00l3-7227/90/1262-0927$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 2 Printed in U.S.A.
Acute Effect of Basic Fibroblast Growth Factor on Secretion of Prolactin as Assessed by the Reverse Hemolytic Plaque Assay* GREGG H. LARSON, ROBERT D. KOOS, MARIA ANGELA SORTINO, AND PHYLLIS M. WISEt Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
lation and dopamine inhibition of PRL secretion. PRL secretion was maximally stimulated (P < 0.01) by 10"7 M TRH. Basic FGF blocked the TRH-induced increase in PRL secretion. PRL secretion was maximally reduced (P < 0.001) by 10"6 M dopamine. Coincubation of bFGF with dopamine reduced (P < 0.01) the mean plaque area to the same extent as dopamine alone. In each experimental situation changes in mean plaque area reflected a shift in the frequency distribution of the plaque area. Neither bFGF, TRH, dopamine, nor the combined treatments influenced the percentage of pituitary cells secreting PRL compared to basal conditions. We have demonstrated that 1) bFGF reduces the basal secretion of PRL in an acute manner; 2) bFGF blocks the TRHinduced increase in PRL; and 3) bFGF does not potentiate the inhibitory effect of dopamine on PRL secretion and, therefore, may act in part through the same inhibitory pathway as dopamine. We conclude from these data that bFGF, or related factors, could play a role in the regulation of PRL secretion. (Endocrinology 126: 927-932, 1990)
ABSTRACT. The effect of basic fibroblast growth factor (bFGF) on acute secretion of PRL by pituitary lactotrophs was examined under basal conditions and after treatment with TRH or dopamine. We used the reverse hemolytic plaque assay (RHPA) to determine the amount of PRL secreted per lactotroph and the percentage of pituitary cells secreting PRL. Young (2- to 3-month-old) female Sprague-Dawley rats were ovariectomized and 1 week later implanted with a Silastic capsule containing 180 ng/ml estradiol in sesame oil. Three days later, rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. In Exp I, time and dose responses to bFGF were determined using the RHPA. Basic FGF reduced (P < 0.0001) the mean basal secretion of prolactin per lactotroph. The effect was similar at 30, 60, 120, and 240 min of incubation. The reduction in PRL was greatest at 3.36 X 10"6 M, while lesser reductions were observed at 1.68 x 106 and 5.60 X 10 7 M. A dose of 3.36 x 106 M (60 ng/ml) and an incubation time of 60 min were subsequently used in Exp II. In Exp II, we examined the effects of bFGF on TRH stimu-
B
bFGF of all organs tested to date (6), with the greatest concentration present in the pars tuberalis (7). The major source of bFGF within the pars tuberalis appears to be folliculo-stellate cells (7), glia-like cells derived from neuroectoderm (8). The precise role of bFGF in the regulation of anterior pituitary function is unclear at this time. Secretagogue-induced secretion (synthesis and/or release) of PRL is suppressed after acute coincubation of folliculo-stellate cells with anterior pituitary cell aggregates (9, 10). In contrast, treatment of long term cultures of anterior pituitary (11), GH3 (5), or Ch4Ci (12) cells with bFGF for a minimum of 48 h enhances basal PRL secretion. Similarly, pretreatment with bFGF for 48 h increases TRH-induced secretion of PRL by anterior pituitary cells (11) and synergistically enhances estradiol-induced secretion of PRL by GH3 cells (5).We used the reverse hemolytic plaque assay (RHPA) to assess the acute effect of bFGF on the amount of hormone secreted per individual lactotroph and the percentage of cells secreting PRL. In this assay the hemolytic
ASIC fibroblast growth factor (bFGF), extracted from the bovine pituitary, is a peptide composed of 146 amino acids (1). Since the initial demonstration of a mitogenic effect on BALB/c3T3 fibroblasts (2), many functions have been ascribed to bFGF, including effects on cell morphology, differentiation, proliferation, transformation, and senescence (3). Consequently, bFGF is thought to be involved in such processes as embryonic development, limb and lens regeneration, angiogenesis, development of the central nervous system, wound healing, and modulation of anterior pituitary hormone synthesis and secretion (3-5). Pituitary glands have the highest concentration of
Received August 9,1989. Address all correspondence and requests for reprints to: Gregg H. Larson, Ph.D., Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. * This work was supported by NIH Grants AG-02224, HD-15955 (to P.M.W.), CA-45055 (to R.D.K.), and Training Grant HD-07170. t NIH Merit Awardee.
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928
ACUTE EFFECT OF FGF ON PRL SECRETION
plaque area around an individual cell correlates directly with the concentration of hormone secreted from that cell (reviewed in Ref. 13). The objectives of this study were to determine if the amount of hormone secreted per individual cell or the percentage of cells secreting PRL was altered by bFGF 1) during basal conditions, 2) during stimulation with TRH, or 3) during inhibition by dopamine. Materials and Methods Animals Young (2- to 3-month-old) female Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, PA) were maintained in a 14-h light, 10-h dark cycle (lights on, 0400-1800 h) at 23 ± 1 C. Rats were fed Purina rat chow (Ralston-Purina, St. Louis, MO) and water ad libitum. Estrous cycticity was recorded by daily vaginal lavage. Only those rats displaying normal estrous cycles were used in this study. All rats were ovariectomized under ether anesthesia, and 1 week later (day 0) they were implanted with Silastic capsules containing 180 Mg/ml estradiol in sesame oil. On the morning of day 3, rats were killed, and the pituitaries were rapidly removed. Serum was collected and stored frozen until RIA for estradiol and PRL. Preparation of anterior pituitary cells and RHPA Pituitary cells were isolated and prepared for use in the RHPA by the procedure described by Neill and co-workers (13) with modifications by Sortino and Wise (14). Briefly, anterior pituitary cells were incubated with trypsin (0.1%; Gibco, Grand Island, NY) in a spinner flask at 37 C. After 80 min, pituitary cells were washed, resuspended in 1 ml Minimum Essential Medium for Suspension (Sigma, St. Louis, MO), and gently triturated. Ox erythrocytes (Colorado Serum Co., Denver, CO) were coupled with Stapholococcus aureus protein-A (Sigma) in the presence of 0.9% chromium chloride (Sigma) in normal saline. Pituitary cells (0.5 ml 5 X 105 cells/ml) were combined with an equal volume of a 12% solution of protein-A-coupled erythrocytes in Dulbecco's Modified Eagle's Medium (Sigma) containing 0.1% BSA, 20 mM HEPES (Sigma), and antibiotics and infused into Cunningham chambers. After a 60-min equilibration period, the plaque reaction was initiated by infusing antirat PRL antibody into the chamber in the presence or absence of bFGF (recombinant bFGF, bovine; Amgen Biologicals, Thousand Oaks, CA), TRH, dopamine, or combinations of these treatments. The exact doses and times of exposure used in specific experiments are outlined below. At the end of the incubation period, guinea pig serum (1:50; Colorado Serum Co.), as a source of complement, was added for 30 min to induce lysis of erythrocytes in the area surrounding the cells secreting PRL. The cells were then fixed overnight with 1% glutaraldehyde in Dulbecco's Modified Eagle's Medium, stained with methyl green pyronin (Eastman Kodak, Rochester, NY), and mounted with Permount (Fisher Scientific Co., Fairlawn, NJ) and a glass coverslip. The antirat PRL antibody was generated in our laboratory, and validation was previously described by Sortino etal. (15).
Endo • 1990 Vol 126 • No 2
Expl Responses of pituitary cells to various doses of bFGF for different times of incubation were determined. Individual anterior pituitaries from three to five rats were used for each experimental group. Antirat PRL antibody was infused into a Cunningham chamber in the presence or absence of 1 (0.56 X 10"7 M), 5 (2.80 x 10"7 M), 10 (5.60 x 10"7 M), 30 (1.68 x 10"6 M), or 60 (3.36 x 10~6 M) ng/ml bFGF for 30, 60, 120, or 240 min.
ExpII Responses of pituitary cells to various doses of TRH or dopamine, and the interaction of these treatments with bFGF were determined. Individual anterior pituitaries from four rats were included in each experimental group. Antirat PRL antibody was infused into a Cunningham chamber in the presence or absence of 1) TRH (10-9-10-7 M) or 2) dopamine (lO'-lO 5 M) for 60 min. The interaction of bFGF with dopamine was tested by adding antibody in the presence or absence of 60 ng/ml bFGF with or without 10"5 M dopamine for 60 min. The interaction of bFGF with TRH was tested by the addition of antibody in the presence or absence of 60 ng/ml bFGF with or without 10"7 M TRH for 60 min. Quantitation In both experiments, treatments were applied to duplicate Cunningham chambers prepared with cells from each pituitary. Hemolytic plaque area was quantitated using a computerized image analysis system equipped with a digitizing pad (Image Technology Corp., Deer Park, NY). The mean area of 100-150 plaques was quantitated per slide. The percentage of anterior pituitary cells that secreted PRL was defined as the number of plaque-forming cells per total number of pituitary cells multiplied by 100. A light microscope was used to count 300-400 cells/slide. Frequency distribution Frequency distributions of mean plaque area were constructed by determining the number of lactotrophs with a mean plaque area within a given range and then expressing this number as a percentage of the total number of lactotrophs. The comparison of frequency distributions of various treatments is a more sensitive measure of differences between treatments than the comparison of differences between overall means. RIA Concentrations of serum estradiol were determined using a RIA kit from Radioassay Systems Laboratory, Inc. (Carson, CA), with minor modifications (16) by our laboratory. Concentrations of serum PRL were determined using a RIA kit provided by the NIDDK (17), using rPRL RP-2 as a standard, as described previously (18). Statistical analysis All data were statistically analyzed and then converted to a percentage of the control value for graphic presentation. The
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ACUTE EFFECT OF FGF ON PRL SECRETION Fmax test was used to test for homogeneity of variance, and data exhibiting heterogeneous variance were log transformed before Statistical analysis. In Exp I, time and dose responses for bFGF and TRH were examined by analysis of variance for a splitplot design with repeated measures in time (19), using the Statistical Analysis Systems (SAS), General Linear Model (GLM) procedures (20). Differences between treatment groups were determined by the Ryan-Einot-Gabriel-Welsch multiple range test (SAS). In Exp II, data were analyzed by analysis of variance for a randomized complete block design using the SAS, GLM procedures (20). In both experiments, frequency distributions of mean plaque area were analyzed by x2 test.
Results Concentration of hormones The animal model used for this study was chosen in order to optimize the synthesis and secretion of PRL. The estradiol treatment protocol resulted in mean serum concentrations of estradiol and PRL of 12.1 ± 2.54 pg/ ml and 20.9 ± 3.27 ng/ml, respectively. These values are comparable to those found during the rat estrous cycle.
929
bFGF DOSE RESPONSE 140 CD CC 1—
CD
120
100
u_
80
-?r
60
iipH&T^
CD
LU C_3 GC
40 201 0
10
1
Basic FGF reduced (P < 0.0001) the mean plaque area similarly at all incubation times (30, 60, 120, and 240 min) in a dose-dependent manner (Fig. 1). Subsequently, a more thorough dose-response study was completed (Fig. 2) using 60-min incubations. Reduction in mean plaque area was maximal at 60 ng/ml, while intermediate reductions were observed at 10 and 30 ng/ml.
60
DOSE (NG/ML) FIG. 2. Effects of various doses of bFGF on mean plaque area as a percentage of the control value (60-min incubation). Treatment with bFGF reduced (P < 0.0001) mean plaque area in a dose-dependent manner.
RELATIONSHIP BETWEEN TRH AND bFGF
TRH DOSE RESPONSE
Exp I
30
180
T
160 140 120 100 80 60
Exp II
T
The effects of bFGF on TRH stimulation or dopamine inhibition of PRL secretion were examined in Exp II. bFGF DOSE AND TIME RESPONSE
o cc o C_3
LU O CC LU D.
3060 120 240 30 60 120 240 30 60 120 240
TIME (MINUTES) FlG. 1. Effects of various doses and times of exposure to bFGF on mean plaque area as a percentage of the control value. Treatment with bFGF reduced (P < 0.0001) mean plaque area similarly at all incubation times in a dose-dependent manner.
TRH FGF (iO-'M) . (60).
TRH
FlG. 3. Effects of various doses of TRH (A) and the relationship between TRH and bFGF (B) on mean plaque area as a percentage of the control value. Treatment with TRH increased (P < 0.01) mean plaque area in a dose-dependent manner. Coincubation of bFGF with TRH (10~7 M) completely blocked the TRH-stimulated increase in PRL secretion.
Since a maximal reduction in mean plaque area was achieved at 60 ng/ml bFGF in Exp I, this dose of bFGF was used in Exp II. Basic FGF alone reduced (P < 0.05) mean plaque area (Figs. 3B and 4B) as in Exp I. Basic FGF did not influence the percentage of total pituitary cells that secreted PRL compared to the value in the basal state (43 ± 3.3% vs. 43 ± 3.1%, respectively). TRH alone increased (P < 0.01) mean plaque area in a dose-dependent manner (Fig. 3A); the maximal increase was observed at 10~7 M TRH. Coincubation of bFGF with TRH (10"7 M) completely blocked the TRHstimulated increase in PRL secretion (Fig. 3B). Neither TRH nor coincubation of bFGF with TRH influenced
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ACUTE EFFECT OF FGF ON PRL SECRETION
930 DOPAMINE DOSE RESPONSE
DOPAMINE DOSE RESPONSE
10"7M 10- 6 M 10"5M
FIG. 4. Effects of various doses of dopamine (DA) (A) and the relationship between DA and bFGF (B) on mean plaque area as a percentage of the control value. Treatment with DA reduced (P < O.OO1) mean plaque area in a dose-dependent manner. Coincubation of DA with bFGF reduced (P < 0.01) PRL secretion to the same extent as incubation with DA alone.
the percentage of total pituitary cells that secreted PRL compared to that in the basal state (45 ± 2.1% or 48 ± 3.1% vs. 43 ± 3.1%, respectively). Dopamine reduced (P < 0.001) mean plaque area in a dose-dependent manner, with the maximal reduction observed at a concentration of 10~5 M dopamine (Fig, 4A). Coincubation of bFGF with dopamine (10~5 M) reduced (P < 0.01) mean plaque area to the same extent as dopamine alone (Fig. 4B). Neither dopamine alone nor the coincubation of bFGF with dopamine influenced the percentage of total pituitary cells that secreted PRL compared to the value in the basal state (35 ± 1.1% or 37 ± 1.6% vs. 43 ± 3.1%, respectively). The effects of bFGF, TRH, dopamine, or combinations of these factors on mean plaque area reflected a shift in the frequency distribution of plaque areas (Figs. 5 and 6). For example, bFGF reduced the percentage of lactotrophs secreting greater quantities (larger plaque areas) of PRL and increased the percentage of cells secreting lesser quantities (smaller plaque areas) of PRL (Figs. 5A and 6A). The dominant plaque area in controls and bFGF-treated cells was approximately between 4,0008,000 /xm2. It is important to note that the distribution of plaque areas was not bimodal. The last bin classification for plaque areas includes all lactotrophs with areas larger than 24,000 nm2, (extending up to 100,000 nm2).
Discussion It has been shown previously that coincubation of folliculo-stellate cells with anterior pituitary cell aggregates reduces secretagogue-induced PRL secretion (10). Folliculo-stellate cells produce a number of substances, including bFGF (7), S-100 protein (21), and interleukin6 (22). We would suggest from the present results that a
Endo • 1990 Voll26«No2
possible explanation for the inhibitory effect of folliculostellate cells is that bFGF suppresses PRL secretion. Using the RHPA, we demonstrated that acute incubation of lactotrophs with bFGF inhibits the secretion of PRL per cell, but does not influence the percentage of pituitary cells that secrete PRL. Previous studies in our laboratory show that TRH and dopamine also do not alter the percentage of pituitary cells that secrete PRL (14). bFGF reduced PRL release in a dose-dependent manner, and the effect was rapid, with maximal inhibition observed at the first time interval examined (30 min). The effects of both bFGF and dopamine were similar in that 1) the reduction in average plaque area was due to a shift in the frequency distribution toward smaller plaque areas; and 2) the effects of bFGF with dopamine were not additive, as evidenced by the lack of a further reduction in PRL or a greater shift in the frequency distribution of plaque areas compared to those after treatment with dopamine alone. Therefore, it appears that bFGF may act in part through the same inhibitory pathway as dopamine. bFGF also blocked the TRH-induced increase in PRL. This effect of bFGF is similar to the ability of dopamine to prevent an increase in PRL after coincubation with TRH in a pituitary cell culture (23). In the present study, low doses (1 or 5 ng/ml) of bFGF did not alter PRL secretion per cell. However, higher doses (10, 30, or 60 ng/nl) of bFGF reduced PRL secretion. Although the doses of bFGF required to modulate PRL secretion may appear relatively high, similar molar concentrations of dopamine (10~6 M) and TRH (10~7 M) are required to suppress or stimulate, respectively, the secretion of PRL in the RHPA. Furthermore, folliculostellate cells within the pituitary gland contain the highest concentration of bFGF (500 /xg/kg tissue) (6, 7). Therefore, it is possible that the doses of bFGF used in the present study are physiologically relevant. In contrast to these results, it has been reported that pretreatment of long term pituitary cell cultures with bFGF for a minimum of 48 h increases both basal and TRH-stimulated secretion of PRL. This effect of bFGF is similar after the addition of the cell growth inhibitor 5-fluorodeoxyuridine, indicating that the effect is not due to the mitogenic activity of bFGF. Comparable results were observed after chronic pretreatment of long term cultures of GH3 cells with bFGF under basal (5,12) and estradiol-stimulated (5) conditions. It is difficult, however, to evaluate the effects of inhibitory factors on tumor-derived GH3 cells, which respond inconsistently to dopamine (reviewed in Ref. 24). Also, it is not known whether the response of pituitary cells to bFGF after long term culture is physiologically similar to the response in vivo or during the relatively short RHPA procedure. Whether and/or how bFGF is secreted has been an
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ACUTE EFFECT OF FGF ON PRL SECRETION
BASAL AND TRH PLAQUE AREA DISTRIBUTIONS
BASAL AND bFGF PLAQUE AREA DISTRIBUTIONS
2 4 6 8 10 12 14 16 IB 20 22 24 >24
2
PLAQUE AREA (//M. X1000)
4 6 8 10 12 14 16 18 20 22 24 >24
PLAQUE AREA (//M
2
X1000)
931 BASAL AND TRH PLUS bFGF PLAQUE AREA DISTRIBUTIONS
2 4 6 8 10 12 14 16 18 20 22 24 >24
PLAQUE AREA (tfM2 XiOOO)
FIG. 5. Frequency distributions of mean plaque area under basal conditions compared to incubations with TRH or bFGF alone or compared to coincubations with TRH and bFGF. The effects of bFGF, TRH, or combinations of these factors on mean plaque area reflected a shift in the frequency distribution of plaque areas.
BASAL AND bFGF PLAQUE AREA DISTRIBUTIONS
BASAL AND DOPAMINE PLAQUE AREA DISTRIBUTIONS
BASAL AND DOPAMINE PLUS bFGF PLAQUE AREA DISTRIBUTIONS
A ^BASM.
• bFGF cr LU
• n .
r
l
n
2 4 6 8 10 12 14 16 IB 20 22 24 >24 2
PLAQUE AREA (//M . X1000)
2 4 6 8 10 12 14 16 18 20 22 24 >24
PLAQUE AREA (//M
2
X1000)
2 4 6 8 10 J2 14 16 IB 20 22 24 >24
PLAQUE AREA (//M 2 X1000)
FIG. 6. Frequency distributions of mean plaque area under basal conditions compared to incubations with dopamine or bFGF alone or compared to coincubations with dopamine and bFGF. The effects of bFGF, dopamine, or combinations of these factors on mean plaque area reflected a shift in the frequency distribution of plaque areas.
area of active discussion, since no signal or leader sequence is present in the predicted primary translation product of the bovine bFGF message (25). Although bFGF is found in the extracellular matrix both in vitro (26, 27) and in vivo (28), most studies indicate that little (29-32) or no (33-35) bFGF is secreted into culture medium, or if secreted, it is rapidly bound to cells or extracellular matrix or inactivated. Nevertheless, several studies demonstrate that bFGF influences cell proliferation and functions in an autocrine manner (36-38). Folliculo-stellate cells have been observed to send cytoplasmic processes between secretory pituitary cells (21, 39, 40). It has been suggested (reviewed in Ref. 3) that bFGF could be secreted from these processes in association with the extracellular matrix component heparan sulfate. Subsequent liberation of the biologically active bFGF could result from hydrolysis of the extracellular matrix (6). Therefore, it is possible that bFGF could act as a paracrine regulator of PRL secretion.
In summary, we have demonstrated that bFGF acts acutely to reduce the secretion of PRL. Further studies are necessary to determine the mechanism of bFGF's inhibitory effect, what factors (hypothalamic, hypophysial, or ovarian) might interact with FGF in this acute inhibitory action, and if FGF affects the function of other anterior pituitary cells.
References 1. Moscatelli D, Joseph-Silverstein J, Manejias R, Rifkin DB 1987 Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5778 2. Gospodarowicz D 1974 Localization of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature 249:123 3. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G 1987 Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95 4. Thomas KA 1987 Fibroblast growth factors. FASEB J 1:434 5. Baird A, Esch F, Mormede P, Ueno N, Ling N, Bohlen P, Ying S-
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ACUTE EFFECT OF FGF ON PRL SECRETION Y, Wehrenberg WB, Guillemin R 1986 Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues. Recent Prog Horm Res 42:143 Gospodarowicz D, Neufeld G, Schweigerer L 1986 Fibroblast growth factor. Mol Cell Endocrinol 46:187 Ferrara N, Schweigerer L, Neufeld G, Mitchell R, Gospodarowicz D 1987 Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5773 Nakajima T, Yamaguchi H, Takahashi K 1980 S-100 protein in folliculostellate cells of the rat pituitary anterior lobe. Brain Res 191:523 Allaerts W, Denef C 1989 Regulatory activity and topological distribution of folliculo-stellate cells in rat anterior pituitary cell aggregates. Neuroendocrinology 49:409 Baes M, Allaerts W, Denef C 1987 Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary cell aggregates. Endocrinology 120:685 Baird A, Mormede P, Ying S-Y, Wehrenberg WE, Ueno N, Ling N, Guillemin R 1985 A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci USA 82:5545 Schonbrunn A, Krasnoff M, Westendorf JM, Tashjian Jr AH 1980 Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhibition of cell proliferation. J Cell Biol 85:786 Neill JD, Smith PF, Luque EH, Munoz de Toro M, Nagy G, Mulchahey JJ 1987 Detection and measurement of hormone secretion from individual pituitary cells. Recent Prog Horm Res 43:175 Sortino MA, Wise PM 1989 Effects of age and long term ovariectomy on prolactin secretion, as assessed by the reverse hemolytic plaque assay. Endocrinology 124:90 Sortino MA, Cronin MJ, Wise PM 1989 Relaxin stimulates prolactin secretion from anterior pituitary cells. Endocrinology 124:2013 DePaolo LV, Shander D, Wise PM, Barraclough CA, Channing CP 1979 Identification of inhibin-like activity in ovarian venous plasma of rats during the estrous cycle. Endocrinology 105:647 Niswender GD, Chen CL, Midgley AR, Meites J, Ellis S 1969 Radioimmunoassay for rat prolactin. Proc Soc Exp Biol Med 130:793 Wise PM 1986 Effects of hyperprolactinemia on estrous cyclicity, serum luteinizing hormone, prolactin, estradiol, and progesterone concentrations, and catecholamine activity in microdissected brain areas. Endocrinology 118:1237 Gill JL, Hafs HD 1971 Analysis of repeated measurements of animals. J Anim Sci 33:331 Barr AJ, Goodnight JH, Stall JP, Welwig JT 1979 Statistical Analysis System. SAS Institute, Raleigh, NC Lloyd RV, Mailloux J 1988 Analysis of S-100 protein positive folliculo-stellate cells in rat pituitary tissues. Am J Pathol 133:338 Vankelecom H, Carmeliet P, Van Damme J, Billiau A, Denef C 1989 Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 49:102 Ray KP, Wallis M 1984 Studies of TRH-induced prolactin secretion and its inhibition by dopamine, using ovine pituitary cells. Mol Cell Endocrinol 36:131
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24. Gourdji D, Tougard C, Tixier-Vidal A 1982 Clonal prolactin strains as a tool in neuroendocrinology. In: Ganong WF, Martini L (eds) Frontiers in Neuroendocrinology. Raven Press, New York, vol 7:317-357 25. Abraham IA, Mergia A, Whang JL, Tumolo A, Friedman J, Hjerrild HA, Gospodarowicz D, Fiddes JC 1986 Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233:545 26. Baird A, Ling N 1987 Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 142:428 27. Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaeli R, Sasse J, Klagsbrun M 1987 Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci USA 84:2292 28. Dimario J, Buffinger N, Yamada S, Strohman RC 1989 Fibroblast growth factor in the extracellular matrix of dystrophic (mdx) mouse muscle. Science 244:688 29. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D 1987 Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 325:257 30. Neufeld G, Mitchell R, Ponte P, Gospodarowicz D 1988 Expression of human basic fibroblast growth factor cDNA in baby hamster kidney-derived cells results in autonomous cell growth. J Cell Biol 106:1385 31. Blam SB, Mitchell R, Tischer E, Rubin JS, Silva M, Silver S, Fiddes JC, Abraham JA, Aaronson SA 1988 Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein. Oncogene 3:129 32. Sato YU, Murphy PR, Sato R, Friesen HG 1989 Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent. Mol Endocrinol 3:744 33. Moscatelii D, Presta M, Joseph-Silverstein J, Rifkin DB 1986 Both normal and tumor cells produce basic fibroblast growth factor. J Cell Physiol 129:273 34. Schweigerer L, Ferrara N, Haaparanta T, Neufeld G, Gospodarowicz D 1988 Basic fibroblast growth factor: expression in cultured cells derived from corneal endothelium and lens epithelium. Exp Eye Res 46:71 35. Rogelj S, Weinberg RA, Fanning P, Klagsbrun M 1988 Basic fibroblast growth factor fused to a signal peptide transforms cells. Nature 331:173 36. Sakaguchi M, Kajio T, Kawahara K, Kato K 1988 Antibodies against basic fibroblast growth factor inhibit the autocrine growth of pulmonary artery endothelial cells. FEBS Lett 233:163 37. Sasada R, Kurokawa T, Iwane M, Igarashi K 1988 Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA. Mol Cell Biol 8:588 38. Sato YA, Rifkin DB 1988 Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol 107:1199 39. Rinehart JF, Farquhar MG 1955 The fine vascular organization of the anterior pituitary gland. An electron microscopic study with histochemical correlations. Anat Rec 121:207 40. Heath E 1970 Cytology of the pars anterior of the bovine adenohypophysis. Am J Anat 127:131
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Vol. 126, No. 2 Printed in U.S.A.
Acute Effect of Basic Fibroblast Growth Factor on Secretion of Prolactin as Assessed by the Reverse Hemolytic Plaque Assay* GREGG H. LARSON, ROBERT D. KOOS, MARIA ANGELA SORTINO, AND PHYLLIS M. WISEt Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
lation and dopamine inhibition of PRL secretion. PRL secretion was maximally stimulated (P < 0.01) by 10"7 M TRH. Basic FGF blocked the TRH-induced increase in PRL secretion. PRL secretion was maximally reduced (P < 0.001) by 10"6 M dopamine. Coincubation of bFGF with dopamine reduced (P < 0.01) the mean plaque area to the same extent as dopamine alone. In each experimental situation changes in mean plaque area reflected a shift in the frequency distribution of the plaque area. Neither bFGF, TRH, dopamine, nor the combined treatments influenced the percentage of pituitary cells secreting PRL compared to basal conditions. We have demonstrated that 1) bFGF reduces the basal secretion of PRL in an acute manner; 2) bFGF blocks the TRHinduced increase in PRL; and 3) bFGF does not potentiate the inhibitory effect of dopamine on PRL secretion and, therefore, may act in part through the same inhibitory pathway as dopamine. We conclude from these data that bFGF, or related factors, could play a role in the regulation of PRL secretion. (Endocrinology 126: 927-932, 1990)
ABSTRACT. The effect of basic fibroblast growth factor (bFGF) on acute secretion of PRL by pituitary lactotrophs was examined under basal conditions and after treatment with TRH or dopamine. We used the reverse hemolytic plaque assay (RHPA) to determine the amount of PRL secreted per lactotroph and the percentage of pituitary cells secreting PRL. Young (2- to 3-month-old) female Sprague-Dawley rats were ovariectomized and 1 week later implanted with a Silastic capsule containing 180 ng/ml estradiol in sesame oil. Three days later, rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. In Exp I, time and dose responses to bFGF were determined using the RHPA. Basic FGF reduced (P < 0.0001) the mean basal secretion of prolactin per lactotroph. The effect was similar at 30, 60, 120, and 240 min of incubation. The reduction in PRL was greatest at 3.36 X 10"6 M, while lesser reductions were observed at 1.68 x 106 and 5.60 X 10 7 M. A dose of 3.36 x 106 M (60 ng/ml) and an incubation time of 60 min were subsequently used in Exp II. In Exp II, we examined the effects of bFGF on TRH stimu-
B
bFGF of all organs tested to date (6), with the greatest concentration present in the pars tuberalis (7). The major source of bFGF within the pars tuberalis appears to be folliculo-stellate cells (7), glia-like cells derived from neuroectoderm (8). The precise role of bFGF in the regulation of anterior pituitary function is unclear at this time. Secretagogue-induced secretion (synthesis and/or release) of PRL is suppressed after acute coincubation of folliculo-stellate cells with anterior pituitary cell aggregates (9, 10). In contrast, treatment of long term cultures of anterior pituitary (11), GH3 (5), or Ch4Ci (12) cells with bFGF for a minimum of 48 h enhances basal PRL secretion. Similarly, pretreatment with bFGF for 48 h increases TRH-induced secretion of PRL by anterior pituitary cells (11) and synergistically enhances estradiol-induced secretion of PRL by GH3 cells (5).We used the reverse hemolytic plaque assay (RHPA) to assess the acute effect of bFGF on the amount of hormone secreted per individual lactotroph and the percentage of cells secreting PRL. In this assay the hemolytic
ASIC fibroblast growth factor (bFGF), extracted from the bovine pituitary, is a peptide composed of 146 amino acids (1). Since the initial demonstration of a mitogenic effect on BALB/c3T3 fibroblasts (2), many functions have been ascribed to bFGF, including effects on cell morphology, differentiation, proliferation, transformation, and senescence (3). Consequently, bFGF is thought to be involved in such processes as embryonic development, limb and lens regeneration, angiogenesis, development of the central nervous system, wound healing, and modulation of anterior pituitary hormone synthesis and secretion (3-5). Pituitary glands have the highest concentration of
Received August 9,1989. Address all correspondence and requests for reprints to: Gregg H. Larson, Ph.D., Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. * This work was supported by NIH Grants AG-02224, HD-15955 (to P.M.W.), CA-45055 (to R.D.K.), and Training Grant HD-07170. t NIH Merit Awardee.
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928
ACUTE EFFECT OF FGF ON PRL SECRETION
plaque area around an individual cell correlates directly with the concentration of hormone secreted from that cell (reviewed in Ref. 13). The objectives of this study were to determine if the amount of hormone secreted per individual cell or the percentage of cells secreting PRL was altered by bFGF 1) during basal conditions, 2) during stimulation with TRH, or 3) during inhibition by dopamine. Materials and Methods Animals Young (2- to 3-month-old) female Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, PA) were maintained in a 14-h light, 10-h dark cycle (lights on, 0400-1800 h) at 23 ± 1 C. Rats were fed Purina rat chow (Ralston-Purina, St. Louis, MO) and water ad libitum. Estrous cycticity was recorded by daily vaginal lavage. Only those rats displaying normal estrous cycles were used in this study. All rats were ovariectomized under ether anesthesia, and 1 week later (day 0) they were implanted with Silastic capsules containing 180 Mg/ml estradiol in sesame oil. On the morning of day 3, rats were killed, and the pituitaries were rapidly removed. Serum was collected and stored frozen until RIA for estradiol and PRL. Preparation of anterior pituitary cells and RHPA Pituitary cells were isolated and prepared for use in the RHPA by the procedure described by Neill and co-workers (13) with modifications by Sortino and Wise (14). Briefly, anterior pituitary cells were incubated with trypsin (0.1%; Gibco, Grand Island, NY) in a spinner flask at 37 C. After 80 min, pituitary cells were washed, resuspended in 1 ml Minimum Essential Medium for Suspension (Sigma, St. Louis, MO), and gently triturated. Ox erythrocytes (Colorado Serum Co., Denver, CO) were coupled with Stapholococcus aureus protein-A (Sigma) in the presence of 0.9% chromium chloride (Sigma) in normal saline. Pituitary cells (0.5 ml 5 X 105 cells/ml) were combined with an equal volume of a 12% solution of protein-A-coupled erythrocytes in Dulbecco's Modified Eagle's Medium (Sigma) containing 0.1% BSA, 20 mM HEPES (Sigma), and antibiotics and infused into Cunningham chambers. After a 60-min equilibration period, the plaque reaction was initiated by infusing antirat PRL antibody into the chamber in the presence or absence of bFGF (recombinant bFGF, bovine; Amgen Biologicals, Thousand Oaks, CA), TRH, dopamine, or combinations of these treatments. The exact doses and times of exposure used in specific experiments are outlined below. At the end of the incubation period, guinea pig serum (1:50; Colorado Serum Co.), as a source of complement, was added for 30 min to induce lysis of erythrocytes in the area surrounding the cells secreting PRL. The cells were then fixed overnight with 1% glutaraldehyde in Dulbecco's Modified Eagle's Medium, stained with methyl green pyronin (Eastman Kodak, Rochester, NY), and mounted with Permount (Fisher Scientific Co., Fairlawn, NJ) and a glass coverslip. The antirat PRL antibody was generated in our laboratory, and validation was previously described by Sortino etal. (15).
Endo • 1990 Vol 126 • No 2
Expl Responses of pituitary cells to various doses of bFGF for different times of incubation were determined. Individual anterior pituitaries from three to five rats were used for each experimental group. Antirat PRL antibody was infused into a Cunningham chamber in the presence or absence of 1 (0.56 X 10"7 M), 5 (2.80 x 10"7 M), 10 (5.60 x 10"7 M), 30 (1.68 x 10"6 M), or 60 (3.36 x 10~6 M) ng/ml bFGF for 30, 60, 120, or 240 min.
ExpII Responses of pituitary cells to various doses of TRH or dopamine, and the interaction of these treatments with bFGF were determined. Individual anterior pituitaries from four rats were included in each experimental group. Antirat PRL antibody was infused into a Cunningham chamber in the presence or absence of 1) TRH (10-9-10-7 M) or 2) dopamine (lO'-lO 5 M) for 60 min. The interaction of bFGF with dopamine was tested by adding antibody in the presence or absence of 60 ng/ml bFGF with or without 10"5 M dopamine for 60 min. The interaction of bFGF with TRH was tested by the addition of antibody in the presence or absence of 60 ng/ml bFGF with or without 10"7 M TRH for 60 min. Quantitation In both experiments, treatments were applied to duplicate Cunningham chambers prepared with cells from each pituitary. Hemolytic plaque area was quantitated using a computerized image analysis system equipped with a digitizing pad (Image Technology Corp., Deer Park, NY). The mean area of 100-150 plaques was quantitated per slide. The percentage of anterior pituitary cells that secreted PRL was defined as the number of plaque-forming cells per total number of pituitary cells multiplied by 100. A light microscope was used to count 300-400 cells/slide. Frequency distribution Frequency distributions of mean plaque area were constructed by determining the number of lactotrophs with a mean plaque area within a given range and then expressing this number as a percentage of the total number of lactotrophs. The comparison of frequency distributions of various treatments is a more sensitive measure of differences between treatments than the comparison of differences between overall means. RIA Concentrations of serum estradiol were determined using a RIA kit from Radioassay Systems Laboratory, Inc. (Carson, CA), with minor modifications (16) by our laboratory. Concentrations of serum PRL were determined using a RIA kit provided by the NIDDK (17), using rPRL RP-2 as a standard, as described previously (18). Statistical analysis All data were statistically analyzed and then converted to a percentage of the control value for graphic presentation. The
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ACUTE EFFECT OF FGF ON PRL SECRETION Fmax test was used to test for homogeneity of variance, and data exhibiting heterogeneous variance were log transformed before Statistical analysis. In Exp I, time and dose responses for bFGF and TRH were examined by analysis of variance for a splitplot design with repeated measures in time (19), using the Statistical Analysis Systems (SAS), General Linear Model (GLM) procedures (20). Differences between treatment groups were determined by the Ryan-Einot-Gabriel-Welsch multiple range test (SAS). In Exp II, data were analyzed by analysis of variance for a randomized complete block design using the SAS, GLM procedures (20). In both experiments, frequency distributions of mean plaque area were analyzed by x2 test.
Results Concentration of hormones The animal model used for this study was chosen in order to optimize the synthesis and secretion of PRL. The estradiol treatment protocol resulted in mean serum concentrations of estradiol and PRL of 12.1 ± 2.54 pg/ ml and 20.9 ± 3.27 ng/ml, respectively. These values are comparable to those found during the rat estrous cycle.
929
bFGF DOSE RESPONSE 140 CD CC 1—
CD
120
100
u_
80
-?r
60
iipH&T^
CD
LU C_3 GC
40 201 0
10
1
Basic FGF reduced (P < 0.0001) the mean plaque area similarly at all incubation times (30, 60, 120, and 240 min) in a dose-dependent manner (Fig. 1). Subsequently, a more thorough dose-response study was completed (Fig. 2) using 60-min incubations. Reduction in mean plaque area was maximal at 60 ng/ml, while intermediate reductions were observed at 10 and 30 ng/ml.
60
DOSE (NG/ML) FIG. 2. Effects of various doses of bFGF on mean plaque area as a percentage of the control value (60-min incubation). Treatment with bFGF reduced (P < 0.0001) mean plaque area in a dose-dependent manner.
RELATIONSHIP BETWEEN TRH AND bFGF
TRH DOSE RESPONSE
Exp I
30
180
T
160 140 120 100 80 60
Exp II
T
The effects of bFGF on TRH stimulation or dopamine inhibition of PRL secretion were examined in Exp II. bFGF DOSE AND TIME RESPONSE
o cc o C_3
LU O CC LU D.
3060 120 240 30 60 120 240 30 60 120 240
TIME (MINUTES) FlG. 1. Effects of various doses and times of exposure to bFGF on mean plaque area as a percentage of the control value. Treatment with bFGF reduced (P < 0.0001) mean plaque area similarly at all incubation times in a dose-dependent manner.
TRH FGF (iO-'M) . (60).
TRH
FlG. 3. Effects of various doses of TRH (A) and the relationship between TRH and bFGF (B) on mean plaque area as a percentage of the control value. Treatment with TRH increased (P < 0.01) mean plaque area in a dose-dependent manner. Coincubation of bFGF with TRH (10~7 M) completely blocked the TRH-stimulated increase in PRL secretion.
Since a maximal reduction in mean plaque area was achieved at 60 ng/ml bFGF in Exp I, this dose of bFGF was used in Exp II. Basic FGF alone reduced (P < 0.05) mean plaque area (Figs. 3B and 4B) as in Exp I. Basic FGF did not influence the percentage of total pituitary cells that secreted PRL compared to the value in the basal state (43 ± 3.3% vs. 43 ± 3.1%, respectively). TRH alone increased (P < 0.01) mean plaque area in a dose-dependent manner (Fig. 3A); the maximal increase was observed at 10~7 M TRH. Coincubation of bFGF with TRH (10"7 M) completely blocked the TRHstimulated increase in PRL secretion (Fig. 3B). Neither TRH nor coincubation of bFGF with TRH influenced
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ACUTE EFFECT OF FGF ON PRL SECRETION
930 DOPAMINE DOSE RESPONSE
DOPAMINE DOSE RESPONSE
10"7M 10- 6 M 10"5M
FIG. 4. Effects of various doses of dopamine (DA) (A) and the relationship between DA and bFGF (B) on mean plaque area as a percentage of the control value. Treatment with DA reduced (P < O.OO1) mean plaque area in a dose-dependent manner. Coincubation of DA with bFGF reduced (P < 0.01) PRL secretion to the same extent as incubation with DA alone.
the percentage of total pituitary cells that secreted PRL compared to that in the basal state (45 ± 2.1% or 48 ± 3.1% vs. 43 ± 3.1%, respectively). Dopamine reduced (P < 0.001) mean plaque area in a dose-dependent manner, with the maximal reduction observed at a concentration of 10~5 M dopamine (Fig, 4A). Coincubation of bFGF with dopamine (10~5 M) reduced (P < 0.01) mean plaque area to the same extent as dopamine alone (Fig. 4B). Neither dopamine alone nor the coincubation of bFGF with dopamine influenced the percentage of total pituitary cells that secreted PRL compared to the value in the basal state (35 ± 1.1% or 37 ± 1.6% vs. 43 ± 3.1%, respectively). The effects of bFGF, TRH, dopamine, or combinations of these factors on mean plaque area reflected a shift in the frequency distribution of plaque areas (Figs. 5 and 6). For example, bFGF reduced the percentage of lactotrophs secreting greater quantities (larger plaque areas) of PRL and increased the percentage of cells secreting lesser quantities (smaller plaque areas) of PRL (Figs. 5A and 6A). The dominant plaque area in controls and bFGF-treated cells was approximately between 4,0008,000 /xm2. It is important to note that the distribution of plaque areas was not bimodal. The last bin classification for plaque areas includes all lactotrophs with areas larger than 24,000 nm2, (extending up to 100,000 nm2).
Discussion It has been shown previously that coincubation of folliculo-stellate cells with anterior pituitary cell aggregates reduces secretagogue-induced PRL secretion (10). Folliculo-stellate cells produce a number of substances, including bFGF (7), S-100 protein (21), and interleukin6 (22). We would suggest from the present results that a
Endo • 1990 Voll26«No2
possible explanation for the inhibitory effect of folliculostellate cells is that bFGF suppresses PRL secretion. Using the RHPA, we demonstrated that acute incubation of lactotrophs with bFGF inhibits the secretion of PRL per cell, but does not influence the percentage of pituitary cells that secrete PRL. Previous studies in our laboratory show that TRH and dopamine also do not alter the percentage of pituitary cells that secrete PRL (14). bFGF reduced PRL release in a dose-dependent manner, and the effect was rapid, with maximal inhibition observed at the first time interval examined (30 min). The effects of both bFGF and dopamine were similar in that 1) the reduction in average plaque area was due to a shift in the frequency distribution toward smaller plaque areas; and 2) the effects of bFGF with dopamine were not additive, as evidenced by the lack of a further reduction in PRL or a greater shift in the frequency distribution of plaque areas compared to those after treatment with dopamine alone. Therefore, it appears that bFGF may act in part through the same inhibitory pathway as dopamine. bFGF also blocked the TRH-induced increase in PRL. This effect of bFGF is similar to the ability of dopamine to prevent an increase in PRL after coincubation with TRH in a pituitary cell culture (23). In the present study, low doses (1 or 5 ng/ml) of bFGF did not alter PRL secretion per cell. However, higher doses (10, 30, or 60 ng/nl) of bFGF reduced PRL secretion. Although the doses of bFGF required to modulate PRL secretion may appear relatively high, similar molar concentrations of dopamine (10~6 M) and TRH (10~7 M) are required to suppress or stimulate, respectively, the secretion of PRL in the RHPA. Furthermore, folliculostellate cells within the pituitary gland contain the highest concentration of bFGF (500 /xg/kg tissue) (6, 7). Therefore, it is possible that the doses of bFGF used in the present study are physiologically relevant. In contrast to these results, it has been reported that pretreatment of long term pituitary cell cultures with bFGF for a minimum of 48 h increases both basal and TRH-stimulated secretion of PRL. This effect of bFGF is similar after the addition of the cell growth inhibitor 5-fluorodeoxyuridine, indicating that the effect is not due to the mitogenic activity of bFGF. Comparable results were observed after chronic pretreatment of long term cultures of GH3 cells with bFGF under basal (5,12) and estradiol-stimulated (5) conditions. It is difficult, however, to evaluate the effects of inhibitory factors on tumor-derived GH3 cells, which respond inconsistently to dopamine (reviewed in Ref. 24). Also, it is not known whether the response of pituitary cells to bFGF after long term culture is physiologically similar to the response in vivo or during the relatively short RHPA procedure. Whether and/or how bFGF is secreted has been an
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ACUTE EFFECT OF FGF ON PRL SECRETION
BASAL AND TRH PLAQUE AREA DISTRIBUTIONS
BASAL AND bFGF PLAQUE AREA DISTRIBUTIONS
2 4 6 8 10 12 14 16 IB 20 22 24 >24
2
PLAQUE AREA (//M. X1000)
4 6 8 10 12 14 16 18 20 22 24 >24
PLAQUE AREA (//M
2
X1000)
931 BASAL AND TRH PLUS bFGF PLAQUE AREA DISTRIBUTIONS
2 4 6 8 10 12 14 16 18 20 22 24 >24
PLAQUE AREA (tfM2 XiOOO)
FIG. 5. Frequency distributions of mean plaque area under basal conditions compared to incubations with TRH or bFGF alone or compared to coincubations with TRH and bFGF. The effects of bFGF, TRH, or combinations of these factors on mean plaque area reflected a shift in the frequency distribution of plaque areas.
BASAL AND bFGF PLAQUE AREA DISTRIBUTIONS
BASAL AND DOPAMINE PLAQUE AREA DISTRIBUTIONS
BASAL AND DOPAMINE PLUS bFGF PLAQUE AREA DISTRIBUTIONS
A ^BASM.
• bFGF cr LU
• n .
r
l
n
2 4 6 8 10 12 14 16 IB 20 22 24 >24 2
PLAQUE AREA (//M . X1000)
2 4 6 8 10 12 14 16 18 20 22 24 >24
PLAQUE AREA (//M
2
X1000)
2 4 6 8 10 J2 14 16 IB 20 22 24 >24
PLAQUE AREA (//M 2 X1000)
FIG. 6. Frequency distributions of mean plaque area under basal conditions compared to incubations with dopamine or bFGF alone or compared to coincubations with dopamine and bFGF. The effects of bFGF, dopamine, or combinations of these factors on mean plaque area reflected a shift in the frequency distribution of plaque areas.
area of active discussion, since no signal or leader sequence is present in the predicted primary translation product of the bovine bFGF message (25). Although bFGF is found in the extracellular matrix both in vitro (26, 27) and in vivo (28), most studies indicate that little (29-32) or no (33-35) bFGF is secreted into culture medium, or if secreted, it is rapidly bound to cells or extracellular matrix or inactivated. Nevertheless, several studies demonstrate that bFGF influences cell proliferation and functions in an autocrine manner (36-38). Folliculo-stellate cells have been observed to send cytoplasmic processes between secretory pituitary cells (21, 39, 40). It has been suggested (reviewed in Ref. 3) that bFGF could be secreted from these processes in association with the extracellular matrix component heparan sulfate. Subsequent liberation of the biologically active bFGF could result from hydrolysis of the extracellular matrix (6). Therefore, it is possible that bFGF could act as a paracrine regulator of PRL secretion.
In summary, we have demonstrated that bFGF acts acutely to reduce the secretion of PRL. Further studies are necessary to determine the mechanism of bFGF's inhibitory effect, what factors (hypothalamic, hypophysial, or ovarian) might interact with FGF in this acute inhibitory action, and if FGF affects the function of other anterior pituitary cells.
References 1. Moscatelli D, Joseph-Silverstein J, Manejias R, Rifkin DB 1987 Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5778 2. Gospodarowicz D 1974 Localization of a fibroblast growth factor and its effect alone and with hydrocortisone on 3T3 cell growth. Nature 249:123 3. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G 1987 Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95 4. Thomas KA 1987 Fibroblast growth factors. FASEB J 1:434 5. Baird A, Esch F, Mormede P, Ueno N, Ling N, Bohlen P, Ying S-
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ACUTE EFFECT OF FGF ON PRL SECRETION Y, Wehrenberg WB, Guillemin R 1986 Molecular characterization of fibroblast growth factor: distribution and biological activities in various tissues. Recent Prog Horm Res 42:143 Gospodarowicz D, Neufeld G, Schweigerer L 1986 Fibroblast growth factor. Mol Cell Endocrinol 46:187 Ferrara N, Schweigerer L, Neufeld G, Mitchell R, Gospodarowicz D 1987 Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5773 Nakajima T, Yamaguchi H, Takahashi K 1980 S-100 protein in folliculostellate cells of the rat pituitary anterior lobe. Brain Res 191:523 Allaerts W, Denef C 1989 Regulatory activity and topological distribution of folliculo-stellate cells in rat anterior pituitary cell aggregates. Neuroendocrinology 49:409 Baes M, Allaerts W, Denef C 1987 Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary cell aggregates. Endocrinology 120:685 Baird A, Mormede P, Ying S-Y, Wehrenberg WE, Ueno N, Ling N, Guillemin R 1985 A nonmitogenic pituitary function of fibroblast growth factor: regulation of thyrotropin and prolactin secretion. Proc Natl Acad Sci USA 82:5545 Schonbrunn A, Krasnoff M, Westendorf JM, Tashjian Jr AH 1980 Epidermal growth factor and thyrotropin-releasing hormone act similarly on a clonal pituitary cell strain. Modulation of hormone production and inhibition of cell proliferation. J Cell Biol 85:786 Neill JD, Smith PF, Luque EH, Munoz de Toro M, Nagy G, Mulchahey JJ 1987 Detection and measurement of hormone secretion from individual pituitary cells. Recent Prog Horm Res 43:175 Sortino MA, Wise PM 1989 Effects of age and long term ovariectomy on prolactin secretion, as assessed by the reverse hemolytic plaque assay. Endocrinology 124:90 Sortino MA, Cronin MJ, Wise PM 1989 Relaxin stimulates prolactin secretion from anterior pituitary cells. Endocrinology 124:2013 DePaolo LV, Shander D, Wise PM, Barraclough CA, Channing CP 1979 Identification of inhibin-like activity in ovarian venous plasma of rats during the estrous cycle. Endocrinology 105:647 Niswender GD, Chen CL, Midgley AR, Meites J, Ellis S 1969 Radioimmunoassay for rat prolactin. Proc Soc Exp Biol Med 130:793 Wise PM 1986 Effects of hyperprolactinemia on estrous cyclicity, serum luteinizing hormone, prolactin, estradiol, and progesterone concentrations, and catecholamine activity in microdissected brain areas. Endocrinology 118:1237 Gill JL, Hafs HD 1971 Analysis of repeated measurements of animals. J Anim Sci 33:331 Barr AJ, Goodnight JH, Stall JP, Welwig JT 1979 Statistical Analysis System. SAS Institute, Raleigh, NC Lloyd RV, Mailloux J 1988 Analysis of S-100 protein positive folliculo-stellate cells in rat pituitary tissues. Am J Pathol 133:338 Vankelecom H, Carmeliet P, Van Damme J, Billiau A, Denef C 1989 Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 49:102 Ray KP, Wallis M 1984 Studies of TRH-induced prolactin secretion and its inhibition by dopamine, using ovine pituitary cells. Mol Cell Endocrinol 36:131
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24. Gourdji D, Tougard C, Tixier-Vidal A 1982 Clonal prolactin strains as a tool in neuroendocrinology. In: Ganong WF, Martini L (eds) Frontiers in Neuroendocrinology. Raven Press, New York, vol 7:317-357 25. Abraham IA, Mergia A, Whang JL, Tumolo A, Friedman J, Hjerrild HA, Gospodarowicz D, Fiddes JC 1986 Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233:545 26. Baird A, Ling N 1987 Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 142:428 27. Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaeli R, Sasse J, Klagsbrun M 1987 Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci USA 84:2292 28. Dimario J, Buffinger N, Yamada S, Strohman RC 1989 Fibroblast growth factor in the extracellular matrix of dystrophic (mdx) mouse muscle. Science 244:688 29. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D 1987 Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 325:257 30. Neufeld G, Mitchell R, Ponte P, Gospodarowicz D 1988 Expression of human basic fibroblast growth factor cDNA in baby hamster kidney-derived cells results in autonomous cell growth. J Cell Biol 106:1385 31. Blam SB, Mitchell R, Tischer E, Rubin JS, Silva M, Silver S, Fiddes JC, Abraham JA, Aaronson SA 1988 Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein. Oncogene 3:129 32. Sato YU, Murphy PR, Sato R, Friesen HG 1989 Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent. Mol Endocrinol 3:744 33. Moscatelii D, Presta M, Joseph-Silverstein J, Rifkin DB 1986 Both normal and tumor cells produce basic fibroblast growth factor. J Cell Physiol 129:273 34. Schweigerer L, Ferrara N, Haaparanta T, Neufeld G, Gospodarowicz D 1988 Basic fibroblast growth factor: expression in cultured cells derived from corneal endothelium and lens epithelium. Exp Eye Res 46:71 35. Rogelj S, Weinberg RA, Fanning P, Klagsbrun M 1988 Basic fibroblast growth factor fused to a signal peptide transforms cells. Nature 331:173 36. Sakaguchi M, Kajio T, Kawahara K, Kato K 1988 Antibodies against basic fibroblast growth factor inhibit the autocrine growth of pulmonary artery endothelial cells. FEBS Lett 233:163 37. Sasada R, Kurokawa T, Iwane M, Igarashi K 1988 Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA. Mol Cell Biol 8:588 38. Sato YA, Rifkin DB 1988 Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol 107:1199 39. Rinehart JF, Farquhar MG 1955 The fine vascular organization of the anterior pituitary gland. An electron microscopic study with histochemical correlations. Anat Rec 121:207 40. Heath E 1970 Cytology of the pars anterior of the bovine adenohypophysis. Am J Anat 127:131
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