Failure of dopamine and bromocriptine to affect prolactin release and cell growth in the dopamine receptor-deficient 235-1 clone

kiolenrlar and CeHukzr Endocrinology, 28 ( 1982) 229-246 Elsevier Scientific Publishers Ireland, Ltd.

229

FAILURF. OF DOPAMINE AND BROMOCRIPTINE TO AFFECT PROLACTIN RELEASE AND CELL GROWTH IN THE DOPAMINE RECEF’TOR-DEFICIENT 235-l CLONE

M.J. CRONTN, S.N. PERKINS, D.A. KEEFER a and R.M. MacLEOD b (with technical assistance from C. Valdenegro b, L. Dabney, J. Howe a, V. Sleep a and S. O’Dell b, Departments of Physiology, Anatomy n and Medicine ‘, lJniz;ersity of Virginia School Medicine, Charlottesville, VA 22908 (U.S.A.) Received

23 March

1982; revision

received

14 June 1982; accepted

of

15 June 1982

The 235-I clone was recently derived from the 7315a transplantable pituitary tumor and continues to secrete rat prolactin. The cells have a prominent Ciolgi apparatus which can be stained immunocytochemically for prolactin, but there were no 600-900 nm granules which are characteristic of normal mammotrophs. in a perfused cell-column apparatus, prolactin retease from the clone was unchanged by dopaminergic agonists, th~otropin-releasing hormone and estradiol but stirn~at~ by dibutyryl cyclic AMP. Cellufar cyclic AMP content was also not changed by dopamine but was dramatically enhanced by prostaglandin E,, indicating that at least one hormone-adenylate cyclase coupling mechanism was functional. In radioligand binding studies using the dopamine antagonist 13H]spiperone, no evidence of a dopamine receptor was obtained. The [ 3H]spiperone binding present was not stereoselective, and exceedingly high concentrations of other ligands were required to displace the binding. In addition, the induction of a prolactin-secreting hard tumor in rats by subcutaneous innoculation of the 235-l cells failed to induce measurable dopamine receptors associated with the tumor cells. In order to address the possibility that there were functional dopamine receptors on these cells, but that they could not be resolved with either the cell column and cyclic AMP studies or the radioreceptor assay, the clone cells were incubated with 0.1-100 nM bromocriptine for up to 8 days. Bromocriptine had no effect on the growth rate or prolactin secretion of the 235-l clone but inhibited prolactin reIease from anterior pituitary cells by over 73% in control studies. We conclude that the 235-l clone does not express dopamine receptors and that the presence of dopamine receptors is obligatory for the typical inhibitory effects of bromocriptine on prolactin release and pituitary cell growth. Keywords:

dopamine receptors; 235-l clone; tine; cell growth; pituitary tumor.

prolactin;

Send reprint requests to: DF. M. Cronin, Department of Physiology, Virginia School of Medicine, Charlottesville, VA 22908 (U.S.A.). 0303-7207/82/~-~0/$02.75

0 Elsevier Scientific

Publishers

cyclic

AMP;

bromocrip-

Box 449, University

Ireland,

Ltd.

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M.J. Cronin et al.

230

The catecholamine dopamine (DA) is released from neurons in the basal hypothalamus and is considered to be a prolactin inhibitory hormone at the level of the anterior pituitary (reviewed in Cronin, 1982). The ability to biochemically dissect this inhibitory mechanism is severely compromised at present by the technical limitations inherent in isolating pure mammotrophs cells from the normal anterior pituitary. Consequently, prolactin-secreting cells which grow in culture and which were originally derived from the anterior pituitary may offer a utilitarian model system until pure normal cells can be obtained routinely. Indeed, such proiactin-secreting clones have been utilized to develop recombinant DNA techniques for rat prolactin (Cooke et al., 1980) and to study polypeptide and steroid receptors (reviewed in Gourdji et al., 1982). In searching for prolactin-secreting clones or cell lines which maintain a differentiated DA receptor-response phenotype, we found that GH, cells were refractory to DA inhibition of prolactin release (Faure et al., 1980) and did not express measurable DA receptors (Cronin et al., 1980). Another prolactin-secreting clone has recently been developed by Dr. John Porter’s group (Reymond et al., 1981). These 235-i cells were derived from the 7315a transplantable pituitary tumor, which is also refractory to dopaminergic inhibition of prolactin release but retains the DA receptor (Cronin et al., 1981). We studied acute prolactin release from the 235-l clone in an isolated cell column and challenged the release with various agents including dopamine and dibutyryl cyclic AMP. To determine whether the observed lack of a secretory response to the DA agonists was due to a DA receptor defect, the DA receptor content of the 235-l cells was determined directly with a radioligand assay. No receptors were measured. These negative findings could be due to limitations in the sensitivity of the assays. Thus, to confirm and extend these studies, we measured one of the effects of DA receptor activation, the inhibition of the cyclic AMP system (Barnes et al., 1978: Gi~nattasio et al., 1981; Labrie et al., 1980; Maurer, 198 1; MacLeod et al., 1980; Ray and Wallis, 198 1). In addition, the clone was maintained with the potent DA agonist bromocriptine over a period of days to determine if the sensitive indices of cell growth and prolactin release were modified, perhaps by a small population of functional DA receptors.

MATERIALS

AND METHODS

Clone maintenance The 235-l clone was obtained at passage 39 from Dr. John Porter

235-l clone, prohctin

release and DA receptors

231

(Reymond et al., 1981). The cells were grown under sterile conditions in 75-mm’ T-flasks (Corning) with RPMI-1640 medium supplemented with 2.5% fetal calf serum, 7.5% horse serum (heat-inactivated, Gibco) and kanamycin (130 pg/ml), hereafter termed complete RPMI. The cells were maintained at 37°C in a humidified atmosphere of 95% sir/5% CO, and passaged at or near confluence. Cells were detached from the plates with Puck’s basic salt solution containing 0.1 mM EDTA. We purposefully avoided trypsin because of its potential effect on crucial cellular components (Patti110 et al., 1979) such as receptors. Once detached, the cell suspension was poured into 30 ml of complete RPM1 and the cells were pelleted by centrifugation (300 X g, 10 min). The pellet was resuspended in the appropriate solution and an aliquot was taken for a cell count and an estimate of their viability with the trypan-blue exclusion test. We also kept a repository of frozen 235-l cells which was used once when the culture became contaminated. Electron microscopy and immunocytochemistry Monolayers of cells, suspensions obtained from monolayers, or subcutaneous tumors of clone cells induced in female rats were fixed either in Karnovsky’s fixative (4% paraformaldehyde-2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2) or in .2% acrolein in 0.1 M phosphate buffer, pH 7.4 (Smith and Keefer, 1982). Karnovsky’s-fixed cells were post-fixed in 1% osmium tetroxide. Cell monolayers were processed according to Kuhn (1981). Pellets of suspended cells were dehydrated and embedded in Epon-Araldite resin (Ladd Research Ind.). Thin sections were cut and examined on a Phillips 300 electron microscope. I-pm thick sections of acrolein-fixed cells were immunocytochemically stained with anti-rPr1 (a gift from Dr. A.F. Parlow, NIAMDD) at a dilution of 1: 10000 using the unlabeled antibody enzyme method (Mason et al., 1969; Sternberger et al., 1970) Perfused cell-column apparatus In order to test their biological responsiveness, these prolactin-secreting cells were studied in perfused cell columns as previously described (Yeo et al., 1979). In brief, about 20 million 235-l cells were mixed with 0.5 g of Bio-Gel P-2 (Bio-Rad) which was pre-swelled in physiological saline. This mixture was drawn up into a sterile 3-ml syringe and then connected to a perfusion apparatus functioning at 37’C. The plunger of the syringe had a 21-gauge needle through the septum and a nylon pad at the septum to prevent cells from escaping with the effluent. By means of an Ismatec peristaltic pump, the cell columns were perfused with 9 parts of Medium 199 with 0.05% bovine serum albumin (Fraction V, Sigma)

232

M.J. Cronin ef al.

and 1 part of physiological saline which contained the test compound or acted as the vehicle control. This mixture was gassed with 95% oxygen/5% CO, and then debubbled prior to reaching the cells. A fraction collector allowed specimens to be obtained at 6-min intervals at a flow rate of 0.5 ml/mm. Prolactin and growth hormone was determined with a double antibody radioimmunoassay using materials and protocols supplied by the NIAMDD Rat Pituitary Hormone Distribution Program; the results are expressed in terms of NIAMDD rat prolactin RP-2 and rat growth hormone RP-1 standards. Extraction of cellular prolactin and growth hormone was done overnight at 2°C with a 1% solution of Triton-X 100 in water. Dopamine receptor binding The clone cells were removed from the plates by either EDTA treatment or scraping with a rubber policeman, counted, pelleted by centrifugation, and resuspended in the salt buffer (ice-cold) used throughout the assay (15 mM Tris, 120 mM NaCl, 5 mM KCl, 1 mM MgCl,, 1 mM CaCl,, 0.1 mM EDTA, 15 FM nialamide, 0.1% ascorbate, pH 7.3). The cells were broken with a 15-set Polytron burst (40% speed) and the homogenate was cent~fuged at 70~0 Xg for 30 min at 0-2°C. The pellet was resuspended in buffer by Polytron (15 see, 40% speed) and an aliquot was taken for protein determination (Bradford, 1976). The DA receptor assay was performed at 22OC, essentially as previously described (Cronin and Weiner, 1979), using [3H]spiperone (spiroperidol: I -phenyl-4-labelled: SA 39-5 1 Ci/mmole: New England Nuclear), the most potent DA antagonist known at the anterior pituitary (Denef and Follebouckt, 1978). The [3H]spiperone was diluted in 5% ethanol buffer; the final ethanol concentration in the assay was 0.8% which had no effect on binding. Bromocriptine (Sandoz), butaclamol (Ayerst), haloperidol {Janssen), and spiperone (Janssen) were generous gifts. Other ligands were purchased from commercial sources. The radioactivity trapped on the Whatman GFjC filters was measured by liquid-scintillation spectroscopy at a machine efficiency of 37-43%. In vivo inoculation of clone Mature female Buffalo rats (Simonsen, Gilroy, CA) were inoculated subcutaneously near the scapulla with a suspension of 30 million 235-l cells each month for 3 months. By day 138 after the first inoculation, tumors measuring 4 X 3 X 3 cm had appeared. The rats were sacrificed by decapitation, trunk blood was collected for prolactin determination, and the tumors were excised, cleaned and prepared for binding as described above.

235-l clone, prolactin release and DNA receptors

233

Cyclic AMP cell content determinations The 235-l cells were transferred to Linbro plates (Flo Laboratories), 10000-20000 cells/well/2 ml of complete RPM1 medium, 10 days prior to a cyclic AMP experiment. The complete RPM1 was changed every 2-3 days and on the day prior to an experiment. At the start of a study, the cells were washed with serum-free medium for 7 min at 37°C. The wash medium was quickly aspirated at timed intervals and 1 ml of serum-free medium with the appropriate drug concentration was immediately added. The incubation proceeded for 5 min at 37’C and was then stopped by the rapid removal of the medium and the immediate addition of 0.5 ml of 0.1 N HCl for 10 min at 37°C. The acid containing the cyclic AMP was then transferred to a conical tube, any cellular debris was pelleted by centrifugation at 2-4’C and the supernatant containing the cyclic AMP was stored at -20°C until assayed within a week. If a pellet was present, it was resuspended in 200 ~1 of 0.2 N NaOH and added to the original well to extract protein (10 mm, 37°C) if desired. The cyclic AMP assay was performed in the Diabetes Research and Training Center Radioimmunoassay Core Laboratory according to the routine procedures described for a Gammaflo Automated RIA system (Harper and Brooker, 1975; Brooker et al., 1976). Briefly, an antibody to cyclic AMP was generated in goats and used at a final titer of 1 : 20000. Cyclic AMP was purified by a 2-step process involving paper chromatography and a Sephadex QAE25 column. The purified cyclic nucleotide was iodinated to a specific activity of approx. 20 pCi/ml. Samples were acetylated directly at room temperature by adding triethylamine and acetic anhydride, 2.5 : 1, and were assayed at a dilution which gave values between 15-85% binding on the standard curve. Standard curves were prepared for every assay using the following concentrations of acetylated cyclic AMP: 0, 0.625, 1.25, 5, 10, 20 pmoles/ml. Percent variability within a set of triplicate values was between 5 and 10%; the sample was repeated when a greater variability was obtained. Standard curves with correlation coefficients lower than 0.99 were rejected and redone. Serially diluted experimental samples generated straight lines that were parallel to each other as well as to the standard curve. Bromocriptine effect on growth rate andprolactin release The 235-l cells were plated in 24-well Linbro plates at a concentration (20000- 1OOOOO/we11/2 ml) such that the cells were in the logarithmic phase of growth throughout each study. Each well contained complete RPM1 medium with either no drug or various concentrations (0.1-100 nM) of bromocriptine (gift of Sandoz). The bromocriptine was initially dissolved in ethanol and then diluted in complete medium so that the

234

M.J. Cronin et al.

cells were exposed to less than 0.001% ethanol. In several studies that extended for more than 4 days, the appropriate concentration of bromocriptine was present for the first 3 days, after which freshly dissolved bromocriptine in new medium was added to the older medium with drug. We determined earlier in normal prolactin cells that the inhibitory bromocriptine effect on prolactin secretion began to wane after 4 days of continuous exposure to the drug. The medium was never changed in order to prevent cell loss. On predetermined days after plating, cells from quadruplicate wells at each bromocriptine concentration were detached with the EDTA solution described above after the medium was removed. To ensure complete recovery of the cells, the medium was centrifuged at 4°C; any cells recovered were pooled with the cells removed from the given well and recentrifuged. An aliquot of the cell-free medium was taken during this centrifugation and frozen until determination of prolactin concentration by radioimmunoassay. After the cells were centrifuged, the supernatant was decanted, the side of the tube dried, and the cell pellet resuspended in a known volume of complete medium. The cell density was determined by hand with the aid of a hemacytometer. Usually 2 aliquots from each well were counted and the counts averaged. In some experiments, cell viability was estimated with the trypan blue exclusion test. Care was taken not to introduce artifactual differences among the drug treatments. Wells were plated across treatment groups to prevent unequal seeding due to settling of the parent cell suspension. Wells were similarly harvested and counted across treatment groups because elapsed time after EDTA exposure slightly decreased the total counts, especially when trypan blue was used. As a control, enzymatically dispersed anterior pituitary cells from normal male rats (Sprague Dawley, 180-220 g, Hilltop, PA) were plated in the same manner as the clone at a seeding concentration of 330000 cells/well. For this experiment, the complete RPM1 medium was supplemented with more antibiotics than used with the clone (i.e., 100 pg/ml gentamycin, 6.3 pg/ml penicillin, 2.5 pg/ml streptomycin, 190 ng/ml fungizone). After the cells had attached to the plastic, they were exposed to either 100 nM bromocriptine or no bromocriptine and aliquots of medium were taken for prolactin determination. Cells were not counted because the adult endocrine cells of the anterior pituitary do not divide rapidly, if at all, in culture; one would be primarily measuring fibroblast growth in such a case. Statistics Groups were compared with a one-way analysis of variance with a

235-l clone, proldctin release and DNA receptors

Neuman-Keuls test for significance. considered significant.

235

A ‘P’ value of less than 0.05 was

RESULTS The 235-l cells had a mean diameter of 10.72 -C 0.16 pm (mean * SEM; n = 75) and viability on passage ranged from 84 to 99%. Passages 45-54 were used in these studies. The prolactin content of the 235-l clone was 205 i 11 fg/cell (n = 4) while the growth hormone content was 2.3 * 0.5 fg/cell in the same samples. The doubling time of the cells was 1.24 -C 0.03 days. Electron microscopy and immunocytochemistry Cultured 235- 1 cells appeared to comprise a morphologically homogeneous population. Pelleted cells had mean diameters of 12 pm with irregularly shaped nuclei and prominent nucleoli (Fig. la, b). The cytoplasm contained an abundance of free polyribosomes while moderate to sparse amounts of rough endoplasmic reticulum were present, generally arranged in short parallel stacks. Profiles of many cells revealed a prominent Golgi apparatus. There was no evidence for cytoplasmic granules characteristic of normal mammotrophs (i.e., 600-900 nm in diameter), and only very rarely were a few granules approx. 100 nm in diameter seen. Cytoplasmic lipid inclusions were evident in every cell and occasional mitotic figures were seen. Immunocytochemical staining of sections with anti-rPr1 revealed antigenicity to be associated with the particular region of cytoplasm corresponding with the Golgi apparatus (Fig. lc, d, e). Cell column 9 different cell columns with the 235-l cells were successfully completed. As illustrated in Figs. 2 and 3, 500 nM DA and 25 ng/ml TRH (Fig. 3) had no obvious effect on the rate of prolactin secretion whereas 3 mM dibutyryl cyclic AMP rapidly stimulated prolactin release. Other drugs which had no effect on secretion during a 30-min exposure included: apomorphine (5 yM); bromocriptine (0.05 pM); dopamine (0.5-5 PM, alone or in the presence of dibutyryl cyclic AMP); norepinephrine (10 PM); ergotamine (0.05 ,uM); ergocryptine (0.05 ELM); haloperidol (0.1 yM, alone or in the presence of ergotamine or ergocryptine). A periodic secretory pattern was observed in some (Fig. 3), but not all (Fig. 2), of the cell column experiments. We have not yet determined the cause of this phenomenon.

236

M.J. Cronin et al.

237

235-I clone, prolactin release and DNA receptors

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2

3

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Fig. 2. The lack of response of the 235-l clone to 2 pulses of 500 nM DA and the stimulation induced by 3 mM dibutyryl cyclic AMP is shown in a cell-column experiment. These clone cells were approx. 60% confluent and 99% viable when they were harvested. Fig. 3. A cell column study of the prolactin secretion of 235-l cells that were confluent at the time of harvesting is shown. Notice the episodic secretion pattern over the first 2.5 h.

DA receptor binding As Fig. 4 shows, there was no [3H]spiperone binding to the 235-l clone that was displacable by 5 PM (+)-butaclamol over a range of 0.5-2.5 mg protein/600 ~1. In the same experiment, 80% of total [3H]spiperone binding to the pig anterior pituitary homogenate was displaced by (+)-butaclamol, which verified that the assay worked in a tissue known to have DA receptors (Cronin, 1981). In another experiment, with increasing concentration of [3H]spiperone, again there was no binding displacable by 5 PM (+)-butaclamol (Fig. 5). Both of these experiments were repeated. The possibility existed that intrinsic factors in the 235-l cells denatured DA receptors. Normal anterior pituitary cells from rats were dispersed (Thorner et al., 1980) and equal numbers of pituitary and 235-l Fig. 1. The ultrastructure and prolactin immunoreactivity of 235-l clone cells in culture is shown. a and b: Electron micrographs of 2 different 235-l cells treated with Karnovsky’s fixative in suspension. Golgi apparatus is indicated with arrows. Bar= 1.0 pm. c, d, e: Photomicrographs of 3 different 235-l cells fixed in acrolein-phosphate buffer. c and d were immunocytochemically stained with anti-rPr1. Reaction product is associated with Golgi apparatus (arrows in c, dark stain in d). The nucleus and nucleolus is also evident in c, as in b. e shows the immunocytochemical control stained with normal goat serum in place of anti-rPr1. 1800 X

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Fig. 4. ‘I%ere was no dispiaceable binding (as defined by (~)-buta~lamo1) (0.4 nM) by increasing 235-l particulate protein concentration.

of [ ‘Hjspiperone

cells were mixed. Controls included a group of both pituitary and clone cells alone, each group containing half as many total cells as the mixed groups. Each of the 3 groups was prepared as described above and tested for binding with 0.4 nM [3H]spiperone. The pituitary alone contained 7.3 2 0.4 fmoles (n = 3); the 235-l cells contained 0.9 2 0.4 fmoles (n = 3); the mixture contained 8.4 t- 1.0 fmoles (n = 3) of displacable [ 3H]spiperone binding, indicating that the clone did not destroy anterior pituitary DA receptors. The binding to the clone alone was significantly less than the other 2 groups (PC 0.01) but not different from zero displacable binding (P > 0.05). The pituitary alone and the mixture groups were not different from each other (P > 0.05). This experiment

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CONCENTRATION 3H-SPlPERONE (nM)

Fig. 5. In homogenates of the 235-l clone (0.2 mg protein), no displaceable or specific [ 3Hlspiperone binding was defined over a concentration range appropriate for D.A receptor binding in the anterior pituitary (Cronin and Weiner, 1979).

235-l clone, prolactin release and DNA receptors

239

was repeated and similar results were obtained. Another plausible explanation for the negligible dopamine receptor activity was that a serum factor not present in the in vitro incubation conditions was required for the expression of this phenotype. This was tested by generating tumors in Buffalo rats from the clone (passage 50). It took 3.5 months for these tumors to grow to the size of the clone’s parent 7315a tumor. The tumor derived from the 235-l cells continued to secrete prolactin in vivo as demonstrated by the serum prolactin levels of 5.97 ? 1.53 pg/ml (n = 5), but the 235-l tumor was more collagenous than the 73 15a tumor. In contrast, circulating prolactin concentration in non-tumor bearing Buffalo rats was 15.6 k 0.5 ng/ml (n = 5). The prolactin clone cells were ultrastructurally similar to the cells in culture, with only very rare small granules, a prominent nucleolus and prominent Golgi (not shown). As with the 235-l cells maintained in vitro, no high affinity specific binding was observed in the induced 235-l tumor. Rather, a low affinity interaction was defined by competition studies (Fig. 6). Compounds studied included apomorphine (n = 3), DA (n = 2), norepinephrine (n = 2), epinephrine (n = 2), spiperone (n = 2), ( +)-butaclamol (n = 3), haloperidol (n = 3) and (-)-butaclamol (n = 3). The rank order and the actual level of potency of these drugs did not correspond to a dopaminergic interaction. Furthermore, the binding sites did not discriminate between the stereoisomers of butaclamol. The competition experiments were not quantitated because nondisplacable binding could

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CONCENTRATION (log Ml Fig. 6. The binding of [3H]spiperone various ligands. The (+)-butaclamol this binding.

to homogenates and (-)-butaclamol

of 235-l tumor was displaced with were equipotent in competing for

fA4.J. Cronin et al.

240

not be defined. This situation is reminiscent of the low affinity interaction observed in the GH, clone (Cronin et al., 1980). The [“Hlspiperone binding in both situations has no relationship to the high affinity DA receptors measured in the normal anterior pituitary (Cronin, 1981). Cyclic AMP

cell content

Table 1 lists the response of the 235-l clone to DA, the phosphodiesterase inhibitor isobutylmethylxanthine (MIX) and prostaglandin E, (PGE,). Neither 0.1 PM nor 10 PM DA modified cyclic AMP cell content significantly. MIX as well as dopamine plus MIX had no significant effect. PGE, induced a significant increase in cyclicAMP which was not modified by concurrent exposure to DA.

As indicated in Fig. 7 and Tables 2 and 3, bromocriptine had no effect on either 235-l cell growth or prolactin accumulation in the medium. In 2 studies, bromocriptine did not affect the average cell viability, with a viability range/treatment of 95-98s. In an experiment in which the cells were studied for 8 days, bromocriptine again had no significant effect on growth or prolactin accumulation (data not shown). In several studies,

Table 1 The cyclic AMP content Treatment

in the 235-l cells Concentration

Cyclic AMP a

na

(@f) Expt.

1b

None Dopamine MIX PGE, Expt. 2 b None Dopamine MIX MIX i dopamine PGE, PGE, + dopamine

0

10 200 10 0

0.1 200 200-k0.1 10 1O’rO.l

1.fzt0.21 1.46t0.11 2.321028 21.17~1.76

2.25 r0.07 2.31 r0.30 6.28i0.55 6.5920.57 36.50~4.43 44.05r2.20

a pmoles/well/106 cells: meaniSEM: Therewere 1.46-tO.13X IO6 235-l cells/well (n = 3) in Expt. 1 and 0.54*0.06X lo6 cells/well (n =3) in Expt. 2. b The PGE, cyclic AMP is different from all other groups in both experiments (p ~0.01). PGE I and PGE, + DA are not different (p > 0.05).

241

235-1 clone, prolacrin release and DNA receptors Table 2 Growth rate constants bromocriptine a Study

1 2 3

of plated

Number of days

3 4 3

Mean 2 SEN

235-l

cells in the presence

Bromocriptine

concentration

of various

concentrations

(nM)

0

0.1

I

f0

100

0.663 0.555 0.572

ND ND 0.559

0.587 0.501 0.591

0.581 0.508 ND

0.537 0.515 0.571

0.597~0.034

of

0.56020.029

0,541*0.016

a Growth rate constants for each treatment were calculated by a linear, least-squares fit on the ln(mean total cells/well) versus time after plating. The number of days on which cells were counted in each study is denoted by ‘number of days.’ The correlation coefficients of the regressions were 0.986 or greater. No significant differences among treatments were found. ND, no determined.

small (13-22% less than control) but statistically significant differences were found among drug treatments in cell counts or prolactin concentrations an a given day. However, these differences did not exhibit either a consistent trend from day to day within the experiment or a true dose response to bromocriptine. In the control experiments in which plated normal anterior pituitary cells were treated with 100 nM bromocriptine, the drug inhibited prolactin accumulation by 88-91% over the 4 days of one study (Table2) and 73-90% over the 3 days of another study (data not shown).

Table 3 Bromocriptine culture a Day No.

1 2 4

effect on proiactin

release from

either 235-I cells or anterior pituitary

235-l cells

Anterior

pituitary

cells in

eelIs

Control

Bromocriptine

Control

Bromocriptine

233-t 5 501*22 2480t7ti

2422 5 503* 34 2 940 * 200

7930-f 150 174401-1010 36100b

846i 168 1640* 140 4320 b

a Values are the mean*SEM, concentration was 100 nM. ’ Mean of 2 wells.

n =4,

expressed

as ng prolactin/well.

The bromocriptine

‘I 1

I

23456’ DAYS

AFTER

PLATING

Fig. 7. The individual value of study No. 3 in Table 2 are depicted. Bromocriptine had no effect on 235-1 cell growth (closed symbols) or prolactin accumulation in the medium (open symbols). Each point is the mean of 3-4 wells:tSEM.

DISCUSSION These studies demonstrate that the 235-l prolatin-secreting clone, obtained from pituitary tumor 7315a, is biolo~caI~y and biochemically unresponsive to DA agonists and contains no DA receptors. We reported similar findings in the GH, clone (Cronin et al., 1980; Faure et al., 1980) which also secretes rat prolactin. Several explanations for such behavior include: (1) The prolactin-secreting clones may have been derived from a single cell which did not express the DA receptor. A corollary to this is that since the various GH clones as well as the 235-1 cells contain both prolactin and growth hormone, the surface receptor phenotype expressed by this dedifferentiated cell may be typical of the normal somatotroph and not of the normal mammotroph. One would then have to propose that the 73 f5a tumor harbors a variety of altered endocrine cells types, some with and some without DA receptors, because DA receptors were present in this 7315a tumor (Cronin et al., 1981) and were indistinguishable from those in the anterior pituitary. (2) The 235-l clone cells may have originally expressed DA receptors, but repeated trypsinization may have permanently damaged or removed the expression of the DA receptor as has happened in human BeWo trophoblastic cells. Patti110 et al. (1979) showed that cultures passaged for 12 years with either mechanical or proteolytic techniques retained or lost {respectively) their ability to

235-l clone, prolactin release and DNA receptors

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secrete human chorionic gonadotropin. Although we routinely passaged the cells with EDTA, trypsin-EDTA was used for 39 passages prior to our obtaining the 235-l clone. (3) The preservation of the DA receptor phenotype may be particularly fragile in vitro, requiring serum constituents that are absent in the fetal calf and horse serum. This has been suggested by the observation that when GH, cells are grown in rats, the clone partially regains the property of DA inhibition of prolactin release (Melmed et al., 1980). Direct measurements of DA receptors were not made, nor was the pharmacological specificity proven in this study. In our 235-l experiments, the reestablishment of a hard tumor in rats did not reinduce a measurable population of DA receptors (Fig. 7). On the other hand, micromolar concentrations of bromocriptine significantly inhibited of prolactin release from the 235-l cells ‘in vitro’ after the cells were grown ‘in viva’ (Reymond et al., 1981). (4) Cell division may be incompatible with the expression of the DA receptor gene. Although mammotrophs surely divide slowly to create a prolactin adenoma, the DA receptors measured in human prolactinomas may be on the great majority of mammotrophs that are not dividing. We treated the 235-l cells chronically with bromocriptine to address several important questions. It is possible that these cells express DA receptors but in numbers too small either to be detected by the acute effects of dopaminergic agents on prolactin secretion or to be measured directly with out radioligand binding assay. Alternatively, these cells may express DA receptors but only briefly between cell divisions. Treatment with bromocriptine at concentrations that markedly inhibited prolactin secretion by normal pituitary cells had no effect on prolactin secretion by the 235-l cells, even with 8 days of exposure. These results suggest no expression, however small or transient, of functional DA receptors by the 235-l cells. In addition to decreasing serum prolactin levels in humans, bromocriptine is effective in reducing the size of prolactin-secreting tumors (Thorner et al., 1981), which contain DA receptor binding (Bression et al., 1980; Cronin et al., 1980). Bromocriptine decreases prolactin release and DNA synthesis (an index of cell division) in rat pituitary tumors (Kalbermann et al., 1980; Prysor-Jones and Jenkins, 1981) as well as in the normal gland (Davies et al., 1974; Lloyd et al., 1975; Jacobi and Lloyd, 1981). In contrast, bromocriptine has no effect on the growth or prolactin secretion of the MtTW 15 and 73 15a transplantable rat pituitary tumors (Lamberts and MacLeod, 1979) although both tumors contain DA receptors (Cronin et al., 1981a, b). Nor did bromocriptine affect hormone release or DNA synthesis in the GH, clone (Prysor-Jones and Jenkins, 1980) which lacks high-affinity DA

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receptors (Cronin et al., 1980b). In a recent communication (Melmed, 1981), bromocriptine was tested in the in vitro clonogenic assay and was found to depress colony formation by the rat GH, cells, but not by a rat lymphoma cell line or human endometrial cancer cells after 8 days of incubation. However, no significant differences in colony formation were found at a bromocriptin concentration of 10 nM, and the concentrations at which significant differences were found (100 and 300 nM) were about 2 orders of magnitude greater than the peak therapeutic serum levels of bromocriptine (1.1 nM) measured in hyperprolactinemic women (Thorner et al., 1980b). In our studies with the 235-l cells, bromocriptine had no effect on cell numbers or growth rate up to 8 days of exposure to a maximum of 100 nM bromocriptine. Our results support the hypothesis that functional DA receptors are required for bromocriptine to reduce prolactin release and prolactin adenoma size. Prolactin release by the 235-l clone was modified only in the presence of dibutyryl cyclic AMP (Figs. 2 and 3). It is apparent that DA acts to reduce basal or stimulated cyclic AMP cell content (Barnes et al., 1978; Labrie et al., 1980; Ray and Wallis, 1981), cyclic AMP secretion (Adams et al., 1979; Maurer, 1981) and adenylate cyclase activity (Giannattasio et al., 1981) in normal anterior pituitary glands. This in turn may contribute to the inhibition of prolactin release observed within 5 min of DA addition to normal mammotrophs (Thorner et al., 1980a). In our studies (Table l), DA had no effect on the cyclic AMP content of the 235-l cells over a 5-min period, a time appropriate to produce the effects seen in the above studies of the normal anterior pituitary mammotroph (Giannattasio et al., 1981; Labrie et al., 1980; Thorner et al., 1980). These data enhance the probability that the DA receptor is absent in this clone. If the [3H]spiperone DA receptor assay was not sensitive enough to measure a small population of DA receptors, and if these few receptors could still inhibit the adenylate cyclase, one might have expected that the cyclic AMP content would be reduced by the DA treatment. In conclusion, the 235-l clone secretes rat prolactin independent of any potential control by the physiological DA inhibitory mechanism. This functional lesion is at the level of the DA receptor at the least, because of the failure to directly label a DA receptor with the potent DA antagonist [3H]spiperone as well as the inability both of DA to modify cyclic AMP cell content and of bromocriptine to alter cell growth and prolactin release. This clone may be particularly useful in the elucidation of the mechanisms of prolactin synthesis and release independent of the influence of several physiological hormones (e.g. dopamine, estrogens, TRH), or for cell fusion studies as a receptor-negative recipient cell in which the normal second messenger mechanisms are intract. Finally, the

failure of bromocriptine to modify either prolactin release or 235-l cell growth rate argues strongly that the effects of this drug on reducing prolactin release and pituitary adenoma size are mediated by a functional DA receptor on the adematous mammotroph.

ACKNOWLEDGEMENTS This work was supported by USPIIS P6OAM-22125 (MJC, DAK), HD12173 (DAK), CA-7535-17 (RMM); RCDA’s lKO4N500601 to MJC and I-ID 00243 to DAK; and a NSF fellowship to SNP. Many thanks to Dr. Alan Rogol for measuring the growth hormone content in the clone and to Ms. Susan Perkins and Mrs. Susan O’Dell for perfor~ng the prolactin RIA.

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