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Comp. Biochem. Physiol., Vol. 65B, pp. 705 to 709 0305-0491/80/0401-0705502.

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© 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 pro-
nounced when dopamine was also present.

INTRODUCTION et al. (1979) have demonstrated that dopamine also


inhibits the release of human prolactin. In the same
It is now well established that a number of protein study they demonstrated that this inhibition was
hormones are first synthesized as a larger precursor accompanied by a decrease in c-AMP levels in the
molecule (Steiner et al., 1967; Rigopoulou et al., 1970; lactotrophs which suggests that this action of
Yalow & Berson, 1971; Kemper et al., 1972; Jenks dopamine involved the adenylate cyclase system. This
et al., 1979). With regard to prolactin the situation is last notion is in agreement with the observation that
controversial. There has been a number of recent c-AMP causes the release of prolactin in mammals
studies in mammals employing cell-free systems that (see McCann et al., 1978).
have indicated that prolactin is initially synthesized as The investigations reported here are part of our
a "short-lived" higher molecular weight protein called program on internal regulatory mechanisms in salmo-
pre-prolactin (Dannies & Tashjian, 1976; Mauer nid fishes and were designed for two purposes. Firstly,
et al., 1976; Lingappa et al., 1977). Recently, the pres- to study whether the biosynthesis of prolactin in rain-
ence of this pre-prolactin was demonstrated in intact bow trout involves the production of a precursor. In
cells and these cells can convert this product directly addition, experiments were conducted to study the
to prolactin (Mauer & McKean, 1978). On the other role of dopamine and c-AMP on the release of prolac-
hand, neither in the studies just mentioned, nor in an tin in the trout and whether such effects also influence
in vitro analysis of prolactin biosynthesis in the rat the synthesis of this hormone.
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). MATERIALS AND METHODS
The form of rat or mouse prolactin secreted in vitro
from the lactotrophs appears to be the same as the Animals
form in which the hormone is extracted from these Rainbow trout, Salmo gairdneri (15-20cm) were
cells (Farmer et al., 1976; Shoer et al., 1978). In man obtained from a local hatchery, transported to the labora-
however, there appears to be a size-heterogeneity of tory and maintained in a flow-through 1001. white porce-
circulating prolactin species (Rogol & Rosen, 1974; lain aquarium. The trout were in water of 14°C, under a
Suh & Frantz, 1974; Guyda, 1975). The smallest of natural photoperiod and were fed commercial trout pellets
these forms is the same as the intra-cellular form. In daily ad libitum.
this connection it is of interest that Jordan & Kendall Pulse experiments
(1978) isolated prolactin from human cerebro-spinal Biosynthesis and secretion of newly synthesized proteins
fluid and found it to be in a small form which, in the were determined by incubating the rostral pars distalis of
presence of plasma, was converted to a larger form. the pituitary gland in [3H]lysine-containing media for
There is increasing evidence that there exists a various pulse times. Incubations were performed in order
complex interrelationship of a number of factors con- to establish rates of synthesis and release for future pulse-
trolling the release of prolactin. This is the case in chase experiments.
mammals (see Lancranjan & Friesen, 1978; Robyn & After trout were killed by severing the spinal cord, the
Harter, 1978) as well as in teleost fish (Nagahama et pituitary was immediately removed and placed in a petri
dish containing incubation medium. The rostral pars dis-
al., 1975; Peter & McKeown, 1975; McKeown &
talis was then dissected away and placed in a siliconized
Brewer, 1978). The release of prolactin in teleosts incubation vial containing 500/A incubation medium. The
appears to be under the control of an inhibitory fac- composition of the incubation medium was: l l2mM
tor from the hypothalamus which may be dopamine NaCI, 2mM KC1, 2mM CaClz.2H20 and 15mM
(see Peter & McKeown, 1975). Recently, De Camilli HEPES, the pH was adjusted to 7.38 with 5 N NaOH.
705
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 Radioactive counts


(Experiment 1) CPM(%)
~o control RPD Experiment 1 Experiment 2
Treatment RPD Medium Total RPD Medium Total RPD Medium

Control 100.0 9.1 27,800 27,301 579 36,055 34,452 1603


(100) (97.9) (2.1) (100) (95.6) (4.4)
Dopamine (10 -5) 100.9 1.8 28,888 28,656 232 ---- --
(100) (99.2) (0.8)
c-AMP (6 mM) 109.8 12.5 65,855 62,730 3125 54,707 47,954 6753
(100) (95.3) (4.7) (100) (87.7) (12.3)
c-AMP (6 m/Vi) and dopamine
(10 -5 M) 107.1 11.4 44,771 42,950 1821 43,759 40,508 3251
(100) (95.9) (4.1) (100) (92.6) (7.4)

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 lactin synthesis and release as well as for determinations of
95% 02:5 % CO2 followed by the additional of bovine R I values. Sodium duodecyl sulfate (SDS) gels were used
serum albumin (Sigma Fraction V, final concentration for pulse-chase analyses since very large proteins could
0.3 mg/ml) and glucose (final concentration 2.5mg/ml). enter this gel. SDS gels were also used for molecular weight
Four rostral pars distalis lobes were added to each vial and determinations.
just prior to incubation, 10 #Ci[3H]lysine Acid electrophoretic analysis was performed on an 11~o
(L-[4,5-3H(N)]lysine, New England Nuclear Co., sp. act. acid-urea polyacrylamide gel (Davis et al., 1972) using a
60Ci/mmol) was added. The lobes were incubated in a vertical slab gel (Biorad model 220, slab thickness 1.5 mm,
Dubnoff metabolic shaker (40Hz) with gassing (95~o 20 sample wells per slab). Basic electrophoretic analysis
O2:5% CO2) for 0.5, 1, 2, 4 and 6hr at 22°C. was performed on a 7.5% Tris-glycine (pH 8.3) polyacryl-
At the end of the incubation periods, each group of four amide slab gel (Davis, 1964). SDS electrophoretic analysis
lobes was removed and homogenized in 500 #1 fresh media. was performed on a 13% Tris-glycine (pH 8.8) polyacryl-
The homogenized lobes and incubation media were trans- amide slab gel (Laemmli, 1970).
ferred to separate plastic vials, 5 #1 removed from each for Analysis for radioactivity in the gels was performed by
scintillation counting to assess total incorporation and cutting the slab gel into strips and then cutting each strip
then 500/~1 ice-cold 20~o trichloroacetic acid (TCA), con- into 2 mm slices. Each slice was dissolved in 500 #1 30%
taining 5 mM L-lysine, was added. Precipitation occurred H20 2 overnight at 60°C, 4 ml Aqua Luma (Lumac) added
overnight at 4°C and then the tubes were centrifuged for and then counted on a liquid scintillation analyzer (Philips,
5 min at 10,000g (Beckman microfuge). The supernatant model DW4540). The results were expressed as counts per
was aspirated, the pellet washed several times with diethyl min since the counting efficiency was similar for all
ether and then air-dried. The pellet was then resuspended samples.
in 20 ~1 of sample buffer for electrophoretic analysis.
Effects of dopamine and c-AMP
Pulse-chase experiments In one experiment (Exp. 1), four groups of four lobes
The conditions for the pulse-chase experiments were each were incubated for 5 hr at 22°C. The osmolality of the
identical to those for the pulse experiments. The pulse time incubation medium was 340 mOsm/kg. One group served
was 1 h and then one group of lobes and its medium were as a control and the other two experimental groups had
processed for electrophoretic analysis. The remaining control incubation media with either 10 -5 M dopamine,
groups had fresh incubation media added containing 5 mM 6 mM N 6, O2'-dibutyryl-adenosine-Y:5'-cyclic monophos-
e-lysine and the incubation continued for varying times up phoric acid (dB-c-AMP) or a combination of I0 5
to 6 hr. The lobes and media were then processed as de- dopamine and 6 mM dB-c-AMP. At the end of the incuba-
scribed before. tion period both the lobes and the media were processed
for electrophoresis. After electrophoresis the gel was
Immunoprecipitation stained overnight with a 0.25% Coomassie Blue solution
Four rostral pars distalis lobes were incubated for 6 hr in (methanol:acetic acid:water, 2:3:35). After destaining in
1.2ml incubation media containing 20gCi[3H]lysine in methanol:acetic acid :water (2:3:35) for 1 week, the density
order to identify the prolactin peak. At the end of the of tlae protein bands was measured on a gel scanner (Isco,
incubation time the medium was subdivided into three model 659). The gels were then sliced and processed for
equal vol of 400gl each. One portion was TCA-precipi- scintillation counting. As the staining procedure of the gels
tated at 4°C for 96 hr. The remaining two portions had 8 gl might cause some loss of labelled peptides, the experiment
of either anti-pollack prolactin serum or normal rabbit was repeated without densitometric analysis (Exp. 2).
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 RESULTS
immunoprecipitate was centrifuged, washed and prepared
for electrophoretic analysis. There was only one major peak of newly synthe-
sized [3H]lysine-containing protein in lobe extracts
Electrophoresis or media in all electrophoretic systems used. This
Three types of polyacrylamide gel electrophoresis were peak had R s values of 0.27 and 0.32 in the acid and
performed. Acid and basic gels were used for densitometric basic gels respectively. Anti-pollack prolactin precipi-
as well as liquid scintillation analyses. These gels were used tated only this newly synthesized product. It had a
to investigate the effects of dopamine and c-AMP on pro- molecular weight of approx 21,000 as determined by
Biosynthesis and release of trout prolactin 707

CPM (x 10-2 I
lh PULSE
t2

10 CPM(x 10-31

(,

2 /
o,

I -O.o"-''°-- -II---o
r i i I

o[/ 7 3 t,
~
7
Hours

l i , ,

12 4Stain CHASE 12 3h CHASE


I~8/,
10 10

8 8

6 G

t, 4

2 2

' , , t

26f . . . .

0 "" , , , i , i

12 90 min CHASE 12 6h CHASE

10 10

8 8

G G

t, t,

2 2

:I ....
0
0 10 20 30 (,0 o 20 30 ,,.0
GeL slice no Gel slice no

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 Kendall (1978) and Garnier et al. (1978), these "big"
we consider this peak to be newly synthesized prolac- prolactins are likely formed after release from the lac-
tin. totrophic cells and therefore, should not be con-
sidered pro-prolactin but either aggregations of the
Pulse and pulse-chase experiments hormone or protein-bound prolactin. Also of interest
The incorporation of [3H]lysine into TCA-precipi- is the fact that these larger forms are biologically less
table proteins in the lobes plus media was approxi- active than the small prolactin (Garnier et al., 1978).
mately linear during the pulse times (Fig. 1, insert). The biological activity of the main biosynthetic
All peaks increased with increased pulse time but product of the trout lactotrophic cells and its possible
there was a selective release of the prolactin peak interactions with plasma remain to be determined.
(data not shown). The amount of newly synthesized There is a number of factors that are known to
prolactin in the medium as a percentage of that in the stimulate the release of prolactin in mammals as well
lobes was 4.5, 7.3 and 20.1 after a pulse time of 1, 2 as in fish. The mechanism of action of some of these
and 4 hr respectively. factors might be mediated by c-AMP as a second
The total newly synthesized protein in the lobes messenger (McCann et al., 1978). Our investigation
plus medium remained unchanged during the chase also showed that c-AMP was a potent stimulator of
periods of the pulse-chase experiment (Fig. 1, insert). release and moreover, seems to promote synthesis as
The prolactin peak in the tissue decreased with well. It is well documented that dopamine inhibits
increasing time of chase whereas this peak increased prolactin release in a number of vertebrates and it
in the medium (Fig. 1). There were no significant may be that this inhibition is due to a blocking of
changes in peak pattern either during the pulse or c-AMP production or enhanced c-AMP breakdown
during the chase periods. (De Camilli et al., 1979). This may also be the case in
rainbow trout as c-AMP stimulated and dopamine
Effects o f dopamine and c - A M P inhibited prolactin release. Although levels of c-AMP
Addition of dopamine to the medium inhibited the are not known following incubations with dopamine
release of both total prolactin and that of newly syn- in this investigation, there may be a relationship
thesized hormone but had no apparent effect on tissue between these two factors since dopamine can de-
levels of total or newly synthesized prolactin. The crease the c-AMP stimulated increase in prolactin
addition of dB-c-AMP increased total prolactin and synthesis and release. The interrelationship of the dif-
newly synthesized prolactin in both medium and tis- ferent factors studied in this investigation that control
sue. When dopamine in combination with dB-c-AMP prolactin synthesis and release in rainbow trout are
was added, an increase occurred in total and newly subjects for further investigations.
synthesized prolactin in both the medium and the tis-
sue but the changes were not as drastic as with Acknowledgements We would like to thank Messrs
dB-c-AMP alone. R. A. C. Lock and R. J. C. Engels for help in procuring and
maintaining fish. We also appreciate the excellent secretar-
ial assistance of Mrs M. H. B. M. van Bakelen-Suurmeijer.
DISCUSSION
In view of the fact that there was no evidence for a
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