Proc. Nati. Acad. Sci. USA Vol. 74, No. 1, pp. 358-359, January 1977
Medical Sciences
f3-Endorphin: Stimulation of growth hormone release in vivo (endogenous opiate/pituitary-brainfrat/methionine-enkephalin/somatotropin) ANDRE DUPONT*, LIONEL CUSAN*, MARIE GARON*, FERNAND LABRIE*, AND CHOH HAO LIt * Laboratory of Molecular Endocrinology, Le Centre Hospitalier de l'Universit6 Laval, Quebec G1V 4 G2, Canada; and tHormone Research Laboratory, University of California, San Francisco, Calif. 94143
Contributed by Choh Hao Li, October 15, 1976
trajugular catheter with 0.2 ml of sheep somatostatin antiserum (kindly supplied by Dr. A. Arimura) 5 min before injection of the indicated doses of f3-endorphin, 400 pg of Met-enkephalin, or the vehicle alone (40 ,l of 0.9% NaCl) over a 3-min period. W-Endorphin was prepared by the solid-phase method as described (13). Met-enikephalin was also synthesized by solid-phase synthesis and kindly supplied by Dr. D. H. Coy. Blood samples (0.7 ml) were then withdrawn into heparinized syringes at the indicated time intervals Saline (0.7 ml) containing heparin was injected after each blood sampling to minimize extracellular volume changes. Plasma was separated by centrifugation at 1200 X g for 40 min at 2-4° and kept at -20° until assayed. Plasma GH was measured in duplicate by double-antibody radioimmunoassay (19, 20) using rat hormones (GH-I-2 and GH-RP-1) and rabbit antiserum (anti-GH-S-2) kindly provided by Dr. A. F. Parlow for the Nationpl Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program. Purified goat anti-rabbit gamma globulins were a product of Endocrinolab Ltd, Quebec. Radioimmunoassay data were analyzed with a HewlettPackard desk-top calculator using a program written in our laboratory and based on model II of Rodbard and Lewald (21). Data are expressed as mean ±SEM. Statistical significance was evaluated according to the multiple-range test of DuncanKramer (22). RESULTS As illustrated in Fig. 1, doses of 2 pig and higher of f3-endorphin led to a significant stimulation of plasma GH release. With the 2-,pg dose, a 6 to 10-fold stimulation of the plasma GH concentration was measured 10 and 20 min after injection of flendorphin, with a progressive decrease to basal levels reached at 45 min. The two higher doses (5 and 25 ,ug) of (3-endorphin led to a 20- to 30-fold stimulation of plasma GH levels measured 20 and 30 min after injection of the peptide. As seen in Table 1, the. NH2-terminal pentapeptide of ftendorphin (Met-enkephalin) has much lower activity in this system. In fact, at the 400-pg dose, an approximately (i-fold stimulation of plasma GH is observed 10 min after the injection, with a rapid return to basal levels at later time intervals. Using a more active analog, [D-Ala2]Met-enkephalin, we have found a maximal stimulatory effect of the peptide at 10 min (A. Dupont, L. Cusan, F. Labrie, and D. H. Coy, unpublished data) while fl-endorphin has a somewhat delayed action, a maximal effect being usually observed between 20 and 30 min after its administration.
ABSTRACT Two micrograms of f-endorphin (f3-lipotroPin61 il) injected intraventricularly in rats that had been treated with antiserum against somatostatin led to a 6- and 10-fold stimulation of the concentration of plasma growth hormone (somatotropin) measured 10 and 20 min after injection of the peptide, whereas-400 ug of methionine-enkephalin led to a 4to 6-fold increase of levels of plasma growth hormone at 10 min with a rapid return to basal levels at later time intervals. At doses of 5 and 25 pg, A-endorphin led to a 20- to 30-fold stimulation-of levels of plasma growth hormone, the maximal effect being measured between 20 and 30 min after injection. These data su 'est the possible role of the endogenous opiate-like peptides in the control of growth hormone'secretion. Morphine is well known to be a potent stimulus of growth hormone (GH; somatotropin) release in the rat (1-3). This effect of the opiate is likely to be mediated by increased release of GH-releasing activity from the hypothalamus (4). Following reports of the presence of endogenous opiate activity in the brain (5-7), the pentapeptide H-Tyr-Gly-GlyPhe-Met-OH (Met-enkephalin) has been isolated from porcine brain (8). This peptide has'been shown to have potent opiate activity (8) and to bind to the opiate receptor (9, 10). The amino-acid sequence of Met-enkephalin is the same as that of the NH-terminal pentapeptide of the COOH-fragment [f3-LPH(61-91)] of 13-lipotropin first isolated from sheep pituitaries (11). This peptide, called f3-endorphin, has been isolated and characterized from camel pituitary glands (12) and shown to be a potent analgesic agent, as assayed by both in vitro (12-15) and in vivo (16,17) methods. These findings raised the possibility whether fl-endorphin could, like morphine (1-4), induce GH release in vivo. Changes of plasma GH levels could then become a useful in vivo test of biological activity of the endogenous opioid peptides and their analogs.
MATERIALS AND METHODS Adult male Sprague-Dawley rats (obtained from Canadian Breeding Farms, St. Constant, Quebec), weighing 250-275 g upon arrival, were kept in a sound-attenuated and temperature-controlled room (24 +.2O) illuminated from 0500 to 1900 hr. Purina rat chow and water were available ad libitum. In order to minimize stress-induced inhibition of GH secretion, we handled the animals twice a day for 7-10 days before inserting a catheter (Venocath no. 18, Abbott) into the right superior vena cava under Surital (50 mg/kg, intraperitoneally) anesthesia. A metallic cannula (gauge 25) was then implanted stereotaxically in the left lateral ventricle of the animals according to the coordinates (A-5.4, L-2, D-3) described by De Groot (18). The cannula was fixed to the skull with a polymerizing acrylate (Yates Flash Acrylic). This chronic technique permits a minimum of manipulations of the animal at the time of the experiment performed 2 days after implantation of the cannulae. Animals were then injected intravenously through the in-
DISCUSSION data demonstrate clearly that fl-endorphin adThe present ministrated intraventricularly can be a potent stimulus of GH release in the rat, while the activity of Met-enkephalin is much lower. As calculated from the areas under the plasma GH response curves, (3-endorphin. is, on a molar basis, approximately 2000 times more potent than Met-enkephalin.
Abbreviation: GH, growth hormone (somatotropin).
358
Medical Sciences: Dupont et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
359
Table 1. Effect of 400 ,g of Met-enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) injected intraventricularly on plasma GH levels in the rat Min after injection
Control Met-enkephalin
-5
0
5
10
20
30
45
19 ± 4 25 ± 6
13 ± 1 31 ± 8
18 ± 6 51 ± 30
GH (ng/ml plasma) 18 ± 4 119 ± 34*
24 ± 6 44 ± 17
26 ± 6 28 ± 6
20 ± 6 21 ± 7
The experiment was performed as described in the legend of Fig. 1. P < 0.05.
This difference of biological activity is in marked contrast to the relative affinities of the two peptides for the opiate receptor. In fact, when tested for their ability to displace [3H] etorphine (14, 15) and [3H]Met-enkephalin (0. Morin, M. G. Caron, and F. Labrie, unpublished data), fl-endorphin was found to be only two to six times more potent than Met-enkephalin. When injected centrally in the mouse, the analgesic activity of fl-endorphin was 18 to 33 times greater than morphine sulfate on a molar basis, whereas Met-enkephalin was relatively inactive (16). These findings would suggest that the higher biological activity of fl-endorphin, when injected intracerebrally, is partly due to its higher resistance to proteolytic degradation. We have recently found that intravenous administration of somatostatin antiserum leads to a rapid rise of plasma GH levels in the rat, thus indicating that somatostatin plays an important o....o Control . v- *8-Endorphin 0.5pg 0--^o , 2 pg
M---a.P 25
350
pg
300
7E N,
200i
0L 150I
-M
A
50 -
5
0
5
10
20
30
45
60
MINUTES AFTER INJECTION FIG. 1. Effect of increasing doses of fl-endorphin on plasma GH levels in the rat. Male rats bearing intraventricular and intrajugular cannulae were injected intravenously with 0.2 ml of sheep somatostatin antiserum 5 min before the intraventricular injection of the indicated amounts of synthetic fl-endorphin. GH concentrations were measured at the indicated time intervals after administration of flendorphin. Data are presented as mean + SEM.
physiological role in the control of GH secretion (3). Using this model, we found that the combined administration of somatostatin antiserum and morphine led to an additive effect on the plasma GH concentration. Since circulating somatostatin was expected to be neutralized by the antibody, it was suggested that morphine stimulates GH secretion through an increased release of GH-releasing activity from the hypothalamus. The present data suggest the possible role of the endogenous opiate-like peptides, ,-endorphin and Met-enkephalin, in the control of GH secretion; this effect is likely mediated by increased release of GH-releasing activity. Preliminary studies also showed that 83-endorphin caused the release of prolactin when injected intraventricularly in the rat. Thus, fl-endorphin may play an important role in the control of many neuroendocrine functions. 1. Kokka, N., Garcia, J. F., George, R. & Elliott, H. W. (1972) Endocrinology 90, 735-743. 2. Ferland, L., Labrie, F., Coy, D. H., Arimura, A. & Schally, A. V. (1976) J. Mol. Cell. Endocrinol. 4, 79-88. 3. Ferland, L., Labrie, F., Jobin, M., Arimura, A. & Schally, A. V. (1976) Biochem. Biophys. Res. Commun. 68, 149-156. 4. Ferland, L., Labrie, F., Arimura, A. & Schally, A. V. (1976) J. Mol. Cell. Endocrinol., in press. 5. Terenius, L. & Wahlstr6m, A. (1975) Acta Physiol. Scand. 94, 74-81. 6. Hughes, J., Smith, T. W., Morgan, B. & Fothergill, L. A. (1975) Life Sci. 16, 1753-1758. 7. Pasternak, G. W., Goodman, R. & Snyder, S. H. (1975) Life Sci. 16, 1765-1769. 8. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. & Morris, H. R. (1975) Nature 258,577-579. 9. Bradbury, A. F., Smyth, D. G. & Snell, C. R. (1976) Nature 260, 793-795. 10. Morin, O., Caron, M. G., De Lean, A. & Labrie, F. (1976) Biochem. Blophys. Res. Commun., in press. 11. Li, C. H., Barnafi, L., Chretien, M. & Chung, D. (1965) Nature 208,1093-1094. 12. Li, C. H. & Chung, D. (1976) Proc. Natl. Acad. Sci. USA 73, 1145-1148. 13. Li, C. H., Lemaire, S., Yamashiro, D. & Doneen, B. A. (1976) Biochem. Biophys. Res. Commun. 71, 19-25. 14. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Natl. Acad. Sci. USA 73, 1821-1823. 15. Lazarus, L. H., Ling, N. & Guilleniin, R. (1976) Proc. Natl. Acad. Sci. USA 73,2156-2159. 16. Loh, H. H., Tseng, L. F., Wei, E. & Li, C. H. (1976) Proc. Natl. Acad. Sci. USA 73,2895-2898. 17. Tseng, L. F., Loh, H. H. & Li, C. H. (1976) Nature 263,239. 18. De Groot, J. (1959) Trans. R. Neth. Acad. Sci. 52, 1-40. 19. Birge, C. A., Peake, G. T., Mariz, I. K. & Daughaday, W. H. (1967) Endocrinology 81, 195-200. 20. Odell, W. D., Rayford, P. L. & Ross, G. T. (1967) J. Lab. Clin. Med. 70, 973-980. 21. Rodbard, D. & Lewald, J. E. (1970) in Karolinska Symposia on
Research Methods in Reproductive Endocrinology, 2nd, ed. Diczfalusy, E., pp. 79-103. 22. Kramer, C. Y. (1956) Biometrics 12,307-310.
Medical Sciences
f3-Endorphin: Stimulation of growth hormone release in vivo (endogenous opiate/pituitary-brainfrat/methionine-enkephalin/somatotropin) ANDRE DUPONT*, LIONEL CUSAN*, MARIE GARON*, FERNAND LABRIE*, AND CHOH HAO LIt * Laboratory of Molecular Endocrinology, Le Centre Hospitalier de l'Universit6 Laval, Quebec G1V 4 G2, Canada; and tHormone Research Laboratory, University of California, San Francisco, Calif. 94143
Contributed by Choh Hao Li, October 15, 1976
trajugular catheter with 0.2 ml of sheep somatostatin antiserum (kindly supplied by Dr. A. Arimura) 5 min before injection of the indicated doses of f3-endorphin, 400 pg of Met-enkephalin, or the vehicle alone (40 ,l of 0.9% NaCl) over a 3-min period. W-Endorphin was prepared by the solid-phase method as described (13). Met-enikephalin was also synthesized by solid-phase synthesis and kindly supplied by Dr. D. H. Coy. Blood samples (0.7 ml) were then withdrawn into heparinized syringes at the indicated time intervals Saline (0.7 ml) containing heparin was injected after each blood sampling to minimize extracellular volume changes. Plasma was separated by centrifugation at 1200 X g for 40 min at 2-4° and kept at -20° until assayed. Plasma GH was measured in duplicate by double-antibody radioimmunoassay (19, 20) using rat hormones (GH-I-2 and GH-RP-1) and rabbit antiserum (anti-GH-S-2) kindly provided by Dr. A. F. Parlow for the Nationpl Institute of Arthritis and Metabolic Diseases, Rat Pituitary Hormone Program. Purified goat anti-rabbit gamma globulins were a product of Endocrinolab Ltd, Quebec. Radioimmunoassay data were analyzed with a HewlettPackard desk-top calculator using a program written in our laboratory and based on model II of Rodbard and Lewald (21). Data are expressed as mean ±SEM. Statistical significance was evaluated according to the multiple-range test of DuncanKramer (22). RESULTS As illustrated in Fig. 1, doses of 2 pig and higher of f3-endorphin led to a significant stimulation of plasma GH release. With the 2-,pg dose, a 6 to 10-fold stimulation of the plasma GH concentration was measured 10 and 20 min after injection of flendorphin, with a progressive decrease to basal levels reached at 45 min. The two higher doses (5 and 25 ,ug) of (3-endorphin led to a 20- to 30-fold stimulation of plasma GH levels measured 20 and 30 min after injection of the peptide. As seen in Table 1, the. NH2-terminal pentapeptide of ftendorphin (Met-enkephalin) has much lower activity in this system. In fact, at the 400-pg dose, an approximately (i-fold stimulation of plasma GH is observed 10 min after the injection, with a rapid return to basal levels at later time intervals. Using a more active analog, [D-Ala2]Met-enkephalin, we have found a maximal stimulatory effect of the peptide at 10 min (A. Dupont, L. Cusan, F. Labrie, and D. H. Coy, unpublished data) while fl-endorphin has a somewhat delayed action, a maximal effect being usually observed between 20 and 30 min after its administration.
ABSTRACT Two micrograms of f-endorphin (f3-lipotroPin61 il) injected intraventricularly in rats that had been treated with antiserum against somatostatin led to a 6- and 10-fold stimulation of the concentration of plasma growth hormone (somatotropin) measured 10 and 20 min after injection of the peptide, whereas-400 ug of methionine-enkephalin led to a 4to 6-fold increase of levels of plasma growth hormone at 10 min with a rapid return to basal levels at later time intervals. At doses of 5 and 25 pg, A-endorphin led to a 20- to 30-fold stimulation-of levels of plasma growth hormone, the maximal effect being measured between 20 and 30 min after injection. These data su 'est the possible role of the endogenous opiate-like peptides in the control of growth hormone'secretion. Morphine is well known to be a potent stimulus of growth hormone (GH; somatotropin) release in the rat (1-3). This effect of the opiate is likely to be mediated by increased release of GH-releasing activity from the hypothalamus (4). Following reports of the presence of endogenous opiate activity in the brain (5-7), the pentapeptide H-Tyr-Gly-GlyPhe-Met-OH (Met-enkephalin) has been isolated from porcine brain (8). This peptide has'been shown to have potent opiate activity (8) and to bind to the opiate receptor (9, 10). The amino-acid sequence of Met-enkephalin is the same as that of the NH-terminal pentapeptide of the COOH-fragment [f3-LPH(61-91)] of 13-lipotropin first isolated from sheep pituitaries (11). This peptide, called f3-endorphin, has been isolated and characterized from camel pituitary glands (12) and shown to be a potent analgesic agent, as assayed by both in vitro (12-15) and in vivo (16,17) methods. These findings raised the possibility whether fl-endorphin could, like morphine (1-4), induce GH release in vivo. Changes of plasma GH levels could then become a useful in vivo test of biological activity of the endogenous opioid peptides and their analogs.
MATERIALS AND METHODS Adult male Sprague-Dawley rats (obtained from Canadian Breeding Farms, St. Constant, Quebec), weighing 250-275 g upon arrival, were kept in a sound-attenuated and temperature-controlled room (24 +.2O) illuminated from 0500 to 1900 hr. Purina rat chow and water were available ad libitum. In order to minimize stress-induced inhibition of GH secretion, we handled the animals twice a day for 7-10 days before inserting a catheter (Venocath no. 18, Abbott) into the right superior vena cava under Surital (50 mg/kg, intraperitoneally) anesthesia. A metallic cannula (gauge 25) was then implanted stereotaxically in the left lateral ventricle of the animals according to the coordinates (A-5.4, L-2, D-3) described by De Groot (18). The cannula was fixed to the skull with a polymerizing acrylate (Yates Flash Acrylic). This chronic technique permits a minimum of manipulations of the animal at the time of the experiment performed 2 days after implantation of the cannulae. Animals were then injected intravenously through the in-
DISCUSSION data demonstrate clearly that fl-endorphin adThe present ministrated intraventricularly can be a potent stimulus of GH release in the rat, while the activity of Met-enkephalin is much lower. As calculated from the areas under the plasma GH response curves, (3-endorphin. is, on a molar basis, approximately 2000 times more potent than Met-enkephalin.
Abbreviation: GH, growth hormone (somatotropin).
358
Medical Sciences: Dupont et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
359
Table 1. Effect of 400 ,g of Met-enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) injected intraventricularly on plasma GH levels in the rat Min after injection
Control Met-enkephalin
-5
0
5
10
20
30
45
19 ± 4 25 ± 6
13 ± 1 31 ± 8
18 ± 6 51 ± 30
GH (ng/ml plasma) 18 ± 4 119 ± 34*
24 ± 6 44 ± 17
26 ± 6 28 ± 6
20 ± 6 21 ± 7
The experiment was performed as described in the legend of Fig. 1. P < 0.05.
This difference of biological activity is in marked contrast to the relative affinities of the two peptides for the opiate receptor. In fact, when tested for their ability to displace [3H] etorphine (14, 15) and [3H]Met-enkephalin (0. Morin, M. G. Caron, and F. Labrie, unpublished data), fl-endorphin was found to be only two to six times more potent than Met-enkephalin. When injected centrally in the mouse, the analgesic activity of fl-endorphin was 18 to 33 times greater than morphine sulfate on a molar basis, whereas Met-enkephalin was relatively inactive (16). These findings would suggest that the higher biological activity of fl-endorphin, when injected intracerebrally, is partly due to its higher resistance to proteolytic degradation. We have recently found that intravenous administration of somatostatin antiserum leads to a rapid rise of plasma GH levels in the rat, thus indicating that somatostatin plays an important o....o Control . v- *8-Endorphin 0.5pg 0--^o , 2 pg
M---a.P 25
350
pg
300
7E N,
200i
0L 150I
-M
A
50 -
5
0
5
10
20
30
45
60
MINUTES AFTER INJECTION FIG. 1. Effect of increasing doses of fl-endorphin on plasma GH levels in the rat. Male rats bearing intraventricular and intrajugular cannulae were injected intravenously with 0.2 ml of sheep somatostatin antiserum 5 min before the intraventricular injection of the indicated amounts of synthetic fl-endorphin. GH concentrations were measured at the indicated time intervals after administration of flendorphin. Data are presented as mean + SEM.
physiological role in the control of GH secretion (3). Using this model, we found that the combined administration of somatostatin antiserum and morphine led to an additive effect on the plasma GH concentration. Since circulating somatostatin was expected to be neutralized by the antibody, it was suggested that morphine stimulates GH secretion through an increased release of GH-releasing activity from the hypothalamus. The present data suggest the possible role of the endogenous opiate-like peptides, ,-endorphin and Met-enkephalin, in the control of GH secretion; this effect is likely mediated by increased release of GH-releasing activity. Preliminary studies also showed that 83-endorphin caused the release of prolactin when injected intraventricularly in the rat. Thus, fl-endorphin may play an important role in the control of many neuroendocrine functions. 1. Kokka, N., Garcia, J. F., George, R. & Elliott, H. W. (1972) Endocrinology 90, 735-743. 2. Ferland, L., Labrie, F., Coy, D. H., Arimura, A. & Schally, A. V. (1976) J. Mol. Cell. Endocrinol. 4, 79-88. 3. Ferland, L., Labrie, F., Jobin, M., Arimura, A. & Schally, A. V. (1976) Biochem. Biophys. Res. Commun. 68, 149-156. 4. Ferland, L., Labrie, F., Arimura, A. & Schally, A. V. (1976) J. Mol. Cell. Endocrinol., in press. 5. Terenius, L. & Wahlstr6m, A. (1975) Acta Physiol. Scand. 94, 74-81. 6. Hughes, J., Smith, T. W., Morgan, B. & Fothergill, L. A. (1975) Life Sci. 16, 1753-1758. 7. Pasternak, G. W., Goodman, R. & Snyder, S. H. (1975) Life Sci. 16, 1765-1769. 8. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. & Morris, H. R. (1975) Nature 258,577-579. 9. Bradbury, A. F., Smyth, D. G. & Snell, C. R. (1976) Nature 260, 793-795. 10. Morin, O., Caron, M. G., De Lean, A. & Labrie, F. (1976) Biochem. Blophys. Res. Commun., in press. 11. Li, C. H., Barnafi, L., Chretien, M. & Chung, D. (1965) Nature 208,1093-1094. 12. Li, C. H. & Chung, D. (1976) Proc. Natl. Acad. Sci. USA 73, 1145-1148. 13. Li, C. H., Lemaire, S., Yamashiro, D. & Doneen, B. A. (1976) Biochem. Biophys. Res. Commun. 71, 19-25. 14. Cox, B. M., Goldstein, A. & Li, C. H. (1976) Proc. Natl. Acad. Sci. USA 73, 1821-1823. 15. Lazarus, L. H., Ling, N. & Guilleniin, R. (1976) Proc. Natl. Acad. Sci. USA 73,2156-2159. 16. Loh, H. H., Tseng, L. F., Wei, E. & Li, C. H. (1976) Proc. Natl. Acad. Sci. USA 73,2895-2898. 17. Tseng, L. F., Loh, H. H. & Li, C. H. (1976) Nature 263,239. 18. De Groot, J. (1959) Trans. R. Neth. Acad. Sci. 52, 1-40. 19. Birge, C. A., Peake, G. T., Mariz, I. K. & Daughaday, W. H. (1967) Endocrinology 81, 195-200. 20. Odell, W. D., Rayford, P. L. & Ross, G. T. (1967) J. Lab. Clin. Med. 70, 973-980. 21. Rodbard, D. & Lewald, J. E. (1970) in Karolinska Symposia on
Research Methods in Reproductive Endocrinology, 2nd, ed. Diczfalusy, E., pp. 79-103. 22. Kramer, C. Y. (1956) Biometrics 12,307-310.
