Naloxone Attenuates Serum Corticosterone and Augments Serum ...

Naloxone Attenuates Serum Corticosterone and Augments Serum Glucose Concentrations in Broilers Stimulated with Adrenocorticotropin1,2 E. DAVID PEEBLES,* A. L. POND,† J. R. THOMPSON,* C. D. MCDANIEL,* N. M. COX,‡ and M. A. LATOUR3,4 *Poultry Science Department, †Center for Environmental Health Sciences, and ‡Animal and Dairy Sciences Department Mississippi State University, Mississippi State, Mississippi 39762 cantly reduced subsequent ACTH-stimulated increases in serum corticosterone; however, when followed by saline, naloxone elevated corticosterone by 90 min after the final injection of saline. Glucose levels were significantly elevated at 60 min in birds receiving ACTH i.v., but remained elevated through 90 min in birds pretreated with naloxone. Naloxone pretreatment attenuated serum corticosterone but augmented serum glucose concentrations in ACTH-stimulated broilers.

ABSTRACT The effects of exogenous naloxone and adrenocorticotropin (ACTH) on circulating concentrations of corticosterone and glucose in broilers were determined. Birds were injected i.m. at 0 and 2 h with either saline or naloxone, then i.v. at 2.5 h with either saline or ACTH. Control birds received saline at each injection. Blood samples were taken before the experiment started (0 min) and 30, 60, and 90 min after the last injection. Intramuscular injections of naloxone signifi-

(Key words: adrenocorticotropin, broiler, corticosterone, glucose, naloxone) 1997 Poultry Science 76:511–515

ACTH, a peptide associated with POMC. Administration of naloxone, a general opiate antagonist, which can likewise block the binding of b-endorphins, has been shown to significantly increase circulating b-endorphin concentrations in humans (Plewe et al., 1987). Researchers have also previously reported that naloxone affects the hypothalamic-pituitary-adrenocortical axis by increasing ACTH secretion (Meites et al., 1979; Pfeiffer and Herz, 1984), augmenting hypothalamic corticotropinreleasing hormone secretion (Eisenberg, 1984), or possibly by increasing the adrenal’s responsiveness to ACTH (Lymangrover et al., 1981). However, the precise mechanisms of action of opioids and their antagonists on the hypothalamic-pituitary-adrenal axis are still unknown (Zerani and Gobbetti, 1992). It is suggested that increased ACTH may be a result of an increase in POMC synthesis in response to lower perceived concentrations of b-endorphins. Blood glucose levels would be expected to increase in response to ACTH-stimulated elevations in glucocorticoids (Compton et al., 1991). Increases in blood concentrations of glucocorticoids in response to naloxone have been demonstrated in a number of species, including humans (Guo et al., 1991), cynomolgus monkeys (McCubbin et al., 1993), pigs (Rushen and Ladewig, 1991), the newt, Triturus carnifex (Zerani and Gobbetti, 1992), the frog, Rana esculenta (Zerani and Gobbetti, 1991), the rat (Tanaka et al., 1983), dehydrated sheep (Thornton and Parrott, 1989), and normal sheep (Przekop et al., 1990). Studies of the effects of naloxone in birds have been limited to its control of ingestive

INTRODUCTION Adrenocorticotropin (ACTH) and b-lipotropins (bLPH) are two of several peptides produced from a single precursor molecule called proopiomelanocortin (POMC; Nakanishi et al., 1979). Upon entering the blood stream, ACTH can stimulate the production of glucocorticoids and b-LPH can be broken down into a number of smaller peptides, one of which is b-endorphin, an endogenous opioid. Boscaro et al. (1990) reported that under most physiological and pathological conditions, stimuli that alter ACTH secretion also alter b-LPH in a parallel manner. Morley (1981) has reported that endogenous opioids appear to inhibit the secretion of ACTH in humans. Furthermore, exogenous aendorphins have been found to depress cortisol secretion in sheep under stress conditions (Przekop et al., 1990) and in frogs (Zerani and Gobbetti, 1992). Therefore, if b-endorphin receptors are blocked, then further production of b-endorphin might occur with possible concomitant increases in related molecules such as

Received for publication July 18, 1996. Accepted for publication November 29, 1996. 1This is Journal Article Number J-8412 from the Mississippi Agricultural and Forestry Experiment Station supported by MIS-2985. 2Use of trade names in this publication does not imply endorsement by Mississippi Agriculture and Forestry Experiment Station of these products, nor similar ones mentioned. 3To whom correspondence should be addressed. 4Current address: Purdue University, Poultry Building, West Lafayette, IN 47907-1026.

511

512

PEEBLES ET AL.

behavior. The attenuation of drinking and feeding behavior in response to naloxone has been noted in chickens (McCormack and Denbow, 1987; Firman and Volmert, 1991). However, the effects of naloxone on circulating corticosterone and glucose concentrations in the chicken are unknown; therefore, the present study was conducted in order to investigate the relationship between exogenous ACTH and naloxone and subsequent circulating concentrations of corticosterone and glucose in chickens.

MATERIALS AND METHODS Forty broilers (Arbor Acres × Arbor Acres), 2 wk of age, were randomly assigned to one of four treatment groups with five replicates per group and two birds per replicate. Birds were housed in Petersime brooder batteries,5 exposed to continuous lighting, and offered a standard diet that met or exceeded NRC (1995) recommendations. Feed and water were available for ad libitum consumption. Birds were allowed to adapt to cages and to handling for 1 wk prior to testing. Blood samples were obtained from the brachial vein for baseline data (0 min) and at 30, 60, and 90 min after the last treatment injection. Blood (500 mL) was collected into nonheparinized tubes at each designated time and then immediately centrifuged at 1,000 × g for 10 min to express serum. Individual serum samples were pooled within each replicate prior to storage. All pooled serum samples remained fluid prior to freezing, and all five samples per treatment and sampling time were assayed. Serum samples for all treatments were stored at –20 C after centrifugation.

Treatments Beginning at 1300 h on 1 d, birds were given intramuscular (i.m.) injections at 0 and 2 h and i.v. injections at 2.5 h. At 0, 2, and 2.5 h, one replicate pen from each treatment was selected at random for injection. This procedure was continued five times until birds in all treatment replicates were injected. Control birds received saline at each injection (SAL-SAL). Other treatments consisted of: 1) naloxone i.m. at 0 and 2 h then saline i.v. at 2.5 h (NAL-SAL); 2) saline i.m. at 0 and 2 h then ACTH i.v. at 2.5 h (SAL-ACTH); and 3) naloxone i.m. at 0 and 2 h then ACTH i.v. at 2.5 h (NAL-ACTH). Naloxone and ACTH were administered at 5 mg/kg (Firman and Volmert, 1991; McCormack and Denbow, 1987) and 10 IU/kg of BW, respectively. The dosage of naloxone was chosen based on dosages known to affect drinking and feeding behavior in chickens.

5Petersime Incubator Co., Gettysburg, OH 45328. 6Endocrine Sciences, Calabasas, CA 91301. 7Eastman Kodak Co., Rochester, NY 14650.

RIA Serum corticosterone was determined by radioimmunoassay according to the procedure of Satterlee et al. (1980). The corticosterone antibody was supplied by Endocrine Sciences.6 Five samples within each treatment and sampling time were assayed in duplicate. All samples were run within one assay to avoid interassay variation. The intra-assay coefficient of variation was 9.2% with the 80 samples assayed in duplicate. The r2 value of the standard curve was 0.9863.

Glucose Analysis The glucose analysis was based on the application of 10 mL of serum on a Kodak Ektachem slide7 specific for glucose. This procedure was performed according to the method of Elliot (1984), as described by Latour et al. (1996).

Statistical Analysis The experimental units were arranged in a completely randomized design. The data were subjected to a 2 (i.m. injection of either saline or naloxone) × 2 (i.v. injection of either saline or ACTH) factorial arrangement of treatments split in time (0, 30, 60, and 90 min). Tests for differences between the main effects of i.m. injection with either saline or naloxone at 0 and 2 h and i.v. injection with either saline or ACTH at 2.5 h, and between the means from interactions of the main effects and sampling time were performed. Interaction means were partitioned by Student-Newman-Keuls test (Steel and Torrie, 1980). The data were analyzed using the General Linear Models procedure of SAS (1995). Statements of significance were based on P ≤ 0.05 unless otherwise noted.

RESULTS AND DISCUSSION The birds treated with saline at each injection had baseline corticosterone concentrations that were considered to be in the normal physiological range at all time periods (Satterlee et al., 1980) and were not different from the concentrations in all birds prior to treatment at 0 min (Figure 1). Mean corticosterone concentration in all birds at 0 min was 3.41 ng/mL. There were significant (P ≤ 0.0001) main effects due to treatment for both the i.m. and i.v. injections (Table 1). Corticosterone concentrations were decreased across time of sampling in birds injected i.m. with naloxone when compared with those injected i.m. with saline. Conversely, circulating corticosterone concentrations were increased across time in birds injected i.v. with ACTH in comparison to those injected i.v. with saline. There were also significant i.m. by i.v. injection (P ≤ 0.0001) and i.m. by i.v. injection by sampling time (P ≤ 0.0001) interactions for serum corticosterone concentration. Data for the three-way interaction are provided in Figure 1. Despite the overall depressing effect of naloxone on corticosterone, previous i.m. injections with naloxone led to elevated corticoster-

513

NALOXONE AND CORTICOSTERONE IN BROILERS

FIGURE 1. Serum corticosterone concentrations for different injection treatments at 0, 30, 60, and 90 min after the last injection in 2-wk-old broilers. Values ± SEM with different letters among the 0, 30, 60, and 90 min sampling periods differ significantly (P ≤ 0.05) by Student-Newman-Keuls test. Individual SEM were based on a pooled estimate of variance. ♦ = Saline at 0 and 2 h (i.m.) and at 2.5 h (i.v.). π = Naloxone at 0 and 2 h (i.m.) and saline at 2.5 h (i.v.). ⁄ = Saline at 0 and 2 h (i.m.) and adrenocorticotropin (ACTH) at 2.5 h (i.v.). ÿ = Naloxone at 0 and 2 h (i.m.) and ACTH at 2.5 h (i.v.). 1n = 5 samples within each treatment and sampling time.

one concentrations by 90 min after a final i.v. injection of saline. The delay in corticosterone increase may be partly attributed to a slower absorption of naloxone due to i.m. injection. The effects of naloxone on corticosterone in the NAL-ACTH birds were evident by 30 min after the last injection of ACTH; however, the delayed response until 90 min in the NAL-SAL birds might have been necessary to allow enough time for significant concentrations of ACTH to be reached after stimulation of its synthesis and secretion. This response would also not have manifested itself until sufficient numbers of bendorphin receptors were blocked by naloxone in NALSAL birds. That is, the sporadic increase in corticosterone at this later time may be viewed as a response to a compensatory elevation in ACTH elicited by the perception of a lowered b-endorphin concentration in association with the blocking of specific b-endorphin receptors by naloxone. An increase in corticosterone or cortisol in response to naloxone has also been observed in studies with other species (Morley, 1981; Thornton and Parrott, 1989; Guo et al., 1991; Rushen and Ladewig, 1991; Zerani and Gobbetti, 1991, 1992; McCubbin et al., 1993). Naloxone injected subcutaneously at 1.5 mg/g BW to adult frogs led to a 1.5-fold increase in corticosterone at 60 min after injection (Zerani and Gobbetti, 1992), whereas a 5.5-fold increase was noted at 2 h after an i.m. injection of naloxone in the present study. Sheep that have not experienced dehydration are an exception to this, in that they do not exhibit an increase in cortisol after naloxone treatment (Przekop et al., 1990).

Birds treated with saline followed by ACTH exhibited elevated corticosterone concentrations compared to control animals at all sampling periods after the last i.v. injection. These results are similar to those of Beuving and Vonder (1978), who observed an increase in plasma corticosterone in young and old laying hens and roosters within several minutes after the i.v. injection of ACTH. It is interesting that the increase was more rapid in younger than older birds, but that the increase lasted longer in the older birds. The response to ACTH in the young broilers in this study would, likewise, be expected to be rapid. An approximate 17-fold increase occurred at 40 min after injection in layers of both sexes in the previous study compared to a 35-fold increase at 60 min in the birds of the present study. The duration of increase, which lasted through 90 min in the broilers, was also longer than that observed in the young laying hens. Corticosterone concentrations between 30 and 90 min in birds treated with naloxone prior to ACTH changed similarly to those treated with saline followed by ACTH. Nevertheless, NAL-ACTH birds exhibited significantly lower corticosterone concentrations than SAL-ACTH birds at 30, 60, and 90 min. Naloxone decreased ACTH-stimulated corticosterone secretion within each of the sampling times. Comparison of these latter two treatments also suggests that naloxone pretreatment dampens the corticosterone response to ACTH as early as 30 min after ACTH injection and that this effect persists for at least another 60 min. Because naloxone’s effects were limited to a depression in corticosterone after exogenous ACTH-stimulated secretion, it is suggested that naloxone may only modify the responsiveness of the adrenal gland to ACTH. Opioids can affect in vitro adrenal steroidogenesis (Guaza et al., 1986), and specific naloxone-sensitive opioid receptors have been found to mediate the effects of opioids in the hypothalamus and adrenal gland (Lotti et al., 1969; Guaza and Borrell, 1984; Al-Damluji et al., 1990). There was a significant (P ≤ 0.0007) main effect on serum glucose due to i.v. injection treatment (Table 1). Serum glucose was elevated across time of sampling in

TABLE 1. Main effects of injection treatments [i.m. saline or naloxone at 0 and 2 h and i.v. saline or adrenocorticotropin (ACTH) at 2.5 h] on serum corticosterone and glucose concentrations across sampling time in 2-wk-old broilers Injection

Treatment

Corticosterone1

Glucose1

i.m.

Saline Naloxone Saline ACTH SEM2

(ng/mL) 45.56a 26.61b 5.89b 66.28a 0.890

(mg/dL) 231.3 236.1 225.1b 242.3a 2.68

i.v.

a,bFor each parameter, means within each type of injection with no common superscript differ significantly (P < 0.05) by StudentNewman-Keuls test. 1n = 40 samples within each treatment group. 2SEM based on pooled estimate of variance.

514

PEEBLES ET AL.

FIGURE 2. Serum glucose concentrations for different injection treatments at 0, 30, 60, and 90 min after the last injection in 2-wk-old broilers. Values ± SEM with different letters among the 0, 30, 60, and 90 min sampling periods differ significantly (P ≤ 0.05) by Student-Newman-Keuls test. Individual SEM were based on a pooled estimate of variance. ♦ = Saline at 0 and 2 h (i.m.) and at 2.5 h (i.v.). π = Naloxone at 0 and 2 h (i.m.) and saline at 2.5 h (i.v.). ⁄ = Saline at 0 and 2 h (i.m.) and adenocorticotropin (ACTH) at 2.5 h (i.v.). ÿ = Naloxone at 0 and 2 h (i.m.) and ACTH at 2.5 h (i.v.). 1n = 5 samples within each treatment and sampling time.

naloxone’s influence on the pituitary. Edens and Parkhurst (1994) have provided data indicating that opioid agonists caused transitory increases in plasma growth hormone and prolactin concentrations 30 min after i.m. injection in broiler cockerels. In addition, Paolisso et al. (1987) have suggested that the effects of b-endorphins on blood glucose are mediated through the pancreas. Naloxone has been reported to inhibit the release of insulin (el-Tayeb et al., 1986a), and exogenous bendorphins have been shown to give rise to significant increases in glucagon (el-Tayeb et al., 1986b). Furthermore, the secretion of b-endorhpins has been shown to be stimulated by insulin (Imura et al., 1982). In conclusion, naloxone elevated corticosterone in broilers as in other species, and subsequently augmented serum glucose concentrations. This study also provides initial evidence that a known b-blocker, naloxone, may attenuate the corticosterone response to ACTH. Recent evidence (Latour et al., 1996) has shown that elevated corticosterone levels predispose birds to higher than expected levels of low (LDL) and high (HDL) density lipoprotein-cholesterol. Therefore, it would be interesting to test whether naloxone could indirectly effect the circulating LDL and HDL, a study that has never been performed in avian species.

REFERENCES birds treated with ACTH when compared to those given saline i.v. The ACTH would be expected to increase glucose as a result of a response to elevated glucocorticoids. However, as was found for corticosterone concentration, there was also a significant (P ≤ 0.01) i.m. by i.v. injection by sampling time interaction for serum glucose concentration. These data are provided in Figure 2. There was no significant main effect (i.m. by i.v. injection) interaction for serum glucose. Serum glucose was unaffected in all treatments at 30 min after the last injection and values were all within the normal physiological range (Latour et al., 1994). However, both i.v. ACTH-treated groups had significantly higher serum glucose concentrations than controls and NAL-SAL treated birds by 60 min. In addition, the glucose concentrations of the NAL-ACTH treated birds remained higher than the other treatment groups by 90 min. Similar to its effect on corticosterone, naloxone led to greater increases in glucose when it was injected prior to ACTH rather than saline. The increase in glucose by 60 min in the ACTH-treated birds may be indirectly attributed to an ACTH-induced increase in corticosterone (Latour et al., 1993) by 30 min. Bisbis et al. (1994) have demonstrated that exogenous corticosterone effectively induced insulin resistance in chickens. At 90 min in the NAL-ACTH treatment, glucose was still elevated but corticosterone was lowered, perhaps indicating that the naloxone indirectly affected the production of factors other than corticosterone which promoted an increase in serum glucose. This effect may have occurred through

Al-Damluji, S., P. Bouloux, A. White, and M. Besser, 1990. The role of alpha-2-adrenoceptors in the control of ACTH secretion; interactions with the opioid system. Neuroendocrinology 51:76–81. Beuving, G., and G.M.A. Vonder, 1978. Effect of stressing factors on corticosterone levels in the plasma of laying hens. Gen. Comp. Endocrinol. 35:153–159. Bisbis, S., M. Taouis, M. Derouet, B. Chevalier, and J. Simon, 1994. Corticosterone-induced insulin resistance is not associated with alterations of insulin receptor number and kinase activity in chicken kidney. Gen. Comp. Endocrinol. 96:370–377. Boscaro, M., A. Paoletta, P. Giacomazzi, F. Fallo, and N. Sonino, 1990. Inhibition of pituitary b-endorphin by ACTH and glucocorticoids. Neuroendocrinology 51:561–564. Compton, M. M., L. R. Johnson, and P. S. Gibbs, 1991. Activation of thymocyte deoxyribonucleic acid degradation by endogenous glucocorticoids. Poultry Sci. 70: 521–529. Edens, F. W., and C. R. Parkhurst, 1994. Plasma growth hormone and prolactin response to FK 33-824, a synthetic opioid agonist, in broiler chickens. Poultry Sci. 73: 1746–1754. Eisenberg, R. M., 1984. Effects of naltrexone on plasma corticosterone in opiate-naive rats: a central action. Life Sci. 34:1185–1191. Elliot, R. J., 1984. Physicians and Computers: Ektachem DT-60 analyzer. Physician’s Leading Computer J. 2:1–3. el-Tayeb, K. M., P. L. Brubaker, H. L. Lickley, E. Cook, and M. Uranic, 1986a. Effect of opiate-receptor blockade on normoglycemic and hypoglycemic glucoregulation. Am. J. Physiol. 250(3):E236–242. el-Tayeb, K. M., C. J. Gauthier, P. L. Brubaker, H. L. Lickley, and M. Uranic, 1986b. Hormonal and metabolic responses

NALOXONE AND CORTICOSTERONE IN BROILERS to intracarotid and intrajugular infusion of beta-endorphin in normal dogs. Can. J. Physiol. Pharmacol. 64(3):306–310. Firman, J. D., and R. D. Volmer, 1991. Research note: Naloxone inhibits drinking in the chick induced by angiotensin II. Poultry Sci. 70:2010–2012. Guaza, C., and J. Borrell, 1984. The Met-enkephalin analog DAla2-Met-enkephalinamide decreases the adrenocortical response to ACTH in dispersed rat adrenal cells. Peptides 5:895–897. Guaza, C., M. Zubair, and J. Borrell, 1986. Corticosteroidogenesis modulation by b-endorphin and dynorphin1–17 in isolated rat adrenocortical cells. Peptides 7:237–240. Guo, A. L., C. L. Lu, Y. F. Shi, and G. Marion-Landais, 1991. Effect of naloxone on the responses of ACTH and cortisol to corticotropin-releasing factor in normal subjects and patients with Cushing’s disease. Current Therapeutic Res. 49(3):497–506. Imura, H., Y. Nakai, and K. Nakao, 1982. Control of biosynthesis and secretion of ACTH, endorphins, and related peptides. Page 137 in: Neuroendocrine Perspectives. E. E. Muller and R. M. MacLeod, ed. Elsevier, Amsterdam, The Netherlands. Latour, M. A., S. A. Laiche, J. R. Thompson, E. D. Peebles, and J. D. May, 1993. Effects of continuous infusion of adrenocorticotropin on plasma corticosterone and lipids in broilers. Poultry Sci. 72(Suppl. 1):60. (Abstr.) Latour, M. A., S. A. Laiche, J. R. Thompson, A. L. Pond, and E. D. Peebles, 1996. Continuous infusion of adrenocorticotropin elevates circulating lipoprotein cholesterol and corticosterone concentrations in chickens. Poultry Sci. 75: 1428–1432. Latour, M. A., E. D. Peebles, C. R. Boyle, and J. D. Brake, 1994. The effects of dietary fat on growth performance, carcass composition, and feed efficiency in the broiler chick. Poultry Sci. 73:1362–1369. Latour, M. A., E. D. Peebles, C. R. Boyle, S. M. Doyle, T. Pansky, and J. D. Brake, 1996. Effects of breeder hen age and dietary fat on embryonic and neonatal broiler serum lipids and glucose. Poultry Sci. 75:695–701. Lotti, V. J., N. Kokka, and R. George, 1969. Pituitary-adrenal activation following intra-hypothalamic microinjection of morphine. Neuroendocrinology 4:326–332. Lymangrover, J. R., L. A. Dokas, A. Kong, R. Martin, and M. Saffran, 1981. Naloxone has a direct effect on the adrenal cortex. Endocrinology 109:1132–1137. McCormack, J. F., and D. M. Denbow, 1987. Ingestive behavior of meat and egg-type chickens: Equal sensitivity to naloxone. Poultry Sci. 66:1714–1720. McCubbin, J. A., J. R. Kaplan, S. B. Manuck, and M. R. Adams, 1993. Opioidergic inhibition of circulatory and endocrine stress responses in cynomolgus monkeys: A preliminary study. Psychosomatic Med. 55(1):23–28.

515

Meites, J., I. F. Bruni, D. A. van Vogt, and A. F. Smith, 1979. Relation of endogenous opioids, peptides and morphine to neuroendocrine functions. Life Sci. 24:1325–1336. Morley, I. E., 1981. The endocrinology of opiates and opioid peptides. Metabolism 39:195–209. Nakanishi, S., A. Inove, T. Kita, M. Nakamura, A. Chang, A. Cohen, and A. Numa, 1979. Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature 278:423–427. National Research Council, 1995. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Paolisso, G., D. Giuglinno, A. J. Scheen, P. Franchimont, F. D’Onofrio, and P. J. Lefebvre, 1987. Primary role of glucagon release in the effect of beta-endorphin on glucose homeostasis in normal man. Acta Endocrinol. 115:161–169. Pfeiffer, A., and A. Herz, 1984. Endocrine actions of opioids. Horm. Metab. Res. 16:386–397. Plewe, G., U. Schneider, U. Kranse, and J. Beyer, 1987. Naloxone increases the response of growth hormone and prolactin to stimuli in obese humans. J. Endocrinol. Invest. 10(2):137–141. Przekop, F., K. Mateusiak, E. Stupnicka, K. Romanowicz, and E. Domanski, 1990. Suppressive effect of b-endorphin and naloxone on the secretion of cortisol under stress conditions in sheep. Exp. Clin. Endocrinol. 95(2):210–216. Rushen, J., and J. Ladewig, 1991. Stress-induced hypoalgesia and opioid inhibition of pigs’ responses to restraint. Physiol. Behav. 50(6):1093–1096. SAS Institute, 1995. SAS/STAT User’s Guide: Release 6.10 Edition. SAS Institute Inc., Cary, NC. Satterlee, D. G., R. B. Abdullah, and R. P. Gildersleeve, 1980. Plasma corticosterone radioimmunoassay and levels in the neonate chick. Poultry Sci. 59:900–905. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw Hill Book Co., New York, NY. Tanaka, M., Y. Kohno, A. Tsuda, R. Nakagawa, Y. Ida, K. Simori, Y. Hoaki, and N. Nagasaki, 1983. Differential effects of morphine on noradrenaline release in brain regions of stressed and non-stressed rats. Brain Res. 275: 105–115. Thornton, S. N., and R. F. Parrott, 1989. Naloxone affects the release of cortisol, but not of vasopressin or oxytocin, in dehydrated sheep. Acta Endocrinol. (Copenh) 120:50–54. Zerani, M., and A. Gobbetti, 1991. Effects of b-endorphin and naloxone on corticosterone and cortisol release in the newt (Triturus carnifex): Studies in vivo and in vitro. J. Endocrinol. 131(2):295–302. Zerani, M., and A. Gobbetti, 1992. In vivo and in vitro effects of b-endorphin and naloxone on corticosterone and cortisol release in male and female water frog, Rana esculenta. Comp. Biochem. Physiol. 102C(3):537–542.