), as described before. 1 1• 13 Protein content of hepatocytes was measured with Folin phenol reagent, as described by Lowry et al. 14 Activity of replicating DNA synthesis (disintegrations per minute per hour per milligram of protein) was calculated by subtracting the value with aphidicolin. Determination of number of nuclei. After culture of fetal hepatocytes for 24 hours, 10- 5 mol/L ritodrine was added to the fresh basal medium as described above, and incubation was continued for 10, 24, or 48 hours. The medium was changed daily. The n~mber of nuclei was measured as described by Nakamura et al. 12 Briefly, Triton X-100 was added, and the cells were harvested. The cell suspension was stirred and centrifuged for 2 minutes at 750g. The precipitate was suspended in 10 ml of phosphate-buffered saline solution, and the number of nuclei was counted in a Coulter counter. The recovery of nuclei was consistently > 90%. Fluorometric assay of nuclear DNA. After culture of fetal hepatocytes for 24 hours, 10-"mol!L ritodrine was added to the fresh basal medium as described above, and incubation was continued for 12 hours. Then the cells were washed twice with 0.1 % sodium citrate at 4° C and incubated in 0.1 % sodium citrate and 0.2% Nonidet P-40 containing 50 fLg/mlpropidium iodide

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for 30 minutes at 4° C. A suspension containing nuclei was obtained by strong shaking with a pipette. The suspension was stored in the dark for 15 minutes and was filtered through nylon mesh (40 u.m). Then the fluorescence of > 10,000 nuclei was measured and the cell cycle was analyzed in the Epics Profile (Coulter, Hialeah, Fla.). Statistical analysis. Statistical analysis was performed by the two-tailed Student t test. A p value of < 0.05 was considered to be significant. Results

Hepatocytes adhered to collagen-coated dishes within 2 hours. This relatively short attachment time allowed almost complete removal of nonparenchymal cells that were not removed during the isolation procedure. In the resultant cultures hepatocytes could be distinguished from other cell types on the basis of their characteristic morphologic features. Within 24 hours they formed aggregates of closely packed polygonal cells with large round nuclei, prominent nucleoli, and granular cytoplasm. Our experiments were always performed under constant conditions with serum- and hormone-free Williams' medium E. Moreover, we added only 5% fetal calf serum (0 to 3 hours) and 10- 9 mol/L dexamethasone (3 to 24 hours) before the experiments. DNA synthesis was determined by tritiated thymidine incorporation in 24 hours. Ritodrine (10-" to 10- 4 mol/L) caused dose-dependent stimulation of DNA synthesis of fetal hepatocytes (Fig. 1). DNA synthesis compared with control was 263% (p < 0.0l) and 173% (p < 0.05) in the presence of 10- 5 and 10- 7 mol/L ritodrine, respectively. The enhancement in DNA synthesis was also seen in neonatal rat hepatocytes. DNA synthesis compared with control was 218% (p < 0.01) and 160% (p < 0.05) in the presence of 10- 5 and 10- 7 mol/L ritodrine, respectively. However, adult rat hepatocytes did not exhibit ritodrine-induced DNA synthesis. DNA synthesis compared with control was 98% and 94% (not significant) in the presence of 10- 5 and 10- 7 mol/L ritodrine, respectively. Table I shows that DNA synthesis offetal hepatocytes stimulated by 10- 5 mol/L ritodrine was inhibited by 3 x 10 - 5 mol/L propranolol. The effect of 10 - 5 mol/L ritodrine on DNA synthesis was equivalent to that of 10- 6 mol/L EGF. The effects ofritodrine and EGF were synergistic. To understand exactly the effect of ritodrine on cell proliferation; we measured the number of nuclei instead of the cell number, because it is difficult to make a single-cell suspension by digestion with trypsin or collagenase after formation of a monolayer. The number was significantly increased at 48 hours after the addition of 10- 5 mol/L ritodrine in primary cultures of fetal hepatocytes (Fig. 2). Fig. 3 shows that under the current culture conditions

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Volume 168, ~ umber 2 Am J Obstet (;ynecol

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DNA content in most cells was 2N. The height of the base of the trough between the 2N and 4N peaks indicates the amount of S-phase nuclei. Cell cycle ·analysis revealed that S-phase fraction was 56% at 12 hours after the addition of 10-" mol/L ritodrine, whereas that of control culture (no addition of ritodrine) was 15%. These data suggest that the initiation of DNA synthesis would be induced in about 40% of hepatocytes by the addition of 10- 5 mol/L ritodrine. However, 10- 7 mol/L ritodrine did not show the remarkable change in the distribution of DNA content as 10- 5 mol/L of ritodrine did (data not shown).

Comment We showed that ritodrine promoted the proliferation of fetal hepatocytes in serum- and hormone-free primary culture (Fig. 2). Analysis with flow cytometry revealed that cells in G] phase seem to be stimulated to enter S phase by ritodrine (Fig. 3). DNA synthesis in terms of tritiated thymidine incorporation was increased in a dose-dependent manner (Fig. 1), and the effect ofritodrine on DNA synthesis was antagonized by propranolol (Table I). These results indicate that the induction of DNA synthesis by ritodrine was mediated by 132-adrenergic receptor stimulation. However, we failed to show a growth-promoting effect of dibutyryl cyclic adneosine 5' -monophosphate on fetal hepatocytes (data not shown). Bronstad et al. 15 reported that dibutyryl cyclic adenosine 5' -monophosphate can both inhibit and

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Table I. Effect of various hormones on DNA synthesis of fetal rat hepatocytes in primary culture

Hormones

No addition 10- 7 mol/L ritodrine 10 -" mol/L ritodrine 10- 6 mol/I. EGF 1O- 5mol/L ritodrine plus 10- 6 mol/L EGF 10- 5 mol/L ritodrine plus 3 x 10-" mol/L propranolol

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stimulate DNA synthesis in adult rat hepatocytes. Intracellular mechanisms after the stimulation of 132-adrenergic receptors must be further investigated, including the effect of dibutyryl cyclic adenosine 5' -monophosphate on DNA synthesis.

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The stimulatory effect of ritodrine on DNA synthesis was observed in the hepatocytes from fetal and neonatal rats; however, hepatocytes from adult rats did not exhibit the enhancement of DNA synthesis by ritodrine. Therefore our results may have relation to the rich expression of ~2-adrenergic receptors in the fetal livers.'; It is known that there are two types of intrauterine growth retardation: symmetric and asymmetric. In the asymmetric type .the size of the liver is remarkably small." 5 Although we used rat hepatocyte cultures in this investigation, we have recently observed enhanced DNA synthesis in primary-cultured hepatocytes from human fetuses (unpublished data). There could be another possibility, that ritodrine may stimulate DNA synthesis by increasing the production of EGF or other growth factors, such as insulin-like growth factor-I, locally. However, we did not examine the production of growth factors, In our culture conditions 10- 6 mol/L EGF enhanced DNA synthesis as much as 10- 5 mol/L ritodrine did (Table I), Further investigation must be required for this interesting possibility, Before our current report there were only in vivo studies of ~2-sympathomimeticagents on fetal growth, Katz et al.!" reported a significant increase in the weight of fetal heart, liver, and kidneys in rabbits treated with terbutaline in utero when compared with control animals. Johnson et al., 17 however, found that fetuses born to ritodrine-treated rhesus monkeys were not associated with an increase in fetal body weight or weight of fetal brain, heart, lung, kidneys, liver, or adrenal glands, Further studies will be necessary for the clarification of these discrepant data and the relationship between our current data and clinical data,

We conclude that ~2-adrenergic stimulation (and its receptors) may playa major role in fetal liver growth, We thank Professor Akira Ichihara (Tokushima University) for his advice on primary cultures of rat hepatocytes and Mr. Yoshiki Yamakawa for his help with flow cytometry analysis, REFERENCES

1. Nuwayhid B, Rajabi M, Beta-sympathomimetic agents: use in perinatal obstetrics, Clin Perinatol 1987;14:757-82, 2, Lin CC, Intrauterine growth retardation, Obstet Gynecol Annu 1985;14:127-221. 3, Caritis SN, Shei LL, Toig G, Wong LK. Pharmacodynamics of ritodrine in pregnant women during preterm labor. AM J OBSTET GYNECOL 1983;147:752-9, 4, Gross TL, Kuhnert BR, Kuhnert PM, Rosen MG, Kazzi NJ, Maternal and fetal plasma concentrations of ritodrine. Obstet Gynecol 1985;65:793-7, 5, Winick M, Brasel JA, Velasco EG, Effects of prenatal nutrition upon pregnancy risk. Clin Obstet Gynecol 1973; 16:184-98. 6. Yeow G. Enzymes and plasma proteins in cultures of fetal hepatocytes. In: Guillouzo A, Guguen-Guillouzo C, eds. Isolated and cultured hepatocytes. London: John Libbey, 1986:171-86. 7. Brenstad G, Christoffersen T. Increased effect of adrenaline on cyclic AMP formation and positive ~2-adrenergic modulation of DNA-synthesis in regenerating hepatocytes. FEBS Lett 1980; 120:89-93. 8. Tanaka K, Sato M, Tomita Y, Ichihara A. Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. J Biochem 1978;84:937-46. 9, Guguen-Guillouzo C, Tichonicky L, Szajnert MF, Kruh J. Changes in some chromatin and cytoplasmic enzymes of perinatal rat hepatocytes during culture. In Vitro 1980;16: 1-10. 10. Seglen PO. Preparation of isolated rat liver cells. In: Prescott DM, ed. Methods in cell biology. New York: Academic Press, 1976:29-83. 11. Tomita Y, Nakamura T, Ichihara A. Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes. Exp Cell Res 1981; 135:363-71.

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12. Nakamura T, Tomita Y, Ichihara A. Density-dependent growth control of adult rat hepatocytes in primary culture. J Biochem 1983;94: 1029-35. 13. Shiota K, Nakamura T, Ichihara A. Distinct effects of transforming growth factor-B, on EGF receptors and EGFinduced DNA synthesis in primary cultured rat hepatocytes. Biochem Int 1986;13:893-901. 14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Bioi Chern 1951; 193:265-75. 15. Brenstad GO, Sand TE, Christoffersen T. Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes. Biochim Biophys Acta 1983;763:58-83.

16. Katz M, Block BS, Heymann MA, et al. Fetal organ weight changes after intravenous terbutaline (I') administration in the rabbit [Abstract 10]. In: Proceedings of the twentyninth annual meeting of the Society for Gynecologic Investigation, Dallas, Texas, March 24-27, 1982. Dallas: Society for Gynecologic Investigation, 1982:7. 17. Johnson JWC, Beck JC, Lee P, et al. Ritodrine hydrochloride effects on the rhesus fetus: carbohydrate metabolism and organ growth [Abstract 324]. In: Proceedings of the thirtieth annual meeting of the Society for Gynecologic Investigation, Washington, DC, March 17-20, 1983. Washington: Society for Gynecologic Investigation, 1983: 176.

Interaction of myogenic and adrenergic mechanisms in isolated, pressurized uterine radial arteries from latepregnant and nonpregnant rats George 0501, PhD, and Marilyn Cipolla, BS

Burlington, Vermont OBJECTIVE: The purpose of this study was to examine how myogenic and adrenergic mechanisms interact in controlling the lumen diameter of small uterine arteries from nonpregnant and late-pregnant rats. STUDY DESIGN: Radial arteries (150 to 250 urn lumen diameter) from nonpregnant (n = 28) and late-pregnant (n = 18) rats were studied in vitro under conditions of varying transmural pressure and agonist concentrations. RESULTS: (1) Arteries from late-pregnant rats were significantly (p < 0.05) larger in diameter and, unlike nonpregnant vessels, developed a stable intrinsic tone at transmural pressures > 25 mm Hg. (2) Vessels from late-pregnant rats displayed a threefold increase in sensitivity to the constrictor effects of phenylephrine; 50% of maximal constriction, nonpregnant = 691 ± 148 nrnol/l, and late-pregnant = 229 ± 32 nmol/L (p < 0.01). (3) There was no difference in sensitivity to potassium depolarization. (4) Arteries from late-pregnant rats actively constricted to changes in transmural pressure, whereas those from nonpregnant did not unless preactivated beforehand with phenylephrine or K' . (5) After preconstriction the autoregulatory effectiveness of late-pregnant arteries in physiologic saline solution versus phenylephrine or K+, or of nonpregnant in K+ and phenylephrine, appeared to be equal in terms of absolute micrometers but not relative percent change in lumen diameter. CONCLUSION: Pregnancy is associated with significant changes in the active contractile properties of uterine resistance artery function, specifically heightened a-adrenergic sensitivity, intrinsic (pressure-dependent) tone, and myogenic reactivity. (AM J OBSTET GVNECOL 1993;168:697-705.)

Key words: Adrenergic, myogenic, pregnancy, radial arteries, rats, uterine circulation From the Division of Research, Department of Obstetrics and Gynecology, University of Vermont College of Medicine. Supported by American Heart Association Grant-In-Aid 523421. G.O. is an Established Investigator ofthe American Heart Association. Received for publication February 6, 1992; revised September 22, 1992; accepted September 25, 1992. Reprint requests: George 0.101, PhD, Department of Obstetrics and Gynecology, Given Building, Room C-213, University of Vermont College of Medicine, Burlington, IT 05405. Copyright © 1993 Mosby-Year Book, Inc. ()()D2-9378/93 $I.DO + .20 6/1/42965

Systemic arterial pressure normally decreases during pregnancy. By definition, gestational increments in uterine flow can therefore only be achieved by reducing the overall resistance of the vascular bed. This is accomplished in part by vascular growth and remodeling, which increases arterial diameter and alters the passive mechanical properties of the vascular wall. 1-3 The uterine circulation normally operates with a substantial

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