OF 17, 1980 m 11 Dopaminergic Inhibition of Prolactin ... Inhibition of Prolactin Synthesis and...

Tns J O U R N A L OF BIOLOGICAL CHEMISTRY Vol. 255. No 17, Issue of September 10, pp. 8092-8097, 1980 Prmted m 1 1 S.A

Dopaminergic Inhibition of Prolactin Synthesis and Prolactin Messenger RNA Accumulation in Cultured Pituitary Cells*

(Received for publication, March 7, 1980, and in revised form, May 20, 1980)

Richard A. Maurer From the Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242

Prolactin synthesis was investigated in monolayer cultures of dispersed pituitary cells maintained in de- fined medium. Electrophoresis of cell extracts from cultures labeled with [36S]methionine demonstrated that treatment with the dopaminergic drug, ergocryptine, specifically inhibited prolactin synthesis. Analysis of the time course of ergocryptine effects demonstrated that prolactin synthesis decreased sharply after 1 t o 2 days of treatment and appeared to reach a new level of 26 to 28% of control values after 4 t o 6 days of treatment. The concentration of ergocryptine which produced half-maximal inhibition of prolactin synthesis was about 0.3 nm. The inhibition of prolactin synthesis produced by ergocryptine treatment was reversed by removal of the ergocryptine. Dopamine, ergocryptine, and bromoergocryptine (also a dopamine agonist) all inhibited prolactin synthesis while epinephrine and norepinephrine had little effect. The concentration of prolactin mRNA in cultured cells was assayed by hybridization of total cell RNA to prolactin cDNA. Anal- ysis of cultures treated either for varying times or with varying doses of ergocryptine demonstrated that there was a close correspondence between the inhibition of prolactin synthesis and the inhibition of prolactin mRNA levels. These studies demonstrate the ability of dopaminergic stimulation to specifically inhibit prolactin synthesis and to decrease prolactin mRNA levels in primary cultures of pituitary cells maintained in de- fined medium.

The regulation of the pituitary hormone prolactin is somewhat complicated involving the interaction of several different hormones. Both estradiol (1-3) and thyrotropin-releasing hormone (4) have been shown to stimulate prolactin production. Although both of these hormones certainly have a physiolog- ical role to stimulate prolactin, the principal biological regulation of prolactin involves an inhibition of prolactin production. This can be demonstrated by transplantation of the pituitary away from the inhibitory influence of the hypothal- amus which leads to increased prolactin production (5). Sev- eral lines of evidence suggest that dopamine is the hypotha- lamic hormone which is responsible for the inhibition of prolactin. This evidence includes the observation that dopamine and dopamine agonists are able to inhibit prolactin secretion (6, 7 ) . Serum levels of dopamine in the blood supply to the pituitary vary inversely with prolactin secretion (8).

This research was supported by Grant AM 21803 from the Na- tional Institutes of Health. Ergocryptine and bromoergocryptine were a generous gift of Sandoz, Inc., East Hanover, New Jersey. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Also, the pituitary contains dopamine receptors, and the bind- ing affinity of several dopamine agonists correlates with the ability of these agonists to inhibit prolactin secretion (9). Although considerable evidence has shown that dopamine and dopamine agonists inhibit prolactin secretion, only limited data is available concerning the role of dopamine in the regulation of prolactin synthesis (1, 10).

The ability of primary cultures of pituitary cells to alter prolactin secretion in response to dopamine agonists 19) suggests that this might be a useful system to examine the regulation of prolactin synthesis. The present study demonstrates the ability of the dopamine agonist, ergocryptine, to inhibit prolactin synthesis. These experiments utilized monolayer cultures of dispersed pituitary cells maintained in serum- free culture medium. The time course, dose response, and reversibility of the inhibition have been examined. Treatment of pituitary cells with ergocryptine decreased the prolactin mRNA content of cells suggesting a pretranslational site of action.

EXPERIMENTAL PROCEDURES

Cell Culture-Pituitaries from female retired breeder Sprague- Dawley rats (Holtzman) were minced finely and then rinsed with calcium-magnesium free Hanks’ balanced salt solution (Grand Island Biological Co.) containing 20 mM Hepes‘ (pH 7.4), 100 uni ts /d of penicillin, 100 p g / d of streptomycin, and 0.25 pg/ml of fungizone. The tissue was dispersed as described by Vale et at. (11). The dispersed cells were washed twice by centrifugation and resuspension in serum-containing medium. The culture medium used for plating contained D-valine-minimal essential medium (Grand Island Biologi- cal Co.) supplemented with 20 mM Hepes (pH 7.4), 5% fetal calf serum, and the same antibiotics as above. Fetal calf serum used for cell culture was extracted with charcoal (12) and dialyzed against 0.15 M NaCl, l0mM sodium phosphate (pH 7.4) to reduce the concentration

to 2 X lo6 cells/35-mm plastic tissue culture dish. Tissue culture of hormones and amino acids. The cells were plated at a density of 1

dishes were pretreated with an aqueous solution of 10 pg/d of polylysine and then washed several times with Earle’s balanced salt solution (Grand Island Biological (20.). This procedure was found to greatly enhance attachment of pituitary cells to the plate. The average yield was 2 to 3 x lo6 cells/pituitary. Cultures were maintained at 37°C in a humidifed atmosphere of 95% air, 5% Con. The medium was usually replaced with serum-free Dulbecco’s modified Eagle’s medium (Grand Island Biological Co.) containing antibiotics and 20 mM Hepes (pH 7.4) 2 days after the cells were plated. Culture medium was replaced every 2 days. Ergocryptine was dissolved in ethanol and diluted so that the final ethanol concentration did not exceed 0.01%.

Analysis ofProlactin Synthesis-Tissue culture dishes were rinsed with Earle’s balanced salt solution and then incubated for 60 min at 37°C in Eagle’s minimal essential medium (Grand Island Biological Co.) containing 20 mg/Iiter of leucine and IO pCi/ml of [’HHpeucine. After the incubation the cells were removed from the dish and homogenized in 0.3 ml of ice-cold 0.15 M NaCI, 10 mM sodium phosphate (pH 7.4), 10 m~ leucine, 1% Triton X-100, and 0.5% deoxycholate. The homogenate was centrifuged at 10,OOO X g for 10

’ The abbreviation used is: Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid.

8092

Inhibition of Prolactin Synthesis 8093

min, and aliquots of the supernatant were combined with 2.5 pg of internally labeled ["Clprolactin and a monospecific antibody to prolactin. The immunoprecipitate was washed, and radioactivity was determined as described previously (1.13). This immunoprecipitation procedure has been shown to be specific for prolactin (1, 14). AU results were corrected for recovery of the ["Clprolactin. To determine total protein synthesis, aliquots of the cell supernatant (10,000 X g) were combined with 1 ml of water and 1 ml of 10% trichloroacetic acid. The samples were incubated on ice for 5 min, and the precipitate was collected on glass fiber filters. The filters were prepared for scintillation counting in the same manner as the immunoprecipitates. For each sample the radioactivity incorporated into nonprolactin proteins was calculated by subtraction of the radioactivity in prolactin from the radioactivity in total protein. In order to correct for any possible differences in amino acid uptake or extraction of labeled proteins from the cells, the data for prolactin synthesis is expressed as the ratio of radioactivity in prolactin to the radioactivity in nonprolactin proteins (relative prolactin synthbsis).

DNA Determinations-The DNA content of aliquots of cell ho- mogenates was determined using the microfluorometric assay of Puzas and Goodman (15).

Analysis of Radiolabeled Proteins by Electrophoresis-Cell cultures were incubated for l h in minimal essential medium containing 100 @Ci/ml of [?3]methionine. Cells were removed from the plate in 0.1 ml of 10 m~ Tris (pH 7.4). 1% Triton X-100, 0.5% deoxycholate, and lOmm methionine, homogenized, and centrifuged at 10,000 X g for 10 min. Aliquots of the supernatant were electrophoresed on a 12% polyacrylamide sodium dodecyl sulfate-containing slab gel (10 X 10 X 0.15 cm) using the buffers of Laemmli (16). After electrophoresis the gel was stained, destained, and dried before exposure to XR- 1 film (Kodak) for autoradiography.

RNA Isolation-For analysis of prolactin mRNA, pituitary cells were cultured in 60-mm tissue culture dishes. Cells were removed from the dish in 3.5 ml of 4% Sarkosyl, 0.05 M Tris (pH 7.5), 1 mg/ml of heparin, and homogenized with 10 strokes of the tight pestle of a Dounce homogenizer. Then 2.5 g of solid CsCl were added and dissolved, and the mixture was layered over 1.2 ml of 6.0 M CsCl, 0.1 M EDTA. The samples were centrifuged at 33,000 rpm in a Beckman SW 50.1 rotor for 18 h at 15°C. The RNA pellet was dissolved as described by Glisin et al. (17) and then precipitated by addition of 2 volumes of ethanol.

Hybridization of cDNA to RNA-Prolactin cDNA was prepared from purified prolactin mRNA as described previously (18). The cDNA was layered over an alkaline sucrose gradient (5 to 20% sucrose in 0.1 M NaOH, 0.9 M NaCl) and centrifuged at 41,000 rpm in a Beckman SW 41 rotor for 12 h at 4OC. The peak of rapidly sedimenting material was pooled, neutralized by addition of 0.1 volume of 3 M sodium acetate (pH 5), and precipitated with ethanol. Sedimentation of cDNA through an alkaline sucrose gradient was found to lower the amount of cDNA which was resistant to S1 nuclease digestion after an 18-h incubation in hybridization mixtures containing no added RNA. RNA was hybridized to cDNA in 20 pl reactions containing 0.6 M NaCl, 10 mM Hepes (pH 7.4), 1 m~ EDTA, 0.2% sodium dodecyl sulfate, 0.1 mg/ml of yeast tRNA, and 2000 cpm of prolactin cDNA. Hybridization mixtures were incubated for 18 h at 68°C. Digestions with S1 nuclease were performed as described previously (18).

RESULTS

In order to examine possible effects of the dopamine agonist, ergocryptine, on the synthesis of prolactin, pituitary cells were pulse-labeled with [%]methionine, and the soluble proteins were electrophoresed on a sodium dodecyl sulfate containing polyacrylamide gel (Fig. 1). A predominant heavily labeled band with the same mobility as prolactin was found in extracts from control cells (Fig. 1, B and 0. Most of the radioactivity in this band is probably incorporated into prolactin as antiprolactin immunoprecipitation of an extract from control cells results in a band with similar mobility and labeling intensity (Fig. IA). This also demonstrates the specificity of the immunoprecipitation procedure. Thus, cultured pituitary cells retain the ability to synthesize substantial amounts of prolactin even after several days in serum-free medium. Treatment of cells for 4 days with ergocryptine resulted in a relatively specific decrease in the incorporation of radioactivity into this

A B C D E

PRL-,

FIG. 1. Electrophoretic analysis of proteins synthesized by control pituitary cell cultures and cultures treated with 10 n~ ergocryptine for 4 days. Monolayer cultures of pituitary cells were maintained in serum-free medium for 6 days. During the last 4 days in culture, half of the cultures were treated with 10 nM ergocryptine. The cells were then incubated with 100 pCi/ml of [:1'"S]methionine for 1 h, and a soluble cell extract was prepared. An aliquot from control cells was immunoprecipitated (A), and aliquots of total soluble proteins from duplicate control cultures ( B and 0 and from duplicate ergocryptine-treated cultures (D and E ) were electrophoresed on a 12% polyacrylamide sodium dodecyl sulfate-containing slab gel. The gel was stained, destained, dried and exposed to x-ray film. An unlabeled prolactin (PRL) standard was also electrophoresed on the gel; the migration of this standard is indicated by the arrow.

band (Fig. 1, D and E ) . This suggests that ergocryptine has specifically decreased prolactin synthesis.

Quantitative analysis of the time course of ['Hlleucine incorporation into prolactin also suggested that ergocryptine alters prolactin synthesis (Fig. 2). In control cells, radiolabeled prolactin first accumulates within the cell (Fig. 2B) and after a lag of about 1.5 to 2.0 h newly synthesized prolactin is secreted into the medium (Fig. 20. This lag in the secretion of newly synthesized prolactin was previously observed when intact pituitaries were incubated with [3H]leucine (1). In ergocryptine-treated cells the secretion of radiolabeled prolactin into the medium was almost completely blocked (Fig. 2 0 . For both control and ergocryptine-treated cells the total amount of labeled prolactin in the system (cells plus medium) accumulated in an approximately linear manner although at a considerably lower rate in the ergocryptine-treated cells. The apparently linear accumulation of labeled prolactin in control and ergocryptine-treated cells suggests that there is little degradation of prolactin in either group and that the differences in incorporation are likely due to differences in synthesis. Ultrastructural studies (19) have suggested that blockage of prolactin secretion can lead to prolactin degradation. However, pulse-chase studies have shown that although ergocryptine does induce prolactin degradation, the rate of degradation is relatively slow requiring several hours to pro- duce substantial effects (20, 21). Therefore, the use of brief labeling pulses should allow estimation of ergocryptine effects on prolactin synthesis relatively free from effects on prolactin

8094 Inhibition of Prolactin Synthesis

5 '

4 -

3.

2.

li C Medium

I 2 4

Tlme (hours)

FIG. 2. Time course of incorporation of radioactivity into prolactin in control and ergocryptine-treated cells. Control cultures of pituitary cells (t".) or cultures treated with 10 nM ergocryptine (Erg) for 4 days (0- - -0) were incubated with ["HI- leucine for varying times. At the end of the incubations the amount of radioactivity incorporated into prolactin was determined by immunoprecipitation of aliquots of cell extracts and aliquots of the medium. Total prolactin was determined by addition of counts in the cells and the medium. Values are means k S.E. for three determinations per point.

degradation. In all subsequent studies, pituitary cells were labeled by a 1-h incubation in ["Hlleucine.

Ergocryptine effects on prolactin synthesis are time-dependent (Fig. 3A). In control cultures, prolactin synthesis was relatively constant for the duration of the experiment. Prolac- tin synthesis was sharply reduced after 1 to 2 days of ergocryptine treatment and appeared to reach a new level of about 26 to 28% of control after 4 to 6 days of treatment. Ergocryp- tine had no effect on the amount of DNA per culture dish, and the amount of DNA per dish was relatively constant for the duration of the experiment (Fig. 3B). However, attempts to maintain cells in serum-free medium for longer than 8 days often resulted in cell deterioration and the loss of substantial numbers of cells from the plate (data not shown).

Ergocryptine effects on prolactin synthesis are dose-dependent (Fig. 4). Maximal inhibition of prolactin synthesis occurred at an ergocryptine concentration of approximately 3 nM and 50% of maximal inhibition at about 0.3 nM. Ergocryp- tine had relatively little effect on the synthesis of nonprolactin proteins a t any of the concentrations examined. This is consistent with the electrophoretic analysis of pituitary proteins (Fig. l j and demonstrates the specificity of ergocryptine effects.

The ability of several different catecholamines as well as ergocryptine and 2-bromo-a-ergocryptine (CB-154) to inhibit prolactin synthesis was examined (Fig. 5). The affinity of

.4 -

.2 -

\ \ \

I\, \ \

'0 . . Ergocryptme . . . -. . 0 """" 0

a n t

5

0 1 2 4 6

Time (days) FIG. 3. Time course of ergocryptine effects on prolactin syn-

thesis. Pituitary cell cultures were maintained in control medium (t".) or in medium containing 10 m ergocryptine (0- - -0). At the start of the ergocryptine treatment (time 0) and 1,2,4, and 6 days thereafter, cultures were labeled for 1 h by incubation with ['HI- leucine. Prolactin synthesis (A) was determined by immunoprecipitation. Relative prolactin synthesis is the ratio of radioactivity incorporated into prolactin to radioactivity incorporated into nonprolactin proteins. An aliquot of each cell extract was also assayed to determine the DNA content of cultures ( B ) . All values are means k S.E. for three independent determinations per point.

ergocryptine for the dopamine receptor is more than 100 times greater than the affinity of dopamine (9). Therefore, the catecholamines, dopamine, epinephrine, and norepinephrine, were used at a concentration of 1 ,UM while ergocryptine and bromoergocryptine were used at a concentration of 10 nM . At the doses examined, epinephrine and norepinephrine had little effect on prolactin synthesis while dopamine, ergocryptine, and bromoergocryptine produced substantial inhibition of prolactin synthesis. The failure of epinephrine and norepinephrine to inhibit prolactin synthesis is consistent with the observation that pituitary catecholamine receptors have been characterized as dopaminergic rather than a- or 8-adrenergic (9).

The inhibition of prolactin synthesis produced by ergocryptine treatment can be reversed by removal of the ergocryptine (Fig. 6). In this experiment the treatments were arranged so that all cultures were assayed for prolactin synthesis a t the same time. Two days of ergocryptine treatment reduced prolactin synthesis by more than 50%. Removal of the ergocryptine treatment for 1 day resulted in a significant increase in prolactin synthesis, and values returned to near control levels


-1 r

1 4 2 I I Days 0 Treated

Doys -

Withdrawn

2

I

2

2 0 IO IO IO IO 10

- 1 1 -10 -9 -8 -7

Ergocryptine (M) FIG. 4. Effects of varying doses of ergocryptine on the syn-

thesis of prolactin and nonprolactin proteins. Pituitary cell cultures were treated for 4 days with the doses of ergocryptine indicated. The cultures were then incubated with ['Hjleucine for 1 h, and radioactivity incorporated into prolactin was determined by immunoprecipitation. Radioactivity incorporated into nonprolactin proteins was calculated by subtraction of the amount of radioactivity in prolactin from the total amount of radioactivity incorporated into protein. Relative prolactin synthesis is the ratio of radioactivity in prolactin to the radioactivity in nonprolactin proteins. All values are means t S.E. for three determinations per point.

Frc. 6. Effects of withdrawal of ergocryptine on prolactin synthesis. In this experiment treatments were arranged so that all cultures were labeled with [:'Hlleucine at the same time. Cultures were treated with 1.0 nM ergocryptine for 0 or 2 days as indicated. Treatment was removed by replacement of medium with fresh medium containing no ergocryptine. After removal of ergocryptine treatment, medium was changed every day.

I I

1 t

- C

I 200 400

Prolactin mRNa (pg)

20 40

Total RNA (ng)

FIG. 7. Hybridization analysis of prolactin mRNA levels. Prolactin cDNA was synthesized from prolactin mRNA which was purified from immunoprecipitated polysomes (18). The cDNA was hybridized to varying amounts of the purified prolactin mRNA to construct a standard curve ( A ) . To determine prolactin mRNA concentrations in samples from cells, total pituitary RNA from control cultures (W) or cultures treated with 10 n~ ergocryptine (Erg) for 4 days ( G - 4 3 ) was hybridized to prolactin cDNA, and the percentage of hybridization was converted to picograms of prolactin mRNA using the standard curve in A.

- IO6 M IO6 1 8 M 10' M IO' M

EP NE OA ERG CB FIG. 5. Effects of different catecholamines and ergocryptine

and bromoergocryptine on prolactin synthesis. Pituitary cell cultures were treated with the indicated concentrations of epinephrine (EP) , norepinephrine ( N E ) , dopamine (DA), ergocryptine (ERG), or bromoergocryptine (CB) for 4 days, and the cells were then labeled with ["Hlleucine for 1 h. Relative prolactin synthesis was determined as in Fig. 4.

7A). Analysis of RNA from control cells or cells treated with 10 nM ergocryptine for 4 days revealed that ergocryptine induced a considerable decrease in prolactin mRNA concentrations (Fig. 7B) . In this experiment prolactin mRNA ac- counted for 0.42% of total RNA in controls and 0.17% of total RNA in ergocryptine-treated cells.

The cDNA hybridization assay was used to compare the effects of ergocryptine treatment on prolactin synthesis and prolactin mRNA levels. Analysis of the time course of ergocryptine effects showed that the inhibition of prolactin synthesis is accompanied by a very similar inhibition of prolactin mRNA levels (Fig. 8). Treatment of pituitary cells with varying doses of ergocryptine also demonstrated that there is a close correspondence between the inhibition of prolactin synthesis and the inhibition of prolactin mRNA levels (Fig. 9).

after 4 days of withdrawal from ergocryptine. It was not possible to examine the reversibility of longer treatments as cell deterioration occurred after prolonged incubation in serum-free medium.

Prolactin messenger RNA concentrations in total pituitary R N A were determined by analysis of the extent of hybridization of R N A to prolactin cDNA. The amount of prolactin mRNA in each sample was determined through the use of standard curves which were included in each experiment (Fig.

Inhibition of Prolactin Synthesis

0 I 2 4

Ergocryptine Treatment (days) FIG. 8. Comparison of the time course of ergocryptine ef-

fects on prolactin synthesis and prolactin mRNA levels. Pitui- tary cells were treated for 0, 1, 2, or 4 days with 10 n~ ergocryptine and then either labeled with ["H]leucine for analysis of prolactin synthesis or used to prepare total RNA for analysis of prolactin mRNA levels. The treatments were arranged so that all cultures were labeled and RNA prepared at the same time. Relative prolactin synthesis was determined as in Fig. 4. The prolactin mRNA concentration was determined as in Fig. 7, and the results are expressed as the percentage of total cell RNA. All values are means f S.E. for three determinations per point.

0 .3 nM

.... \..... :. ..>: ...". . ..... . - .4 -

U z U - c 0

- . 3 2 .c 0

I nM lOnM

Ergocryptine FIG. 9. Effects of varying concentrations of ergocryptine on

prolactin synthesis and prolactin mRNA levels. Pituitary cell cultures were treated with the indicated concentration of ergocryptine for 4 days, and the relative prolactin synthesis and prolactin mRNA levels were determined as in Fig. 7.

DISCUSSION

These results demonstrate that the dopaminergic drug, ergocryptine, inhibits prolactin synthesis and prolactin mRNA levels in cultured pituitary cells maintained in serum-free medium. The effects of ergocryptine are relatively specific for prolactin as the bulk of other pituitary proteins is not affected. The effects are specific for dopaminergic agents as dopamine, ergocryptine, and bromoergocryptine inhibit prolactin synthesis, but epinephrine and norepinephrine do not. Inhibition of prolactin synthesis induced by ergocryptine is reversed by withdrawal of the ergocryptine. The effects of ergocryptine on

both prolactin synthesis and prolactin mRNA levels are time- and dose-dependent. Previous studies have shown that dopaminergic drugs induce degradation of newly synthesized prolactin by pituitary cells (20, 21). Thus, dopaminergic regulation of prolactin production includes both inhibition of prolactin synthesis and stimulation of prolactin degradation.

Pituitary cells maintained in serum-free medium continue to synthesize large amounts of prolactin for at least a week. This suggests that stimulatory factors may not be required for expression of the prolactin gene. I t is possible that the prolactin gene is expressed constitutively and that regulation involves repression by dopamine. The ability of estrogen and thyrotropin-releasing hormone to stimulate prolactin gene expression may be due to reversal of dopamine repression. Raymond et al. (22) have shown that estradiol treatment is able to reverse the dopaminergic inhibition of prolactin re- lease. Thus, the primary regulation of prolactin gene expression may involve repression by dopamine which is modulated by estrogen and thyrotropin-releasing hormone. Further studies of the interaction of these hormones will be required to test this hypothesis.

Ergocryptine effects on prolactin synthesis are somewhat slow, requiring about 2 days for 50% inhibition of prolactin synthesis. The response of prolactin synthesis to other regu- lators such as estradiol is also quite slow (1). This may indicate that prolactin mRNA has a long half-life. I t is possible that ergocryptine acts quickly to alter the synthesis of prolactin mRNA, but that effects on prolactin mRNA accumulation are delayed due to the long half-life of the messenger.

I t seems likely that changes in prolactin mRNA concentration are the principal mechanism involved in the regulation of prolactin synthesis. The present study suggests that the effects of dopaminergic stimuli to inhibit prolactin synthesis are mediated by changes in prolactin mRNA levels. Similarly, the effects of estrogen to stimulate prolactin synthesis have been shown to involve an accumulation of prolactin mRNA (23- 26). The effects of thyrotropin-releasing hormone on prolactin synthesis also appear to be mediated by changes in prolactin mRNA concentrations(27-29).

The possible mechanisms involved in dopaminergic effects on prolactin mRNA remain to be determined. Dopamine receptors have been identified on the plasma membrane of pituitary cells (9). Brain dopamine receptors are likely coupled to adenylate cyclase (30). Thus, cyclic AMP may be the intracellular mediator of dopamine effects on prolactin synthesis and prolactin mRNA levels. However, studies of the effects of cyclic AMP on prolactin production have yielded inconclusive findings (31, 32).

The ability of dopaminergic treatment to alter the concentration of prolactin mRNA in pituitary cells suggests a pretranslational site of action. This may involve effects on transcription or on post-transcriptional events. Post-transcriptional events might involve removal of intervening sequences, modification of the termini of the transcript, transport of the mature mRNA to the cytoplasm, and degradation of the mature mRNA. Recently a large potential precursor for prolactin mRNA has been detected in pituitary nuclear RNA (33). At the present time, hormonal effects on the transcription of the prolactin gene or the post-transcriptional fate of prolactin transcripts have not been evaluated. The availability of recombinant DNA plasmids containing the prolactin coding sequence should provide a specific hybridization probe for analysis of prolactin mRNA synthesis and degradation (34). The pituitary cell culture system described in this report should provide a useful system for analysis of the dopaminergic regulation of prolactin gene transcription as well as the processing and degradation of the prolactin transcripts.


Acknowledgments-I wish to thank M. Lieberman for supplying the antiprolactin used in these studies and B. Maurer for technical assistance and aid in preparing this manuscript.

REFERENCES 1. Maurer, R. A,, and Gorski, J. (1977) Endocrinology 101, 76-84 2. Lieberman, M. E., Maurer, R. A., and Gorski, J . (1978) Proc.

Natl. Acad. Sci. U. S. A . 75, 5946-5949 3. Haug, E., and Gautvik, K. M. (1976) Endocrinology 99, 1482-

1489 4. Dannies, P. S., and Tashjian, A. H., Jr. (1973) J. Biol. Chem. 248,

6174-6179 5. Chen, C. L., Amenomori, Y., Lu, K. H., Voogt, J. L., and Meites,

J. (1970) Neuroendocrinology 6, 220-227 6. MacLeod, R. M., Fontham, E. H., and Lehmeyer, J . E. (1970)

Neuroendocrinology 6, 283-294 7. Birge, C. A., Jacobs, L. S., Hammer, C. T., and Daughaday, W. H.

(1970) Endocrinology 86, 120-130 8. Ben-Jonathan, N., Oliver, C., Weiner, H. J., Mical, R. S., and

Porter, J. C. (1977) Endocrinology 100,452-458 9. Caron, M. G., Beaulieu, M., Raymond, V., Gagne, B., Drouin, J.,

Lefkowitz, R. J., and Labrie, F. (1978) J. Biol. Chem. 253,2244- 2253

10. MacLeod, R. M., and Lehmeyer, J. E. (1974) Cancer Res. 34,

11. Vale, W., Grant, G., Amoss, M., Blackwell, R., and Guillemin, R.

12. Honvitz, K. B., Costlow, M. E., and McGuire, W. L. (1975)

13. Maurer, R. A. (1979) Mol. Cell. Endocr. 13, 291-300 14. Maurer, R. A,, Stone, R., and Gorski, J . (1976) J . Biol. Chem.

15. Puzas, J . E., and Goodman, D. B. P. (1978) Anal. Biochem. 86,

345-350

(1972) Endocrinology 91, 562-572

Steroids 26, 785-795

251,2801-2807

50-55

16. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 17. Gliiin, V., Crkvenjakov, R., and Byus, C. (1974) Biochemistry 13,

18. Maurer, R. A. (1980) J. Biol. Chem. 255,854-859 19. Smith, R. E., and Farquhar, M. G. (1966) J. Cell Biol. 31, 319-

20. Dannies, P. S., and Rudnick, M. S. (1980) J. Biol. Chem. 255,

21. Maurer, R. A. (1980) Biochemistry, in press 22. Raymond, V., Beaulieu, M., Labrie, F., and Boissier, J. (1978)

23. Stone, R. T., Maurer, R. A,, and Gorski, J. (1977) Biochemistry

24. Ryan, R., Shupnik, M. A., and Gorski, J . (1979) Biochemistry 18,

25. Seo, H., Refetoff, S., Vassart, G., and Brocas, H. (1979) Proc.

26. Shupnik, M. A,, Baxter, L. A., French, L. R., and Gorski, J. (1979)

27. Evans, G . A., and Rosenfeld, M. G. (1976) J . Biol. Chem. 251,

28. Dannies, P. S., and Tashjian, A. H., Jr. (1976) Biochem. Biophys.

29. Evans, G. A., David, D. N., and Rosenfeld. M. G. (1978) Proc.

30. Kehabian, J . W., Petzold, G. L., and Greengard, P. (1972) Proc.

31. Dannies, P. S., Gautvik, K. M., and Tashjian, A. H., J r . (1976)

32. Hill, M. K., MacLeod, R. M., and Orcutt, P. (1976) Endocrinology

33. Maurer, R. A., Guhbins, E. J., Erwin, C. R., and Donelson, J. E.

3 4 . Gubbins, E. J., Maurer, R. A,, Hartley, J. L., and Donelson, J. E.

2633-2637

347

2776-2781

Science 200, I 173- 1 175

16, 4915-4921

2044-2048

Natl. Acad. Sei. U. S. A. 76,824-828

Endocrinology 104, 729-735

2842-2847

Res. Commun. 70, 1180-1189

Natl. Acad. Sci. U. S. A . 75, 1294-1298

Natl. Acad. Sci. U. S. A . 69, 2145-2149

Endocrinology 98, 1147-1159

99, 1612-1616

(1980) J. Biol. Chem. 255, 2243-2246

(1979) Nucleic Acids Res. 6, 915-930

OF 17, 1980 m 11 Dopaminergic Inhibition of Prolactin ... Inhibition of Prolactin Synthesis and...

Documents

Transcript of OF 17, 1980 m 11 Dopaminergic Inhibition of Prolactin ... Inhibition of Prolactin Synthesis and...