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Biosynthesis and release of prolactin from the pituitary gland of the rainbow trout, Salmo gairdneri
Comp. Biochem. Physiol., Vol. 65B, pp. 705 to 709
0305-0491/80/0401-0705502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
BIOSYNTHESIS A N D RELEASE OF PROLACTIN FROM THE PITUITARY G L A N D OF THE RAINBOW TROUT, SALMO G A I R D N E R I B. A. M c K E o w y , B. G. JENKS and A. P. VAN OVERBEEKE Department of Zoology, University Nijmegen, 6525 ED Nijmegen, The Netherlands (Received 24 July 1979)
Abstract--l. A pulse-chase analysis using rostral pars distalis cells from rainbow trout pituitary glands failed to demonstrate the existence of a prolactin precursor molecule. 2. Prolactin secreted in vitro had the same electrophoretic characteristics as prolactin extracted from trout lactotrophic cells. 3. Dopamine (10 -5 M) inhibited the release of prolactin but did not effect synthesis. 4. Prolactin synthesis and release were both stimulated by dB-c-AMP (6 mM) but were not as pronounced when dopamine was also present.
INTRODUCTION
It is now well established that a number of protein hormones are first synthesized as a larger precursor molecule (Steiner et al., 1967; Rigopoulou et al., 1970; Yalow & Berson, 1971; Kemper et al., 1972; Jenks et al., 1979). With regard to prolactin the situation is controversial. There has been a number of recent studies in mammals employing cell-free systems that have indicated that prolactin is initially synthesized as a "short-lived" higher molecular weight protein called pre-prolactin (Dannies & Tashjian, 1976; Mauer et al., 1976; Lingappa et al., 1977). Recently, the presence of this pre-prolactin was demonstrated in intact cells and these cells can convert this product directly to prolactin (Mauer & McKean, 1978). On the other hand, neither in the studies just mentioned, nor in an in vitro analysis of prolactin biosynthesis in the rat using pulse-chase techniques, was there any evidence for the existence of a "longer-lived" storage form of a large prolactin ("pro-prolactin") (Zanini et al., 1974). The form of rat or mouse prolactin secreted in vitro from the lactotrophs appears to be the same as the form in which the hormone is extracted from these cells (Farmer et al., 1976; Shoer et al., 1978). In man however, there appears to be a size-heterogeneity of circulating prolactin species (Rogol & Rosen, 1974; Suh & Frantz, 1974; Guyda, 1975). The smallest of these forms is the same as the intra-cellular form. In this connection it is of interest that Jordan & Kendall (1978) isolated prolactin from human cerebro-spinal fluid and found it to be in a small form which, in the presence of plasma, was converted to a larger form. There is increasing evidence that there exists a complex interrelationship of a number of factors controlling the release of prolactin. This is the case in mammals (see Lancranjan & Friesen, 1978; Robyn & Harter, 1978) as well as in teleost fish (Nagahama et al., 1975; Peter & McKeown, 1975; McKeown & Brewer, 1978). The release of prolactin in teleosts appears to be under the control of an inhibitory factor from the hypothalamus which may be dopamine (see Peter & McKeown, 1975). Recently, De Camilli 705
et al. (1979) have demonstrated that dopamine also inhibits the release of human prolactin. In the same study they demonstrated that this inhibition was accompanied by a decrease in c-AMP levels in the lactotrophs which suggests that this action of dopamine involved the adenylate cyclase system. This last notion is in agreement with the observation that c-AMP causes the release of prolactin in mammals (see McCann et al., 1978). The investigations reported here are part of our program on internal regulatory mechanisms in salmonid fishes and were designed for two purposes. Firstly, to study whether the biosynthesis of prolactin in rainbow trout involves the production of a precursor. In addition, experiments were conducted to study the role of dopamine and c-AMP on the release of prolactin in the trout and whether such effects also influence the synthesis of this hormone.
MATERIALS AND METHODS
Animals
Rainbow trout, Salmo gairdneri (15-20cm) were obtained from a local hatchery, transported to the laboratory and maintained in a flow-through 1001. white porcelain aquarium. The trout were in water of 14°C, under a natural photoperiod and were fed commercial trout pellets daily ad libitum. Pulse experiments Biosynthesis and secretion of newly synthesized proteins were determined by incubating the rostral pars distalis of the pituitary gland in [3H]lysine-containing media for various pulse times. Incubations were performed in order to establish rates of synthesis and release for future pulsechase experiments. After trout were killed by severing the spinal cord, the pituitary was immediately removed and placed in a petri dish containing incubation medium. The rostral pars distalis was then dissected away and placed in a siliconized incubation vial containing 500/A incubation medium. The composition of the incubation medium was: l l2mM NaCI, 2mM KC1, 2mM CaClz.2H20 and 15mM HEPES, the pH was adjusted to 7.38 with 5 N NaOH.
706
B. A. McKEowN, B. G. JENKS and A. P. VAN OVERBEEKE Table 1. The effect of dopamine and c-AMP on synthesis and release of prolactin from the rostral pars distalis (RPD) of rainbow trout Densitometry (Experiment 1) ~o control RPD RPD Medium
Radioactive counts CPM(%) Experiment 1 Experiment 2 Total RPD Medium Total RPD Medium
Control
100.0
9.1
100.9
1.8
34,452 (95.6) --
109.8
12.5
579 (2.1) 232 (0.8) 3125 (4.7)
36,055 (100) ----
c-AMP (6 mM)
27,301 (97.9) 28,656 (99.2) 62,730 (95.3)
1603 (4.4)
Dopamine (10 -5)
27,800 (100) 28,888 (100) 65,855 (100)
54,707 (100)
47,954 (87.7)
6753 (12.3)
c-AMP (6 m/Vi) and dopamine (10 -5 M)
107.1
11.4
44,771 (100)
42,950 (95.9)
1821 (4.1)
43,759 (100)
40,508 (92.6)
3251 (7.4)
Treatment
Each medium contained four rostral pars distalis lobes incubated for 5 hr at 22°C.
Before incubation the medium was gassed for 10 min with 95% 02:5 % CO2 followed by the additional of bovine serum albumin (Sigma Fraction V, final concentration 0.3 mg/ml) and glucose (final concentration 2.5mg/ml). Four rostral pars distalis lobes were added to each vial and just prior to incubation, 10 #Ci[3H]lysine (L-[4,5-3H(N)]lysine, New England Nuclear Co., sp. act. 60Ci/mmol) was added. The lobes were incubated in a Dubnoff metabolic shaker (40Hz) with gassing (95~o O2:5% CO2) for 0.5, 1, 2, 4 and 6hr at 22°C. At the end of the incubation periods, each group of four lobes was removed and homogenized in 500 #1 fresh media. The homogenized lobes and incubation media were transferred to separate plastic vials, 5 #1 removed from each for scintillation counting to assess total incorporation and then 500/~1 ice-cold 20~o trichloroacetic acid (TCA), containing 5 mM L-lysine, was added. Precipitation occurred overnight at 4°C and then the tubes were centrifuged for 5 min at 10,000g (Beckman microfuge). The supernatant was aspirated, the pellet washed several times with diethyl ether and then air-dried. The pellet was then resuspended in 20 ~1 of sample buffer for electrophoretic analysis.
lactin synthesis and release as well as for determinations of R I values. Sodium duodecyl sulfate (SDS) gels were used for pulse-chase analyses since very large proteins could enter this gel. SDS gels were also used for molecular weight determinations. Acid electrophoretic analysis was performed on an 11~o acid-urea polyacrylamide gel (Davis et al., 1972) using a vertical slab gel (Biorad model 220, slab thickness 1.5 mm, 20 sample wells per slab). Basic electrophoretic analysis was performed on a 7.5% Tris-glycine (pH 8.3) polyacrylamide slab gel (Davis, 1964). SDS electrophoretic analysis was performed on a 13% Tris-glycine (pH 8.8) polyacrylamide slab gel (Laemmli, 1970). Analysis for radioactivity in the gels was performed by cutting the slab gel into strips and then cutting each strip into 2 mm slices. Each slice was dissolved in 500 #1 30% H20 2 overnight at 60°C, 4 ml Aqua Luma (Lumac) added and then counted on a liquid scintillation analyzer (Philips, model DW4540). The results were expressed as counts per min since the counting efficiency was similar for all samples.
Effects of dopamine and c-AMP Pulse-chase experiments The conditions for the pulse-chase experiments were identical to those for the pulse experiments. The pulse time was 1 h and then one group of lobes and its medium were processed for electrophoretic analysis. The remaining groups had fresh incubation media added containing 5 mM e-lysine and the incubation continued for varying times up to 6 hr. The lobes and media were then processed as described before.
Immunoprecipitation Four rostral pars distalis lobes were incubated for 6 hr in 1.2ml incubation media containing 20gCi[3H]lysine in order to identify the prolactin peak. At the end of the incubation time the medium was subdivided into three equal vol of 400gl each. One portion was TCA-precipitated at 4°C for 96 hr. The remaining two portions had 8 gl of either anti-pollack prolactin serum or normal rabbit serum added and kept at 4°C for 48 hr. Then 20/A goat anti-rabbit gamma globulin (Calbiochem) was added and the vials were again left at 4°C for 48 hr. The resulting immunoprecipitate was centrifuged, washed and prepared for electrophoretic analysis.
Electrophoresis Three types of polyacrylamide gel electrophoresis were performed. Acid and basic gels were used for densitometric as well as liquid scintillation analyses. These gels were used to investigate the effects of dopamine and c-AMP on pro-
In one experiment (Exp. 1), four groups of four lobes each were incubated for 5 hr at 22°C. The osmolality of the incubation medium was 340 mOsm/kg. One group served as a control and the other two experimental groups had control incubation media with either 10 -5 M dopamine, 6 mM N 6, O2'-dibutyryl-adenosine-Y:5'-cyclic monophosphoric acid (dB-c-AMP) or a combination of I0 5 dopamine and 6 mM dB-c-AMP. At the end of the incubation period both the lobes and the media were processed for electrophoresis. After electrophoresis the gel was stained overnight with a 0.25% Coomassie Blue solution (methanol:acetic acid:water, 2:3:35). After destaining in methanol:acetic acid :water (2:3:35) for 1 week, the density of tlae protein bands was measured on a gel scanner (Isco, model 659). The gels were then sliced and processed for scintillation counting. As the staining procedure of the gels might cause some loss of labelled peptides, the experiment was repeated without densitometric analysis (Exp. 2). RESULTS There was only one major peak of newly synthesized [3H]lysine-containing protein in lobe extracts or media in all electrophoretic systems used. This peak had R s values of 0.27 and 0.32 in the acid and basic gels respectively. Anti-pollack prolactin precipitated only this newly synthesized product. It had a molecular weight of approx 21,000 as determined by
Biosynthesis and release of trout prolactin
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Fig. 1. SDS gel electrophoretic analysis of TCA-precipitable proteins synthesized in rostral pars distalis lobes of rainbow trout (upper graphs) or secreted into the medium (lower graphs) during pulse and pulse-chase incubations using I-3H]lysine. The dye front was at gel slice number 45. Insert, upper right: Data from same experiment to show total incorporation of label (tissue plus medium) as a function of pulse (0-------0) and chase (O O) times.
708
B.A. McKEOWN, B. G. JENKSand A. P. VAN OVERBEEKE
SDS electrophoresis. Because of these characteristics we consider this peak to be newly synthesized prolactin. Pulse and pulse-chase experiments
The incorporation of [3H]lysine into TCA-precipitable proteins in the lobes plus media was approximately linear during the pulse times (Fig. 1, insert). All peaks increased with increased pulse time but there was a selective release of the prolactin peak (data not shown). The amount of newly synthesized prolactin in the medium as a percentage of that in the lobes was 4.5, 7.3 and 20.1 after a pulse time of 1, 2 and 4 hr respectively. The total newly synthesized protein in the lobes plus medium remained unchanged during the chase periods of the pulse-chase experiment (Fig. 1, insert). The prolactin peak in the tissue decreased with increasing time of chase whereas this peak increased in the medium (Fig. 1). There were no significant changes in peak pattern either during the pulse or during the chase periods. Effects o f dopamine and c - A M P
Addition of dopamine to the medium inhibited the release of both total prolactin and that of newly synthesized hormone but had no apparent effect on tissue levels of total or newly synthesized prolactin. The addition of dB-c-AMP increased total prolactin and newly synthesized prolactin in both medium and tissue. When dopamine in combination with dB-c-AMP was added, an increase occurred in total and newly synthesized prolactin in both the medium and the tissue but the changes were not as drastic as with dB-c-AMP alone. DISCUSSION In view of the fact that there was no evidence for a delayed synthesis of prolactin during pulse experiments or, more importantly, no increase in prolactin during chase incubations indicates that the biosynthesis of prolactin is not preceded by the synthesis of a long-lived prohormone. Nonetheless, the possibility that synthesis of prolactin might have been preceded by a short-lived pre-prolactin, such as described by Mauer & McKean (1978) for the rat, cannot be excluded. If such a product existed, it must have been converted within the few minutes required to process the tissue at the end of the incubations. The fact that in rainbow trout the prolactin secreted into the medium in vitro had the same electrophoretic properties, in all three systems used, as the prolactin extracted from the tissue, does not necessarily mean that in this species, in vivo, there is only one form of prolactin occurring in both tissue and plasma. In the rat and in the mouse, in vitro studies revealed that the same prolactin that is present in the lactotrophs is also released into the medium (Farmer et al., 1976; Shoer et al., 1978). However, Jordan & Kendall (1978) reported that in man, prolactin in the presence of plasma is converted to a larger form. Heterogeneity of circulating prolactins has been reported by a number of investigators (see Garnier et al., 1978). In these studies the larger forms of prolactin were found in the plasma but, as pointed out by Jordan &
Kendall (1978) and Garnier et al. (1978), these "big" prolactins are likely formed after release from the lactotrophic cells and therefore, should not be considered pro-prolactin but either aggregations of the hormone or protein-bound prolactin. Also of interest is the fact that these larger forms are biologically less active than the small prolactin (Garnier et al., 1978). The biological activity of the main biosynthetic product of the trout lactotrophic cells and its possible interactions with plasma remain to be determined. There is a number of factors that are known to stimulate the release of prolactin in mammals as well as in fish. The mechanism of action of some of these factors might be mediated by c-AMP as a second messenger (McCann et al., 1978). Our investigation also showed that c-AMP was a potent stimulator of release and moreover, seems to promote synthesis as well. It is well documented that dopamine inhibits prolactin release in a number of vertebrates and it may be that this inhibition is due to a blocking of c-AMP production or enhanced c-AMP breakdown (De Camilli et al., 1979). This may also be the case in rainbow trout as c-AMP stimulated and dopamine inhibited prolactin release. Although levels of c-AMP are not known following incubations with dopamine in this investigation, there may be a relationship between these two factors since dopamine can decrease the c-AMP stimulated increase in prolactin synthesis and release. The interrelationship of the different factors studied in this investigation that control prolactin synthesis and release in rainbow trout are subjects for further investigations. Acknowledgements We would like to thank Messrs R. A. C. Lock and R. J. C. Engels for help in procuring and maintaining fish. We also appreciate the excellent secretarial assistance of Mrs M. H. B. M. van Bakelen-Suurmeijer.
REFERENCES DANNIES P. S. & TASHJIANA. H. JR (1976) Thyrotropinreleasing hormone increases prolactin RNA activity in the cytoplasm of GH-cells as measured by translation in a wheat germ cell-free system. Biochem. biophys. Res. Commun. 70, 118(~ 1189. DAVlS B. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Ann. N. Y Acad. Sci. 121,404-427. DAVIS R. H., COPENHAVER J. H. & CARVER M. J. (1972) Characterization of acetic proteins in cell nuclei from rat brain by high resolution acrylamide gel electrophoresis. J. Neurochem. 19, 473-477. DE CAMILLIP., MACCONID. & SPADAA. (1979) Dopamine inhibits adenylate cyclase in human prolactin-secreting pituitary adenomas. Nature 278, 252-254. FARMER S. W., BEWLEYT. A., RUSSELL S. M. & NICOLL C. S. (1976) Comparison of secreted and extracted forms of rat pituitary prolactin. Biochim. biophys. Acta 437, 562-570. GARNIERP. E., AUBERTM. L., KAPLANS. L. & GRUMBACH M. M. (1978) Heterogeneity of pituitary and plasma prolactin in man: Decreased affinity of "big" prolactin in a radioreceptor assay and evidence for its secretion, d. Clin. Endoer. Metab. 47, 1273-1281. GUYDA H. J. 0975) Heterogeneity of human growth hormone and prolactin secreted in vitro: immunoassay and radioreceptor assay correlations. J. Clin. Endoer. Metab. 41,953 957.
Biosynthesis and release of trout prolactin JENKS B. G., MEULEPASW. J. A. M., SOONS P. J. A. & VAN OVERBEEKE A. P. (1979) Biosynthesis of MSH and related peptides in the pars intermedia of the mouse: A pulse-chase analysis. Mol. Cell. Endocr. 13, 149-158. JORDAN R. M. & KENDALL J. W. (1978) Dissociation of plasma and CSF prolactin heterogeneity. Acta Endocr. 89, 38-47. KEMPER B., HABENER J. F., POTTS J. T. 8~; RICH A. (1972) Proparathyroid hormone: identification of a biosynthetic precursor to parathyroid hormone. Proc. Natn. Acad. Sci. U.S.A. 69, 643 647. LAEMMLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68(~685. LANCRANJAN I. & FRIESEN H. G. (1978) The neural regulation of prolactin secretion. In Current Studies of Hypothalamic Function (Edited by VEALEW. L. & LEDERIS K.) pp. 131 150. Karger, Basel. LINGAPPA V. R., DEVILLERS-THIERVA. & BLOBEL G. (1977) Nascent prehormones are intermediates in the biosynthesis of authentic bovine pituitary growth hormone and prolactin. Proc. Natn. Acad. Sci. U.S.A. 74, 2432 2436. MAUER R. A. & MCKEAN D. J. (1978) Synthesis of preprolactin and conversion to prolactin in intact cells and a cell-free system. J. biol. Chem. 253, 6315 6318. MAUER R. A., STONE R. & GORSK1 J. (1976) Cell-free synthesis of a large translation product of prolactin messenger RNA. J. biol. Chem. 25L 2801-2807. MCCANN S. M., KRULICH L., OJEDA S. R. & VIJAYAN E. (1978) Control of prolactin release by putative synaptic transmitters. In Proyress in Prolactin Physiology and Pathology (Edited by ROBVN C. & HARTER M.) pp. 137 148, Elsevier/North-Holland Biomedical, Amsterdam. MCKEOWN B. A. & BREWER K. J. (1978) Control of prolactin secretion in teleosts with special reference to recent studies in pacific salmon. In Comparative Endocrinolo,qy
709
(Edited by GAILLARD P. J. & BOER H. H.) pp. 357 362, Elsevier/North-Holland Biomedical, Amsterdam. NAGAHAMA Y., NISHIOKA R. S., BERN H. A. & GUNTHER R. L. (1975) Control of prolactin secretion in teleosts, with special reference to Gillichthys mirabilis and Tilapia mossambica. Gen. Comp. Endocr. 25, 166-188. PETER R. E. & McKEOWN B. A. (1975) Hypothalamic control of prolactin and thyrotropin secretion in teleosts, with special reference to recent studies in the goldfish. Gen. Comp. Endocr. 25, 153-165. RIGOPOULOU D., VALVERDE I., MARCO J., FALOONA G. UNGER R. H. (1970) Large glucagon immunoreactivity in extracts of pancreas. J. biol. Chem. 245, 496-501. ROBYN C. 8¢ HARTER M. (1978) Progress in Prolactin Physiology and Patholo.qy. Elsevier/North-Holland Biomedical, Amsterdam. ROGOL A. D. & ROSEN S. W. (1974) Prolactin of apparent large molecular size: the major immunoreactive prolactin component in plasma of a patient with a pituitary tumor. J. Clin. Endocr. Metab. 38, 714-719. SHOER L. F., SHINE N. R. 8¢ TALAMANTES F. (1978) Isolation and partial characterization of secreted mouse pituitary prolactin. Biochim. biophys. Acta 537, 336-347. STE1NER D. F., CUNNINGHAM D., SP1GELMANL. & ATEN B. (1967) Insulin biosynthesis: evidence for a precursor. Science 157, 697 700. SUH H. K. & FRANTZ A. G. (1974) Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocr. Metab. 39, 928 934. YALOW R. S. & BERSON S. A. (1971) Size heterogeneity of immunoreactive human ACTH in plasma and in extracts of pituitary glands and ACTH-producing thymoma. BiDchem. Res. Commun. 44, 439-445. ZANINI A., GIANNATTASIO G. & MELDOLESI J. (1974) Studies on in vitro synthesis and secretion of growth hormone and prolactin If. Evidence against the existence of precursor molecules. Endocrinology 94, 104-111.
0305-0491/80/0401-0705502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
BIOSYNTHESIS A N D RELEASE OF PROLACTIN FROM THE PITUITARY G L A N D OF THE RAINBOW TROUT, SALMO G A I R D N E R I B. A. M c K E o w y , B. G. JENKS and A. P. VAN OVERBEEKE Department of Zoology, University Nijmegen, 6525 ED Nijmegen, The Netherlands (Received 24 July 1979)
Abstract--l. A pulse-chase analysis using rostral pars distalis cells from rainbow trout pituitary glands failed to demonstrate the existence of a prolactin precursor molecule. 2. Prolactin secreted in vitro had the same electrophoretic characteristics as prolactin extracted from trout lactotrophic cells. 3. Dopamine (10 -5 M) inhibited the release of prolactin but did not effect synthesis. 4. Prolactin synthesis and release were both stimulated by dB-c-AMP (6 mM) but were not as pronounced when dopamine was also present.
INTRODUCTION
It is now well established that a number of protein hormones are first synthesized as a larger precursor molecule (Steiner et al., 1967; Rigopoulou et al., 1970; Yalow & Berson, 1971; Kemper et al., 1972; Jenks et al., 1979). With regard to prolactin the situation is controversial. There has been a number of recent studies in mammals employing cell-free systems that have indicated that prolactin is initially synthesized as a "short-lived" higher molecular weight protein called pre-prolactin (Dannies & Tashjian, 1976; Mauer et al., 1976; Lingappa et al., 1977). Recently, the presence of this pre-prolactin was demonstrated in intact cells and these cells can convert this product directly to prolactin (Mauer & McKean, 1978). On the other hand, neither in the studies just mentioned, nor in an in vitro analysis of prolactin biosynthesis in the rat using pulse-chase techniques, was there any evidence for the existence of a "longer-lived" storage form of a large prolactin ("pro-prolactin") (Zanini et al., 1974). The form of rat or mouse prolactin secreted in vitro from the lactotrophs appears to be the same as the form in which the hormone is extracted from these cells (Farmer et al., 1976; Shoer et al., 1978). In man however, there appears to be a size-heterogeneity of circulating prolactin species (Rogol & Rosen, 1974; Suh & Frantz, 1974; Guyda, 1975). The smallest of these forms is the same as the intra-cellular form. In this connection it is of interest that Jordan & Kendall (1978) isolated prolactin from human cerebro-spinal fluid and found it to be in a small form which, in the presence of plasma, was converted to a larger form. There is increasing evidence that there exists a complex interrelationship of a number of factors controlling the release of prolactin. This is the case in mammals (see Lancranjan & Friesen, 1978; Robyn & Harter, 1978) as well as in teleost fish (Nagahama et al., 1975; Peter & McKeown, 1975; McKeown & Brewer, 1978). The release of prolactin in teleosts appears to be under the control of an inhibitory factor from the hypothalamus which may be dopamine (see Peter & McKeown, 1975). Recently, De Camilli 705
et al. (1979) have demonstrated that dopamine also inhibits the release of human prolactin. In the same study they demonstrated that this inhibition was accompanied by a decrease in c-AMP levels in the lactotrophs which suggests that this action of dopamine involved the adenylate cyclase system. This last notion is in agreement with the observation that c-AMP causes the release of prolactin in mammals (see McCann et al., 1978). The investigations reported here are part of our program on internal regulatory mechanisms in salmonid fishes and were designed for two purposes. Firstly, to study whether the biosynthesis of prolactin in rainbow trout involves the production of a precursor. In addition, experiments were conducted to study the role of dopamine and c-AMP on the release of prolactin in the trout and whether such effects also influence the synthesis of this hormone.
MATERIALS AND METHODS
Animals
Rainbow trout, Salmo gairdneri (15-20cm) were obtained from a local hatchery, transported to the laboratory and maintained in a flow-through 1001. white porcelain aquarium. The trout were in water of 14°C, under a natural photoperiod and were fed commercial trout pellets daily ad libitum. Pulse experiments Biosynthesis and secretion of newly synthesized proteins were determined by incubating the rostral pars distalis of the pituitary gland in [3H]lysine-containing media for various pulse times. Incubations were performed in order to establish rates of synthesis and release for future pulsechase experiments. After trout were killed by severing the spinal cord, the pituitary was immediately removed and placed in a petri dish containing incubation medium. The rostral pars distalis was then dissected away and placed in a siliconized incubation vial containing 500/A incubation medium. The composition of the incubation medium was: l l2mM NaCI, 2mM KC1, 2mM CaClz.2H20 and 15mM HEPES, the pH was adjusted to 7.38 with 5 N NaOH.
706
B. A. McKEowN, B. G. JENKS and A. P. VAN OVERBEEKE Table 1. The effect of dopamine and c-AMP on synthesis and release of prolactin from the rostral pars distalis (RPD) of rainbow trout Densitometry (Experiment 1) ~o control RPD RPD Medium
Radioactive counts CPM(%) Experiment 1 Experiment 2 Total RPD Medium Total RPD Medium
Control
100.0
9.1
100.9
1.8
34,452 (95.6) --
109.8
12.5
579 (2.1) 232 (0.8) 3125 (4.7)
36,055 (100) ----
c-AMP (6 mM)
27,301 (97.9) 28,656 (99.2) 62,730 (95.3)
1603 (4.4)
Dopamine (10 -5)
27,800 (100) 28,888 (100) 65,855 (100)
54,707 (100)
47,954 (87.7)
6753 (12.3)
c-AMP (6 m/Vi) and dopamine (10 -5 M)
107.1
11.4
44,771 (100)
42,950 (95.9)
1821 (4.1)
43,759 (100)
40,508 (92.6)
3251 (7.4)
Treatment
Each medium contained four rostral pars distalis lobes incubated for 5 hr at 22°C.
Before incubation the medium was gassed for 10 min with 95% 02:5 % CO2 followed by the additional of bovine serum albumin (Sigma Fraction V, final concentration 0.3 mg/ml) and glucose (final concentration 2.5mg/ml). Four rostral pars distalis lobes were added to each vial and just prior to incubation, 10 #Ci[3H]lysine (L-[4,5-3H(N)]lysine, New England Nuclear Co., sp. act. 60Ci/mmol) was added. The lobes were incubated in a Dubnoff metabolic shaker (40Hz) with gassing (95~o O2:5% CO2) for 0.5, 1, 2, 4 and 6hr at 22°C. At the end of the incubation periods, each group of four lobes was removed and homogenized in 500 #1 fresh media. The homogenized lobes and incubation media were transferred to separate plastic vials, 5 #1 removed from each for scintillation counting to assess total incorporation and then 500/~1 ice-cold 20~o trichloroacetic acid (TCA), containing 5 mM L-lysine, was added. Precipitation occurred overnight at 4°C and then the tubes were centrifuged for 5 min at 10,000g (Beckman microfuge). The supernatant was aspirated, the pellet washed several times with diethyl ether and then air-dried. The pellet was then resuspended in 20 ~1 of sample buffer for electrophoretic analysis.
lactin synthesis and release as well as for determinations of R I values. Sodium duodecyl sulfate (SDS) gels were used for pulse-chase analyses since very large proteins could enter this gel. SDS gels were also used for molecular weight determinations. Acid electrophoretic analysis was performed on an 11~o acid-urea polyacrylamide gel (Davis et al., 1972) using a vertical slab gel (Biorad model 220, slab thickness 1.5 mm, 20 sample wells per slab). Basic electrophoretic analysis was performed on a 7.5% Tris-glycine (pH 8.3) polyacrylamide slab gel (Davis, 1964). SDS electrophoretic analysis was performed on a 13% Tris-glycine (pH 8.8) polyacrylamide slab gel (Laemmli, 1970). Analysis for radioactivity in the gels was performed by cutting the slab gel into strips and then cutting each strip into 2 mm slices. Each slice was dissolved in 500 #1 30% H20 2 overnight at 60°C, 4 ml Aqua Luma (Lumac) added and then counted on a liquid scintillation analyzer (Philips, model DW4540). The results were expressed as counts per min since the counting efficiency was similar for all samples.
Effects of dopamine and c-AMP Pulse-chase experiments The conditions for the pulse-chase experiments were identical to those for the pulse experiments. The pulse time was 1 h and then one group of lobes and its medium were processed for electrophoretic analysis. The remaining groups had fresh incubation media added containing 5 mM e-lysine and the incubation continued for varying times up to 6 hr. The lobes and media were then processed as described before.
Immunoprecipitation Four rostral pars distalis lobes were incubated for 6 hr in 1.2ml incubation media containing 20gCi[3H]lysine in order to identify the prolactin peak. At the end of the incubation time the medium was subdivided into three equal vol of 400gl each. One portion was TCA-precipitated at 4°C for 96 hr. The remaining two portions had 8 gl of either anti-pollack prolactin serum or normal rabbit serum added and kept at 4°C for 48 hr. Then 20/A goat anti-rabbit gamma globulin (Calbiochem) was added and the vials were again left at 4°C for 48 hr. The resulting immunoprecipitate was centrifuged, washed and prepared for electrophoretic analysis.
Electrophoresis Three types of polyacrylamide gel electrophoresis were performed. Acid and basic gels were used for densitometric as well as liquid scintillation analyses. These gels were used to investigate the effects of dopamine and c-AMP on pro-
In one experiment (Exp. 1), four groups of four lobes each were incubated for 5 hr at 22°C. The osmolality of the incubation medium was 340 mOsm/kg. One group served as a control and the other two experimental groups had control incubation media with either 10 -5 M dopamine, 6 mM N 6, O2'-dibutyryl-adenosine-Y:5'-cyclic monophosphoric acid (dB-c-AMP) or a combination of I0 5 dopamine and 6 mM dB-c-AMP. At the end of the incubation period both the lobes and the media were processed for electrophoresis. After electrophoresis the gel was stained overnight with a 0.25% Coomassie Blue solution (methanol:acetic acid:water, 2:3:35). After destaining in methanol:acetic acid :water (2:3:35) for 1 week, the density of tlae protein bands was measured on a gel scanner (Isco, model 659). The gels were then sliced and processed for scintillation counting. As the staining procedure of the gels might cause some loss of labelled peptides, the experiment was repeated without densitometric analysis (Exp. 2). RESULTS There was only one major peak of newly synthesized [3H]lysine-containing protein in lobe extracts or media in all electrophoretic systems used. This peak had R s values of 0.27 and 0.32 in the acid and basic gels respectively. Anti-pollack prolactin precipitated only this newly synthesized product. It had a molecular weight of approx 21,000 as determined by
Biosynthesis and release of trout prolactin
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Fig. 1. SDS gel electrophoretic analysis of TCA-precipitable proteins synthesized in rostral pars distalis lobes of rainbow trout (upper graphs) or secreted into the medium (lower graphs) during pulse and pulse-chase incubations using I-3H]lysine. The dye front was at gel slice number 45. Insert, upper right: Data from same experiment to show total incorporation of label (tissue plus medium) as a function of pulse (0-------0) and chase (O O) times.
708
B.A. McKEOWN, B. G. JENKSand A. P. VAN OVERBEEKE
SDS electrophoresis. Because of these characteristics we consider this peak to be newly synthesized prolactin. Pulse and pulse-chase experiments
The incorporation of [3H]lysine into TCA-precipitable proteins in the lobes plus media was approximately linear during the pulse times (Fig. 1, insert). All peaks increased with increased pulse time but there was a selective release of the prolactin peak (data not shown). The amount of newly synthesized prolactin in the medium as a percentage of that in the lobes was 4.5, 7.3 and 20.1 after a pulse time of 1, 2 and 4 hr respectively. The total newly synthesized protein in the lobes plus medium remained unchanged during the chase periods of the pulse-chase experiment (Fig. 1, insert). The prolactin peak in the tissue decreased with increasing time of chase whereas this peak increased in the medium (Fig. 1). There were no significant changes in peak pattern either during the pulse or during the chase periods. Effects o f dopamine and c - A M P
Addition of dopamine to the medium inhibited the release of both total prolactin and that of newly synthesized hormone but had no apparent effect on tissue levels of total or newly synthesized prolactin. The addition of dB-c-AMP increased total prolactin and newly synthesized prolactin in both medium and tissue. When dopamine in combination with dB-c-AMP was added, an increase occurred in total and newly synthesized prolactin in both the medium and the tissue but the changes were not as drastic as with dB-c-AMP alone. DISCUSSION In view of the fact that there was no evidence for a delayed synthesis of prolactin during pulse experiments or, more importantly, no increase in prolactin during chase incubations indicates that the biosynthesis of prolactin is not preceded by the synthesis of a long-lived prohormone. Nonetheless, the possibility that synthesis of prolactin might have been preceded by a short-lived pre-prolactin, such as described by Mauer & McKean (1978) for the rat, cannot be excluded. If such a product existed, it must have been converted within the few minutes required to process the tissue at the end of the incubations. The fact that in rainbow trout the prolactin secreted into the medium in vitro had the same electrophoretic properties, in all three systems used, as the prolactin extracted from the tissue, does not necessarily mean that in this species, in vivo, there is only one form of prolactin occurring in both tissue and plasma. In the rat and in the mouse, in vitro studies revealed that the same prolactin that is present in the lactotrophs is also released into the medium (Farmer et al., 1976; Shoer et al., 1978). However, Jordan & Kendall (1978) reported that in man, prolactin in the presence of plasma is converted to a larger form. Heterogeneity of circulating prolactins has been reported by a number of investigators (see Garnier et al., 1978). In these studies the larger forms of prolactin were found in the plasma but, as pointed out by Jordan &
Kendall (1978) and Garnier et al. (1978), these "big" prolactins are likely formed after release from the lactotrophic cells and therefore, should not be considered pro-prolactin but either aggregations of the hormone or protein-bound prolactin. Also of interest is the fact that these larger forms are biologically less active than the small prolactin (Garnier et al., 1978). The biological activity of the main biosynthetic product of the trout lactotrophic cells and its possible interactions with plasma remain to be determined. There is a number of factors that are known to stimulate the release of prolactin in mammals as well as in fish. The mechanism of action of some of these factors might be mediated by c-AMP as a second messenger (McCann et al., 1978). Our investigation also showed that c-AMP was a potent stimulator of release and moreover, seems to promote synthesis as well. It is well documented that dopamine inhibits prolactin release in a number of vertebrates and it may be that this inhibition is due to a blocking of c-AMP production or enhanced c-AMP breakdown (De Camilli et al., 1979). This may also be the case in rainbow trout as c-AMP stimulated and dopamine inhibited prolactin release. Although levels of c-AMP are not known following incubations with dopamine in this investigation, there may be a relationship between these two factors since dopamine can decrease the c-AMP stimulated increase in prolactin synthesis and release. The interrelationship of the different factors studied in this investigation that control prolactin synthesis and release in rainbow trout are subjects for further investigations. Acknowledgements We would like to thank Messrs R. A. C. Lock and R. J. C. Engels for help in procuring and maintaining fish. We also appreciate the excellent secretarial assistance of Mrs M. H. B. M. van Bakelen-Suurmeijer.
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(Edited by GAILLARD P. J. & BOER H. H.) pp. 357 362, Elsevier/North-Holland Biomedical, Amsterdam. NAGAHAMA Y., NISHIOKA R. S., BERN H. A. & GUNTHER R. L. (1975) Control of prolactin secretion in teleosts, with special reference to Gillichthys mirabilis and Tilapia mossambica. Gen. Comp. Endocr. 25, 166-188. PETER R. E. & McKEOWN B. A. (1975) Hypothalamic control of prolactin and thyrotropin secretion in teleosts, with special reference to recent studies in the goldfish. Gen. Comp. Endocr. 25, 153-165. RIGOPOULOU D., VALVERDE I., MARCO J., FALOONA G. UNGER R. H. (1970) Large glucagon immunoreactivity in extracts of pancreas. J. biol. Chem. 245, 496-501. ROBYN C. 8¢ HARTER M. (1978) Progress in Prolactin Physiology and Patholo.qy. Elsevier/North-Holland Biomedical, Amsterdam. ROGOL A. D. & ROSEN S. W. (1974) Prolactin of apparent large molecular size: the major immunoreactive prolactin component in plasma of a patient with a pituitary tumor. J. Clin. Endocr. Metab. 38, 714-719. SHOER L. F., SHINE N. R. 8¢ TALAMANTES F. (1978) Isolation and partial characterization of secreted mouse pituitary prolactin. Biochim. biophys. Acta 537, 336-347. STE1NER D. F., CUNNINGHAM D., SP1GELMANL. & ATEN B. (1967) Insulin biosynthesis: evidence for a precursor. Science 157, 697 700. SUH H. K. & FRANTZ A. G. (1974) Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocr. Metab. 39, 928 934. YALOW R. S. & BERSON S. A. (1971) Size heterogeneity of immunoreactive human ACTH in plasma and in extracts of pituitary glands and ACTH-producing thymoma. BiDchem. Res. Commun. 44, 439-445. ZANINI A., GIANNATTASIO G. & MELDOLESI J. (1974) Studies on in vitro synthesis and secretion of growth hormone and prolactin If. Evidence against the existence of precursor molecules. Endocrinology 94, 104-111.