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PMA-sensitive protein kinase C is not necessary in TRH-stimulated prolactin release from female rat primary pituitary cells
L i f e S c i e n c e s , Vol. P r i n t e d in the U S A
51,
pp.
1957-1967
Pergamon
Press
PMA-SENSITIVE PROTEIN KINASE C IS NOT NECESSARY IN TRH-STIMULATED PROLACTIN RELEASE FROM FEMALE RAT PRIMARY PITUITARY CELLS Kang Cheng, Wanda W.-S. Chan, Robert Arias, Albert Barreto, Jr., and Bridget Buffer Department of Growth Biochemistry and Physiology Merck Research Laboratories P. O. Box 2000 Rahway, N.J. 07065 (Received
in final
form O c t o b e r
9,
1992)
Summilrv In GH3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca 2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRHstimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10 -6 M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary ceils with TRH (10 -7 M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin, sn-l,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent responses to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca 2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C. Thyrotropin releasing hormone (TRH) has been shown to stimulate the release of prolactin from both anterior pituitary cells and pituitary tumor cell lines. Numerous studies in elucidating the molecular mechanism of action of TRH on prolactin release were performed with pituitary tumor cell lines in culture. The effect of TRH on prolactin release in GH3 cells is mediated, at least in part, via elevation of intracellular Ca 2÷ (1-4). The increase of intracellular Ca 2÷ in response to TRH was a result of mobilization of a nonmitochondrial Ca 2÷ pool (3) and enhancement of extracellular Address all correspondence to: Dr. Kang Cheng, Merck Research Laboratories, R80T-138, P.O. Box 2000, Rahway, NJ 07065 Copyright
© 1992
0 0 2 4 - 3 2 0 5 / 9 2 $5.00 + .00 P e r g a m o n Press Ltd All r i g h t s
reserved.
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Ca 2+ influx (2). Phorbol esters and synthetic diacylglycerols, activators of protein kinase C, have been demonstrated to stimulate prolactin release from pituitary cells (5-8). Furthermore, it has been shown that TRH caused a rapid translocation (within 15 seconds) of protein kinase C from cytosol to plasma membrane in GH4C1 cells (7). Elevations of inositol trisphosphate and 1,2diacylglycerol in GH3 cells in response to TRH have also been reported (8-10). These results suggest that phospholipase C mediates the effect of TRH on prolactin release in pituitary tumor cell lines. In contrast, it has recently been demonstrated that the effect of TRH on prolactin release from a rat pituitary cell perfusion system is not dependent upon the activation of protein kinase C (11). In fact, the release of other pituitary hormones, such as GH in response to GRF (12) and LH in response to GnRH (13-16) also do not require protein kinase C activation. In this study we examined the role of protein kinase C and Ca 2+ in TRH-stimulated prolactin release from female rat primary pituitary cells in static incubations. A direct comparison of the results on prolactin release in response to TRH was made between pituitary mammotrophs and GH3 cells which have been pretreated with either PMA or TRH. A preliminary report of these findings has been presented (17). Mat~ri~ll~ and Me|hods
Materials Dulbecco's Modified Eagle Medium (DMEM), Ham F-10, horse and fetal bovine sera, glutamine (100-fold concentrated), non-essential amino acids (100-fold concentrated), nystatin (10,000 U/ml), gentamycin (50 mg/ml), penicillin (5,000 U/ml)-streptomycin (5,000 Ilg/ml), Hank's Balanced Salt Solution and collagenase were purchased from Gibco (Grand Island, NY). Hyaluronidase and phorbol- 12-myristate- 13-acetate were obtained from Sigma (St. Louis, MO). TRH was from Peninsula Laboratories (San Carlos, CA). Forskolin was purchased from Calbiochem-Behring Corp. (La Jolla, CA). GH3 cells was obtained from American Type Culture Collection. The Wistar lactating female rats were obtained from Charles River Laboratories (Wilmington, MA). Rats were maintained at a constant temperature (25°C) on a 14-hr light, 10-hr dark cycle. Rat chow and water were available ad libitum. Rat Di|uitarv c$11 culture Rat pituitary cells were isolated from pituitaries by enzymatic digestion with 0.2% collagenase and 0.2% hyaluronidase in Hank's Balanced Salt Solution as described previously (18). For culture, the cells were suspended in culture medium and adjusted to 1.5 x 105 cells/ml, and 1.0 ml of this suspension was placed in each well of a 24-well tray (Costar, Cambridge, MA). Cells were maintained in a humidified 5% CO2-95% air atmosphere at 37°C for 3 to 4 days. The culture medium consisted of DMEM containing 0.37% NaHCO3, 10% horse serum, 2.5% fetal bovine serum, 1% non-essential amino acids, 1% glutamine, 1% nystatin, and 0.1% gentamycin. i~xoeriments for orolactin release On the day of an experiment, cells were washed twice 1.5 hrs prior to and once more immediately before the start of the experiment with the above culture medium containing 25 mM HEPES, pH 7.4. Prolactin release was initiated by adding 1 ml of fresh medium containing test agents to each well in quadruplicate. Incubation was carried out at 37°C for 15 minutes or 2 hours as indicated. After incubation, medium was removed and centrifuged at 2,000 x g for 15 minutes to remove any cellular material. The supernatant fluid was removed and assayed for prolactin content. GHa cell culture IffH3 cells were plated at a density of 3 x 105 cells/well in 12-well cell culture plates (Costar) and maintained in Ham's F-10 medium containing 15% horse serum, 2.5% fetal bovine serum, 0.1% gentamycin and 0.1% penicillin-streptomycin for 3 to 4 days. Before experiment, cells were washed as described for primary pituitary cells with Ham's F-10 medium and the experiment was carried out as described in the figure legend.
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DoWll-regulation of orotein kinase C To down-regulate protein kinase C, rat pituitary cells were incubated with 10-6 M PMA in the culture medium containing 25 mM HEPES, pH 7.4, for 24 hours at 37°C. After the pretreatment, the cells were washed three times with fresh culture medium and the experiments were carried out as described in the figure legends. Under these conditions, it has been demonstrated that cellular protein kinase C content in rat pituitary cells and GH3 cells was decreased by more than 90% (13,19,20). RIAs Rat prolactin in culture medium was measured by a double antibody RIA procedure using materials obtained from Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA) and expressed in terms of the standard rat prolactin RP-3.
60
+PMA (10 6M)
E
40
20
0
101 lO
I
10 .9
J
10 .8
[
10 .7
I
10 6
TRH (M)
FIG. 1 Effect of TRH on prolactin release from rat pituitary cells in the presence and absence of PMA. Cells were incubated with various concentrations of TRH in the presence (o) or absence (o) of 10 -6 M PMA at 37°C for 15 min. Each point represents the mean + SEM of quadruplicate incubations. In this and subsequent figures, if no SEM is shown it was smaller than the symbol representing the mean. Data oresentation The results presented in figures are from a single experiment in which quadruplicate determinations were performed and in each case are representative of at least three separate experiments. Statistical analysis was performed with Student's t test. Results and Discussion Effects of T R H and PMA on Prolactin Release from Rat Pituitarv Cells As shown in Figure 1, TRH stimulated prolactin release in a dose-dependent manner. Maximal stimulation was observed at a concentration of 10 -7 M of TRH. PMA (10 -6 M) increased prolactin
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release by 250% and further increased the prolactin release even in the presence of maximal concentrations of TRH. However, the combined effect of TRH and PMA on prolactin release was slightly less than additive but certainly was greater than that of either one of the compounds alone. In Figure 2, similar results were obtained with a different experimental protocol from that used in Figure 1. Namely, maximal concentrations of TRH and PMA together produced a prolactin response which was almost equal to the sum of individual effects. These results suggest that the effects of TRH and PMA on prolacfin release are probably mediated via different mechanisms.
60
c E to
40
.E
20
I
[
I
I
[
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0
10 .9
10 .8
10 7
10 6
10 5
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FIG. 2 Effect of PMA on prolactin release from rat pituitary cells in the presence and absence of TRH. Cells were incubated with various concentrations of PMA in the presence (o) or absence (o) of 10 .7 M TRH at 37°C for 15 rain. Each point represents the mean + SEM of quadruplicate incubations. Effects o f P M A and T R H P r e t r e a t m e n t on Prolactin R e l e a s e f r o m Rat
Pituitarv
Cells Phorbol esters have been reported to down-regulate protein kinase C activity in a number of cell types including rat primary pituitary cells and GH3 cells (13,19-22). To further evaluate the role of protein kinase C activation in TRH-induced prolactin release, the PMA-pretreated pituitary cells were used in the next series of studies. As shown in Figure 3, rat pituitary cells pretreated with 10 -6 M PMA for 24 hours did not release prolactin in response to a second dose of PMA at a concentration as high as 10-5 M. This result indicates that all of the PMA-sensitive protein kinase C had been removed during the 24 hour PMA pretreatment period. Interestingly, PMA-pretreated cells responded equally well to TRH when compared to control cells (Figure 4), suggesting that PMA-sensitive protein kinase C may not be involved in TRH-induced prolactin release. Pituitary cells pretreated with 10 .7 M TRH for 24 hours had a lower basal prolactin release as shown in Figures 3 and 4. The effect of TRH on prolactin release from these cells was almost completely inhibited while the PMA effect was normal as compared to untreated control cells.
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Effect
of TRH on P r o l a c t i n
Release
1961
The effect of TRH on prolactin release from clonal rat pituitary cells has been shown to be biphasic (23-25). The first phase is dependent upon the release of Ca 2+ from intracellular stores and the second phase requires the activation of protein kinase C and Ca 2+ influx. Thus, in one experiment the incubation time of TRH with PMA-pretreated cells was extended from 15 minutes to 2 hours in order to be certain that most of the medium prolactin was secreted during the second phase in which protein kinase C plays a major role in mediating the effect of TRH. As shown in Figure 5, the dose-response curves for TRH stimulation of prolactin release from control and PMA-pretreated cells are superimposable. However, TRH-pretreated cells responded poorly to a second dose of TRH. This is probably the result of down-regulation of TRH receptors.
100 E" 80 I
Non-Pretreated
60 /
40
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EL
20
~ ~ I
0
'~' I
I
~11A-Tr~%e4d h~rsells P
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109 108 107 106 105 PMA(M) FIG. 3
Effects of TRH and PMA pretreatments on PMA-stimulated prolactin release from rat pituitary cells. Cells were pretreated with either TRH (10 -7 M) or PMA (10 .6 M) at 37°C for 24 hours. After pretreatment, cells were washed three times with fresh medium and incubated with various concentrations of PMA at 37°C for 15 min, non-pretreated cells (o), TRHtreated cells (.) and PMA-treated cells (A). Each point represents the mean + SEM of quadruplicate incubations. PMA Pretreatment inhibited the Svner~istic Action of Forskolin and PMA ~but not TRH~ on Prolactin Release from Rat P~tuitarv Cells Forskolin increases intracellular cyclic AMP by activating adenylate cyclase and stimulates prolactin release in rat pituitary cells (26). Maximal concentrations of PMA and forskolin, when added together, produced a synergistic effect on prolactin release as shown in Figure 6. This synergistic effect was completely inhibited by down-regulation of PMA-sensitive protein kinase C. As expected, down-regulation of PMA-sensitive protein kinase C had no effect on forskolininduced prolactin release, since the effect of forskolin was mediated via cyclic AMP. TRH together with forskolin elicited a prolactin response which was slightly greater than additive. However, their effect was not inhibited by down-regulation of PMA-sensitive protein kinase C. Effects of s n - l . 2 - D i o c t a n v l ~ l v c e r o l Pretreatment on T R H - I n d u c e d Prolactin Release from Rat Pituit~Irv C¢ll~ In GH3 cells, it has been shown that depletion of protein kinase C by phorbol diesters does not completely inhibit the effects of diacylglycerols (27). Thus, in the next several experiments we studied the effects of TRH on prolactin release from rat pituitary cells which had been pretreated
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with sn-l,2-dioctanylglycerol (DOG), a synthetic diacylglycerol, for 24 hours. As depicted in Figure 7, DOG stimulated prolactin release in a dose-dependent manner. However, at a concentration of 500 gM, the DOG stimulation of prolactin release still did not reach a plateau. Pretreatment of cells with 250 I.tM of DOG for 24 hours attenuated the subsequent response to a second dose of DOG. Interestingly, PMA pretreatment totally eliminated the effect of DOG on prolactin release in rat primary pituitary cells. DOG pretreatment partially inhibited the effect of PMA on prolactin release but had no effect on that induced by TRH (Figure 8).
120
100
80
60
PMA-Treated Cells (10-6 M, 24 hrs) / /
~/
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Non-Pretreated Cells
40 //
TRH-Treated Cells
20
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0
I
I
10 10
I
10 9 10 8 TRH (M)
l
I
10 7
106
FIG. 4 Effects of TRH and PMA pretreatments on TRH-stimulated prolactin release from rat pituitary cells. Cells were pretreated with either TRH (10 -7 M) or PMA (10 -6 M) at 37°C for 24 hours. After pretreatment, cells were washed three times with fresh medium and incubated with various concentrations of TRH at 37°C for 15 min, non-pretreated cells (o), TRHtreated cells (o) and PMA-treated cells (A). Each point represents the mean + SEM of quadruplicate incubations. Differential Effects of Extracellular Calcium on TRH and PMA-Stimulated Prolactin Release from Rat Pituitarv Cells It has been demonstrated that extracellular calcium is essential for TRH-induced prolactin release in the second phase in GH3 cells(23). Thus, in the next experiment we evaluated the effects of removing extracellular calcium on TRH and PMA-induced prolactin release from rat pituitary cells. As shown in Table I, basal, TRH-, PMA- and forskolin-induced prolactin releases were decreased by 50%, 90%, 10% and 57%, respectively, by the removal of extracellular calcium. The combined effect of forskolin and TRH on prolactin release was also markedly inhibited in the absence of extracellular calcium. On the contrary, the synergistic effect between forskolin and PMA was not dependent upon extracellular calcium at all. The decrease of basal prolactin release in calcium-free medium was probably due to the presence of 1 mM EGTA which might remove some of the intracellular calcium during the incubation. Effects of TRH and PMA Pretreatments on TRH-. PMA- and Forskolin-Stimulated Prolactin Release from GH 2 Cells It has previously been demonstrated that PMA pretreatment inhibits the subsequent effect of
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100
1963
Non-PretreatedC e l ~
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FIG. 5 Effects of TRH and PMA pretreatments on TRH-stimulated prolactin release from rat pituitary cells. Experimental conditions were the same as in Figure 4 legend, except after the pretreatments cells were incubated with various concentrations of TRH at 37°C for 2 hours, non-pretreated cells (o), TRH-treated cells (e) and PMA-treated cells (A). Each point represents the mean + SEM of quadruplicate incubations. TRH on prolactin release in GH3 cells (28). Results from our laboratory and others indicate that the effect of TRH on prolactin release from rat pituitary cells is independent of protein kinase C activation. However, the effects of protein kinase C down-regulation on TRH-induced prolactin release in these systems were never directly compared in the same laboratory under the same conditions. Thus, GH3 cells were pretreated with 10-6 M of TRH or PMA for 24 hrs and the release of prolactin from these cells in response to TRH, PMA and forskolin were examined. As shown in Figure 9, TRH (10 -7 M), PMA (10 -7 M) and forskolin (10 .5 M) stimulated prolactin release from non-pretreated GH 3 cells by 70%, 240% and 80%, respectively. TRH pretreatment inhibited the subsequent prolactin response to TRH by 70% and had very little or no effect on that stimulated by PMA or forskolin. PMA pretreatment almost completely inhibited the effects of TRH and PMA on prolactin release. Again, in our control experiment the forskolin-induced prolactin release was not affected by the protein kinase C down-regulation. Conclusions The data presented in this report provide additional evidence that the effect of TRH on prolactin release from female rat primary pituitary cell culture is probably not mediated via PMA-sensitive
1964
Effect
._= E ub
of TRH
on Prolactin
[]
Non-Pretreated Cells
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PMA-Pretreated celfs (10-6M, 24 hrs)
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1992
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TRH 10 8M + FSK 10-5M
FSK 10-5M
PMA 10-6M + FSK 10 5M
FIG. 6 Effect of protein kinase C down-regulation on TRH, PMA and forskolin-induced prolactin release from rat pituitary cells. Protein kinase C was down-regulated as described in Materials and Methods. Incubations were carried out at 37°C for 15 min in the presence of various agents as indicated. Data represent the mean + SEM of quadruplicate incubations. 60
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FIG. 7 Effect of DOG and PMA pretreatment on prolactin release induced by DOG in rat pituitary cells. Cells were pretreated with either DOG (0.25 raM) or PMA (10 -6 M) at 37°C for 24 hours. After pretreatment, cells were washed three times with fresh medium and incubated with various concentrations of DOG at 37°C for 15 min, non-pretreated cells (1~), DOGtreated cells (m) and PMA-treated cells (o). Each point represents the mean + SEM of quadruplicate incubations.
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E f f e c t of T R H o n P r o l a c t i n R e l e a s e
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protein kinase C. In support of this conclusion, PMA-pretreated rat pituitary cells which did not respond to a second dose of PMA had a normal response to TRH as did the non-pretreated cells. On the other hand, GH3 cells which had been pretreated with PMA responded very poorly to a subsequent TRH stimulation (Figure 9 and Ref 28). Since it has been reported that some of the effects elicited by synthetic diacylglycerol in GH3 cells are not mimicked by PMA (27,29), it is possible that the effect of TRH on prolactin release might still be mediated via the activation of protein kinase C by endogenous diacylglycerol. To test this hypothesis, we studied the effect of DOG pretreaunent on the subsequent prolactin response to TRH. Cells pretreated with DOG for 24 hours had an attenuated response to DOG and PMA, but they responded normally to TRH (Figures 7 and 8). These results indicate that DOG pretreatment only removed part of cellular protein kinase C. The decreased efficacy of DOG in down-regulating protein kinase C relative to the efficacy of PMA is probably due to the fact that the plasma membrane is less permeable to DOG than PMA. 70
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~
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0L ! i i L i 0 1010109108 107 106 TRH(M)
FIG. 8 Effect of DOG pretreatment on prolactin release induced by PMA and TRH in rat pituitary cells. Cells were pretreated with DOG (0.25 mM) at 37°C for 24 hours. After pretreatment, cells were washed twice with fresh medium and incubated with various concentrations of (a) PMA or (b) TRH at 37°C for 15 min; non-pretreated cells (e-,) and DOG-treated cells ( I ) . Each point represents the mean + SEM of quadruplicate incubations. TABLE I Requirement of Extracellular Calcium for the Action of TRH, PMA and Forskolin on Prolactin Release from Rat Pituitary Cells.
Conditions Control TRH (10 .8 M) PMA (10 .6 M) Forskolin (10 -5 M) Forskolin (10 .5 M) + TRH (10 -8 M) Forskolin (10 -5 M) + PMA (10 -6 M)
Pr01aqtin (nWwell/l 5 min) Ca2+-free Medium Normal Medium + 1 mM EGTA 13.1 53.3 96.7 35.5
+ + + +
0.2 0.2 4.9 0.6
6.9 + 1.1 11.2 + 0.5 83.6 + 9.8 16.6 + 0.5
95.1 + 9.1
19.3 + 0.8
168.5 + 7.1
144.6 + 4.3
Cells were incubated with various agents as indicated at 37°C for 15 min. Data represent the mean + SEM of quadruplicate incubations.
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300 [] Non Pretreated Cells [ ] TRH-Treated Cells B PMA Treated Oells 200
cE
~
100
TRH(0.1pM)
PMA(0 1p M)
FSK(10~tM)
FIG. 9 Effects of TRH and PMA pretreatments on prolactin release from GH3 cells. Cells were pretreated with TRH (10 -6 M) or PMA (10 -6 M) at 37 ° C for 24 hours. After pretreatment, cells were washed twice with fresh medium and incubated with 10-7 M TRH, l0 n M PMA or 10-5 M forskolin at 37 ° C for 15 min. Data represent the mean + SEM of three different experiments. *, p < 0.01 (vs. non-pretreated cells). This is also evident from the results which show that much higher concentrations of DOG are needed to maximally stimulate prolactin release. Interestingly, PMA-pretreated cells did not respond to DOG at all, suggesting that the protein kinase C(s) which mediates the effect of DOG on prolactin release in rat primary pituitary cells is also sensitive to PMA. Thus, it is unlikely that protein kinase C plays a significant role in mediating the effect of TRH on prolactin release in rat primary pituitary cells. This is consistent with the findings from a recent study using a pituitary cell perfusion system (11). In this study we further demonstrated that the effect of TRH was very much dependent upon the presence of extracellular calcium, while PMA stimulated prolactin release responded almost equally well in the presence or absence of extracellular calcium (Table I). In addition to a number of differences already known to exist between the primary pituitary cells and tumor cell lines, we have demonstrated the effect of TRH on prolactin release is mediated via different mechanisms in these two cell types.
Acknowledgement The authors would like to thank Theresa Timko for her help in manuscript preparation.
I~¢ferences M.C. GERSHENGORN and K. THAW, Endocrinology 113 1522-1524 (1983). P.R. ALBERT and A.H. TASHJIAN, J Biol Chem 259 5827-5832 (1984). M.C. GERSHENGORN, E. GERAS, V. SPINA PURELLO and M.J. REBECCHI, J Biol Chem 259 10675-10681 (1984). 4. K.W. SNOWDOWNE and A.B. BORLE, Am J Physiol 246 E198-E201 (1984). 5. S.T. SUMMERS, P.L. CANONICO, R.M. MACLEOD, A.D. ROGOL and M.J. CRONIN, Eur J Pharmacol 111 371-376 (1985). 6. A. NEGRO-VILAR and E.G. LAPETINA, Endocrinology 117 1559-1564 (1985). 7. C.W. FEARON and A.H. TASHJIAN, J Biol Chem 260 8366-8371 (1985). 8. T.F.J. MARTIN, J Biol Chem 2,58 14816-14822 (1983). 9. M.J. REBECCHI and M.C. GERSHENGORN, Biochem J 216 287-294 (1983). 10. C.H. MACPHEE and A.H. DRUMMOND, Mol Pharmacol 25 193-200 (1984). 1. 2. 3.
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11. A.M. JUDD, I.S. LOGIN and R.M. MACLEOD, Endocrinology 125 1134-1141 (1989). 12. M.B. FRENCH, B.C. MOOR, B.T. LUSSIER and J. KRAICER, Endocrinology 124 22352244 (1989). 13. C.A. MCARDLE, W.R. HUCKLE and P.M. CONN, J. Biol Chem 202 5028-5035 (1987). 14. M.S. JOHNSON, R. MITCHELL and G.J. FINK, Endocrinology 110 231-239 (1988). 15. M.J. BEGGS and W.L. MILLER, Endocrinology 124 667-674 (1989). 16. P.A. VAN DER MERWE, R.P. MILLAR and J.S. DAVIDSON, Biochem. J. 208 493-498 (1990). 17. K. CHENG, W.W.S.CHAN, A. BARRETO and B. BUTLER, 72rid Annual Meeting of The Endocrine Society, Atlanta, GA, p 83 (Abstract), 1990. 1 8. K. CHENG, W.W.S.CHAN, A. BARRETO, E.M. CONVEY and R.G. SMITH, Endocrinology 124 2791-2798 (1989). 19. R. BALLESTER and O.A. ROSEN, J Biol Chem 260 15194-15199 (1985). 20. S.S. STOJIKOVIC, J.P. CHANG, D. NGO and K.J. CATT, J Biol Chem 203 1730717311 (1988). 21. P.M. TAPLEY and A.W. MURRAY, EurJ Biochem 151 419-423 (1985). 22. E. MELLONI, S. PONTREMOLI, M. MICHETI'I, O. SACCO, B. SPARATORE and B.L. HORECKER, J. Biol Chem 201 4101-4105 (1986). 23. T.F.J. MARTIN and J.A. KOWALCHYK, Endocrinology 115 1527-1536 (1984). 24. P.R. ALBERT and A.H. TASHJIAN, J Biol Chem 259 15350-15363 (1984). 25. T. AIZAWA and P.M. HINKLE, Endocrinology 116 73-82 (1985). 26. D. DELBEKE, J.G. SCAMMELL and P.S. DANNIES, Endocrinology 114 1433-1440 (1983). 27. R.N. KOLESNICK and A.E. PALEY, J Biol Chem 262 9204-9210 (1987). 28. R. RODRIGUEZ, A. IMAI and M.C. GERSHENGORN, Mol Endocrinol / 802-807 (1987). 29. R.N. KOLESNICK and S. CLEGG, J Biol Chem 263 6534-6537 (1988).
51,
pp.
1957-1967
Pergamon
Press
PMA-SENSITIVE PROTEIN KINASE C IS NOT NECESSARY IN TRH-STIMULATED PROLACTIN RELEASE FROM FEMALE RAT PRIMARY PITUITARY CELLS Kang Cheng, Wanda W.-S. Chan, Robert Arias, Albert Barreto, Jr., and Bridget Buffer Department of Growth Biochemistry and Physiology Merck Research Laboratories P. O. Box 2000 Rahway, N.J. 07065 (Received
in final
form O c t o b e r
9,
1992)
Summilrv In GH3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca 2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRHstimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10 -6 M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary ceils with TRH (10 -7 M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin, sn-l,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent responses to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca 2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C. Thyrotropin releasing hormone (TRH) has been shown to stimulate the release of prolactin from both anterior pituitary cells and pituitary tumor cell lines. Numerous studies in elucidating the molecular mechanism of action of TRH on prolactin release were performed with pituitary tumor cell lines in culture. The effect of TRH on prolactin release in GH3 cells is mediated, at least in part, via elevation of intracellular Ca 2÷ (1-4). The increase of intracellular Ca 2÷ in response to TRH was a result of mobilization of a nonmitochondrial Ca 2÷ pool (3) and enhancement of extracellular Address all correspondence to: Dr. Kang Cheng, Merck Research Laboratories, R80T-138, P.O. Box 2000, Rahway, NJ 07065 Copyright
© 1992
0 0 2 4 - 3 2 0 5 / 9 2 $5.00 + .00 P e r g a m o n Press Ltd All r i g h t s
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Ca 2+ influx (2). Phorbol esters and synthetic diacylglycerols, activators of protein kinase C, have been demonstrated to stimulate prolactin release from pituitary cells (5-8). Furthermore, it has been shown that TRH caused a rapid translocation (within 15 seconds) of protein kinase C from cytosol to plasma membrane in GH4C1 cells (7). Elevations of inositol trisphosphate and 1,2diacylglycerol in GH3 cells in response to TRH have also been reported (8-10). These results suggest that phospholipase C mediates the effect of TRH on prolactin release in pituitary tumor cell lines. In contrast, it has recently been demonstrated that the effect of TRH on prolactin release from a rat pituitary cell perfusion system is not dependent upon the activation of protein kinase C (11). In fact, the release of other pituitary hormones, such as GH in response to GRF (12) and LH in response to GnRH (13-16) also do not require protein kinase C activation. In this study we examined the role of protein kinase C and Ca 2+ in TRH-stimulated prolactin release from female rat primary pituitary cells in static incubations. A direct comparison of the results on prolactin release in response to TRH was made between pituitary mammotrophs and GH3 cells which have been pretreated with either PMA or TRH. A preliminary report of these findings has been presented (17). Mat~ri~ll~ and Me|hods
Materials Dulbecco's Modified Eagle Medium (DMEM), Ham F-10, horse and fetal bovine sera, glutamine (100-fold concentrated), non-essential amino acids (100-fold concentrated), nystatin (10,000 U/ml), gentamycin (50 mg/ml), penicillin (5,000 U/ml)-streptomycin (5,000 Ilg/ml), Hank's Balanced Salt Solution and collagenase were purchased from Gibco (Grand Island, NY). Hyaluronidase and phorbol- 12-myristate- 13-acetate were obtained from Sigma (St. Louis, MO). TRH was from Peninsula Laboratories (San Carlos, CA). Forskolin was purchased from Calbiochem-Behring Corp. (La Jolla, CA). GH3 cells was obtained from American Type Culture Collection. The Wistar lactating female rats were obtained from Charles River Laboratories (Wilmington, MA). Rats were maintained at a constant temperature (25°C) on a 14-hr light, 10-hr dark cycle. Rat chow and water were available ad libitum. Rat Di|uitarv c$11 culture Rat pituitary cells were isolated from pituitaries by enzymatic digestion with 0.2% collagenase and 0.2% hyaluronidase in Hank's Balanced Salt Solution as described previously (18). For culture, the cells were suspended in culture medium and adjusted to 1.5 x 105 cells/ml, and 1.0 ml of this suspension was placed in each well of a 24-well tray (Costar, Cambridge, MA). Cells were maintained in a humidified 5% CO2-95% air atmosphere at 37°C for 3 to 4 days. The culture medium consisted of DMEM containing 0.37% NaHCO3, 10% horse serum, 2.5% fetal bovine serum, 1% non-essential amino acids, 1% glutamine, 1% nystatin, and 0.1% gentamycin. i~xoeriments for orolactin release On the day of an experiment, cells were washed twice 1.5 hrs prior to and once more immediately before the start of the experiment with the above culture medium containing 25 mM HEPES, pH 7.4. Prolactin release was initiated by adding 1 ml of fresh medium containing test agents to each well in quadruplicate. Incubation was carried out at 37°C for 15 minutes or 2 hours as indicated. After incubation, medium was removed and centrifuged at 2,000 x g for 15 minutes to remove any cellular material. The supernatant fluid was removed and assayed for prolactin content. GHa cell culture IffH3 cells were plated at a density of 3 x 105 cells/well in 12-well cell culture plates (Costar) and maintained in Ham's F-10 medium containing 15% horse serum, 2.5% fetal bovine serum, 0.1% gentamycin and 0.1% penicillin-streptomycin for 3 to 4 days. Before experiment, cells were washed as described for primary pituitary cells with Ham's F-10 medium and the experiment was carried out as described in the figure legend.
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DoWll-regulation of orotein kinase C To down-regulate protein kinase C, rat pituitary cells were incubated with 10-6 M PMA in the culture medium containing 25 mM HEPES, pH 7.4, for 24 hours at 37°C. After the pretreatment, the cells were washed three times with fresh culture medium and the experiments were carried out as described in the figure legends. Under these conditions, it has been demonstrated that cellular protein kinase C content in rat pituitary cells and GH3 cells was decreased by more than 90% (13,19,20). RIAs Rat prolactin in culture medium was measured by a double antibody RIA procedure using materials obtained from Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA) and expressed in terms of the standard rat prolactin RP-3.
60
+PMA (10 6M)
E
40
20
0
101 lO
I
10 .9
J
10 .8
[
10 .7
I
10 6
TRH (M)
FIG. 1 Effect of TRH on prolactin release from rat pituitary cells in the presence and absence of PMA. Cells were incubated with various concentrations of TRH in the presence (o) or absence (o) of 10 -6 M PMA at 37°C for 15 min. Each point represents the mean + SEM of quadruplicate incubations. In this and subsequent figures, if no SEM is shown it was smaller than the symbol representing the mean. Data oresentation The results presented in figures are from a single experiment in which quadruplicate determinations were performed and in each case are representative of at least three separate experiments. Statistical analysis was performed with Student's t test. Results and Discussion Effects of T R H and PMA on Prolactin Release from Rat Pituitarv Cells As shown in Figure 1, TRH stimulated prolactin release in a dose-dependent manner. Maximal stimulation was observed at a concentration of 10 -7 M of TRH. PMA (10 -6 M) increased prolactin
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release by 250% and further increased the prolactin release even in the presence of maximal concentrations of TRH. However, the combined effect of TRH and PMA on prolactin release was slightly less than additive but certainly was greater than that of either one of the compounds alone. In Figure 2, similar results were obtained with a different experimental protocol from that used in Figure 1. Namely, maximal concentrations of TRH and PMA together produced a prolactin response which was almost equal to the sum of individual effects. These results suggest that the effects of TRH and PMA on prolacfin release are probably mediated via different mechanisms.
60
c E to
40
.E
20
I
[
I
I
[
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10 .9
10 .8
10 7
10 6
10 5
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FIG. 2 Effect of PMA on prolactin release from rat pituitary cells in the presence and absence of TRH. Cells were incubated with various concentrations of PMA in the presence (o) or absence (o) of 10 .7 M TRH at 37°C for 15 rain. Each point represents the mean + SEM of quadruplicate incubations. Effects o f P M A and T R H P r e t r e a t m e n t on Prolactin R e l e a s e f r o m Rat
Pituitarv
Cells Phorbol esters have been reported to down-regulate protein kinase C activity in a number of cell types including rat primary pituitary cells and GH3 cells (13,19-22). To further evaluate the role of protein kinase C activation in TRH-induced prolactin release, the PMA-pretreated pituitary cells were used in the next series of studies. As shown in Figure 3, rat pituitary cells pretreated with 10 -6 M PMA for 24 hours did not release prolactin in response to a second dose of PMA at a concentration as high as 10-5 M. This result indicates that all of the PMA-sensitive protein kinase C had been removed during the 24 hour PMA pretreatment period. Interestingly, PMA-pretreated cells responded equally well to TRH when compared to control cells (Figure 4), suggesting that PMA-sensitive protein kinase C may not be involved in TRH-induced prolactin release. Pituitary cells pretreated with 10 .7 M TRH for 24 hours had a lower basal prolactin release as shown in Figures 3 and 4. The effect of TRH on prolactin release from these cells was almost completely inhibited while the PMA effect was normal as compared to untreated control cells.
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Effect
of TRH on P r o l a c t i n
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The effect of TRH on prolactin release from clonal rat pituitary cells has been shown to be biphasic (23-25). The first phase is dependent upon the release of Ca 2+ from intracellular stores and the second phase requires the activation of protein kinase C and Ca 2+ influx. Thus, in one experiment the incubation time of TRH with PMA-pretreated cells was extended from 15 minutes to 2 hours in order to be certain that most of the medium prolactin was secreted during the second phase in which protein kinase C plays a major role in mediating the effect of TRH. As shown in Figure 5, the dose-response curves for TRH stimulation of prolactin release from control and PMA-pretreated cells are superimposable. However, TRH-pretreated cells responded poorly to a second dose of TRH. This is probably the result of down-regulation of TRH receptors.
100 E" 80 I
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109 108 107 106 105 PMA(M) FIG. 3
Effects of TRH and PMA pretreatments on PMA-stimulated prolactin release from rat pituitary cells. Cells were pretreated with either TRH (10 -7 M) or PMA (10 .6 M) at 37°C for 24 hours. After pretreatment, cells were washed three times with fresh medium and incubated with various concentrations of PMA at 37°C for 15 min, non-pretreated cells (o), TRHtreated cells (.) and PMA-treated cells (A). Each point represents the mean + SEM of quadruplicate incubations. PMA Pretreatment inhibited the Svner~istic Action of Forskolin and PMA ~but not TRH~ on Prolactin Release from Rat P~tuitarv Cells Forskolin increases intracellular cyclic AMP by activating adenylate cyclase and stimulates prolactin release in rat pituitary cells (26). Maximal concentrations of PMA and forskolin, when added together, produced a synergistic effect on prolactin release as shown in Figure 6. This synergistic effect was completely inhibited by down-regulation of PMA-sensitive protein kinase C. As expected, down-regulation of PMA-sensitive protein kinase C had no effect on forskolininduced prolactin release, since the effect of forskolin was mediated via cyclic AMP. TRH together with forskolin elicited a prolactin response which was slightly greater than additive. However, their effect was not inhibited by down-regulation of PMA-sensitive protein kinase C. Effects of s n - l . 2 - D i o c t a n v l ~ l v c e r o l Pretreatment on T R H - I n d u c e d Prolactin Release from Rat Pituit~Irv C¢ll~ In GH3 cells, it has been shown that depletion of protein kinase C by phorbol diesters does not completely inhibit the effects of diacylglycerols (27). Thus, in the next several experiments we studied the effects of TRH on prolactin release from rat pituitary cells which had been pretreated
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with sn-l,2-dioctanylglycerol (DOG), a synthetic diacylglycerol, for 24 hours. As depicted in Figure 7, DOG stimulated prolactin release in a dose-dependent manner. However, at a concentration of 500 gM, the DOG stimulation of prolactin release still did not reach a plateau. Pretreatment of cells with 250 I.tM of DOG for 24 hours attenuated the subsequent response to a second dose of DOG. Interestingly, PMA pretreatment totally eliminated the effect of DOG on prolactin release in rat primary pituitary cells. DOG pretreatment partially inhibited the effect of PMA on prolactin release but had no effect on that induced by TRH (Figure 8).
120
100
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Non-Pretreated Cells
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TRH-Treated Cells
20
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FIG. 4 Effects of TRH and PMA pretreatments on TRH-stimulated prolactin release from rat pituitary cells. Cells were pretreated with either TRH (10 -7 M) or PMA (10 -6 M) at 37°C for 24 hours. After pretreatment, cells were washed three times with fresh medium and incubated with various concentrations of TRH at 37°C for 15 min, non-pretreated cells (o), TRHtreated cells (o) and PMA-treated cells (A). Each point represents the mean + SEM of quadruplicate incubations. Differential Effects of Extracellular Calcium on TRH and PMA-Stimulated Prolactin Release from Rat Pituitarv Cells It has been demonstrated that extracellular calcium is essential for TRH-induced prolactin release in the second phase in GH3 cells(23). Thus, in the next experiment we evaluated the effects of removing extracellular calcium on TRH and PMA-induced prolactin release from rat pituitary cells. As shown in Table I, basal, TRH-, PMA- and forskolin-induced prolactin releases were decreased by 50%, 90%, 10% and 57%, respectively, by the removal of extracellular calcium. The combined effect of forskolin and TRH on prolactin release was also markedly inhibited in the absence of extracellular calcium. On the contrary, the synergistic effect between forskolin and PMA was not dependent upon extracellular calcium at all. The decrease of basal prolactin release in calcium-free medium was probably due to the presence of 1 mM EGTA which might remove some of the intracellular calcium during the incubation. Effects of TRH and PMA Pretreatments on TRH-. PMA- and Forskolin-Stimulated Prolactin Release from GH 2 Cells It has previously been demonstrated that PMA pretreatment inhibits the subsequent effect of
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1963
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FIG. 5 Effects of TRH and PMA pretreatments on TRH-stimulated prolactin release from rat pituitary cells. Experimental conditions were the same as in Figure 4 legend, except after the pretreatments cells were incubated with various concentrations of TRH at 37°C for 2 hours, non-pretreated cells (o), TRH-treated cells (e) and PMA-treated cells (A). Each point represents the mean + SEM of quadruplicate incubations. TRH on prolactin release in GH3 cells (28). Results from our laboratory and others indicate that the effect of TRH on prolactin release from rat pituitary cells is independent of protein kinase C activation. However, the effects of protein kinase C down-regulation on TRH-induced prolactin release in these systems were never directly compared in the same laboratory under the same conditions. Thus, GH3 cells were pretreated with 10-6 M of TRH or PMA for 24 hrs and the release of prolactin from these cells in response to TRH, PMA and forskolin were examined. As shown in Figure 9, TRH (10 -7 M), PMA (10 -7 M) and forskolin (10 .5 M) stimulated prolactin release from non-pretreated GH 3 cells by 70%, 240% and 80%, respectively. TRH pretreatment inhibited the subsequent prolactin response to TRH by 70% and had very little or no effect on that stimulated by PMA or forskolin. PMA pretreatment almost completely inhibited the effects of TRH and PMA on prolactin release. Again, in our control experiment the forskolin-induced prolactin release was not affected by the protein kinase C down-regulation. Conclusions The data presented in this report provide additional evidence that the effect of TRH on prolactin release from female rat primary pituitary cell culture is probably not mediated via PMA-sensitive
1964
Effect
._= E ub
of TRH
on Prolactin
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Non-Pretreated Cells
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PMA-Pretreated celfs (10-6M, 24 hrs)
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Release
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1992
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PMA 10 6M
TRH 10 8M
TRH 10 8M + FSK 10-5M
FSK 10-5M
PMA 10-6M + FSK 10 5M
FIG. 6 Effect of protein kinase C down-regulation on TRH, PMA and forskolin-induced prolactin release from rat pituitary cells. Protein kinase C was down-regulated as described in Materials and Methods. Incubations were carried out at 37°C for 15 min in the presence of various agents as indicated. Data represent the mean + SEM of quadruplicate incubations. 60
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FIG. 7 Effect of DOG and PMA pretreatment on prolactin release induced by DOG in rat pituitary cells. Cells were pretreated with either DOG (0.25 raM) or PMA (10 -6 M) at 37°C for 24 hours. After pretreatment, cells were washed three times with fresh medium and incubated with various concentrations of DOG at 37°C for 15 min, non-pretreated cells (1~), DOGtreated cells (m) and PMA-treated cells (o). Each point represents the mean + SEM of quadruplicate incubations.
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E f f e c t of T R H o n P r o l a c t i n R e l e a s e
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protein kinase C. In support of this conclusion, PMA-pretreated rat pituitary cells which did not respond to a second dose of PMA had a normal response to TRH as did the non-pretreated cells. On the other hand, GH3 cells which had been pretreated with PMA responded very poorly to a subsequent TRH stimulation (Figure 9 and Ref 28). Since it has been reported that some of the effects elicited by synthetic diacylglycerol in GH3 cells are not mimicked by PMA (27,29), it is possible that the effect of TRH on prolactin release might still be mediated via the activation of protein kinase C by endogenous diacylglycerol. To test this hypothesis, we studied the effect of DOG pretreaunent on the subsequent prolactin response to TRH. Cells pretreated with DOG for 24 hours had an attenuated response to DOG and PMA, but they responded normally to TRH (Figures 7 and 8). These results indicate that DOG pretreatment only removed part of cellular protein kinase C. The decreased efficacy of DOG in down-regulating protein kinase C relative to the efficacy of PMA is probably due to the fact that the plasma membrane is less permeable to DOG than PMA. 70
120[
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~ ~o
~
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N o
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i J i i 109 108 107 106 105 PMA(M)
0L ! i i L i 0 1010109108 107 106 TRH(M)
FIG. 8 Effect of DOG pretreatment on prolactin release induced by PMA and TRH in rat pituitary cells. Cells were pretreated with DOG (0.25 mM) at 37°C for 24 hours. After pretreatment, cells were washed twice with fresh medium and incubated with various concentrations of (a) PMA or (b) TRH at 37°C for 15 min; non-pretreated cells (e-,) and DOG-treated cells ( I ) . Each point represents the mean + SEM of quadruplicate incubations. TABLE I Requirement of Extracellular Calcium for the Action of TRH, PMA and Forskolin on Prolactin Release from Rat Pituitary Cells.
Conditions Control TRH (10 .8 M) PMA (10 .6 M) Forskolin (10 -5 M) Forskolin (10 .5 M) + TRH (10 -8 M) Forskolin (10 -5 M) + PMA (10 -6 M)
Pr01aqtin (nWwell/l 5 min) Ca2+-free Medium Normal Medium + 1 mM EGTA 13.1 53.3 96.7 35.5
+ + + +
0.2 0.2 4.9 0.6
6.9 + 1.1 11.2 + 0.5 83.6 + 9.8 16.6 + 0.5
95.1 + 9.1
19.3 + 0.8
168.5 + 7.1
144.6 + 4.3
Cells were incubated with various agents as indicated at 37°C for 15 min. Data represent the mean + SEM of quadruplicate incubations.
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300 [] Non Pretreated Cells [ ] TRH-Treated Cells B PMA Treated Oells 200
cE
~
100
TRH(0.1pM)
PMA(0 1p M)
FSK(10~tM)
FIG. 9 Effects of TRH and PMA pretreatments on prolactin release from GH3 cells. Cells were pretreated with TRH (10 -6 M) or PMA (10 -6 M) at 37 ° C for 24 hours. After pretreatment, cells were washed twice with fresh medium and incubated with 10-7 M TRH, l0 n M PMA or 10-5 M forskolin at 37 ° C for 15 min. Data represent the mean + SEM of three different experiments. *, p < 0.01 (vs. non-pretreated cells). This is also evident from the results which show that much higher concentrations of DOG are needed to maximally stimulate prolactin release. Interestingly, PMA-pretreated cells did not respond to DOG at all, suggesting that the protein kinase C(s) which mediates the effect of DOG on prolactin release in rat primary pituitary cells is also sensitive to PMA. Thus, it is unlikely that protein kinase C plays a significant role in mediating the effect of TRH on prolactin release in rat primary pituitary cells. This is consistent with the findings from a recent study using a pituitary cell perfusion system (11). In this study we further demonstrated that the effect of TRH was very much dependent upon the presence of extracellular calcium, while PMA stimulated prolactin release responded almost equally well in the presence or absence of extracellular calcium (Table I). In addition to a number of differences already known to exist between the primary pituitary cells and tumor cell lines, we have demonstrated the effect of TRH on prolactin release is mediated via different mechanisms in these two cell types.
Acknowledgement The authors would like to thank Theresa Timko for her help in manuscript preparation.
I~¢ferences M.C. GERSHENGORN and K. THAW, Endocrinology 113 1522-1524 (1983). P.R. ALBERT and A.H. TASHJIAN, J Biol Chem 259 5827-5832 (1984). M.C. GERSHENGORN, E. GERAS, V. SPINA PURELLO and M.J. REBECCHI, J Biol Chem 259 10675-10681 (1984). 4. K.W. SNOWDOWNE and A.B. BORLE, Am J Physiol 246 E198-E201 (1984). 5. S.T. SUMMERS, P.L. CANONICO, R.M. MACLEOD, A.D. ROGOL and M.J. CRONIN, Eur J Pharmacol 111 371-376 (1985). 6. A. NEGRO-VILAR and E.G. LAPETINA, Endocrinology 117 1559-1564 (1985). 7. C.W. FEARON and A.H. TASHJIAN, J Biol Chem 260 8366-8371 (1985). 8. T.F.J. MARTIN, J Biol Chem 2,58 14816-14822 (1983). 9. M.J. REBECCHI and M.C. GERSHENGORN, Biochem J 216 287-294 (1983). 10. C.H. MACPHEE and A.H. DRUMMOND, Mol Pharmacol 25 193-200 (1984). 1. 2. 3.
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11. A.M. JUDD, I.S. LOGIN and R.M. MACLEOD, Endocrinology 125 1134-1141 (1989). 12. M.B. FRENCH, B.C. MOOR, B.T. LUSSIER and J. KRAICER, Endocrinology 124 22352244 (1989). 13. C.A. MCARDLE, W.R. HUCKLE and P.M. CONN, J. Biol Chem 202 5028-5035 (1987). 14. M.S. JOHNSON, R. MITCHELL and G.J. FINK, Endocrinology 110 231-239 (1988). 15. M.J. BEGGS and W.L. MILLER, Endocrinology 124 667-674 (1989). 16. P.A. VAN DER MERWE, R.P. MILLAR and J.S. DAVIDSON, Biochem. J. 208 493-498 (1990). 17. K. CHENG, W.W.S.CHAN, A. BARRETO and B. BUTLER, 72rid Annual Meeting of The Endocrine Society, Atlanta, GA, p 83 (Abstract), 1990. 1 8. K. CHENG, W.W.S.CHAN, A. BARRETO, E.M. CONVEY and R.G. SMITH, Endocrinology 124 2791-2798 (1989). 19. R. BALLESTER and O.A. ROSEN, J Biol Chem 260 15194-15199 (1985). 20. S.S. STOJIKOVIC, J.P. CHANG, D. NGO and K.J. CATT, J Biol Chem 203 1730717311 (1988). 21. P.M. TAPLEY and A.W. MURRAY, EurJ Biochem 151 419-423 (1985). 22. E. MELLONI, S. PONTREMOLI, M. MICHETI'I, O. SACCO, B. SPARATORE and B.L. HORECKER, J. Biol Chem 201 4101-4105 (1986). 23. T.F.J. MARTIN and J.A. KOWALCHYK, Endocrinology 115 1527-1536 (1984). 24. P.R. ALBERT and A.H. TASHJIAN, J Biol Chem 259 15350-15363 (1984). 25. T. AIZAWA and P.M. HINKLE, Endocrinology 116 73-82 (1985). 26. D. DELBEKE, J.G. SCAMMELL and P.S. DANNIES, Endocrinology 114 1433-1440 (1983). 27. R.N. KOLESNICK and A.E. PALEY, J Biol Chem 262 9204-9210 (1987). 28. R. RODRIGUEZ, A. IMAI and M.C. GERSHENGORN, Mol Endocrinol / 802-807 (1987). 29. R.N. KOLESNICK and S. CLEGG, J Biol Chem 263 6534-6537 (1988).