0O13-7227/78/O102-OOO2$O2.OO/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 102, No. 2

Printed in U.S.A.

CATHERINE RIVIER,f JEAN RIVIER, AND WYLIE VALE Laboratories for Neuroendocrinology, The Salk Institute, La Jolla, California 92037 diphenhydramine or the opiate antagonist naloxone. By contrast, naloxone reverses the GH-releasing activity of bombesin, suggesting an opiate-dependent mechanism of action of this peptide on GH secretion. The absence of an in vitro effect of bombesin and related peptides on PRL and GH secretion and a minimal effective dose of approximately 30 ng in bombesin and alytesin administered in steroid-primed rats, make them the most active peptides reported so far to act on the brain to modify PRL and GH secretion. (Endocrinology 102: 519, 1978)

ABSTRACT. Bombesin and other related peptides isolated from the skin of anuran species and found in mammalian brain stimulate PRL and GH release in steroid-primed male rats. On a molar basis, bombesin and alytesin are the most active peptides in this assay system. Their PRL- and GH-releasing activity is not affected by the type of anesthetics used, but their minimal effective dose is lower after intravenous administration as compared with intracisternal administration. The in vivo stimulatory effect of bombesin on PRL secretion is not modified by the histamine antagonist

S

EVERAL NATURALLY occurring peptides, neurotensin (NT), substance P (SP), and /?-endorphin, have recently been shown to stimulate the secretion of PRL and GH in normal or steroid-primed rats (1-3). Since these peptides do not release PRL and GH from cultured anterior pituitary cells, we suggested that they have a central nervous system site of action (1, 2). The tetradecapeptide bombesin, and several related peptides originally isolated and characterized from the skin of various anuran species (4), and more recently found in mammalian brain (5), have been found to be highly potent in lowering body temperature in rats (6). Since NT also lowers body temperature (6, 7), we have studied the effects of these peptides on the secretion of PRL and GH.

mg/100 g BW) or ether. A control blood sample was removed from the jugular vein, followed by injection of the test material into the same vein (iv) or intracisternally (ic). A second blood sample was obtained 5, 10, or 15 min later. Heparin was added to the blood, which was centrifuged at 4 C and the plasma was kept at —20 C until assayed. The peptides were diluted in saline for iv injections and in artificial cerebrospinal fluid for ic injections. Diphenhydramine and naloxone were administered iv. PRL and GH determinations were performed by double antibody methods as previously described (1). All peptides were synthesized by solid phase methodology (8). Diphenhydramine was purchased from Parke, Davis and Company, Detroit, MI, and naloxone was a gift from Endo Laboratories, Inc., Garden City, NY.

Results and Discussion

Materials and Methods Male Holtzmann rats (42-44 days old) received estrogen (50 jug) and progesterone (25 mg sc) in corn oil daily for 3 days before the assay. On the 4th day, they were anesthetized with urethane (150 July 13,1977. * This research was supported by NIH Grants AM18811, HD-09690, and National Foundation 1-411. f Requests for reprints should be addressed to: Dr. Catherine Rivier, The Salk Institute, Laboratories for Neuroendocrinology, P.O. Box 1809, San Diego, California 92112.

The sequences of the peptides tested are presented in Table 1. Figure 1 illustrates the elevation of PRL plasma level after iv administration of several doses of alytesin, bombesin, ranatensin, and litorin, as compared with NT and SP. On a molar basis, bombesin and alytesin are around 100 times more potent than NT and SP, and approximately 50 times more potent than ranatensin and litorin, which are 11- and 9-amino acid peptides, respectively, isolated from the skin of other frog species and which share the 8 C-terminal

519

Downloaded from https://academic.oup.com/endo/article-abstract/102/2/519/2618233 by The Australian National University user on 06 December 2018

The Effect of Bombesin and Related Peptides on Prolactin and Growth Hormone Secretion in the Rat*

520

Kndo • 1978 Voll02»No2

RIVIER ET AL.

TABLE 1. Sequences of bombesin, alytesin, substance P, neurotensin, ranatensin, and litorin pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-AlaVal-Gly-His-Leu-Met-NH2 Alytesin: pGlu-Gly-Arg-Leu-Gly-Thr-Gln-Trp-AlaVal-Gly-His-Leu-Met-NH2 Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-Met-NH2 Neurotensin: pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-ArgPro-Tyr-Ile-Leu-OH Ranatensin: pGlu-Val-Pro-Gln-Trp-Ala-Val-Gly-HisPhe-Met-NH2 Litorin: pGlu-Gln-Trp-Ala-Val-Gly-His-Phe-MetNH2

-1000

-800

-600

0

5

15

0

5

15

MINUTES

FIG. 2. Comparison of the influence of urethane and ether anesthesia on the PRL- and GH-releasing activity of bombesin in steroid-primed rats as a function of time

after administration of the peptide. , Plasma hormone levels of control rats; , plasma hormone levels of bombesin-injected rats. Each point represents the mean ± SEM for groups of five rats.

- 1000

800

FIG. 1. Effect of graded doses of iv administered peptides on plasma PRL levels in steroid-primed rats. Each point represents the mean ± SEM for groups of five rats.

amino acids of bombesin (9-11). Since our original observation of the PRL-releasing activity of /?-endorphin (2), we have found (not shown) that its minimal active dose is approximately 0.3 fig, which makes it 10 times less active than bombesin. As has been previously shown for NT, SP, and /?-endorphin (1, 2), bombesin and alytesin do not modify PRL or GH secretion by cultured pituitary cells (not shown). Since a number of anesthetics affect PRL and GH plasma levels (12,13), we have investigated the possibility that the sensitivity of our test animals might be modified by the anesthetic employed. As illustrated in Fig. 2, comparison of the PRL and GH responses to bombesin show similar elevations of plasma

•600

0.01 0.1

10

0.01 0.1

fiq BOMBESIN

FIG. 3. Influence of the route of administration on the PRL- and GH-releasing activity of several doses of bombesin. c, Control. Each point represents the mean ± SEM for groups of five rats. *, P < 0.05; **, P < 0.01.

hormone levels under urethane- or ether-induced anesthesia. Similarly, Lawson and Gala (13) have observed no effect of anesthetics on the TRF-induced stimulation of PRL secretion. Using rats under urethane anesthesia, we then studied the influence of the route of administration of bombesin on PRL and GH secretion (Fig. 3). After iv administration, 0.01

Downloaded from https://academic.oup.com/endo/article-abstract/102/2/519/2618233 by The Australian National University user on 06 December 2018

Bombesin:

PEPTIDE EFFECTS ON PRL AND GH SECRETIONS

20

CON

TABLE 2. Interaction of iv injected bombesin and pharmacological blockers on PRL and GH secretion in vivo" % Stimulator alone Treatment Bombesin 0.5 Mg 0.5 jug + Nal* 0.25 mg 0.5 fig + DIPH 0.5 mg

n

PRL

P

GH

P

5 5 5

100 110 122

>0.05 >0.05

100 55 118

0.05

" In these experiments, the rats were sacrificed 5 min after injection of the test materials. h Nal: naloxone.

dependent mechanism (1). By contrast, we have observed that DIPH does not interfere with the PRL- or GH-releasing activity of bombesin (Table 2). However, the opiate antagonist naloxone reverses the action of bombesin on GH secretion [as it reverses its hypothermic effects (17)], without seeming to modify the ability of bombesin to stimulate PRL secretion, thus probably reflecting an opiatedependent mechanism of action of bombesin on GH release. As in the case of other secretagogues that stimulate the in vivo secretion of PRL and GH, the physiological role of bombesin and related peptides remains to be determined. Bombesin and alytesin, however, represent the most active peptides so far reported capable of acting on the brain to modify PRL and GH secretion. Acknowledgment The authors thank E. Tucker, C. Douglas, A. Wolfe, W. Schaber, and R. Kaiser for excellent technical assistance. We are indebted to the National Institute of Arthritis, Metabolism and Digestive Diseases, Rat Pituitary Hormone programs, for supplying the rat PRL and GH radioimmunoassay kits.

FIG. 4. Interaction of SS on the PRL- (A) and GH-releasing (B) activity of several doses of bombesin in steroid-primed rats. The open bars represent the plasma hormone levels of five rats injected with bombesin only, and the solid bars the plasma hormone levels of rats injected simultaneously with SS and bombesin. Both peptides were injected iv.

Downloaded from https://academic.oup.com/endo/article-abstract/102/2/519/2618233 by The Australian National University user on 06 December 2018

jug bombesin does not consistently elevate plasma PRL and GH levels, but 0.1 jug always results in a significantly increased hormonal secretion. Intracisternal injection seems to be a less efficient method, since no less than 1.0-3.0 jug bombesin routinely stimulates PRL and GH secretion. Thus, these results are in contrast with those observed on thermoregulation, in which ic administration only is effective (6). They probably reflect differences in the sites of action of these peptides to modulate these various biological parameters. With a minimal effective dose of approximately 30 ng iv, bombesin and alytesin are the most potent peptides acting on the brain to affect PRL and GH secretion. Somatostatin (SS) acts at the pituitary level to inhibit the spontaneous release of GH, as well as GH secretion mediated by a variety of secretagogues such as hypothalamic extracts, cAMP derivatives, and prostaglandins (14, 15). As is illustrated in Fig. 4, 10 /xg SS lowers basal GH secretion by about 50%, and completely abolishes the GH-releasing activity of bombesin. In accordance with previous observations (3, 16) we also observed some (but not statistically significant) effect of SS on basal and stimulated PRL secretion. Previous studies have suggested the involvement of histamine (1) or opiate (2) receptors in the centrally mediated PRL- and GHreleasing activity of some peptides. In particular, concomitant administration of diphenhydramine (DIPH), an Hi-histamine blocker, prevents the NT-induced PRL secretion (1). These results had led us to propose that neurotensin action is mediated via a histamine-

521

522

RIVIER ET AL.

References

of a hypothalamic peptide, neurotensin, J Biol Chem 250: 1907, 1975. 10. Von Euler, U. S., and T. H. Gaddum, An unidentified depressor substance in certain tissue extracts, J Physiol (London) 72: 74, 1931. 11. Chang, M. M., and S. E. Leeman, Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P, J Biol Chem 245: 4734, 1970. 12. Lawson, D. M., and R. R. Gala, The influence of surgery, time of day, blood volume reduction and anesthetics on plasma prolactin in ovariectomized rats, J Endocrinol 62: 75, 1974. 13. Lawson, D. M., and R. R. Gala, Influence of anesthetics on basal, perphenazine-induced and thyrotropin releasing hormone-induced prolactin secretion in ovariectomized, estrogentreated rats, J Endocrinol 66:151, 1975. 14. Vale, W., P. Brazeau, G. Grant, A. Nussey, R. Burgus, J. Rivier, N. Ling, and R. Guillemin, Premieres observations sur le mode d'action de la somatostatine, un facteur hypothalamique qui inhibe la secretion de l'hormone de croissance, C R Acad Sci (Paris) 275: 2913, 1972. 15. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier, and R. Guillemin, Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science ( Washington) 179: 77, 1973. 16. Vale, W., P. Brazeau, C. Rivier, M. Brown, B. Boss, J. Rivier, R. Burgus, N. Ling, and R. Guillemin, Somatostatin, Recent Prog Horm Res 31: 365, 1975. 17. Brown, M., J. Rivier, and W. Vale, Actions of bombesin, thyrotropin releasing factor, prostaglandin E_> and naloxone on thermoregulation in the rat, Life Sci 20: 1681, 1977.

Downloaded from https://academic.oup.com/endo/article-abstract/102/2/519/2618233 by The Australian National University user on 06 December 2018

1. Rivier, C, M. Brown, and W. Vale, Effect of neurotensin, substance P and morphine sulfate on the secretion of PRL and GH in the rat, Endocrinology 100: 751, 1977. 2. Rivier, C, W. Vale, N. Ling, M. Brown, and R. Guillemin, Stimulation in vivo of the secretion of prolactin and growth hormone by /8-endorphin, Endocrinology 100: 238, 1977. 3. Vale, W., C. Rivier, and M. Brown, Regulatory peptides of the hypothalamus, Ann Rev Physiol 39: 473, 1977. 4. Anastasia, A., V. Erspamer, and H. Bucci, Isolation and structure of bombesin and alytesin, two analogous active peptides from the skin of the European amphibians Bombina and Alytes, Experientia 27: 166, 1971. 5. Brown, M., J. Rivier, R. Kobayashi, and W. Vale, Neurotensin-like and Bombesin-like peptides: CNS distribution and action, International Gut Hormone Symposium, Lausanne, Switzerland, 1977 (in press). 6. Brown, M., J. Rivier, and W. Vale, Bombesin: Potent effects on thermoregulation in the rat, Science (Washington) 196: 998, 1977. 7. Bissette, G., C. Nemeroff, A. Prange, and M. Lipton, Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin, Nature (London) 262: 607, 1976. 8. Rivier, J., M. Brown, C. Rivier, N. Ling, and W. Vale, Hypothalamic hypophysiotropic hormones: review on the design of synthetic analogs, In Loffet, A. (ed.), Peptides '76, Editions de l'Universite de Bruxelles, 1976, p. 427. 9. Carraway, R., and S. E. Leeman, The amino acid sequence

Endo • 1978 Vol 102 • No 2