- Email: [email protected]
Stimulation of corticosterone release in the fowl by recombinant DNA-derived chicken growth hormone
GENERAL
AND
COMPARATIVE
Stimulation
ENDOCRINOLOGY
69, 128132 (1988)
of Corticosterone Release in the Fowl by Recombinant DNA-Derived Chicken Growth Hormone A. CHEUNG,~ T.R.
HALL,AND
S. HARVEY
Wolfson Institute, University of Hull, Hull HU6 7RX, England Accepted September 14, 1987 The effects of recombinant DNA-derived chicken growth hormone (rcGH) on plasma corticosterone in young broiler cockerels were investigated. A single injection of 200 uglkg rcGH significantly increased plasma corticosterone concentrations 2 hr (but not 20 or 40 mm) after treatment. Administration of 10 or 100 kg/kg rcGH also signiticantly increased plasma corticosterone levels after 2 hr, with the higher dose eliciting greater responses. Chronic treatment with seven daily injections of the same doses of rcGH gave similar increases in plasma concentrations of corticosterone. No obvious difference in magnitude of plasma corticosterone was observed between acute and chronic exposure to rcGH. In a further experiment in which serial blood sampling was performed after a single injection or five daily injections of vehicle or 200 ug/kg rcGH, there were significant increases in plasma corticosterone concentrations 40 min after acute rcGH treatment and 40 and 80 min after chronic treatment when compared with plasma corticosterone concentrations of vehicleinjected controls. However, the increases could have incorporated a stress response due to repeated sampling because the control birds also showed elevated plasma corticosterone concentrations. The corticosterone response did not diminish with repeated GH challenge. These results suggest that GH may play a role in the acute regulation of corticosterone secretion in intact chickens. 0 1988 Academic Press. Inc.
Adrenal corticosterone secretion in birds is under the control of a hypothalamo (corticotrophin releasing factor, CRF)hypophyseal (adrenocorticotrophin, ACTH)-adrenal axis (Stainer and Holmes, 1969; Salem et al., 1970). It is becoming increasingly apparent that many other signals, such as catecholamines (Freeman and Manning, 1979; Rees et al., 1985) and serotonin (Cheung et al., 1987), participate in the regulation of corticosteroidogenesis in birds. Recent studies with CRF have shown that this peptide may also have effects on growth hormone (GH) secretion since CRF reduces spontaneous GH secretion in the rat when injected in vivo (Rivier and Vale, 1984) and stimulates release of somatostatin from the central nervous tissues in vitro
(Peterfreund and Vale, 1982). Conversely, it is feasible that GH may affect adrenal function, either directly or indirectly, though there is a paucity of data on this subject. Implantation of a GH-secreting tumor enhances corticosterone response to ACTH stimulation in intact rats (Coyne et al., 1981) and GH administration increases corticosterone secretion in hypophysectomized rats (Colby et al., 1973; Kramer et al., 1977). Recently, it has been shown that GH replacement increases ACTH induced maxi,mal corticosterone production from adrenocortical cells i.solated from hypophysectomized chickens (Carsia et al., 1985). The present experiments were designed to test whether GH could affect corticosterone levels in normal, intact chickens.
’ To whom correspondence should be addressed at Department of Anatomy, University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong.
MATERIALS
One-day-old Ross broiler cockerets were pnrchased 128
0016-6480188 $1.50 Copyright All rights
Q 1988 by Academic Press, Inc. of reproduction in any form reserved.
AND METHOD
GH
EFFECTS
ON
and raised under conditions of continuous light (to prevent photoperiodic effects on hypothalamic, pituitary, and adrenal activity) with food and water available ad /i&urn. Three experiments were performed: Experiment 1. Six-week-old birds were injected once intramuscularly (im) either with 200 kg/kg body weight recombinant DNA-derived chicken growth hormone (rcGH), a polypeptide differing from native chicken GH only in its terminal amino acid (Souza et al., 1984), or with 1 ml/kg vehicle (0.25% NaHCO,, 0.2% mannitoi, pH 8.5). Blood samples were withdrawn from the brachial vein 20.40, and 120 min later. Experiment 2. Five-week-old cockerels were injected im once daily with 10 or 100 Kg/kg rcGH or with vehicle for 8 days. On the last day, a further three groups of untreated birds were also given a single injection of rcGH or vehicle. Blood samples were collected from a wing vein immediately before and 2 hr following the last injection. Experiment 3. Five-week-old cockerels were injected im daily for 5 days with either 200 wgikg rcGH or with vehicle. On the day of the last injection, a blood sample was withdrawn immediately prior to, and 5, 10, 20, 40, 80. 120, and 240 min after the injection. Further groups of birds were bled in a similar fashion after only a single injection of rcGH or vehicle. Plasma was obtained from the blood samples after centrifugation and separation and stored at - 20” until assayed. Corticosterone concentrations in plasma were determined in duplicate by specific radioimmunoassay (Harvey et al.. 1980). The assay has a minimum detection limit of 30 pmoliliter and an intraassay coefficient of variability of 3.2%. All results are expressed as means 4 SEM. Statistical differences in the results were determined by Student’s t test, with a levei of significance of P < 0.05.
RESULTS Experiment 1. A significant rise (P < 0.01) in plasma corticosterone concentrations was observed 120 min after a single injection of rcGH (Fig. 1). Although the mean plasma corticosterone concentration in the rcGH-treated birds appeared to be higher at an earlier time (40 min after injection), it was not significantly different from that of the control group. Experiment 2. Figure 2 shows that 2 hr after acute treatment with 10 or 100 pgikg rcGH, there were significant increases (P < 0.001) in plasma corticosterone levels compared with preinjection values. For the higher dose, there was a more than fivefold increase in plasma corticosterone concen-
129
CORTICOSTERONE
Time after
injection
FIG. 1. Effects of an im injection of 200 wgikg recombinant DNA-derived chicken GH (r&H) or vehicle on plasma corticosterone concentrations in 5week-old cockerels. Results are means i: SEM (~1varied between 5 and 7). *P < 0.01 compared with vehicle-treated control.
tration. Chronic treatment wit rcGH increased (P < 0.05) plasma corticosterone relative to preinjection level but was not significantly different from that of vehicle control. The higher dose of rcGH promoted a more marked increase in corticosterone response (P < 0.01 compared with preinjection level) which was also significantly (P < 0.05) higher than that of vehicle-injected control. There is no obvious difference in magnitude of plasma corticosterone with respect to the number of injections. In addition, the preinjection levels of corticosterone did not differ significantly between vehicle and rcGH-injected birds. Experiment 3. Figure 3 shows the plasma corticosterone concentrations withes the or the first 80 min following a singie ( rcGW fifth (chronic) injection of 200 or vehicle. In both acute and chronic experiments, the stress imposed by repeated sampling caused the plasma ~~~~cos%~ro~~ concentrations of vehicle-injected control < O.OS), group to increase significant1 after the reaching a maximum level 20 injection. Thereafter, the cor%~~os%~rone concentrations gradually dec~~~~~ but re-
CHEUNG.
HALL,
AND
HARVEY
of rcGH can affect plasma corticosterone concentration in immature chickens. The q 2h after injection action of rcGH in intact chickens is transitory and relatively modest, and the corticosterone response appears to revert to normal within 24 hr since chronic treatment did *r” not create a higher basal level of corticosterone (Fig. 3). Corticosterone response in i birds is known to adapt to a number of chronic stressors (El-Halawani et al., 1973; Harvey and Phillips, 1982). The response to rcGH did not seem to habituate to repeated challenge since the corticosterone response was not blunted after eight daily injections of rcGH (Fig. 2). Similarly, corticosterone responses to handling and bleeding (Harvey and Phillips, 1982) and to daily exogenous 0 ACTH treatment (Freeman et al., 1979; Vehicle GH GH Vehicle OH lO/~glKg 1OOQglKg 1OlrglKg Rees et al., 1983) also do not show habituAcute treatment Chronic treatment ation. The purity of the GH preparation FIG. 2. Effects of acute (single injection) or chronic was >9.5% (Souza et al., 1984). The possi(eight daily injections) administration of vehicle or response was rcGH on plasma corticosterone concentrations in 5- bility that the corticosterone due to the stress of a possible endotoxin week-old cockerels. Blood samples were collected imcontaminant can be discounted since such mediately before and 2 hr after injection. Results show means i: SEM (N varied between 5 and 7). *P -C0.05, responses are rapidly onsetting and termi**P < 0.01, ***P < 0.001 compared with preinjection nated (Harvey et al., 1984), whereas the relevel. sponse to the GH preparation lasted for up to 2 hr (Fig. 2). Therefore, GH probably mained significantly (I’ < 0.05) higher than participates in the acute regulation of corthe preinjection level. A much greater ele- ticosterone secretion. vation in corticosterone concentrations was There were no apparent dose-dependent observed in the rcGH-treated birds. Cortieffects since the lowest dose of rcGH emcosterone concentrations were significantly ployed (10 kg/kg) was not much less effec(P < 0.01) higher than vehicle-injected tive than the highest dose (200 pg/kg). group 40 min after the acute rcGH treatThese doses were chosen to represent the ment. For chronically treated birds, cortiminimal with biological activity up to a costerone concentrations were found to be dose with pronounced activity (e.g., see significantly (P < 0.05) higher than vehicleSouza et al., 1984). The reason why there treated group both 40 and 80 min after the was no clear-cut dose-related effects may last injection. At 120 and 240 min, there be due to the experimental protocol. In the were no significant differences between ve- preliminary experiment (Fig. l), blood samhicle- and rcGH-treated animals. However, ples were removed at a lesser frequency there was a very large variability in cortithan the extended confirmatory experiment costerone levels in control birds, possibly (Fig. 3), and more frequently than in experdue to inadvertent stress (disturbance), and iment 2. Since handling and bleeding stress accordingly these data were rejected. markedly affect corticosterone rhythms (Harvey et al., 1984), these responses may DISCUSSION be superimposed on the responses to GH. Our data show that in vivo administration However, it is abundantly clear that in 0 pre-injection
GH EFFECTS
Time
ON CORTICOSTERONE
after
injection
131
(mh)
3. Effects of a single injection (a, acute response) or the fifth injection (b, chronic treatment) of vehicle (0) or 200 p,g/kg rcGH (0) into 6-week-old cockerels on plasma corticosterone concentrations. Results are means -+ SEM (n varied between 5 and 7). *P < 0.05, **P < 0.01 compared with vehicle-injected control; +P < 0.05 compared with preinjection concentration. FIG.
three separate experiments exogenous GH was able to increase circulating corticosterone concentrations in young cockerels. Administration of GH alone to hypophysectomized rats fails to maintain corticosterone secretion by adrenal tissue both in vivo (Colby et al., 1973; Kramer et al., 1977) and in vitro (Kramer et al., 1977). However, when given simultaneously with ACTH, the effects of ACTH on corticosterone production both in vivo (Colby et al., 1973; Kramer e2 al., 1977) and in vitro (Kramer et al., 1977) are augmented, indicating that GH acts synergistically with ACTH in stimulating corticosterone release. Although corticosteroidogenesis in chicken is regulated primarily by the CRFACTH-adrenal axis, the adrenal of the chicken differs from that of mammalian species in having a certain degree of autonomy so that plasma corticosterone does not decrease drastically after hypophysectomy (Carsia et al., 1985). In addition, unlike in
hypophysectomized rats, @El replacement alone can maintain and elevate the ACTHinduced maximal corticosterone pro from isolated adrenocortical cells from hypophysectomized chickens (Carsia et al., 1985). Our study has demonstrated for the first time that GH also stimulates carticosterone secretion in intact chickens. This is in agreement with the results from mammalian studies in which implantation of the GH-secreting tumor StW5 was shown to increase basal concentration of corticosterone and to enhance corticosterone response to ACTH and stress in intact rats (Coyne et al., 1981). The exact mechanism of action of GH on corticosterone release is not fully understood but it has been suggested that GII may act at the steroidogenic enzyme level by inhibiting the action of adrenal Sa-reductase, an enzyme involved in the degradation of corticosterone (Carsia eit al. I 1985). The control of corticosterone’ secre-
132
CHEUNG,
HALL,
tion is rather complex and involves a variety of neuronal and extra-neuronal signals (Harvey et al., 1984). Growth hormone may be one of the factors participating in this integrated regulatory process. ACKNOWLEDGMENTS This work was supported by a grant from Imperial Chemical lndustries, Billingham. The authors are grateful to Keith Langley of Amgen (Thousand Oaks, California) for the generous gift of rcGH. A. Cheung was supported by a fellowship from the Croucher Foundation.
REFERENCES Carsia, R. V., Weber, H., King, D. B., and Scanes, C. G. (1985). Adrenocortical cell function in the hypophysectomized domestic fowl: Effects of growth hormone and 3,5,3’-triiodothyronine replacement. Endocrinology 117, 928-933. Cheung, A., Hall, T. R., and Harvey, S. (1987). Serotoninergic regulation of corticosterone secretion in domestic fowl. J. Endocrinol., 113,159-165. Colby, H. D., Caffrey, J. L., and Kitay, J. I. (1973). Interaction of growth hormone and ACTH in the regulation of adrenocortical secretion in rats. Endocrinology 93, 188-192. Coyne, M. D., Alpert, L. C., Harter, K. C., and Nunez, A. (1981). Effect of growth hormonesecreting tumor StW5 on pituitary and adrenal gland function in rats. Horm. Res. 14, 3k46. El-Halawani, M. E., Waibel, P. E., Appel, J. R., and Good, A. L. (1973). Effects of temperature stress on catecholamines and corticosterone of male turkeys. Amer. J. Physiol. 224, 384-388. Freeman, B. M., and Manning, A. C. C. (1979). The effects of repeated injections of adrenaline on the response of the fowl to further alarm stimulations. Res. Vet. Sci. 27, 76-81. Freeman, B. M., Manning, A. C. C., and Flack, I. H. (1979). Habituation by the immature fowl in re-
AND
HARVEY
sponse to repeated injections of corticotrophin. Brit. Poult. Sci. 20, 391-399. Harvey, S., Merry, B. J., and Phillips, J. G. (1980). Influence of stress on corticosterone secretion in the duck (Anus plutyrhynchos). J. Endocrinof. 87, 161-171. Harvey, S., and Phillips, J. G. (1982). Adrenocortical responses of ducks to treadmill exercise. J. Endocrinol. 94, 141-146. Harvey, S., Phillips, J. G., Rees, A., and Hall, T. R. (1984). Stress and adrenal function. J. Exp. Zool. 232, 633-645. Kramer, R. E., Greiner, J. W., and Colby, H. D. (1977). Site of action of growth hormone on adrenocortical steroidogenesis in rats. Endocrinology 101, 297-303.
Peterfreund, R. A., and Vale, W. W. (1982). Ovine corticotropin releasing factor stimulates somatostatin secretion from cultured brain cells. Endocrinology 112, 1275-1278. Rees, A., Harvey, S., and Phillips, J. G. (1983). Habituation of the corticosterone response of ducks (Anus platyrhynchos) to daily treadmill exercise. Gen. Comp. Endocrinol. 49, 485-489. Rees. A., Harvey, S., and Phillips, J. G. (1985). Adrenergic stimulation of adrenocortical secretion in immature fowl. Comp. Biochem. Physiol. C 81, 387-389. Rivier, C., and Vale, W. (1984). Corticotropinreleasing factor (CRF) acts centrally to inhibit growth hormone secretion in the rat. Endocrinology 114, 2409-2411. Salem, W. N., Norton, H. W., and Nalbandov, A. V. (1970). A study of ACTH and CRF in chickens. Gen. Comp. Endocrinol. 14, 270-280. Souza, L. M., Boone, T. C., Murdock, D., Langley, K., Wypych, J., Fenton, D., Johnson, S., Lai, P. J., Everett, R., Hsu, R.-Y., and Bosselman; R. (1984). Application of recombinant DNA technologies to studies on chicken growth hormone. J. Exp. 2001. 232, 465-473. Stainer, I. M., and Holmes, W. N. (1969). Some evidence for the presence of a corticotropin releasing factor (CRF) in the duck (Anus platyrhynchos). Gen. Comp. Endocrinol. 12, 350-359.
AND
COMPARATIVE
Stimulation
ENDOCRINOLOGY
69, 128132 (1988)
of Corticosterone Release in the Fowl by Recombinant DNA-Derived Chicken Growth Hormone A. CHEUNG,~ T.R.
HALL,AND
S. HARVEY
Wolfson Institute, University of Hull, Hull HU6 7RX, England Accepted September 14, 1987 The effects of recombinant DNA-derived chicken growth hormone (rcGH) on plasma corticosterone in young broiler cockerels were investigated. A single injection of 200 uglkg rcGH significantly increased plasma corticosterone concentrations 2 hr (but not 20 or 40 mm) after treatment. Administration of 10 or 100 kg/kg rcGH also signiticantly increased plasma corticosterone levels after 2 hr, with the higher dose eliciting greater responses. Chronic treatment with seven daily injections of the same doses of rcGH gave similar increases in plasma concentrations of corticosterone. No obvious difference in magnitude of plasma corticosterone was observed between acute and chronic exposure to rcGH. In a further experiment in which serial blood sampling was performed after a single injection or five daily injections of vehicle or 200 ug/kg rcGH, there were significant increases in plasma corticosterone concentrations 40 min after acute rcGH treatment and 40 and 80 min after chronic treatment when compared with plasma corticosterone concentrations of vehicleinjected controls. However, the increases could have incorporated a stress response due to repeated sampling because the control birds also showed elevated plasma corticosterone concentrations. The corticosterone response did not diminish with repeated GH challenge. These results suggest that GH may play a role in the acute regulation of corticosterone secretion in intact chickens. 0 1988 Academic Press. Inc.
Adrenal corticosterone secretion in birds is under the control of a hypothalamo (corticotrophin releasing factor, CRF)hypophyseal (adrenocorticotrophin, ACTH)-adrenal axis (Stainer and Holmes, 1969; Salem et al., 1970). It is becoming increasingly apparent that many other signals, such as catecholamines (Freeman and Manning, 1979; Rees et al., 1985) and serotonin (Cheung et al., 1987), participate in the regulation of corticosteroidogenesis in birds. Recent studies with CRF have shown that this peptide may also have effects on growth hormone (GH) secretion since CRF reduces spontaneous GH secretion in the rat when injected in vivo (Rivier and Vale, 1984) and stimulates release of somatostatin from the central nervous tissues in vitro
(Peterfreund and Vale, 1982). Conversely, it is feasible that GH may affect adrenal function, either directly or indirectly, though there is a paucity of data on this subject. Implantation of a GH-secreting tumor enhances corticosterone response to ACTH stimulation in intact rats (Coyne et al., 1981) and GH administration increases corticosterone secretion in hypophysectomized rats (Colby et al., 1973; Kramer et al., 1977). Recently, it has been shown that GH replacement increases ACTH induced maxi,mal corticosterone production from adrenocortical cells i.solated from hypophysectomized chickens (Carsia et al., 1985). The present experiments were designed to test whether GH could affect corticosterone levels in normal, intact chickens.
’ To whom correspondence should be addressed at Department of Anatomy, University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong.
MATERIALS
One-day-old Ross broiler cockerets were pnrchased 128
0016-6480188 $1.50 Copyright All rights
Q 1988 by Academic Press, Inc. of reproduction in any form reserved.
AND METHOD
GH
EFFECTS
ON
and raised under conditions of continuous light (to prevent photoperiodic effects on hypothalamic, pituitary, and adrenal activity) with food and water available ad /i&urn. Three experiments were performed: Experiment 1. Six-week-old birds were injected once intramuscularly (im) either with 200 kg/kg body weight recombinant DNA-derived chicken growth hormone (rcGH), a polypeptide differing from native chicken GH only in its terminal amino acid (Souza et al., 1984), or with 1 ml/kg vehicle (0.25% NaHCO,, 0.2% mannitoi, pH 8.5). Blood samples were withdrawn from the brachial vein 20.40, and 120 min later. Experiment 2. Five-week-old cockerels were injected im once daily with 10 or 100 Kg/kg rcGH or with vehicle for 8 days. On the last day, a further three groups of untreated birds were also given a single injection of rcGH or vehicle. Blood samples were collected from a wing vein immediately before and 2 hr following the last injection. Experiment 3. Five-week-old cockerels were injected im daily for 5 days with either 200 wgikg rcGH or with vehicle. On the day of the last injection, a blood sample was withdrawn immediately prior to, and 5, 10, 20, 40, 80. 120, and 240 min after the injection. Further groups of birds were bled in a similar fashion after only a single injection of rcGH or vehicle. Plasma was obtained from the blood samples after centrifugation and separation and stored at - 20” until assayed. Corticosterone concentrations in plasma were determined in duplicate by specific radioimmunoassay (Harvey et al.. 1980). The assay has a minimum detection limit of 30 pmoliliter and an intraassay coefficient of variability of 3.2%. All results are expressed as means 4 SEM. Statistical differences in the results were determined by Student’s t test, with a levei of significance of P < 0.05.
RESULTS Experiment 1. A significant rise (P < 0.01) in plasma corticosterone concentrations was observed 120 min after a single injection of rcGH (Fig. 1). Although the mean plasma corticosterone concentration in the rcGH-treated birds appeared to be higher at an earlier time (40 min after injection), it was not significantly different from that of the control group. Experiment 2. Figure 2 shows that 2 hr after acute treatment with 10 or 100 pgikg rcGH, there were significant increases (P < 0.001) in plasma corticosterone levels compared with preinjection values. For the higher dose, there was a more than fivefold increase in plasma corticosterone concen-
129
CORTICOSTERONE
Time after
injection
FIG. 1. Effects of an im injection of 200 wgikg recombinant DNA-derived chicken GH (r&H) or vehicle on plasma corticosterone concentrations in 5week-old cockerels. Results are means i: SEM (~1varied between 5 and 7). *P < 0.01 compared with vehicle-treated control.
tration. Chronic treatment wit rcGH increased (P < 0.05) plasma corticosterone relative to preinjection level but was not significantly different from that of vehicle control. The higher dose of rcGH promoted a more marked increase in corticosterone response (P < 0.01 compared with preinjection level) which was also significantly (P < 0.05) higher than that of vehicle-injected control. There is no obvious difference in magnitude of plasma corticosterone with respect to the number of injections. In addition, the preinjection levels of corticosterone did not differ significantly between vehicle and rcGH-injected birds. Experiment 3. Figure 3 shows the plasma corticosterone concentrations withes the or the first 80 min following a singie ( rcGW fifth (chronic) injection of 200 or vehicle. In both acute and chronic experiments, the stress imposed by repeated sampling caused the plasma ~~~~cos%~ro~~ concentrations of vehicle-injected control < O.OS), group to increase significant1 after the reaching a maximum level 20 injection. Thereafter, the cor%~~os%~rone concentrations gradually dec~~~~~ but re-
CHEUNG.
HALL,
AND
HARVEY
of rcGH can affect plasma corticosterone concentration in immature chickens. The q 2h after injection action of rcGH in intact chickens is transitory and relatively modest, and the corticosterone response appears to revert to normal within 24 hr since chronic treatment did *r” not create a higher basal level of corticosterone (Fig. 3). Corticosterone response in i birds is known to adapt to a number of chronic stressors (El-Halawani et al., 1973; Harvey and Phillips, 1982). The response to rcGH did not seem to habituate to repeated challenge since the corticosterone response was not blunted after eight daily injections of rcGH (Fig. 2). Similarly, corticosterone responses to handling and bleeding (Harvey and Phillips, 1982) and to daily exogenous 0 ACTH treatment (Freeman et al., 1979; Vehicle GH GH Vehicle OH lO/~glKg 1OOQglKg 1OlrglKg Rees et al., 1983) also do not show habituAcute treatment Chronic treatment ation. The purity of the GH preparation FIG. 2. Effects of acute (single injection) or chronic was >9.5% (Souza et al., 1984). The possi(eight daily injections) administration of vehicle or response was rcGH on plasma corticosterone concentrations in 5- bility that the corticosterone due to the stress of a possible endotoxin week-old cockerels. Blood samples were collected imcontaminant can be discounted since such mediately before and 2 hr after injection. Results show means i: SEM (N varied between 5 and 7). *P -C0.05, responses are rapidly onsetting and termi**P < 0.01, ***P < 0.001 compared with preinjection nated (Harvey et al., 1984), whereas the relevel. sponse to the GH preparation lasted for up to 2 hr (Fig. 2). Therefore, GH probably mained significantly (I’ < 0.05) higher than participates in the acute regulation of corthe preinjection level. A much greater ele- ticosterone secretion. vation in corticosterone concentrations was There were no apparent dose-dependent observed in the rcGH-treated birds. Cortieffects since the lowest dose of rcGH emcosterone concentrations were significantly ployed (10 kg/kg) was not much less effec(P < 0.01) higher than vehicle-injected tive than the highest dose (200 pg/kg). group 40 min after the acute rcGH treatThese doses were chosen to represent the ment. For chronically treated birds, cortiminimal with biological activity up to a costerone concentrations were found to be dose with pronounced activity (e.g., see significantly (P < 0.05) higher than vehicleSouza et al., 1984). The reason why there treated group both 40 and 80 min after the was no clear-cut dose-related effects may last injection. At 120 and 240 min, there be due to the experimental protocol. In the were no significant differences between ve- preliminary experiment (Fig. l), blood samhicle- and rcGH-treated animals. However, ples were removed at a lesser frequency there was a very large variability in cortithan the extended confirmatory experiment costerone levels in control birds, possibly (Fig. 3), and more frequently than in experdue to inadvertent stress (disturbance), and iment 2. Since handling and bleeding stress accordingly these data were rejected. markedly affect corticosterone rhythms (Harvey et al., 1984), these responses may DISCUSSION be superimposed on the responses to GH. Our data show that in vivo administration However, it is abundantly clear that in 0 pre-injection
GH EFFECTS
Time
ON CORTICOSTERONE
after
injection
131
(mh)
3. Effects of a single injection (a, acute response) or the fifth injection (b, chronic treatment) of vehicle (0) or 200 p,g/kg rcGH (0) into 6-week-old cockerels on plasma corticosterone concentrations. Results are means -+ SEM (n varied between 5 and 7). *P < 0.05, **P < 0.01 compared with vehicle-injected control; +P < 0.05 compared with preinjection concentration. FIG.
three separate experiments exogenous GH was able to increase circulating corticosterone concentrations in young cockerels. Administration of GH alone to hypophysectomized rats fails to maintain corticosterone secretion by adrenal tissue both in vivo (Colby et al., 1973; Kramer et al., 1977) and in vitro (Kramer et al., 1977). However, when given simultaneously with ACTH, the effects of ACTH on corticosterone production both in vivo (Colby et al., 1973; Kramer e2 al., 1977) and in vitro (Kramer et al., 1977) are augmented, indicating that GH acts synergistically with ACTH in stimulating corticosterone release. Although corticosteroidogenesis in chicken is regulated primarily by the CRFACTH-adrenal axis, the adrenal of the chicken differs from that of mammalian species in having a certain degree of autonomy so that plasma corticosterone does not decrease drastically after hypophysectomy (Carsia et al., 1985). In addition, unlike in
hypophysectomized rats, @El replacement alone can maintain and elevate the ACTHinduced maximal corticosterone pro from isolated adrenocortical cells from hypophysectomized chickens (Carsia et al., 1985). Our study has demonstrated for the first time that GH also stimulates carticosterone secretion in intact chickens. This is in agreement with the results from mammalian studies in which implantation of the GH-secreting tumor StW5 was shown to increase basal concentration of corticosterone and to enhance corticosterone response to ACTH and stress in intact rats (Coyne et al., 1981). The exact mechanism of action of GH on corticosterone release is not fully understood but it has been suggested that GII may act at the steroidogenic enzyme level by inhibiting the action of adrenal Sa-reductase, an enzyme involved in the degradation of corticosterone (Carsia eit al. I 1985). The control of corticosterone’ secre-
132
CHEUNG,
HALL,
tion is rather complex and involves a variety of neuronal and extra-neuronal signals (Harvey et al., 1984). Growth hormone may be one of the factors participating in this integrated regulatory process. ACKNOWLEDGMENTS This work was supported by a grant from Imperial Chemical lndustries, Billingham. The authors are grateful to Keith Langley of Amgen (Thousand Oaks, California) for the generous gift of rcGH. A. Cheung was supported by a fellowship from the Croucher Foundation.
REFERENCES Carsia, R. V., Weber, H., King, D. B., and Scanes, C. G. (1985). Adrenocortical cell function in the hypophysectomized domestic fowl: Effects of growth hormone and 3,5,3’-triiodothyronine replacement. Endocrinology 117, 928-933. Cheung, A., Hall, T. R., and Harvey, S. (1987). Serotoninergic regulation of corticosterone secretion in domestic fowl. J. Endocrinol., 113,159-165. Colby, H. D., Caffrey, J. L., and Kitay, J. I. (1973). Interaction of growth hormone and ACTH in the regulation of adrenocortical secretion in rats. Endocrinology 93, 188-192. Coyne, M. D., Alpert, L. C., Harter, K. C., and Nunez, A. (1981). Effect of growth hormonesecreting tumor StW5 on pituitary and adrenal gland function in rats. Horm. Res. 14, 3k46. El-Halawani, M. E., Waibel, P. E., Appel, J. R., and Good, A. L. (1973). Effects of temperature stress on catecholamines and corticosterone of male turkeys. Amer. J. Physiol. 224, 384-388. Freeman, B. M., and Manning, A. C. C. (1979). The effects of repeated injections of adrenaline on the response of the fowl to further alarm stimulations. Res. Vet. Sci. 27, 76-81. Freeman, B. M., Manning, A. C. C., and Flack, I. H. (1979). Habituation by the immature fowl in re-
AND
HARVEY
sponse to repeated injections of corticotrophin. Brit. Poult. Sci. 20, 391-399. Harvey, S., Merry, B. J., and Phillips, J. G. (1980). Influence of stress on corticosterone secretion in the duck (Anus plutyrhynchos). J. Endocrinof. 87, 161-171. Harvey, S., and Phillips, J. G. (1982). Adrenocortical responses of ducks to treadmill exercise. J. Endocrinol. 94, 141-146. Harvey, S., Phillips, J. G., Rees, A., and Hall, T. R. (1984). Stress and adrenal function. J. Exp. Zool. 232, 633-645. Kramer, R. E., Greiner, J. W., and Colby, H. D. (1977). Site of action of growth hormone on adrenocortical steroidogenesis in rats. Endocrinology 101, 297-303.
Peterfreund, R. A., and Vale, W. W. (1982). Ovine corticotropin releasing factor stimulates somatostatin secretion from cultured brain cells. Endocrinology 112, 1275-1278. Rees, A., Harvey, S., and Phillips, J. G. (1983). Habituation of the corticosterone response of ducks (Anus platyrhynchos) to daily treadmill exercise. Gen. Comp. Endocrinol. 49, 485-489. Rees. A., Harvey, S., and Phillips, J. G. (1985). Adrenergic stimulation of adrenocortical secretion in immature fowl. Comp. Biochem. Physiol. C 81, 387-389. Rivier, C., and Vale, W. (1984). Corticotropinreleasing factor (CRF) acts centrally to inhibit growth hormone secretion in the rat. Endocrinology 114, 2409-2411. Salem, W. N., Norton, H. W., and Nalbandov, A. V. (1970). A study of ACTH and CRF in chickens. Gen. Comp. Endocrinol. 14, 270-280. Souza, L. M., Boone, T. C., Murdock, D., Langley, K., Wypych, J., Fenton, D., Johnson, S., Lai, P. J., Everett, R., Hsu, R.-Y., and Bosselman; R. (1984). Application of recombinant DNA technologies to studies on chicken growth hormone. J. Exp. 2001. 232, 465-473. Stainer, I. M., and Holmes, W. N. (1969). Some evidence for the presence of a corticotropin releasing factor (CRF) in the duck (Anus platyrhynchos). Gen. Comp. Endocrinol. 12, 350-359.