Failure of naloxone to influence physiological growth hormone and prolactin secretion

210

Brain Research, 168 (1979) 210-215 (') Elsevier/North-Holland Biomedical Press

Failure of naloxone to influence physiological growth hormone and prolactin secretion

JOSEPH B. MARTIN*, GEORGE TOLLS, IVAN WOODS and HARVEY GUYDA Departments of Neurology, Medicine and Pediatrics, McGill University, Montreal (Canada)

(Accepted January 18th, 1979)

Morphine and the opioid peptides, fl-endorphin, [MetZ]enkephalin and [LeuZ]enkephalin are reported to stimulate growth hormone (GH) and prolactin (PRL) secretion in the rat2-5,7, 9,14. Hormone release has been demonstrated after both systemic [intravenous (IV), intraperitoneal (IP) or subcutaneous (SC)] and intracerebral ventricular (ICV) injections; the amounts given have, in general, been large (1-100 mg systemic or 1-100 #g ICV) and anesthetized rats have commonly been used. Shaar et al. is reported that enkephalin analogs administered SC in doses of 10 mg/kg also elicited G H and PRL release in the female rat. The site and mechanism of action of these effects has not been determined. A hypothalamic or other brain locus is suggested by the fact that the substances fail to elicit hormone release from pituitary glands in vitro 1. Martin et al. 1° showed that large hypothalamic lesions failed to completely block morphine-induced G H release and postulated that the effects were mediated at the level of the median eminence. Both enkephalin and fl-endorphin containing cell perikarya occur in the hypothalamus with axons that terminate in the median eminence ~,s. The role of endogenous opioids in the regulation of physiologic G H and PRL secretion is controversial. Previous reports in which single samples of blood were obtained by decapitation suggested a suppression of baseline G H and PRL levels after administration of naloxone, an opioid antagonist 2,7. Stress and suckling-induced PRL responses are also reported to be inhibited by prior administration of opioid antagonists 7. The objective of the present studies was to investigate the effects of naloxone on G H and PRL secretion in unanesthetized rats and in human subjects during sleep. Experiments in our laboratory have documented that physiologic G H secretion in unanesthetized, freely behaving rats is episodic with abrupt surges of secretion occurring at intervals of 3-4 h12, ~9. Basal PRL secretion in such rats is low (less than 10 ng/ml) with infrequent small secretory surges. Sleep-associated changes in G H and PRL secretion are well documented in man, a surge of G H occurring within 2 h of sleep * To whom all correspondence should be sent at: Department of Neurology, Massachusetts General Hospital, Fruit Street, Boston, Mass. 02114, U.S.A.

211 onset, whereas, PRL rises in a series of secretory bursts during the later period of sleep 15. Male Charles River (CD) rats, were prepared with chronic indwelling jugular cannulae as previously describedlL After recovery from surgical procedures, the rats were adapted to sampling cages and blood was removed every 15 rain for 6 h beginning at 1000 h. Naloxone (Narcan) was administered IV in two doses at 1015 and 1230 h to coincide with anticipated GH secretory surges. Control rats received 0.9 ~ saline vehicle. Each blood sample was centrifuged, plasma removed, and red blood cells resuspended in saline were reinjected into the animal to minimize blood volume and hematocrit changes. Plasma GH and PRL were measured by radioimmunoassay using material supplied by Dr. Albert Parlow and the National Institute of Arthritis, Metabolism and Digestive Diseases19.,19. Sleep-associated GH and PRL secretion were studied in 5 female subjects aged 21-28 years. Blood samples were removed every 20-30 rain from an antecubital vein with electroencephalographic recording techniques applied to monitor sleep stage. Each subject was sampled on two separate nights during the early follicular phase of the menstrual cycle. During the control study, each subject received an infusion of normal saline. On the experimental night a continuous intravenous infusion of 0.8 mg/kg naloxone/h was given. Blood samples were centrifuged and GH and PRL levels measured by radioimmunoassay 20. GH secretion in control rats was characterized by rapid surges of release occurring at intervals of 3 4 h (Fig. 1, top). Each burst of GH consisted of a rise from undetectable ( < 5 ng/ml) to levels that exceeded 200 ng/ml, results consistent with previously reported observations 19. Administration ofnaloxone in doses of 0.4, 10 and 20 mg/kg b.w. had no significant effect on individual bursts of GH secretion (Fig. 1, bottom). Mean GH levels as measured by planimetry were unchanged in control (81.9 4- 12.2 ng/ml) versus naloxone-treated rats (0.4 mg/kg-89.4 4- 12.9; 10 mg/kg-76.8 4- 11.2; 20 mg/kg-99.1 4- 11.4 ng/ml, respectively). Plasma PRL levels were low ( < 10 ng/ml) and frequently undetectable ( < 1 ng/ml). Naloxone had no significant effect on PRL levels; elevation of PRL in response to a mild stress (handling for 3 min) at the end of the experimental sampling period was not prevented by the prior administration of the opioid antagonist. Administration of naloxone also failed to influence nocturnal GH or PRL secretion in the human subjects. GH release normally occurs during the early part of night sleep in association with the first episode of Stage Ill-IV sleep (Fig. 2). In contrast, PRL levels rise in episodic bursts during the latter part of sleep to reach highest values immediately prior to awakening (Fig. 2). Peak GH levels on the control night (9.4 4- 3.1 ng/ml) were not significantly different from those on the night that naloxone was administered (9.6 4- 4.8 ng/ml). Similarly, peak levels of PRL were not significantly different (26.2 4- 5.4 vs 28.2 4- 6.4 ng/ml) after saline or naloxone administration. The duration of total sleep (341 4- 17 vs 348 4- 51 min) and per cent of sleep characterized as Stage I and II (68 4- 7 vs 71 4- 3), Stage III and IV (19 :k 6 vs 18 ± 3) and rapid eye movement (REM) (12 4- 3 vs 15 zk 5), were unchanged by the administration of naloxone (Fig. 2).

212

Saline Control 500.0

,', 200 Growth

I

I

10:00

11:00

Hormone

I

I

I

I

I

12:00

13:00 Time in hours

14:00

15:00

16:00

Naloxone 20mg/kg

I

I

J9-1

30C

= ~. 200

~- 100

,¢ 10:0t

I

11:00

I

12:00

I

13..~

I

14:00

!

15:00

I

16:00

Time in hours Fig. 1. Plasma growth hormone (GH) and prolactin (PRL) levels after administration of saline (upper) or naloxone 20 mg/kg (lower) to rats. Injections were given i.v. at times indicated by arrows. Naloxone had no effect on secretion of either hormone.

213 D.M.

24

i 16 8

a.

~

Prolactin GrowthHormone

0

Awake Total Sleep Time: 276 qlir~ Stage ]: 21.4% Stage T[: 52.2% Stage TIT: 7.2% Stage IV: 19.2% REM: 0

8" 111

REM i

,

24:00

I

I

2:00

I

I

4:00

I

I

6:00

Time in hours

D.M.

32 24 L

"4

I Prolactin

16

Z N

E

8

.....o.-

'~'u""J"'Q "t""" ".......... / G r o w t h

Hormone

L,

0

Awake

r

-i[

24:00

i

I

2:00

I

I

4:00

I

i

6:00

Tptal Sleep Time: 356 rain Stage ~ 1.4% Stage 11: 73.0% Stage Ili: 9.3% Stage W': 7.0% REMI 9.3%

I

Time in hours Fig. 2. Plasma growth hormone (GH) and prolactin (PRL) levels during sleep in one subject (D.M.) during control (upper) and naloxone (lower) infusions. In each graph, GH secretion occurs concurrent with first episode of stage III-IV sleep and PRL rises during the morning hours. Naloxone has no apparent effect on secretion of either hormone or on sleep.

214 These data suggest that endogenous opioids have a minor role, if any, in the mediation of physiologic G H and PRL secretion in rat and man. The doses of naloxone used in the present experiments are sufficient to block the effects of morphine and of opioid peptides in several experimental paradigms17, 22. A similar dose of naloxone in man was shown to block the prolactin-stimulating effect of morphine. These results are at variance with the previous reports of Bruni et al. 2 and Shaar et al. is, but are consistent with the observations of Cocchi et al. 4. Bruni et al. 2 reported that naloxone 0.2 mg/kg administered IP resulted in a significant decrease in plasma G H levels in decapitated rats. The marked variability in plasma G H levels which occur as a result of pulsatile hormone secretion make difficult any interpretation of group data obtained from single sampling points 19. in the report of Shaar and coworkers TM, doses of naloxone from 0.02-0.4 mg/kg resulted in inhibition of basal G H levels in female immature rats when the results were expressed as per cent inhibition. Higher doses failed to have any effect. Analysis of saline treated controls indicate that G H levels in these rats were very low [13.6 ÷ 4.8 ng/ml], much less than those reported in adult animals. Cocchi and coworkers 4 who also used infant rats of either sex found no effects of naloxone administered alone when compared to vehicle. The results obtained from the experiments in the female volunteers also suggest that naloxone has no significant effect on the nocturnal secretion of G H and PRL in the human. Furthermore, the data show that endogenous opioid antagonism has no significant effects on total sleep time, or sleep stage in man, an observation that has not been previously reported in the literature. It is important to reconsider the significance of morphine and the endogenous opioids in terms of their postulated neuroendocrine effects. Acute epiteptogenic effects of morphine, as shown by recording electrodes placed in the ventromedial hypothalamic nucleus, brain stem and cerebral cortex have been documented in rats1% Administration of the opioid peptides is reported to induce similar seizure activity in rats 6,22. Thus, G H and PRL responses to either morphine or opioid peptide administration may be mediated by induction of abnormal electrical events in brain. Similar neuroendocrine responses occur in experimental animals after electrical stimulation of either hypothalamic or limbic system areas 1°,13. The present findings do not exclude the possibility that opioid peptides participate in the neural control of pituitary G H secretion. They do suggest, however, that the hypothalamic stimulus that is involved in the abrupt surges of G H secretion, which we have postulated elsewhere to be the result of stimulation by G H releasing factor 12, is not dependent in the physiologic state on release of endogenous opioids. It is more likely that the opioids act to modulate neuroendocrine responses to perturbations of the neuroendocrine axis such as occur during stress as have been reported by othersT, 23.

1 Bloom,F. E., Rossier, J., Battenberg, E. L. F., Bayon, A., French, E., Henriksen, S. J., Siggins, G. R., Segal,D., Browne, R., Ling, N. and Guillemin, R., fl-endorphin : cellular localization, electrophysiological and behavioral effects. In E. Costa and M. Trabucchi (Eds.), The Endorphins: Advances in Biochemical Psychopharmacology, Vol. 18, Raven Press, New York, 1978, pp. 51-70.

215 2 Bruni, J. F., Van Gugt, D., Marshall, S. and Meites, J., Effects of naloxone, morphine and methionine enkephalin on serum prolactin, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone and growth hormone, Life Sci., 21 (1977) 461-466. 3 Chihara, L., Arimura, A., Coy, D. H. and Schally, A. V,, Studies on the interaction of endorphins, substance P, and endogenous somatostatin on growth hormone and prolactin release in rats, Endocrinology, 102 (1978) 281-290. 4 Cocchi, D., Santagostino, A., Gil-ad, I., Ferri, S. and Muller, E., Leu-enkephalin-stimulated growth hormone and prolactin release in the rat: comparison with the effect of morphine, Life Sci., 20 (1977) 2041-2046. 5 Dupont, A., Cusan, L., Caron, M., Labrie, F. and Li, C. H., fl-endorphin stimulation of growth hormone release in vivo, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 358-359. 6 Frenk, H., Urca, G. and Liebeskind, J. C., Epileptic properties of leucine- and methionineenkephalin: comparison with morphine and reversibility by naloxone, Brain Research, 147 (1978) 327-337. 7 Grandison, L. and Guidotti, A., Regulation of prolactin release by endogenous opiaies, Nature (Lond.), (1977) 357-359. 8 Johansson, D., H6kfelt, T., Elde, R. P., Schultzberg, M. and Terenius, L., Immunohistochemical distribution of enkephalin neurons. In E. Costa and M. Trabucchi (Eds.) The Endorphins: Advances in Biochemical Psychopharmacology, Vol. 18, Raven Press, New York, 1978, pp. 51-70. 9 Lien, E. L., Feniehel, R. L., Garsk, V., Sarantakis, D. and Grant, N., Enkephalin-stimulated prolactin release, Life Sci., 19 (1978) 837-840. 10 Martin, J. B., Plasma growth hormone (GH) response to hypothalamic or extrahypothalamic electrical stimulation, Endocrinology, 91 (1972) 107-115. 11 Martin, J. B., Audet, J. and Saunders, A., Effect of somatostatin and hypoihalamic ventromedial lesions on GH release induced by morphine, Endocrirology, 96 (1975) 839-847. 12 Martin, J. B., Durand, D., Gurd, W., Gaille, G., Audet, J. and Brazeau, P., Neuropharmacologic regulation of episodic growth hormone and prolactin secretion in the rat, Endocrinology, 102 (1978) 106-113. 13 Quadri, S. K., Norman, R. L. and Spies, H., Prolactin release following electrical stimulation of the brain in ovariectomized and ovariectomized estrogen-treated rhesus monkeys, Endocrinology, 100 (1977) 325-330. 14 Rivier, C., Vale, W., Ling, M., Brown, M. and Guillemin, R., Stimulation in vivo of the secretion of prolactin and growth hormone by fl-endorphin, Endocrinology, 100 (1977) 238-241. 15 Sassin, J. F., Frantz, A. G., Weitzman, E. D. and Kapen, S., Human prolactin: 24-hour pattern with increased release during sleep, Science, 177 (1972) 1205-1207. 16 Sawyer, C. H., Critchlow, B. V. and Barraclough, C. A., Mechanism of blockade of pituitary activation in the rat by morphine, atropine and barbiturates, Endocrinology, 57 (1955) 345-354. 17 Segal, D. S., Browne, R. G., Bloom, F., Ling, N. and Guillemin, R., fl-endorphin: endogenous opiate or neuroleptic? Science 198 (1977) 411-414. 18 Shaar, C. J., Frederickson, R. C. A., Dininger, N. B. and Jackson, L., Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin--evidence for regulation by an endogenous opioid peptide in brain, Life Sci., 21 (1977) 853-860. 19 Tannenbaum, G. S. and Martin, J. B., Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat, Endocrinology, 98 (1976) 562-570. 20 Tolis, G., Dent, R. and Guyda, H., Opiates, prolactin and the dopamine receptor, J. clin. Erdocr., 47 (1978) 200-203. 21 Tolis, G., Ruggere, D., Pinter, E. and Banovac, K., Naloxone blockade of morphine induced hyperprolactinemia and its ineffectiveness to modify basal prolactin (PRL) secretion or its release by thyrotropin releasing hormone and metochtopramide, Endocrinology, 102, Suppl. 1 (1978) 359. 22 Urea, G., Frenk, H. and Liebeskind, J., Morphine and enkephalin: analgesic and epileptic properties, Science, 177 (1977) 83-86. 23 Van Vugt, D. A., Bruni, J. F. and Meites, J., Naloxone inhibition of stress-induced increase in prolactin secretion, Life Sci., 22 (1978) 85-90.