GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
32, 120-131 (1977)
Factors Affecting in Vitro Activity of Prolactin Cells in the Euryhaline Teleost Sarotherodon mossambicus (Tilapia mossambica) TREVOR
WIGHAM, Department University
RICHARD
S. NISHIOKA,
AND HOWARD
A. BERN
of Zoology and Cancer Research Laboratory, of California, Berkeley, California 94720
Accepted November 15, 1976 The rostraf pars distahs (RPD) of the euryhaline teleost Sarotherodon mossambicus mossambica) was incubated in vitro to study the activity of the prolactin cells. Total prolactin release during 18 hr was measured by disc gel electrophoresis; release of newly synthesised prolactin was followed using a 3H-leucine tracer. Synthetic activity was estimated by calculating the specific activity of the prolactin bands. Prolactin release from the control tissues was always greater in hyposmotic than in hyperosmotic medium, indicating a direct effect of osmotic pressure upon the cells. Doparnine (I pg/ml) inhibited release, but not synthesis, of prolactin in hyposmotic medium. Octopamine (5 pgiml and 10 ngiml) in hyposmotic medium and y-amino-nbutyric acid (GABA) (100 rig/ml) in hyperosmotic medium had no effect on prolactin secretion. Cortisol (1 pg/ml) inhibited prolactin release in hyposmotic medium and, to a lesser extent, in hyperosmotic medium. Prolactin specific activity was increased only in the latter medium. EstradioL17P (100 ngiml) did not alter prolactin release in hyperosmotic medium, but did appear to enhance hormone synthesis. Thyrotropin-releasing hormone (TRH) (100 rig/ml) inhibited prolactin release, but not synthesis, in hyposmotic medium, and did not affect prolactin secretion in hyperosmotic medium. TRH at 100 pg/ml was ineffective in hyperosmotic medium. Somatostatin (300 rig/ml) inhibited prolactin synthesis and release in hyposmotic medium, but inhibited only release in hyperosmotic medium. These results indicate that there may be a complex regulation of the prolactin cells in this teleost. The in vitro responses of teleost prolactin cells to various potential mediators resemble those seen in other vertebrates: however, the differences in detail may have special adaptive significance for the teleost. (Tilapia
Prolactin is considered to be an important osmoregulatory hormone in most euryhaline teleosts (cf. Bern, 1975). In such fish in fresh water, prolactin is actively secreted and enhances ion conservation. However, in a seawater environment, where such conservation would be disadvantageous, the prolactin cells are much less active (cf. Holmes and Ball, 1974; Schreibman and Holtzman, 1975). The regulation of prolactin cell activity in these teleosts may involve a direct effect of ambient osmotic pressure, since these cells have been shown to be more active in low than in high osmotic pressures in vitro in
Poecilia latipinna and Anguilla anguilla (Ingleton et al., 1973) and in Tilapia mossambica (Zambrano et al., 1974; Nagahama et al., 1975). However, the re-
sults of hypothalamic lesions, pituitary transplantations and treatment with such drugs as reserpine, 6hydroxydopamine (6OHDA) and L-P,3-4-dihydroxyphenylalanine (L-dopa) indicate that an inhibitory, possibly catecholaminergic, hypothalamic regulation of prolactin cells exists in at least some teleosts (Ball and Baker, 1969; Olivereau, 1971, 1975; Ball et al., 1972; Peter, 1973; Peter and McKeown, 1974; Zambrano, 1975; Wigham and Ball, 120
Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 00166480
TELEOST
PROLACTIN
1976). Furthermore, in vitro studies have shown that the prolactin cells of P. Zutipinna (Wigham and Ball, 1974; Wigham et al., 1975) and T. mossambica (Nagahama et al., 1975) are inhibited from releasing their hormone by the addition of dopamine to the medium. Thus, the postulated hypothalamic inhibition may involve dopaminergic fibers. Recent experiments with the incubation of mammalian pituitaries or prolactin cells have shown that these cells alter their activity in response to a number of factors including catecholamines (Birge et al., 1970; Macleod and Lehmeyer, 1972, 1974; Shaar and Clemens, 1974), somatostatin (Vale et al., 1974; Grant et al., 1974; Drouin et al., 1976)) thyrotropin-releasing hormone (TRH) (Morin et al., 1975; Smith and Convey, 1975; and others), insulin and human chorionic somatomammotropin (Shenai and Wallis, 1975) and medium osmotic pressure (Labella er al., 1975). Therefore, in our present studies we have incubated the prolactin cell region, the rostral pars distalis (RPD), of Sarotherodon mossambicus (Tilapia mossambica) pituitaries with several agents, both to examine the possible factors which regulate teleost prolactin cells and to compare the results with those obtained from mammalian and other nonmammalian vertebrates. MATERIALS
AND METHODS
Incubation S. mossambicus has been referred to in previous publications as T. mossambica. However, this teleost has been re-named (see Thys van den Audenaerde, 1968; Trewavas, 1973). In this and future publications, the new nomenclature will be used. Fish 9-14 cm long were obtained from Hawaii and maintained, together with a breeding population, at 27 -+ 3” under natural lighting conditions in a freshwater pond at the University of California, Berkeley. Pituitaries were removed and placed in a medium made up in Sarurhero&~~ bicarbonate-Ringer with glucose (500 mg/liter), glutamine (290 mgiliter) and Eagle’s (1959) minimum essential medium organic constituents (MEM: Bio-Rad, Richmond, Calif.) for dissection of the RPD region. Two milliliters of SOxMEM were
CELLS
121
added to 98 ml Ringer solution. The Ringer solution isotonic to freshwater Sarotherodon plasma (approximately 320 mOsm/kg in this species) consisted (in mEq/liter) of NaCl, 135; KC], 2.35; CaCl,, 4.2; MgSO,, 2.8; KH2P04, 1.25; NaHCO,, 25. The pH of the medium was 7.25 after gassing for 10 min. Media of different osmotic pressures were prepared by appropriate changes in NaCl concentration. The RPD, containing prolactin and ACTH cells, were preincubated at 27 2 1” in medium under an atmosphere of 95% 045% CO,. During this and the ensuing incubation, the medium was continuously mixed on a gyrotory platform. After 1 hr the tissues were transferred to a multicompartment culture tray, with two RPD in each of 12 compartments containing 0.2 ml of medium. This final medium included 13.5 LLyml of 4,5-3H-L-leucine (sp act of stock, 45-60 Ci/mmole, concentration 0.5 mCi/ml) (Schwarz-Mann, USA) for incorporation into newly synthesized prolactin. Six compartments were used for control and six for experimental media. The glands were incubated for 18 hr under the same conditions as for preincubation. At termination of incubation the tissues and media were separated for subsequent disc gel electrophoresis.
Gel Electrophoresis The use of polyacrylamide disc gel electrophoresis for the measurement of teleost pituitary prolactin has previously been validated for Anguilla (Knight et al., 1970), Poecilia latipinna (Ingleton ef al., 1973), Salmo gairdneri (Baker and Ingleton, 1975) and for Gillichthys mirabilis and Tilapia mossambica (Nagahama et al., 1975). Polyacrylamide gels were prepared in 3-mm (Ld.) glass tubes from the solutions described by Davis (1964). Each gel consisted of a 5.5-cm running gel surmounted by a l-cm large-pore stacking gel. RPD were homogenized, two per tube, in 0.2 ml of ice cold 1: 15 Tris-glycine buffer (Davis, 1964). The homogenates were kept on ice for 30 min, after which 0.2 ml of large-pore gel solution without sucrose was added to each homogenate. After mixing, the solutions were pipetted into the space above each stacking gel and then allowed to polymerize. Each sample of medium (0.2 ml) was mixed with an equal volume of large-pore gel solution without sucrose, and the mixture was pipetted onto the stacking gels for polymerization. Fifty microliters of 5 x lo-% bromophenol blue tracking dye were pipetted onto each sample gel, and the remaining space was filled with 1:l Tris-glycine electrode buffer. The gel tubes were placed in the electrophoresis apparatus, which was filled with 1: 1 Tris-glycine buffer. The apparatus included a refrigerating water jacket to keep the gels at about 6”, since this was found to produce better band definition. The current was regulated at 1.5 mA/tube supplied from a Buchler 3-l 155 constant current, high voltage power pack. The run was ended
122
WIGHAM,
NISHIOKA
AND
BERN
facilitate the detection of an expected response or to establish the effect of osmotic pressure upon the response to a drug.
when the tracking dye reached an etched mark on the glass tube. The gels were removed, the location of the tracking dye was permanently marked with india ink, and the gels were stained overnight in a solution of 0.1% Coomassie brilliant blue (CBB) in methanol: acetic acid:water (2:1:7). Destaining was carried out in a Bio-Rad diffusion destainer using staining fluid without CBB. After destaining, the gels were stored in 7% acetic acid. The densities of the prolactin bands in the gels were measured on a Photovolt densitometer. The radioactivity of the tritium within the prolactin band was measured by dissolving the excised gel bands in 0.1 ml of 30% H,O,. The solution was mixed with 10 ml of scintillation fluid (8 g BBOT, 160 g napthalene, 1200 ml toluene, 800 ml ethoxyethanol). The mixture was allowed to equilibrate in a refrigerator for 3 days, and the radioactivity was then measured on a Packard Model 3330 liquid scintillation counter.
RESULTS
The percentage release of both total (densitometrically measured) and newly synthesized (tritiated) prolactin by the control tissue was greater by 40 and 41%, respectively, in the hyposmotic (300 mOsm/kg) medium than in the hyperosmotic (360 mOsm/kg) medium. Thus, changes in ambient osmotic pressure alone are sufficient to alter the activity of the prolactin cells. This result agrees with earlier reports on Sarotherodon by Zambrano et al. (1974) and Nagahama et al. (1975). Dopamine at 1 pg/ml inhibited release of both total and newly synthesized prolactin Calculations into an otherwise stimulatory hyposmotic The densitometric measurements of the prolactin medium (Table 2) (cf. Nagahama et al., bands were used to calculate prolactin release into the medium as a percentage of the total present in 1975). However, it did not appear to alter RPD plus medium. Release of newly synthesized pro- prolactin synthesis during the incubation, lactin into the medium was separately calculated as since no significant change in specific activthe percentage of 3H counts present in the tissue plus ity was observed (Table 2). Octopamine at medium. Specific activity of the prolactin was estieither 5 pglml or 10 rig/ml had no effect on mated as the sum of the 3H counts present in tissue release or specific activity in hyposmotic plus medium divided by the sum of densitometric units in the bands from RPD plus those from medium. medium. Addition of y-amino-n-butyric The data were analysed for statistical significance acid (GABA) (100 rig/ml) to a slightly using Student’s t test. hyperosmotic (340 mOsrn/kg) medium was also ineffective in altering release or spePharmacological Studies cific activity (Table 2). Various drugs were tested for their ability to alter Cortisol (1 pg/ml) inhibited total and synthesis and release of prolactin by the RPD (Table 1). The osmotic pressure of the medium used for each tritiated prolactin release into the stimulapharmacological experiment was chosen either to tory hyposmotic medium, but affected only TABLE DRUGS
TESTED
Drug Dopamine (3-hydroxytyramine) D, L-Octopamine HCl
1
IN THE INCUBATION
OF Sarotherodon
RPD
Supplier HCI
y-Amino-n-butyric acid (GABA) Cortisol (A grade) Estradiol- 178 Thyrotropin-releasing hormone (porcine), synthetic (TRH) Somatostatin (ovine), synthetic Albumin (bovine serum), fraction
Sigma (USA) Sigma (USA) Sigma (USA) Calbiochem (USA) Calbiochem (USA) Beckman (USA)
V
Beckman (USA) Calbiochem (USA)
Concentration 1 pg/ml 5 pg/ml 10 rig/ml 100 rig/ml 1 pg/ml 100 rig/ml 100 rig/ml 100 pg/ml 300 rig/ml 1 pg/ml
5.27 2.64 5.27 1.03 2.64 3.67 2.77 2.77 1.83 1.67
x lo+ M x lo-” M x
lO-8
M
x lO-s M x lo+ M x lo-‘M x lo-‘M
x lo-r0 M x lo-’ M x IO-* M
340
(6)
(6) 46.1 k2.61
(9
45.7 k3.29
(9
79.9 k2.4.5 (3) 57.2 k2.94
(6)
57.7 2 3.47 (9 75.4 kO.49 (3) 61.8 +- 1.21
Exp
76.2* k2.24
Cont
(9
54.4 kl.78
82.2* 21.52 (9 84.4 24.2 (3) 63.4 k4.0 (9
Cont
(9
55.3 ~1.52
83.5 k4.0 (3) 66.5 k4.77 (9
(6)
19.9 21.63
Bxp
Newly synthesized prolactin release (%)
ON PROLACTIN CELL ACTIVITY"
a Results are given as the mean plus or minus one standard error; n in parentheses. * P < 0.01 = significance of difference between control (Cont) and experimental (Exp) values.
100 rig/ml
1.03 x 10eB M
GABA
300
5.27 x IO-‘M
10 rig/ml
300
2.64 x 10m5M
5 &ml
300
Octopamine
5.27 x 10mBM
1 pg/ml
Concentration
Dopamine
Neurotransmitter
Medium osmolality (mOsm/kg)
Total prolactin release (%)
TABLE 2 EFFECTS OF NEUROTRANSMITTERS: DOPAMINE, OCTOPAMINE AND GABA,
(9
(9
(6)
2475 2162
(6) 2298 1148
1946 + 437 (3) 1508 +74
(6)
1443 2100
Exp
1539 2609 (3) 1329 -c92
(6)
1342 I?104
Cont
Sp act
E
8
5 2
;;i ii a -I 2 $
124
WIGHAM,
NISHIOKA
the former in the hyperosmotic medium (Table 3). This suggests that the cortisolinduced inhibition of newly synthesized prolactin release was insufficient to appear against a background of inhibition by medium osmotic pressure. The specific activity of the prolactin band was unaffected by cortisol in the hyposmotic medium, but was increased in the hyperosmotic medium (Table 3). The latter experiment was repeated, with similar results. Estradiol-17/3 at 100 rig/ml did not significantly alter prolactin release into a slightly inhibitory (340 mOsm/kg) medium (Table 3), whereas specific activity was increased. Thus, estradiol-17P, as well as cortisol, may increase prolactin synthesis, but not release, into a hyperosmotic medium. It is possible that an increase in release may occur subsequent to stimulation of synthesis by estradiol-17P, and that 18 hr is too short for such an effect to become measurable by gel electrophoresis. However, in some preliminary 5-day incubations with estradiol17p no stimulation of prolactin release was observed. TRH at 100 rig/ml inhibited release of prolactin into hyposmotic medium, but not into hyperosmotic medium (Table 4). Thus, an already inhibitory medium may be capable of masking the action of TRH on the cells. Specific activity of the prolactin band was not significantly altered in either experiment (Table 4). A low concentration of TRH (100 pg/ml) altered neither prolactin release nor specific activity in the slightly hyperosmotic (340 mOsm/kg) medium (Table 4). Thus, TRH is only effective at higher concentrations. Somatostatin (300 rig/ml) markedly inhibited prolactin release into both the hyperosmotic and the hyposmotic media (Table 4). The specific activity of the prolactin band was also reduced in the latter medium, but the result in the hyperosmotic medium was less clear (Table 4). In one experiment, somatostatin appeared to increase prolactin specific activity, even
AND
BERN
though it strongly inhibited release. However, the specific activity of the control tissues was very low compared with that of controls of the other experiments. Also, the results of two similar experiments with hyperosmotic medium gave no indication that somatostatin altered prolactin specific activity. Therefore, we feel that the original result may be anomalous. The ability of somatostatin to reduce hormone release into both hyposmotic and hyperosmotic media suggests that it is a potent inhibitor of prolactin secretion, although a lack of effect on synthesis in the latter medium indicates that the drug is more effective on hormone release than on synthesis. Bovine serum albumin (BSA) at 1 pg/ml had no effect on release or synthesis of prolactin in hyposmotic medium (Table 4). Thus, the simple addition of a large molecule to the medium does not, by itself, appear to alter the activity of the prolactin cells. DISCUSSION
The ability of prolactin cells actively to secrete hormone in vitro has been established in several species of teleost (Ingleton et al., 1973; Zambrano et al., 1974; Nagahama et al., 1975; Baker and Ingleton, 1975; Wigham et al., 1975). These reports have also described the stimulatory action of low osmotic pressure of the medium on prolactin cells. Our present experiments confirm this for S. mossambicus, since incubation in hyposmotic medium produced consistently higher levels of prolactin release than in hyperosmotic medium. Dopamine has been implicated in the regulation.,of prolactin cells in P. latipinna (Wigham and Ball, 1974; Wigham et al., 1975), A. anguilla (Olivereau, 1975; P. M. Ingleton and B. I. Baker, unpublished data) and T. mossambica (Nagahama et al., 1975). Our results confirm the inhibitory effect of dopamine .on prolactin release by Sarotherodon (Tifupia) RPD. In addition,
3.67 x 10-r M
340
360
2.76 x IO-“M
1 pg/ml
100 rig/ml
300
2.76 x 10e6 M
1 pg/ml
Concentration
49.6 21.62 (6)
55.3* ?I.2 (6)
(6)
4 1.36
78.0*
Cont
75.8 23.91
(6)
(6)
(6)
92.9* 21.01 (6) 75.5 22.95
Cont
73.0 +3.48 (5)
85.7 22.32 (6) 64.2 23.95 t.9
Bxp
Newly synthesized prolactin release (%I
51.2 21.22
66.2 k2.72 (6) 47.4 ?I.84 (6)
Exp
a Results are given as the mean plus or minus one standard error; n in parentheses. * P < 0.01. ** P < 0.001 = significance of difference between control (Cont) and experimental (Exp) values.
Estradiol- 17fi
Cortisol
Hormone
Medium osmolality (mOsm/kg)
Total prolactin release (%)
TABLE 3 EFFECTS OF THE STEROIDS, CORTISOL AND ESTRADIOL-17/3, ON PROLACTIN CELL ACTIVITY”
1470** 24.5 (6)
1869 263 (6) 957** 268 (6)
Cont
Sp act
1835 215 (5)
1843 538 (5)
(6)
1923 249
Exp
.
:: cn
5 2 8
2 ?
6 2
1.83 x IO-‘M
300 rig/ml
1.67 x lo-*
1.83 x IO-‘M
300 rig/ml
1 pg/ml
1.83 x IO-‘M
300 rig/ml
2.77 ST IO-“‘M
100 pg/ml
1.83 x IO-‘M
2.17 x lo-’
100 rig/ml
300 rig/ml
2.77 x IO-‘M
100 rig/ml
M
M
HYPOPHYSIOTROPINS
Concentration
OF THE
300
360
360
360
300
340
360
300
Medium osmolality (mOsm/kg)
TRH AND
TABLE
4
values
(6)
(6) (6)
31.7** k4.28 (6) 51.5** k5.10
54.4** 26.2 (5)
91.2** al.64 (6)
(6)
50.7 k2.5
(6)
27.2 23.28
(6)
83.2** k2.82
Cont
73.6 23.66
(Exp)
ACTIVITY”
1945 *131
(6)
(6)
302*” *50 C.9 2034 4141 (6) 1099 ,134
(6)
1834 ?I43
1098 240
(6)
2008 275
1312 +-IO0 (5)
(6)
381 230
(6)
(6) l271*” ?56 (6)
“$
F
zi a i$
E
2
g
i
AND
COMPARATIVE
ENDOCRINOLOGY
32, 120-131 (1977)
Factors Affecting in Vitro Activity of Prolactin Cells in the Euryhaline Teleost Sarotherodon mossambicus (Tilapia mossambica) TREVOR
WIGHAM, Department University
RICHARD
S. NISHIOKA,
AND HOWARD
A. BERN
of Zoology and Cancer Research Laboratory, of California, Berkeley, California 94720
Accepted November 15, 1976 The rostraf pars distahs (RPD) of the euryhaline teleost Sarotherodon mossambicus mossambica) was incubated in vitro to study the activity of the prolactin cells. Total prolactin release during 18 hr was measured by disc gel electrophoresis; release of newly synthesised prolactin was followed using a 3H-leucine tracer. Synthetic activity was estimated by calculating the specific activity of the prolactin bands. Prolactin release from the control tissues was always greater in hyposmotic than in hyperosmotic medium, indicating a direct effect of osmotic pressure upon the cells. Doparnine (I pg/ml) inhibited release, but not synthesis, of prolactin in hyposmotic medium. Octopamine (5 pgiml and 10 ngiml) in hyposmotic medium and y-amino-nbutyric acid (GABA) (100 rig/ml) in hyperosmotic medium had no effect on prolactin secretion. Cortisol (1 pg/ml) inhibited prolactin release in hyposmotic medium and, to a lesser extent, in hyperosmotic medium. Prolactin specific activity was increased only in the latter medium. EstradioL17P (100 ngiml) did not alter prolactin release in hyperosmotic medium, but did appear to enhance hormone synthesis. Thyrotropin-releasing hormone (TRH) (100 rig/ml) inhibited prolactin release, but not synthesis, in hyposmotic medium, and did not affect prolactin secretion in hyperosmotic medium. TRH at 100 pg/ml was ineffective in hyperosmotic medium. Somatostatin (300 rig/ml) inhibited prolactin synthesis and release in hyposmotic medium, but inhibited only release in hyperosmotic medium. These results indicate that there may be a complex regulation of the prolactin cells in this teleost. The in vitro responses of teleost prolactin cells to various potential mediators resemble those seen in other vertebrates: however, the differences in detail may have special adaptive significance for the teleost. (Tilapia
Prolactin is considered to be an important osmoregulatory hormone in most euryhaline teleosts (cf. Bern, 1975). In such fish in fresh water, prolactin is actively secreted and enhances ion conservation. However, in a seawater environment, where such conservation would be disadvantageous, the prolactin cells are much less active (cf. Holmes and Ball, 1974; Schreibman and Holtzman, 1975). The regulation of prolactin cell activity in these teleosts may involve a direct effect of ambient osmotic pressure, since these cells have been shown to be more active in low than in high osmotic pressures in vitro in
Poecilia latipinna and Anguilla anguilla (Ingleton et al., 1973) and in Tilapia mossambica (Zambrano et al., 1974; Nagahama et al., 1975). However, the re-
sults of hypothalamic lesions, pituitary transplantations and treatment with such drugs as reserpine, 6hydroxydopamine (6OHDA) and L-P,3-4-dihydroxyphenylalanine (L-dopa) indicate that an inhibitory, possibly catecholaminergic, hypothalamic regulation of prolactin cells exists in at least some teleosts (Ball and Baker, 1969; Olivereau, 1971, 1975; Ball et al., 1972; Peter, 1973; Peter and McKeown, 1974; Zambrano, 1975; Wigham and Ball, 120
Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 00166480
TELEOST
PROLACTIN
1976). Furthermore, in vitro studies have shown that the prolactin cells of P. Zutipinna (Wigham and Ball, 1974; Wigham et al., 1975) and T. mossambica (Nagahama et al., 1975) are inhibited from releasing their hormone by the addition of dopamine to the medium. Thus, the postulated hypothalamic inhibition may involve dopaminergic fibers. Recent experiments with the incubation of mammalian pituitaries or prolactin cells have shown that these cells alter their activity in response to a number of factors including catecholamines (Birge et al., 1970; Macleod and Lehmeyer, 1972, 1974; Shaar and Clemens, 1974), somatostatin (Vale et al., 1974; Grant et al., 1974; Drouin et al., 1976)) thyrotropin-releasing hormone (TRH) (Morin et al., 1975; Smith and Convey, 1975; and others), insulin and human chorionic somatomammotropin (Shenai and Wallis, 1975) and medium osmotic pressure (Labella er al., 1975). Therefore, in our present studies we have incubated the prolactin cell region, the rostral pars distalis (RPD), of Sarotherodon mossambicus (Tilapia mossambica) pituitaries with several agents, both to examine the possible factors which regulate teleost prolactin cells and to compare the results with those obtained from mammalian and other nonmammalian vertebrates. MATERIALS
AND METHODS
Incubation S. mossambicus has been referred to in previous publications as T. mossambica. However, this teleost has been re-named (see Thys van den Audenaerde, 1968; Trewavas, 1973). In this and future publications, the new nomenclature will be used. Fish 9-14 cm long were obtained from Hawaii and maintained, together with a breeding population, at 27 -+ 3” under natural lighting conditions in a freshwater pond at the University of California, Berkeley. Pituitaries were removed and placed in a medium made up in Sarurhero&~~ bicarbonate-Ringer with glucose (500 mg/liter), glutamine (290 mgiliter) and Eagle’s (1959) minimum essential medium organic constituents (MEM: Bio-Rad, Richmond, Calif.) for dissection of the RPD region. Two milliliters of SOxMEM were
CELLS
121
added to 98 ml Ringer solution. The Ringer solution isotonic to freshwater Sarotherodon plasma (approximately 320 mOsm/kg in this species) consisted (in mEq/liter) of NaCl, 135; KC], 2.35; CaCl,, 4.2; MgSO,, 2.8; KH2P04, 1.25; NaHCO,, 25. The pH of the medium was 7.25 after gassing for 10 min. Media of different osmotic pressures were prepared by appropriate changes in NaCl concentration. The RPD, containing prolactin and ACTH cells, were preincubated at 27 2 1” in medium under an atmosphere of 95% 045% CO,. During this and the ensuing incubation, the medium was continuously mixed on a gyrotory platform. After 1 hr the tissues were transferred to a multicompartment culture tray, with two RPD in each of 12 compartments containing 0.2 ml of medium. This final medium included 13.5 LLyml of 4,5-3H-L-leucine (sp act of stock, 45-60 Ci/mmole, concentration 0.5 mCi/ml) (Schwarz-Mann, USA) for incorporation into newly synthesized prolactin. Six compartments were used for control and six for experimental media. The glands were incubated for 18 hr under the same conditions as for preincubation. At termination of incubation the tissues and media were separated for subsequent disc gel electrophoresis.
Gel Electrophoresis The use of polyacrylamide disc gel electrophoresis for the measurement of teleost pituitary prolactin has previously been validated for Anguilla (Knight et al., 1970), Poecilia latipinna (Ingleton ef al., 1973), Salmo gairdneri (Baker and Ingleton, 1975) and for Gillichthys mirabilis and Tilapia mossambica (Nagahama et al., 1975). Polyacrylamide gels were prepared in 3-mm (Ld.) glass tubes from the solutions described by Davis (1964). Each gel consisted of a 5.5-cm running gel surmounted by a l-cm large-pore stacking gel. RPD were homogenized, two per tube, in 0.2 ml of ice cold 1: 15 Tris-glycine buffer (Davis, 1964). The homogenates were kept on ice for 30 min, after which 0.2 ml of large-pore gel solution without sucrose was added to each homogenate. After mixing, the solutions were pipetted into the space above each stacking gel and then allowed to polymerize. Each sample of medium (0.2 ml) was mixed with an equal volume of large-pore gel solution without sucrose, and the mixture was pipetted onto the stacking gels for polymerization. Fifty microliters of 5 x lo-% bromophenol blue tracking dye were pipetted onto each sample gel, and the remaining space was filled with 1:l Tris-glycine electrode buffer. The gel tubes were placed in the electrophoresis apparatus, which was filled with 1: 1 Tris-glycine buffer. The apparatus included a refrigerating water jacket to keep the gels at about 6”, since this was found to produce better band definition. The current was regulated at 1.5 mA/tube supplied from a Buchler 3-l 155 constant current, high voltage power pack. The run was ended
122
WIGHAM,
NISHIOKA
AND
BERN
facilitate the detection of an expected response or to establish the effect of osmotic pressure upon the response to a drug.
when the tracking dye reached an etched mark on the glass tube. The gels were removed, the location of the tracking dye was permanently marked with india ink, and the gels were stained overnight in a solution of 0.1% Coomassie brilliant blue (CBB) in methanol: acetic acid:water (2:1:7). Destaining was carried out in a Bio-Rad diffusion destainer using staining fluid without CBB. After destaining, the gels were stored in 7% acetic acid. The densities of the prolactin bands in the gels were measured on a Photovolt densitometer. The radioactivity of the tritium within the prolactin band was measured by dissolving the excised gel bands in 0.1 ml of 30% H,O,. The solution was mixed with 10 ml of scintillation fluid (8 g BBOT, 160 g napthalene, 1200 ml toluene, 800 ml ethoxyethanol). The mixture was allowed to equilibrate in a refrigerator for 3 days, and the radioactivity was then measured on a Packard Model 3330 liquid scintillation counter.
RESULTS
The percentage release of both total (densitometrically measured) and newly synthesized (tritiated) prolactin by the control tissue was greater by 40 and 41%, respectively, in the hyposmotic (300 mOsm/kg) medium than in the hyperosmotic (360 mOsm/kg) medium. Thus, changes in ambient osmotic pressure alone are sufficient to alter the activity of the prolactin cells. This result agrees with earlier reports on Sarotherodon by Zambrano et al. (1974) and Nagahama et al. (1975). Dopamine at 1 pg/ml inhibited release of both total and newly synthesized prolactin Calculations into an otherwise stimulatory hyposmotic The densitometric measurements of the prolactin medium (Table 2) (cf. Nagahama et al., bands were used to calculate prolactin release into the medium as a percentage of the total present in 1975). However, it did not appear to alter RPD plus medium. Release of newly synthesized pro- prolactin synthesis during the incubation, lactin into the medium was separately calculated as since no significant change in specific activthe percentage of 3H counts present in the tissue plus ity was observed (Table 2). Octopamine at medium. Specific activity of the prolactin was estieither 5 pglml or 10 rig/ml had no effect on mated as the sum of the 3H counts present in tissue release or specific activity in hyposmotic plus medium divided by the sum of densitometric units in the bands from RPD plus those from medium. medium. Addition of y-amino-n-butyric The data were analysed for statistical significance acid (GABA) (100 rig/ml) to a slightly using Student’s t test. hyperosmotic (340 mOsrn/kg) medium was also ineffective in altering release or spePharmacological Studies cific activity (Table 2). Various drugs were tested for their ability to alter Cortisol (1 pg/ml) inhibited total and synthesis and release of prolactin by the RPD (Table 1). The osmotic pressure of the medium used for each tritiated prolactin release into the stimulapharmacological experiment was chosen either to tory hyposmotic medium, but affected only TABLE DRUGS
TESTED
Drug Dopamine (3-hydroxytyramine) D, L-Octopamine HCl
1
IN THE INCUBATION
OF Sarotherodon
RPD
Supplier HCI
y-Amino-n-butyric acid (GABA) Cortisol (A grade) Estradiol- 178 Thyrotropin-releasing hormone (porcine), synthetic (TRH) Somatostatin (ovine), synthetic Albumin (bovine serum), fraction
Sigma (USA) Sigma (USA) Sigma (USA) Calbiochem (USA) Calbiochem (USA) Beckman (USA)
V
Beckman (USA) Calbiochem (USA)
Concentration 1 pg/ml 5 pg/ml 10 rig/ml 100 rig/ml 1 pg/ml 100 rig/ml 100 rig/ml 100 pg/ml 300 rig/ml 1 pg/ml
5.27 2.64 5.27 1.03 2.64 3.67 2.77 2.77 1.83 1.67
x lo+ M x lo-” M x
lO-8
M
x lO-s M x lo+ M x lo-‘M x lo-‘M
x lo-r0 M x lo-’ M x IO-* M
340
(6)
(6) 46.1 k2.61
(9
45.7 k3.29
(9
79.9 k2.4.5 (3) 57.2 k2.94
(6)
57.7 2 3.47 (9 75.4 kO.49 (3) 61.8 +- 1.21
Exp
76.2* k2.24
Cont
(9
54.4 kl.78
82.2* 21.52 (9 84.4 24.2 (3) 63.4 k4.0 (9
Cont
(9
55.3 ~1.52
83.5 k4.0 (3) 66.5 k4.77 (9
(6)
19.9 21.63
Bxp
Newly synthesized prolactin release (%)
ON PROLACTIN CELL ACTIVITY"
a Results are given as the mean plus or minus one standard error; n in parentheses. * P < 0.01 = significance of difference between control (Cont) and experimental (Exp) values.
100 rig/ml
1.03 x 10eB M
GABA
300
5.27 x IO-‘M
10 rig/ml
300
2.64 x 10m5M
5 &ml
300
Octopamine
5.27 x 10mBM
1 pg/ml
Concentration
Dopamine
Neurotransmitter
Medium osmolality (mOsm/kg)
Total prolactin release (%)
TABLE 2 EFFECTS OF NEUROTRANSMITTERS: DOPAMINE, OCTOPAMINE AND GABA,
(9
(9
(6)
2475 2162
(6) 2298 1148
1946 + 437 (3) 1508 +74
(6)
1443 2100
Exp
1539 2609 (3) 1329 -c92
(6)
1342 I?104
Cont
Sp act
E
8
5 2
;;i ii a -I 2 $
124
WIGHAM,
NISHIOKA
the former in the hyperosmotic medium (Table 3). This suggests that the cortisolinduced inhibition of newly synthesized prolactin release was insufficient to appear against a background of inhibition by medium osmotic pressure. The specific activity of the prolactin band was unaffected by cortisol in the hyposmotic medium, but was increased in the hyperosmotic medium (Table 3). The latter experiment was repeated, with similar results. Estradiol-17/3 at 100 rig/ml did not significantly alter prolactin release into a slightly inhibitory (340 mOsm/kg) medium (Table 3), whereas specific activity was increased. Thus, estradiol-17P, as well as cortisol, may increase prolactin synthesis, but not release, into a hyperosmotic medium. It is possible that an increase in release may occur subsequent to stimulation of synthesis by estradiol-17P, and that 18 hr is too short for such an effect to become measurable by gel electrophoresis. However, in some preliminary 5-day incubations with estradiol17p no stimulation of prolactin release was observed. TRH at 100 rig/ml inhibited release of prolactin into hyposmotic medium, but not into hyperosmotic medium (Table 4). Thus, an already inhibitory medium may be capable of masking the action of TRH on the cells. Specific activity of the prolactin band was not significantly altered in either experiment (Table 4). A low concentration of TRH (100 pg/ml) altered neither prolactin release nor specific activity in the slightly hyperosmotic (340 mOsm/kg) medium (Table 4). Thus, TRH is only effective at higher concentrations. Somatostatin (300 rig/ml) markedly inhibited prolactin release into both the hyperosmotic and the hyposmotic media (Table 4). The specific activity of the prolactin band was also reduced in the latter medium, but the result in the hyperosmotic medium was less clear (Table 4). In one experiment, somatostatin appeared to increase prolactin specific activity, even
AND
BERN
though it strongly inhibited release. However, the specific activity of the control tissues was very low compared with that of controls of the other experiments. Also, the results of two similar experiments with hyperosmotic medium gave no indication that somatostatin altered prolactin specific activity. Therefore, we feel that the original result may be anomalous. The ability of somatostatin to reduce hormone release into both hyposmotic and hyperosmotic media suggests that it is a potent inhibitor of prolactin secretion, although a lack of effect on synthesis in the latter medium indicates that the drug is more effective on hormone release than on synthesis. Bovine serum albumin (BSA) at 1 pg/ml had no effect on release or synthesis of prolactin in hyposmotic medium (Table 4). Thus, the simple addition of a large molecule to the medium does not, by itself, appear to alter the activity of the prolactin cells. DISCUSSION
The ability of prolactin cells actively to secrete hormone in vitro has been established in several species of teleost (Ingleton et al., 1973; Zambrano et al., 1974; Nagahama et al., 1975; Baker and Ingleton, 1975; Wigham et al., 1975). These reports have also described the stimulatory action of low osmotic pressure of the medium on prolactin cells. Our present experiments confirm this for S. mossambicus, since incubation in hyposmotic medium produced consistently higher levels of prolactin release than in hyperosmotic medium. Dopamine has been implicated in the regulation.,of prolactin cells in P. latipinna (Wigham and Ball, 1974; Wigham et al., 1975), A. anguilla (Olivereau, 1975; P. M. Ingleton and B. I. Baker, unpublished data) and T. mossambica (Nagahama et al., 1975). Our results confirm the inhibitory effect of dopamine .on prolactin release by Sarotherodon (Tifupia) RPD. In addition,
3.67 x 10-r M
340
360
2.76 x IO-“M
1 pg/ml
100 rig/ml
300
2.76 x 10e6 M
1 pg/ml
Concentration
49.6 21.62 (6)
55.3* ?I.2 (6)
(6)
4 1.36
78.0*
Cont
75.8 23.91
(6)
(6)
(6)
92.9* 21.01 (6) 75.5 22.95
Cont
73.0 +3.48 (5)
85.7 22.32 (6) 64.2 23.95 t.9
Bxp
Newly synthesized prolactin release (%I
51.2 21.22
66.2 k2.72 (6) 47.4 ?I.84 (6)
Exp
a Results are given as the mean plus or minus one standard error; n in parentheses. * P < 0.01. ** P < 0.001 = significance of difference between control (Cont) and experimental (Exp) values.
Estradiol- 17fi
Cortisol
Hormone
Medium osmolality (mOsm/kg)
Total prolactin release (%)
TABLE 3 EFFECTS OF THE STEROIDS, CORTISOL AND ESTRADIOL-17/3, ON PROLACTIN CELL ACTIVITY”
1470** 24.5 (6)
1869 263 (6) 957** 268 (6)
Cont
Sp act
1835 215 (5)
1843 538 (5)
(6)
1923 249
Exp
.
:: cn
5 2 8
2 ?
6 2
1.83 x IO-‘M
300 rig/ml
1.67 x lo-*
1.83 x IO-‘M
300 rig/ml
1 pg/ml
1.83 x IO-‘M
300 rig/ml
2.77 ST IO-“‘M
100 pg/ml
1.83 x IO-‘M
2.17 x lo-’
100 rig/ml
300 rig/ml
2.77 x IO-‘M
100 rig/ml
M
M
HYPOPHYSIOTROPINS
Concentration
OF THE
300
360
360
360
300
340
360
300
Medium osmolality (mOsm/kg)
TRH AND
TABLE
4
values
(6)
(6) (6)
31.7** k4.28 (6) 51.5** k5.10
54.4** 26.2 (5)
91.2** al.64 (6)
(6)
50.7 k2.5
(6)
27.2 23.28
(6)
83.2** k2.82
Cont
73.6 23.66
(Exp)
ACTIVITY”
1945 *131
(6)
(6)
302*” *50 C.9 2034 4141 (6) 1099 ,134
(6)
1834 ?I43
1098 240
(6)
2008 275
1312 +-IO0 (5)
(6)
381 230
(6)
(6) l271*” ?56 (6)
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zi a i$
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