GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
39, 244-246
(1979)
NOTE Somatostatin
Inhibition
of Teleost
Growth
Hormone
Secretion
Pituitary glands of a teleost, tilapia (Sarorherodon mossambicus). were incubated in vitro in the presence or absence of synthetic somatostatin. After a 4-hr incubation period, the growth hormone (GH) concentration of the incubation medium was determined using a highiy specific homologous radioreceptor assay. The presence of somatostatin in the incubation medium at concentrations of 0.1 to 2.0 &ml produced a dose-dependent inhibition of the spontaneous release of GH. These results suggest that GH secretion in teleosts may be influenced by a somatostatin-like peptide which acts to suppress the release of GH from the pituitary.
Little is known concerning the control of tin would influence GH secretion from a teleost pituitary in an in vitro test system. growth hormone (GH) secretion in teleosts. A role for the hypothalamus in the control MATERIALS AND METHODS of GH secretion has frequently been Pituitary glands were obtained from adult tilapia, suggested, but has not been defined (Peter, Sarothemdon mossambicus, maintained in a heated 1973). Several studies have shown that the outdoor freshwater pool at the University of California GH cells of teleosts are capable of some at Berkeley. Fish were killed by decapitation, the and placed spontaneous activity and continue to syn- pituitaries removed. halved longitudinally, in petri dishes (12 pituitary halves per dish) containing thesize and secrete GH in vitro (Poecilia a bicarbonate-Ringer incubation medium of glucose latipinna: Ingleton et al., 1973; Anguilla (500 mg/liter), glutamine (290 mg/liter), and Eagle’s anguilla and Salmo gairdneri: Baker and minimum essential medium organic constituents Ingleton, 1973). (MEM: Bio-Rad, Richmond, Calif.). Two milliliters of 50 x MEM were added to 98 ml Ringer solution conThe tetradecapeptide somatostatin (growth hormone-release inhibiting factor) stituted as (in meq/liter) NaCI, 135; KCI, 2.35: Car&, 4.2; MgS04, 2.8; KHzP04, 1.25; NaHCOn, 25; and is a potent inhibitor of the spontaneous re- gassed for 10 min with 95% 02/5% CO,. Following a prelease of GH from mammalian pituitaries incubation period of 1 hr, the incubation medium was both in vivo and in vitro (for recent reviews replaced with 1 ml of medium alone (control) or con(Sigma) at concentrasee Vale et al., 1977; Reichlin, 1976). taining synthetic somatostatin tions of 0.1 to 2 Kg/ml. During the preincubation and Dubois et al. (1974), using an immunoperiods, the petri dishes were continuously cytochemical technique, demonstrated the incubation swirled on a gyratory platform at 27 k 1” under an presence of somatostatin-like activity in the atmosphere of 95% OZ/5% CO*. After an incubation pebrain, pars distalis, and pars nervosa of the riod of 4 hr, the incubation medium was removed from rainbow trout. Radioimmunoassays for the petri dishes and stored frozen at -20”. GH released into the incubation medium was meamammalian somatostatin have demonsured using a highly specific homologous radiorecepstrated the presence of somatostatin-like tor assay utilizing the binding of lZ~l-labelled tilapia activity in brain extracts of a catfish, an GH to liver microsomal membrane fractions (Fryer, elasmobranch (Torpedo), and hagfish (Vale 1979). Livers were obtained from adult tilapia collected et al., 1976), and in the spiny dogfish and catfish (Crim et al., 1978). In the light of from the Wahiawa Reservoir, Hawaii. The livers were frozen and transported on dry ice to the Unithese immunological observations of rapidly versity of California at Berkeley where they were somatostatin-like activity in fish brain ma- stored at -20” until fractionation. For the binding asterial, it was of considerable interest to de- says tilapia liver membranes (300 pg of protein) were incubated in the presence of 50,000 cpm Y-labelled termine if synthetic mammalian somatosta244 00166480/79/100244-03$01.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
245
NOTE Incubation
tilapia GH at 4” for 18 hr in the presence of dilutions of unlabelled tilapia GH or 100 ~1 of pituitary incubation medium in a final volume of 0.5 ml. Assays of the pituitary incubation medium were performed in triplicate with 100 ~1 of fresh incubation medium added to the standards. Separation of bound from free labelled tilapia GH was achieved by the addition of 1 ml of assay buffer (25 mM Tris-HCl, 10 mM CaCl,, 0.5% BSA, pH 7.6) followed by centrifugation at 2OOOg for 20 min and decantation of the supernatant. The ““I-labelled tilapia GH to the membrane pellet was counted in an automatic gamma counter (Packard Auto-Gamma scintillation spectrometer). Statistical differences were assessed using a oneway analysis of variance followed by Duncan’s multiple range test.
6.3 I
Medium
12.5 ,
C$
25
50
100
‘“c”
RESULTS
The results of an experiment in which dilutions of pituitary incubation medium were tested for the ability to displace lz5Tlabelled tilapia GH from the tilapia liver GH-binding site are presented in Fig. 1. In this receptor assay dilutions of pituitary incubation medium produced a competition curve parallel to authentic tilapia GH (Farmer et al., 1976) standards. The effect of increasing concentration of somatostatin in the incubation medium on the spontaneous release of GH from tilapia pituitaries during a 4-hr incubation period is summarized in Fig. 2. The data from three separate experiments have been pooled and the GH concentration of the incubation medium in the presence of somatostatin has been expressed as a percentage of the GH concentration in the incubation medium in the absence of somatostatin. Somatostatin in the incubation medium at concentrations of 0.1 to 2.0 pg/ml produced a dosedependent inhibition of the spontaneous release of GH from tilapia pituitaries. The highest concentration of somatostatin tested (2.0 &ml) produced an 80% inhibition of GH release compared with that observed from pituitaries incubated m the absence of somatostatin. DISCUSSION
Details of the development of the radioreceptor assay for tilapia GH and of the specificity of the tilapia liver GH-
a
1
30 50 Unlabelled FIG. 1. Competition tilapia GH and unlabelled tions of pituitary culture binding sites on a tilapia fraction.
I loo
L
250
tGH (ns,
curves between ‘~JI-labelled tilapia GH (circles) or dilumedium (triangles) for GHliver microsomal membrane
100
80
-0 ?I 60 81
b
+I
2
3
40
zp
lhn b
. .
20
Somatortotin
I
7
2
.s
l.0
20
$@rnl)
FIG. 2. Effect of increasing somatostatin on the spontaneous the culture medium during a 4-hr percentage of that released in the statin. Each histogram denotes a, significantly lower (P < 0.05) b, significantly lower than control and 0.1 wg/ml somatostatin (P
AND
COMPARATIVE
ENDOCRINOLOGY
39, 244-246
(1979)
NOTE Somatostatin
Inhibition
of Teleost
Growth
Hormone
Secretion
Pituitary glands of a teleost, tilapia (Sarorherodon mossambicus). were incubated in vitro in the presence or absence of synthetic somatostatin. After a 4-hr incubation period, the growth hormone (GH) concentration of the incubation medium was determined using a highiy specific homologous radioreceptor assay. The presence of somatostatin in the incubation medium at concentrations of 0.1 to 2.0 &ml produced a dose-dependent inhibition of the spontaneous release of GH. These results suggest that GH secretion in teleosts may be influenced by a somatostatin-like peptide which acts to suppress the release of GH from the pituitary.
Little is known concerning the control of tin would influence GH secretion from a teleost pituitary in an in vitro test system. growth hormone (GH) secretion in teleosts. A role for the hypothalamus in the control MATERIALS AND METHODS of GH secretion has frequently been Pituitary glands were obtained from adult tilapia, suggested, but has not been defined (Peter, Sarothemdon mossambicus, maintained in a heated 1973). Several studies have shown that the outdoor freshwater pool at the University of California GH cells of teleosts are capable of some at Berkeley. Fish were killed by decapitation, the and placed spontaneous activity and continue to syn- pituitaries removed. halved longitudinally, in petri dishes (12 pituitary halves per dish) containing thesize and secrete GH in vitro (Poecilia a bicarbonate-Ringer incubation medium of glucose latipinna: Ingleton et al., 1973; Anguilla (500 mg/liter), glutamine (290 mg/liter), and Eagle’s anguilla and Salmo gairdneri: Baker and minimum essential medium organic constituents Ingleton, 1973). (MEM: Bio-Rad, Richmond, Calif.). Two milliliters of 50 x MEM were added to 98 ml Ringer solution conThe tetradecapeptide somatostatin (growth hormone-release inhibiting factor) stituted as (in meq/liter) NaCI, 135; KCI, 2.35: Car&, 4.2; MgS04, 2.8; KHzP04, 1.25; NaHCOn, 25; and is a potent inhibitor of the spontaneous re- gassed for 10 min with 95% 02/5% CO,. Following a prelease of GH from mammalian pituitaries incubation period of 1 hr, the incubation medium was both in vivo and in vitro (for recent reviews replaced with 1 ml of medium alone (control) or con(Sigma) at concentrasee Vale et al., 1977; Reichlin, 1976). taining synthetic somatostatin tions of 0.1 to 2 Kg/ml. During the preincubation and Dubois et al. (1974), using an immunoperiods, the petri dishes were continuously cytochemical technique, demonstrated the incubation swirled on a gyratory platform at 27 k 1” under an presence of somatostatin-like activity in the atmosphere of 95% OZ/5% CO*. After an incubation pebrain, pars distalis, and pars nervosa of the riod of 4 hr, the incubation medium was removed from rainbow trout. Radioimmunoassays for the petri dishes and stored frozen at -20”. GH released into the incubation medium was meamammalian somatostatin have demonsured using a highly specific homologous radiorecepstrated the presence of somatostatin-like tor assay utilizing the binding of lZ~l-labelled tilapia activity in brain extracts of a catfish, an GH to liver microsomal membrane fractions (Fryer, elasmobranch (Torpedo), and hagfish (Vale 1979). Livers were obtained from adult tilapia collected et al., 1976), and in the spiny dogfish and catfish (Crim et al., 1978). In the light of from the Wahiawa Reservoir, Hawaii. The livers were frozen and transported on dry ice to the Unithese immunological observations of rapidly versity of California at Berkeley where they were somatostatin-like activity in fish brain ma- stored at -20” until fractionation. For the binding asterial, it was of considerable interest to de- says tilapia liver membranes (300 pg of protein) were incubated in the presence of 50,000 cpm Y-labelled termine if synthetic mammalian somatosta244 00166480/79/100244-03$01.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
245
NOTE Incubation
tilapia GH at 4” for 18 hr in the presence of dilutions of unlabelled tilapia GH or 100 ~1 of pituitary incubation medium in a final volume of 0.5 ml. Assays of the pituitary incubation medium were performed in triplicate with 100 ~1 of fresh incubation medium added to the standards. Separation of bound from free labelled tilapia GH was achieved by the addition of 1 ml of assay buffer (25 mM Tris-HCl, 10 mM CaCl,, 0.5% BSA, pH 7.6) followed by centrifugation at 2OOOg for 20 min and decantation of the supernatant. The ““I-labelled tilapia GH to the membrane pellet was counted in an automatic gamma counter (Packard Auto-Gamma scintillation spectrometer). Statistical differences were assessed using a oneway analysis of variance followed by Duncan’s multiple range test.
6.3 I
Medium
12.5 ,
C$
25
50
100
‘“c”
RESULTS
The results of an experiment in which dilutions of pituitary incubation medium were tested for the ability to displace lz5Tlabelled tilapia GH from the tilapia liver GH-binding site are presented in Fig. 1. In this receptor assay dilutions of pituitary incubation medium produced a competition curve parallel to authentic tilapia GH (Farmer et al., 1976) standards. The effect of increasing concentration of somatostatin in the incubation medium on the spontaneous release of GH from tilapia pituitaries during a 4-hr incubation period is summarized in Fig. 2. The data from three separate experiments have been pooled and the GH concentration of the incubation medium in the presence of somatostatin has been expressed as a percentage of the GH concentration in the incubation medium in the absence of somatostatin. Somatostatin in the incubation medium at concentrations of 0.1 to 2.0 pg/ml produced a dosedependent inhibition of the spontaneous release of GH from tilapia pituitaries. The highest concentration of somatostatin tested (2.0 &ml) produced an 80% inhibition of GH release compared with that observed from pituitaries incubated m the absence of somatostatin. DISCUSSION
Details of the development of the radioreceptor assay for tilapia GH and of the specificity of the tilapia liver GH-
a
1
30 50 Unlabelled FIG. 1. Competition tilapia GH and unlabelled tions of pituitary culture binding sites on a tilapia fraction.
I loo
L
250
tGH (ns,
curves between ‘~JI-labelled tilapia GH (circles) or dilumedium (triangles) for GHliver microsomal membrane
100
80
-0 ?I 60 81
b
+I
2
3
40
zp
lhn b
. .
20
Somatortotin
I
7
2
.s
l.0
20
$@rnl)
FIG. 2. Effect of increasing somatostatin on the spontaneous the culture medium during a 4-hr percentage of that released in the statin. Each histogram denotes a, significantly lower (P < 0.05) b, significantly lower than control and 0.1 wg/ml somatostatin (P
