- Email: [email protected]
Phorbol esters affect pituitary growth hormone (GH) and prolactin release: The interaction with GH releasing factor, somatostatin and bromocriptine
European Journal of Pharmacology, 111 (1985)371-376
371
Elsevier
P H O R B O L E S T E R S AFFECT P I T U I T A R Y G R O W T H H O R M O N E (GH) AND P R O L A C T I N RELEASE: T H E I N T E R A C T I O N W I T H G H R E L E A S I N G FACTOR, S O M A T O S T A T I N AND BROMOCRIPTINE STEPHEN T. SUMMERS, PIER L. CANONICO *, ROBERT M. MACLEOD *, ALAN D. ROGOL * and MICHAEL J. CRONIN ** Departments of Physiology, * Medicine, * Pediatrics and * Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, U.S.A.
Received 2 October 1984, revised MS received 6 February 1985, accepted 5 March 1985
S.T. SUMMERS, P.L. CANONICO, R.M. MACLEOD, A.D. ROGOL and M.J. CRONIN, Phorbol esters affect pituitary growth hormone (GH) and prolactin release: the interaction with GH releasing factor, somatostatin and bromocriptine, European Journal of Pharmacology 111 (1985) 371-376. Phorbol esters are tumor promotors that directly stimulate protein kinase C activity. We asked whether these agents affect basal or receptor initiated alterations in growth hormone (GH) and prolactin release. In 4 h incubations of anterior pituitary cells, phorbol esters enhanced basal and GH releasing factor (GRF)-induced GH release. Somatostatin reduced by 38% the 4-fold stimulation of GH release induced by phorbol ester. These tumor promoters also reversed the ability of bromocriptine, a dopamine agonist, to inhibit prolactin release, with no apparent effect on basal prolactin secretion. When these agents were applied for 24 h, an increase in the basal release of both GH and prolactin was apparent. These data lead us to suggest that an intact protein kinase C system may be necessary for the full expression of GRF-stimulated GH release and dopaminergic inhibition of prolactin release. Growth hormone releasing hormone Growth hormone
Anterior pituitary Bromocriptine
1. I n t r o d u c t i o n
Phorbol esters exhibit diverse biological effects in addition to tumor promotion (Blumberg, 1980). For example, in perifused anterior pituitary (AP) cells, nanomolar concentrations of phorbol-12myristate-13-acetate (PMA) stimulate the secretion of growth hormone (GH), luteinizing hormone, thyroid hormone and adrenocorticotrophic hormone (Smith and Vale, 1980). In the G H 4 C 1 pituitary cell line that can actively export both prolactin and G H , there are specific, high affinity binding sites for phorbol esters (Jaken et al., 1981a,b). The production and release of prolactin and G H by G H 4 C 1 cells, as well as the progenitor ** To whom all correspondence should be addressed: Department of Physiology, Box 449, University of Virginia School of Medicine, Charlottesville, Virginia 22908, U.S.A. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Protein kinase C Prolactin
Phorbol esters Dopamine
Teleocidin
G H 3 cell line cloned from a rat pituitary tumor, was also affected by phorbol esters (Osborne and Tashijian, 1981). These data would only constitute interesting phenomena were it not for the subsequent report that phorbol ester effects are at least partially mediated by the activation of a protein kinase C (Castagna et al., 1982). Indeed, there is now good evidence that protein kinase C and the phorbol ester receptor are the same molecule (reviewed in Nishizuka, 1984). PMA, phorbol 12,13-dibutyrate (PDB) and the structurally unrelated tumor promoter teleocidin (Horowitz et al., 1983), were applied to cultured AP cells. If enhanced protein kinase C activity was sufficient to activate exocytosis in the AP gland, as suggested for prolactin release from tumor cell lines (Osborne and Tashjian, 1981; Delbeke et al., 1984; Martin and Kowalchyk, 1984) and G H release from AP cells (Smith and Vale, 1980), then
372
we expected pharmacological activation of this enzyme to mimic G H releasing factor (GRF) activity and attenuate dopaminergic activity (i.e., inhibition of prolactin release). Unfortunately, these hormone receptor mediated effects could not be tested in the available cell lines which do not express the dopamine (Cronin et al., 1980, 1982) or G R F (Zeitin et al., 1984; M. Cronin and M. Thorner, unpublished observations) receptor phenotype.
2. Materials and methods 2.1. Materials
Teleocidin B. (Dr. Fujiki, Tokyo, Japan), bromocriptine (Sandoz, Nutley, N J), somatostatin (Drs. Ling and Guillemin, La Jolla, CA) and GRF-40 (Drs. Rivier and Vale, La Jolla, CA) were kind gifts. PMA and PDB were purchased from Sigma (St. Louis, MO). Media, enzymes and antibiotics were from GIBCO (Grand Island, NY) and cell culture plastic was from Falcon (Oxnard, CA). Reagents for the radioimmunoassay of rat G H and prolactin were purchased from the National Hormone and Pituitary Agency. 2.2. Methods
Adult rats of both sexes (180-220 g: SpragueDawley, Hilltop, Scottsdale, PA) were maintained for at least 2 days in a thermally controlled room with a 12:12 light:dark schedule and free access to food and water. The animals were sacrificed by decapitation in the morning and the anterior lobes of the pituitary quickly removed and dispersed as previously described (Cronin et al., 1983a). The cells were plated in either 96-well plates ( 9 0 0 0 0 / w e l l ) or 24-well plates ( 2 0 0 0 0 0 500 000/well) for 3-7 days (unless otherwise noted) in RPMI-1640 medium, supplemented with 7.5% horse serum (Hyclone, Logan, Utah), 2.5% fetal bovine serum (Hyclone) and antibiotics. Neither the seeding density nor the days in culture affected the overall results reported; original findings in older cultures were successfully replicated in younger cultures. On the day of a study, the cells
were washed 3 x with serum-free medium and then incubated for either 4 h (without serum) or 24 h (with serum) with the drug or vehicle (less than 0.01% dimethylsulfoxide or ethanol). Medium was collected for RIA of prolactin and G H and the ceils were terminated. The cells of the studies utilizing 24-well plates were extracted for protein (Bradford, 1976) to ensure that the treatment did not remove cells from the plate or significantly alter the protein content of the cells in a major way. The RIAs were done according to the protocols described by the National Hormone and Pituitary Agency, using RP-2 as the prolactin standard and RP-1 as the G H standard. Between and within assay coefficients of variation were less than 10%. Statistics were performed using an analysis of variance with a Newman Keuls test for significance. Differences are reported as those comparisons with a P value less than 0.05.
3. Results 3.1. GH release
In 4 independent 4 h studies with PMA, there was a concentration-dependent increase in basal G H release that averaged 8.3-fold at 100 nM PMA. GH RELEASE (IJg/well) 6
o CONTROL
*
5 4 3 2 1 0
O
m
0
,
1
,
1
10
,
--
9
8
PMA (log M)
Fig. 1. The step-wise increase in basal and GRF (10 nM)-stimulated GH releaseis shown during a 4 h incubationof male cells in a 96-wellplate (mean + S.E.M.: n = 5-6). Starred values denote a significant differencefrom the respectivecontrol; all GRF values are greater than control values at a given PMA concentration(P < 0.05).
373 TABLE 1 The effect of somatostatin on phorbol ester-stimulated basal G H release. Mean + S.E.M. (n) is represented in these 4 hour studies of male cells plated at 0.5 million cells per well. N D = not done. [Somatostatin] = 30 nM: [GRF] = 100 nM.
PROLACTIN RELEASE
(pg/mg protein) o CONTROL • BROMOCRIPTINE
c] CONTROL
• PMA 4O
40
G H release ( p , g / m g protein) Vehicle
PMA (10 nM)
Experiment 1 Control Somatostatin
16.1 -+ 1.7 (6) 14.8 _+ 1.5 (6)
52.4_+ 3.9(6)* 35.6_+ 3.7(6)*
Experiment 2 7.08_+ 0.93 (6) * 90.8_+ 7.1 (6) b Control 146.0 _+10.0 (6)* 182.0 ___ 8.0 (6)* GRF ND 58.9_+ 12.4 (6) a Somatostatin G R F + somatostatin 72.3 + 3.5 (6)a.b N D * Different from all other groups: a,b differ from all other groups except each other.
Minimally effective concentrations were between 1 and 10 nM, and G H levels produced by 100 nM PMA were equivalent to those obtained with 10 nM G R F (fig. 1). PMA (100 nM) also increased GRF-stimulated G H release by a mean factor of 1.57-fold (P < 0.05). A second, less potent phorbol ester, PDB, also enhanced both basal G H release in a concentration-dependent manner (2.23-fold at 100 nM PDB) and GRF-stimulated G H release (1.64-fold at 100 nM). Somatostatin attenuated the PMA- and GRF-induced G H release (table 1) while having no effect on prolactin release (data now shown). We also measured basal G H release for 24 h without adding peptide hormones (nanomolar concentrations of G R F and somatostatin are degraded in this primary culture system over a 24 h period). As listed in table 2, there was a significant enhancement of G H release by both PMA and teleocidin under these conditions.
30
¢
ao
20
20
10
10
0
r--
0
i
i
i
-9
-8
-7
PMA (log M)
O
l J.
!
0
1
1
i
2
i
4
HOURS
Fig. 2. Reversal by P M A of bromocriptine (10 nM) inhibition of basal prolactin release by P M A is shown in this 4 h study of male cells (left figure). The right figure is a time course for prolactin accumulation with vehicle control or 100 n M PMA. The mean -+_S.E.M. of 5 to 6 wells per group is represented. The starred values in the bromocriptine group indicate that they are different from the zero PMA value (P < 0.05). Only the 100 n M P M A + bromocriptine value is not different from the non-bromocriptine-treated group.
reversed this inhibition in a concentration-dependent manner. Unlike the results obtained with 4 h of incubation, a significant 50% increase in prolactin release could be measured after a 24 h treatment with either PMA or teleocidin (table 2). A given weight of teleocidin (the molecular weight is unknown) produced a greater degree of pro-
TABLE 2 The action of P M A and teleocidin on basal G H and prolactin release over 24 h in A P ceils. Mean _+S.E.M. (n) is represented. 5 n g / m l P M A is 8 nM. The amount of hormone released by the 2 concentrations of either P M A or teleocidin did not differ; 0.2 million cells from female rats were seeded per well. Hormone release (# g / m g protein)
3.2. Prolactin release
In 5 independent 4 h studies, PMA and PDB had no apparent effect on basal prolactin release, even at concentrations as high as 100 nM (0.02 + 0.10-fold change (n = 5): fig. 2). Bromocriptine, a potent dopaminergic agonist, effectively inhibited basal prolactin release (fig. 2); the addition of either PMA (fig. 2) or PDB (data not shown)
Control PMA 8 nM 16nM Teleocidin 5 ng/ml 10 n g / m l
GHrelease
Prolactin release
22.6_.+0.7 (6)
125.0+ 6.0 (6) a
30.3+1.7 (3) a 36.7+2.3(3) a
138.0_+ 7.0 (3) a 165.0_+ 6.0(3) b'¢
50.5 _+3.9 (3) b 50.0 + 3.0 (3) b
195.0 + 16.0 (3) b 182.0 _+10.0 (3) c
a,b.c Differ from all other groups except each other.
374
lactin release than a similar weight of PMA, as was also noted in the results for GH secretion. 4. Discussion
Phorbol esters stimulate secretion from a number of cell types including platelets (Sano et al., 1983), neurons (Peterfreund and Vale, 1983), pancreatic fl cells (Virji et al., 1983), adrenal chromaffin cells (Knight and Baker, 1983) and steroidogenic cells of the adrenal cortex (Kojima et al., 1983). The purpose of this study was to further characterize the effect of phorbol esters on G H release, originally noted by Smith and Vale (1980), and to explore the possibility that these tumor promoters (Blumberg, 1980) could affect basal or dopamine inhibited prolactin release. The effects we observed at nanomolar concentrations of phorbol esters in AP cells are consistent with the hypothesis that these agents exert their effects on secretion through binding to and activating protein kinase C. This contention is based on the effective concentration range for this enzyme. The protein kinase C activation curve in broken ceils extends from about 1 nM to 15 nM PMA (Castagna et al., 1982), whereas serotonin release from platelets is induced from about 1 nM to 50 nM PMA (Kikkawa et al., 1984). Furthermore, 3H-PDB binding to putative protein kinase C in broken cells exhibited dissociation constants in the low nanomolar range (Sando et al., 1983). Teleocidin, a tumor promoter structurally unrelated to phorbol esters, is also capable of activating protein kinase C in vitro (Fujiki et al., 1984). The similar responses to both PMA and teleocidin further support a role of protein kinase C in prolactin and G H secretion and provide an argument against a nonspecific interaction of these phorbol esters (e.g., chaotrophic membrane effects of lipophilic PMA). We have demonstrated that somatostatin, which reduces GRF-induced G H release (referenced in Cronin et al., 1983b), is capable of attenuating the level of PMA-stimulated GH secretion. This suggests that intracellular signals, generated by somatostatin receptor activity, can either inhibit protein kinase C activity directly or act distal to protein kinase C on a mechanism necessary for activated exocytosis.
Phorbol esters also reversed dopaminergic inhibition of prolactin release in a graded manner, with maximal effectiveness noted at 100 nM PMA. This is particularly impressive because of the rapid and irreversible properties of bromocriptine (Cronin et al., 1984), the dopaminergic agonist used in these studies. However, phorbol esters did not apparently change the accumulated release of prolactin over the first 4 h of exposure, in spite of the GH stimulation measured in the same wells. It is possible that there was a brief initial change in prolactin release which could not be discerned after 4 h. However, data from static incubations do not support this (fig. 2). A preferred explanation is that the low calcium content of the medium (0.5 raM) may have been inadequate to allow further stimulation of a disinhibited prolactin cell (mammotrophs cultured for several days are no longer exposed to the tonic dopaminergic inhibition that is characteristic of the in vivo condition). Indeed, phorbol esters can increase basal prolactin release, determined in the hemolytic plaque assay, using media with 1.4 mM calcium (J. Anderson and M. Cronin, unpublished observation). With loriger exposure to tumor promoters, the stimulation of GH release of the magnitude observed at 4 h does not persist for 24 h; however, the cause of this has not yet been explored. Possible explanations for these data include desensitization (Jaken et al., 1981a,b), metabolism of the secretagogue, or depletion of hormone stores without new synthesis. In the case of prolactin release, the basal hormone levels were unchanged after 4 h of exposure to phorbol ester, but an increase was discerned after 24 h. This response may be due to an enhanced prolactin synthesis caused by prolonged treatment with PMA (or teleocidin), as has been shown in an AP tumor cell line (Osborne and Tashjian, 1981). The level(s) at which protein kinase C acts in the pathway from the signal to the secretory event in GH and prolactin cells is now open for investigation. Phorbol esters stimulate G H secretion and overcome dopaminergic inhibition of prolactin secretion, two responses that are correlated with amplified cyclic AMP levels (referenced in Cronin et al., 1982; Cronin et al., 1983a). These results suggest that two signal transducing systems (i.e.,
375 cyclic A M P via p r o t e i n kinase A a n d p r o t e i n k i n a s e C) m a y positively interact or be r e d u n d a n t parallel p a t h w a y s to ensure a c o m p l e t e r e s p o n s e in these cell types. D a t a from the a d r e n a l m e d u l l a s u p p o r t the h y p o t h e s i s that p r o t e i n kinase C c o u l d be active in a late event such as exocytosis. Indeed, p r o t e i n kinase C b i n d s to a d r e n a l secretory vesicle m e m b r a n e s in the presence of calcium (Creutz et al., 1983). Thus, p r o t e i n kinase C could have a role in vesicle t r a n s p o r t a n d / o r fusion, a n a l o g o u s to the r e g u l a t i o n of the a c t i n - m y o s i n i n t e r a c t i o n b y m y o s i n light chain k i n a s e in s m o o t h muscle contraction. A n o t h e r intriguing possibility is that p r o tein kinase C m a y be involved at a level of regul a t i o n m o r e closely associated with the initial signals, either b y c o n t r o l l i n g the response to the s e c o n d messengers generated b y d o p a m i n e a n d G R F r e c e p t o r o c c u p a t i o n , or b y p h o s p h o r y l a t i o n of the r e c e p t o r s themselves, as suggested for the fl-adrenergic r e c e p t o r ( K e l l e h e r et al., 1984; Sibley et al., 1984). In conclusion, activation of p r o t e i n kinase C amplifies G H release a n d reverses d o p a m i n e r g i c i n h i b i t i o n of p r o l a c t i n release in c u l t u r e d p i t u i t a r y cells. These d a t a lead us to p r o p o s e that s o m e G R F s m a y act via this novel k i n a s e to i m p o s e a secretory signal on the s o m a t o t r o p h . In the m a m m o t r o p h , it is suggested that d o p a m i n e r e c e p t o r a c t i v a t i o n m a y require a t t e n u a t i o n of p r o t e i n k i n a s e C activity for the full expression of this i n h i b i t o r y signal. Acknowledgements We thank Dr. Julianne Sando for her most insightful and supportive discussions and Gwen Baber, Suzanne O'DeU, Catherine Cassada and Margaret MacLeod for their expert technical performance. This work was supported by RCDA 1K04NS00601, NS18409, AM32632, AM22125, 149-B from the American Cancer Society, The Upjohn Company (MJC) and CA07535 (RMM). Mr. Summers is supported by NIH Medical Scientist Training Program grant GM07267-08 at the University of Virginia. Dr. Canonico is currently a faculty member at the University of Catania School of Medicine, Department of Pharmacology, Catania, Italy.
References Blumberg, P.M., 1980, In vitro studies on the mode of action of the phorbol esters, potent tumor promoters, C.R.C. Crit. Rev. Tox. 8, 153.
Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72, 248. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y. Nishizuka, 1982, Direct activation of calciumactivated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem. 257, 7847. Creutz, C.E., L.G. Dowling, J.J. Sando, C. Villar-Palasi, J.H. Whipple and W.J. Zaks, •983, Characterization of the chromobindins, J. Biol. Chem. 258, 14664. Cronin, M.J., W.S. Evans and M.O. Thorner, 1984, One minute of bromocriptine irreversibly inhibits prolactin release for hours, European J. Pharmacol. 99, 85. Cronin, M.J., N. Faure, J. Martial and R.I. Weiner, 1980, Absence of high affinity dopamine receptors in GH 3 cells: a prolactin-secreting clone resistant to the inhibitory action of dopamine, Endocrinology 106, 718. Cronin, M.J., G.A. Myers, R.M. MacLeod and E.L. Hewlett, 1983a, Pertussis toxin uncouples dopamine agonist inhibition of prolactin release, Am. J. Physiol. 244, E499. Cronin, M.J., S.N. Perkins, D. Keefer and R.M. MacLeod, 1982, Failure of dopamine and bromocriptine to affect prolactin release and cell growth in the dopamine-receptor deficient 235-1 clone, Mol. Cell. Endocrinol. 28, 229. Cronin, M.J., A.D. Rogol, R.M. MacLeod, D.A. Keefer, I.S. Login, J.L.C. Borges and M.O. Thorner, 1983b, Biological activity of a growth hormone releasing factor secreted by a human tumor, Am. J Physiol. 244, E346. Delbeke, D., I. Kojima, P.S. Dannies and H. Rasmussen, 1984, Synergistic stimulation of prolactin release by phorbol ester, A23187 and forskolin, Biochem. Biophys. Res. Commun. 123, 735. Fujiki, H., Y. Tanaka, R. Miyake, U. Kikkawa, Y. Nishizuka and T. Sugimura, 1984, Activation of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) by new classes of tumor promoters: teleocidin and debromoaplysiatoxin, Biochem. Biophys. Res. Commun. 120, 339. Horowitz, A.D., H. Fujiki, I.B. Weinstein, A. Jeffrey, E. Okin, R.E. Moore and T. Sugimura, 1983, Comparative effects of aplysiatoxin, debromoaplysiatoxin, and teleocidin on receptor binding and phospholipid metabolism, Canc. Res. 43, 1529. Jaken, S., A.H. Tashjian, Jr. and P.M. Bhimberg, 1981a, Characterization of phorbol ester receptors and their down-modulation in GH4C 1 rat pituitary cells, Cancer Res, 41, 2175. Jaken, S., A.H. Tashjian, Jr. and P.M. Blumberg, 1981b, Relationship between biological responsiveness to phorbol esters and receptor levels in GHqC ~ rat pituitary cells, Cancer Res., 41, 4956. Kelleher, D.J., J.E. Pessin, A.E. Ruohoh and G.L. Johnson, 1984, Phorbol esters induce desensitization of adenylate cyclase and phosphorylation of the beta-adrenergicreceptor in turkey erythrocytes, Proc. Natl. Acad. Sci. U.S.A. 81, 4316. Kikkawa, U., R. Miyake, Y. Tanaka, Y. Takai and Y. Nishizuka, 1984, Protein kinase C and mechanism of action of tumor
376 promoters, Cold Spring Harbor Conference on Cell Proliferation, The Cancer Cell (in press). Knight, D.E. and P.F. Baker, 1983, The phorbol ester TPA increases the affinity of exocytosis for calcium in 'leaky' adrenal medullary cells, FEBS Lett. 160, 98. Kojima, I., H. Lippes, K. Kojima and H. Rasmussen, 1983, Aldosterone secretion: Effect of phorbol ester and A23187, Biochem. Biophys. Res. Commun. 116, 555. Martin, T.F.J. and J.A. Kowalchyk, 1984, Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action: involvement of diacylglycerol, Endocrinology 115, 1517. Nishizuka, Y., 1984, The role of protein kinase C in cell surface signal transduction and tumor promotion, Nature 308, 693. Osborne, R. and A.H. Tashjian, Jr., 1981, Tumor-promoting phorbol esters affect production of prolactin and growth hormone by rat pituitary cells, Endocrinology 108, 1164. Peterfreund, R.A. and W.W. Vale, 1983, Phorbol diesters stimulate somatostatin secretion from cultured brain cells, Endocrinology 113, 200. Sando, J.J. and M.C. Young, 1983, Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells.
Requirement for calcium, magnesium, and phospholipids, Proc. Natl. Acad. Sci. U.S.A. 80, 2642. Sano, K., Y. Takai, J. Yamanishi and Y. Nishizuka, 1983, A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation J. Biol. Chem. 258, 2010. Sibley, D.R., P. Nambi, J.R. Peters and R.J. Lefkowicz, 1984, Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes, Biochem. Biophys. Res. Commun. 121, 973. Smith, M.J. and W.W. Vale, 1980, Superfusion of rat anterior pituitary cells attached to cytodex beads: Validation of a technique, Endocrinology 107, 1425. Virji, M.J., M.W. Steffes and R.D. Estensen, 1978, Phorbol myristate acetate: Effect of a tumor promotor on insulin release from isolated rat islets of Langerhans, Endocrinology 102, 706. Zeitin, F.N., G.G. Glick, P. Brazeau, N. Ling, M. McLaughlin and C. Bancroft, 1984, Growth hormone (GH) releasing factor does not regulate GH release or GH mRNA levels in GH 3 cells, Endocrinology 114, 2054.
371
Elsevier
P H O R B O L E S T E R S AFFECT P I T U I T A R Y G R O W T H H O R M O N E (GH) AND P R O L A C T I N RELEASE: T H E I N T E R A C T I O N W I T H G H R E L E A S I N G FACTOR, S O M A T O S T A T I N AND BROMOCRIPTINE STEPHEN T. SUMMERS, PIER L. CANONICO *, ROBERT M. MACLEOD *, ALAN D. ROGOL * and MICHAEL J. CRONIN ** Departments of Physiology, * Medicine, * Pediatrics and * Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, U.S.A.
Received 2 October 1984, revised MS received 6 February 1985, accepted 5 March 1985
S.T. SUMMERS, P.L. CANONICO, R.M. MACLEOD, A.D. ROGOL and M.J. CRONIN, Phorbol esters affect pituitary growth hormone (GH) and prolactin release: the interaction with GH releasing factor, somatostatin and bromocriptine, European Journal of Pharmacology 111 (1985) 371-376. Phorbol esters are tumor promotors that directly stimulate protein kinase C activity. We asked whether these agents affect basal or receptor initiated alterations in growth hormone (GH) and prolactin release. In 4 h incubations of anterior pituitary cells, phorbol esters enhanced basal and GH releasing factor (GRF)-induced GH release. Somatostatin reduced by 38% the 4-fold stimulation of GH release induced by phorbol ester. These tumor promoters also reversed the ability of bromocriptine, a dopamine agonist, to inhibit prolactin release, with no apparent effect on basal prolactin secretion. When these agents were applied for 24 h, an increase in the basal release of both GH and prolactin was apparent. These data lead us to suggest that an intact protein kinase C system may be necessary for the full expression of GRF-stimulated GH release and dopaminergic inhibition of prolactin release. Growth hormone releasing hormone Growth hormone
Anterior pituitary Bromocriptine
1. I n t r o d u c t i o n
Phorbol esters exhibit diverse biological effects in addition to tumor promotion (Blumberg, 1980). For example, in perifused anterior pituitary (AP) cells, nanomolar concentrations of phorbol-12myristate-13-acetate (PMA) stimulate the secretion of growth hormone (GH), luteinizing hormone, thyroid hormone and adrenocorticotrophic hormone (Smith and Vale, 1980). In the G H 4 C 1 pituitary cell line that can actively export both prolactin and G H , there are specific, high affinity binding sites for phorbol esters (Jaken et al., 1981a,b). The production and release of prolactin and G H by G H 4 C 1 cells, as well as the progenitor ** To whom all correspondence should be addressed: Department of Physiology, Box 449, University of Virginia School of Medicine, Charlottesville, Virginia 22908, U.S.A. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Protein kinase C Prolactin
Phorbol esters Dopamine
Teleocidin
G H 3 cell line cloned from a rat pituitary tumor, was also affected by phorbol esters (Osborne and Tashijian, 1981). These data would only constitute interesting phenomena were it not for the subsequent report that phorbol ester effects are at least partially mediated by the activation of a protein kinase C (Castagna et al., 1982). Indeed, there is now good evidence that protein kinase C and the phorbol ester receptor are the same molecule (reviewed in Nishizuka, 1984). PMA, phorbol 12,13-dibutyrate (PDB) and the structurally unrelated tumor promoter teleocidin (Horowitz et al., 1983), were applied to cultured AP cells. If enhanced protein kinase C activity was sufficient to activate exocytosis in the AP gland, as suggested for prolactin release from tumor cell lines (Osborne and Tashjian, 1981; Delbeke et al., 1984; Martin and Kowalchyk, 1984) and G H release from AP cells (Smith and Vale, 1980), then
372
we expected pharmacological activation of this enzyme to mimic G H releasing factor (GRF) activity and attenuate dopaminergic activity (i.e., inhibition of prolactin release). Unfortunately, these hormone receptor mediated effects could not be tested in the available cell lines which do not express the dopamine (Cronin et al., 1980, 1982) or G R F (Zeitin et al., 1984; M. Cronin and M. Thorner, unpublished observations) receptor phenotype.
2. Materials and methods 2.1. Materials
Teleocidin B. (Dr. Fujiki, Tokyo, Japan), bromocriptine (Sandoz, Nutley, N J), somatostatin (Drs. Ling and Guillemin, La Jolla, CA) and GRF-40 (Drs. Rivier and Vale, La Jolla, CA) were kind gifts. PMA and PDB were purchased from Sigma (St. Louis, MO). Media, enzymes and antibiotics were from GIBCO (Grand Island, NY) and cell culture plastic was from Falcon (Oxnard, CA). Reagents for the radioimmunoassay of rat G H and prolactin were purchased from the National Hormone and Pituitary Agency. 2.2. Methods
Adult rats of both sexes (180-220 g: SpragueDawley, Hilltop, Scottsdale, PA) were maintained for at least 2 days in a thermally controlled room with a 12:12 light:dark schedule and free access to food and water. The animals were sacrificed by decapitation in the morning and the anterior lobes of the pituitary quickly removed and dispersed as previously described (Cronin et al., 1983a). The cells were plated in either 96-well plates ( 9 0 0 0 0 / w e l l ) or 24-well plates ( 2 0 0 0 0 0 500 000/well) for 3-7 days (unless otherwise noted) in RPMI-1640 medium, supplemented with 7.5% horse serum (Hyclone, Logan, Utah), 2.5% fetal bovine serum (Hyclone) and antibiotics. Neither the seeding density nor the days in culture affected the overall results reported; original findings in older cultures were successfully replicated in younger cultures. On the day of a study, the cells
were washed 3 x with serum-free medium and then incubated for either 4 h (without serum) or 24 h (with serum) with the drug or vehicle (less than 0.01% dimethylsulfoxide or ethanol). Medium was collected for RIA of prolactin and G H and the ceils were terminated. The cells of the studies utilizing 24-well plates were extracted for protein (Bradford, 1976) to ensure that the treatment did not remove cells from the plate or significantly alter the protein content of the cells in a major way. The RIAs were done according to the protocols described by the National Hormone and Pituitary Agency, using RP-2 as the prolactin standard and RP-1 as the G H standard. Between and within assay coefficients of variation were less than 10%. Statistics were performed using an analysis of variance with a Newman Keuls test for significance. Differences are reported as those comparisons with a P value less than 0.05.
3. Results 3.1. GH release
In 4 independent 4 h studies with PMA, there was a concentration-dependent increase in basal G H release that averaged 8.3-fold at 100 nM PMA. GH RELEASE (IJg/well) 6
o CONTROL
*
5 4 3 2 1 0
O
m
0
,
1
,
1
10
,
--
9
8
PMA (log M)
Fig. 1. The step-wise increase in basal and GRF (10 nM)-stimulated GH releaseis shown during a 4 h incubationof male cells in a 96-wellplate (mean + S.E.M.: n = 5-6). Starred values denote a significant differencefrom the respectivecontrol; all GRF values are greater than control values at a given PMA concentration(P < 0.05).
373 TABLE 1 The effect of somatostatin on phorbol ester-stimulated basal G H release. Mean + S.E.M. (n) is represented in these 4 hour studies of male cells plated at 0.5 million cells per well. N D = not done. [Somatostatin] = 30 nM: [GRF] = 100 nM.
PROLACTIN RELEASE
(pg/mg protein) o CONTROL • BROMOCRIPTINE
c] CONTROL
• PMA 4O
40
G H release ( p , g / m g protein) Vehicle
PMA (10 nM)
Experiment 1 Control Somatostatin
16.1 -+ 1.7 (6) 14.8 _+ 1.5 (6)
52.4_+ 3.9(6)* 35.6_+ 3.7(6)*
Experiment 2 7.08_+ 0.93 (6) * 90.8_+ 7.1 (6) b Control 146.0 _+10.0 (6)* 182.0 ___ 8.0 (6)* GRF ND 58.9_+ 12.4 (6) a Somatostatin G R F + somatostatin 72.3 + 3.5 (6)a.b N D * Different from all other groups: a,b differ from all other groups except each other.
Minimally effective concentrations were between 1 and 10 nM, and G H levels produced by 100 nM PMA were equivalent to those obtained with 10 nM G R F (fig. 1). PMA (100 nM) also increased GRF-stimulated G H release by a mean factor of 1.57-fold (P < 0.05). A second, less potent phorbol ester, PDB, also enhanced both basal G H release in a concentration-dependent manner (2.23-fold at 100 nM PDB) and GRF-stimulated G H release (1.64-fold at 100 nM). Somatostatin attenuated the PMA- and GRF-induced G H release (table 1) while having no effect on prolactin release (data now shown). We also measured basal G H release for 24 h without adding peptide hormones (nanomolar concentrations of G R F and somatostatin are degraded in this primary culture system over a 24 h period). As listed in table 2, there was a significant enhancement of G H release by both PMA and teleocidin under these conditions.
30
¢
ao
20
20
10
10
0
r--
0
i
i
i
-9
-8
-7
PMA (log M)
O
l J.
!
0
1
1
i
2
i
4
HOURS
Fig. 2. Reversal by P M A of bromocriptine (10 nM) inhibition of basal prolactin release by P M A is shown in this 4 h study of male cells (left figure). The right figure is a time course for prolactin accumulation with vehicle control or 100 n M PMA. The mean -+_S.E.M. of 5 to 6 wells per group is represented. The starred values in the bromocriptine group indicate that they are different from the zero PMA value (P < 0.05). Only the 100 n M P M A + bromocriptine value is not different from the non-bromocriptine-treated group.
reversed this inhibition in a concentration-dependent manner. Unlike the results obtained with 4 h of incubation, a significant 50% increase in prolactin release could be measured after a 24 h treatment with either PMA or teleocidin (table 2). A given weight of teleocidin (the molecular weight is unknown) produced a greater degree of pro-
TABLE 2 The action of P M A and teleocidin on basal G H and prolactin release over 24 h in A P ceils. Mean _+S.E.M. (n) is represented. 5 n g / m l P M A is 8 nM. The amount of hormone released by the 2 concentrations of either P M A or teleocidin did not differ; 0.2 million cells from female rats were seeded per well. Hormone release (# g / m g protein)
3.2. Prolactin release
In 5 independent 4 h studies, PMA and PDB had no apparent effect on basal prolactin release, even at concentrations as high as 100 nM (0.02 + 0.10-fold change (n = 5): fig. 2). Bromocriptine, a potent dopaminergic agonist, effectively inhibited basal prolactin release (fig. 2); the addition of either PMA (fig. 2) or PDB (data not shown)
Control PMA 8 nM 16nM Teleocidin 5 ng/ml 10 n g / m l
GHrelease
Prolactin release
22.6_.+0.7 (6)
125.0+ 6.0 (6) a
30.3+1.7 (3) a 36.7+2.3(3) a
138.0_+ 7.0 (3) a 165.0_+ 6.0(3) b'¢
50.5 _+3.9 (3) b 50.0 + 3.0 (3) b
195.0 + 16.0 (3) b 182.0 _+10.0 (3) c
a,b.c Differ from all other groups except each other.
374
lactin release than a similar weight of PMA, as was also noted in the results for GH secretion. 4. Discussion
Phorbol esters stimulate secretion from a number of cell types including platelets (Sano et al., 1983), neurons (Peterfreund and Vale, 1983), pancreatic fl cells (Virji et al., 1983), adrenal chromaffin cells (Knight and Baker, 1983) and steroidogenic cells of the adrenal cortex (Kojima et al., 1983). The purpose of this study was to further characterize the effect of phorbol esters on G H release, originally noted by Smith and Vale (1980), and to explore the possibility that these tumor promoters (Blumberg, 1980) could affect basal or dopamine inhibited prolactin release. The effects we observed at nanomolar concentrations of phorbol esters in AP cells are consistent with the hypothesis that these agents exert their effects on secretion through binding to and activating protein kinase C. This contention is based on the effective concentration range for this enzyme. The protein kinase C activation curve in broken ceils extends from about 1 nM to 15 nM PMA (Castagna et al., 1982), whereas serotonin release from platelets is induced from about 1 nM to 50 nM PMA (Kikkawa et al., 1984). Furthermore, 3H-PDB binding to putative protein kinase C in broken cells exhibited dissociation constants in the low nanomolar range (Sando et al., 1983). Teleocidin, a tumor promoter structurally unrelated to phorbol esters, is also capable of activating protein kinase C in vitro (Fujiki et al., 1984). The similar responses to both PMA and teleocidin further support a role of protein kinase C in prolactin and G H secretion and provide an argument against a nonspecific interaction of these phorbol esters (e.g., chaotrophic membrane effects of lipophilic PMA). We have demonstrated that somatostatin, which reduces GRF-induced G H release (referenced in Cronin et al., 1983b), is capable of attenuating the level of PMA-stimulated GH secretion. This suggests that intracellular signals, generated by somatostatin receptor activity, can either inhibit protein kinase C activity directly or act distal to protein kinase C on a mechanism necessary for activated exocytosis.
Phorbol esters also reversed dopaminergic inhibition of prolactin release in a graded manner, with maximal effectiveness noted at 100 nM PMA. This is particularly impressive because of the rapid and irreversible properties of bromocriptine (Cronin et al., 1984), the dopaminergic agonist used in these studies. However, phorbol esters did not apparently change the accumulated release of prolactin over the first 4 h of exposure, in spite of the GH stimulation measured in the same wells. It is possible that there was a brief initial change in prolactin release which could not be discerned after 4 h. However, data from static incubations do not support this (fig. 2). A preferred explanation is that the low calcium content of the medium (0.5 raM) may have been inadequate to allow further stimulation of a disinhibited prolactin cell (mammotrophs cultured for several days are no longer exposed to the tonic dopaminergic inhibition that is characteristic of the in vivo condition). Indeed, phorbol esters can increase basal prolactin release, determined in the hemolytic plaque assay, using media with 1.4 mM calcium (J. Anderson and M. Cronin, unpublished observation). With loriger exposure to tumor promoters, the stimulation of GH release of the magnitude observed at 4 h does not persist for 24 h; however, the cause of this has not yet been explored. Possible explanations for these data include desensitization (Jaken et al., 1981a,b), metabolism of the secretagogue, or depletion of hormone stores without new synthesis. In the case of prolactin release, the basal hormone levels were unchanged after 4 h of exposure to phorbol ester, but an increase was discerned after 24 h. This response may be due to an enhanced prolactin synthesis caused by prolonged treatment with PMA (or teleocidin), as has been shown in an AP tumor cell line (Osborne and Tashjian, 1981). The level(s) at which protein kinase C acts in the pathway from the signal to the secretory event in GH and prolactin cells is now open for investigation. Phorbol esters stimulate G H secretion and overcome dopaminergic inhibition of prolactin secretion, two responses that are correlated with amplified cyclic AMP levels (referenced in Cronin et al., 1982; Cronin et al., 1983a). These results suggest that two signal transducing systems (i.e.,
375 cyclic A M P via p r o t e i n kinase A a n d p r o t e i n k i n a s e C) m a y positively interact or be r e d u n d a n t parallel p a t h w a y s to ensure a c o m p l e t e r e s p o n s e in these cell types. D a t a from the a d r e n a l m e d u l l a s u p p o r t the h y p o t h e s i s that p r o t e i n kinase C c o u l d be active in a late event such as exocytosis. Indeed, p r o t e i n kinase C b i n d s to a d r e n a l secretory vesicle m e m b r a n e s in the presence of calcium (Creutz et al., 1983). Thus, p r o t e i n kinase C could have a role in vesicle t r a n s p o r t a n d / o r fusion, a n a l o g o u s to the r e g u l a t i o n of the a c t i n - m y o s i n i n t e r a c t i o n b y m y o s i n light chain k i n a s e in s m o o t h muscle contraction. A n o t h e r intriguing possibility is that p r o tein kinase C m a y be involved at a level of regul a t i o n m o r e closely associated with the initial signals, either b y c o n t r o l l i n g the response to the s e c o n d messengers generated b y d o p a m i n e a n d G R F r e c e p t o r o c c u p a t i o n , or b y p h o s p h o r y l a t i o n of the r e c e p t o r s themselves, as suggested for the fl-adrenergic r e c e p t o r ( K e l l e h e r et al., 1984; Sibley et al., 1984). In conclusion, activation of p r o t e i n kinase C amplifies G H release a n d reverses d o p a m i n e r g i c i n h i b i t i o n of p r o l a c t i n release in c u l t u r e d p i t u i t a r y cells. These d a t a lead us to p r o p o s e that s o m e G R F s m a y act via this novel k i n a s e to i m p o s e a secretory signal on the s o m a t o t r o p h . In the m a m m o t r o p h , it is suggested that d o p a m i n e r e c e p t o r a c t i v a t i o n m a y require a t t e n u a t i o n of p r o t e i n k i n a s e C activity for the full expression of this i n h i b i t o r y signal. Acknowledgements We thank Dr. Julianne Sando for her most insightful and supportive discussions and Gwen Baber, Suzanne O'DeU, Catherine Cassada and Margaret MacLeod for their expert technical performance. This work was supported by RCDA 1K04NS00601, NS18409, AM32632, AM22125, 149-B from the American Cancer Society, The Upjohn Company (MJC) and CA07535 (RMM). Mr. Summers is supported by NIH Medical Scientist Training Program grant GM07267-08 at the University of Virginia. Dr. Canonico is currently a faculty member at the University of Catania School of Medicine, Department of Pharmacology, Catania, Italy.
References Blumberg, P.M., 1980, In vitro studies on the mode of action of the phorbol esters, potent tumor promoters, C.R.C. Crit. Rev. Tox. 8, 153.
Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72, 248. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa and Y. Nishizuka, 1982, Direct activation of calciumactivated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem. 257, 7847. Creutz, C.E., L.G. Dowling, J.J. Sando, C. Villar-Palasi, J.H. Whipple and W.J. Zaks, •983, Characterization of the chromobindins, J. Biol. Chem. 258, 14664. Cronin, M.J., W.S. Evans and M.O. Thorner, 1984, One minute of bromocriptine irreversibly inhibits prolactin release for hours, European J. Pharmacol. 99, 85. Cronin, M.J., N. Faure, J. Martial and R.I. Weiner, 1980, Absence of high affinity dopamine receptors in GH 3 cells: a prolactin-secreting clone resistant to the inhibitory action of dopamine, Endocrinology 106, 718. Cronin, M.J., G.A. Myers, R.M. MacLeod and E.L. Hewlett, 1983a, Pertussis toxin uncouples dopamine agonist inhibition of prolactin release, Am. J. Physiol. 244, E499. Cronin, M.J., S.N. Perkins, D. Keefer and R.M. MacLeod, 1982, Failure of dopamine and bromocriptine to affect prolactin release and cell growth in the dopamine-receptor deficient 235-1 clone, Mol. Cell. Endocrinol. 28, 229. Cronin, M.J., A.D. Rogol, R.M. MacLeod, D.A. Keefer, I.S. Login, J.L.C. Borges and M.O. Thorner, 1983b, Biological activity of a growth hormone releasing factor secreted by a human tumor, Am. J Physiol. 244, E346. Delbeke, D., I. Kojima, P.S. Dannies and H. Rasmussen, 1984, Synergistic stimulation of prolactin release by phorbol ester, A23187 and forskolin, Biochem. Biophys. Res. Commun. 123, 735. Fujiki, H., Y. Tanaka, R. Miyake, U. Kikkawa, Y. Nishizuka and T. Sugimura, 1984, Activation of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) by new classes of tumor promoters: teleocidin and debromoaplysiatoxin, Biochem. Biophys. Res. Commun. 120, 339. Horowitz, A.D., H. Fujiki, I.B. Weinstein, A. Jeffrey, E. Okin, R.E. Moore and T. Sugimura, 1983, Comparative effects of aplysiatoxin, debromoaplysiatoxin, and teleocidin on receptor binding and phospholipid metabolism, Canc. Res. 43, 1529. Jaken, S., A.H. Tashjian, Jr. and P.M. Bhimberg, 1981a, Characterization of phorbol ester receptors and their down-modulation in GH4C 1 rat pituitary cells, Cancer Res, 41, 2175. Jaken, S., A.H. Tashjian, Jr. and P.M. Blumberg, 1981b, Relationship between biological responsiveness to phorbol esters and receptor levels in GHqC ~ rat pituitary cells, Cancer Res., 41, 4956. Kelleher, D.J., J.E. Pessin, A.E. Ruohoh and G.L. Johnson, 1984, Phorbol esters induce desensitization of adenylate cyclase and phosphorylation of the beta-adrenergicreceptor in turkey erythrocytes, Proc. Natl. Acad. Sci. U.S.A. 81, 4316. Kikkawa, U., R. Miyake, Y. Tanaka, Y. Takai and Y. Nishizuka, 1984, Protein kinase C and mechanism of action of tumor
376 promoters, Cold Spring Harbor Conference on Cell Proliferation, The Cancer Cell (in press). Knight, D.E. and P.F. Baker, 1983, The phorbol ester TPA increases the affinity of exocytosis for calcium in 'leaky' adrenal medullary cells, FEBS Lett. 160, 98. Kojima, I., H. Lippes, K. Kojima and H. Rasmussen, 1983, Aldosterone secretion: Effect of phorbol ester and A23187, Biochem. Biophys. Res. Commun. 116, 555. Martin, T.F.J. and J.A. Kowalchyk, 1984, Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action: involvement of diacylglycerol, Endocrinology 115, 1517. Nishizuka, Y., 1984, The role of protein kinase C in cell surface signal transduction and tumor promotion, Nature 308, 693. Osborne, R. and A.H. Tashjian, Jr., 1981, Tumor-promoting phorbol esters affect production of prolactin and growth hormone by rat pituitary cells, Endocrinology 108, 1164. Peterfreund, R.A. and W.W. Vale, 1983, Phorbol diesters stimulate somatostatin secretion from cultured brain cells, Endocrinology 113, 200. Sando, J.J. and M.C. Young, 1983, Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells.
Requirement for calcium, magnesium, and phospholipids, Proc. Natl. Acad. Sci. U.S.A. 80, 2642. Sano, K., Y. Takai, J. Yamanishi and Y. Nishizuka, 1983, A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation J. Biol. Chem. 258, 2010. Sibley, D.R., P. Nambi, J.R. Peters and R.J. Lefkowicz, 1984, Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes, Biochem. Biophys. Res. Commun. 121, 973. Smith, M.J. and W.W. Vale, 1980, Superfusion of rat anterior pituitary cells attached to cytodex beads: Validation of a technique, Endocrinology 107, 1425. Virji, M.J., M.W. Steffes and R.D. Estensen, 1978, Phorbol myristate acetate: Effect of a tumor promotor on insulin release from isolated rat islets of Langerhans, Endocrinology 102, 706. Zeitin, F.N., G.G. Glick, P. Brazeau, N. Ling, M. McLaughlin and C. Bancroft, 1984, Growth hormone (GH) releasing factor does not regulate GH release or GH mRNA levels in GH 3 cells, Endocrinology 114, 2054.