Klinische Wochenschrift

Klin Wochenschr (1987) 65:453-457

© Springer-Verlag 1987

Potentiation of the hCRF-Induced Release of ACTH in Man by an Opioid Antagonist* H. Ehrenreich, Ch. Kolmar, O.A. Miiller, and F.-D. Goebel Medizinische Poliklinik and Medizinische Klinik Innenstadt, UniversitS.t Mfinchen

Summary. Administration of synthetic human corticotropin-releasing factor (hCRF; 2 gg/kg body weight) to six normal male subjects produced a significant rise in plasma ACTH, followed by an increase in circulating cortisol. Simultaneous treatment with the opioid antagonist naloxone (1.6 mg i.v. bolus, followed by an infusion at a rate of 1.2 rag/h) significantly potentiated the hCRF-induced rise in ACTH and enhanced the cortisol response to hCRF. It is suggested that naloxone acts by antagonizing an inhibitory ultra-short-loop feedback effect of coreleased fl-endorphin on pituitary corticotrophs, thereby amplifying the net effect of hCRF, i.e., the release of ACTH.

Key words: Human corticotropin-releasing factor (hCRF) - ACTH - Cortisol - Naloxone - Opioid receptor blockade

lanocortin (POMC)-derived peptides, e.g., ACTH and fl-endorphin, followed also by cortisot in man [3, 8, 10, 12, 13]. At several sites and under various conditions, endogenous opioid systems seem to be involved in the hypothalamus-pituitary-adrenal axis. Opioid peptides are co-localized with C R F in rat hypothalamic neurons [6, 15]. Opioids inhibit the release of POMC-related peptides and lower cortisol levels in man [4, 18, 19]. Finally, opioid peptides seem to interfere with corticosteroidogenesis directly at the adrenal level [14]. It was therefore of interest to investigate the effect of the opioid antagonist naloxone on the hCRF-induced release of ACTH and cortisol in man.

Subjects and Methods

The isolation and characterization of a corticotropin-releasing factor (CRF) from sheep hypothalami was first reported in 1981 (20). Recently, the structure of human CRF was demonstrated by analysis of the human prepro-CRF gene [16]. Human CRF (hCRF), like ovine CRF (oCRF), is a polypeptide of 41 amino acids, but differs from oCRF by seven amino acids and, at least, by its pharmacokinetic properties [5, 17]. Both peptides are potent stimulators of the release of proopiome* This study was supported by the Deutsche Forschungsgemeinschaft (Go 299/3-2; M n 585/2-2) A C T H = adrenocorticotropic hormone; C R F = corticotropinreleasing factor; h C R F = h m n a n C R F ; o C R F = o v i n e C R F ; E K G = electrocardiogram ; P O M C = proopiomelanocortin; RIA = radioimmunoassay; S.E.M. = standard error of the mean

Six healthy men (25 to 42 years of age) volunteered to participate in this study after approval of the study protocol by the Committee for Medical Ethics of the Medical Faculty of the University of Munich. Each subject was tested on two to four occasions in random order, at intervals of a least 1 week. The basic protocol remained identical on all occasions: After an overnight fast, subjects had an indwelling intravenous cannuta inserted in an antecubital vein of each forearm. Through one of the cannulas, 0.9% saline solution (with or without naloxone, see below) was infused at a rate to compensate for blood volume withdrawn; the other cannula served for bolus injections and for collecting blood samples for hormone assays. Blood samples were taken at 0800, 0900, 1000, 1010, 1020, 1030, 1040, 1050, 1100, 1110, 1120, J130, 1145, 1200, 1215, 1230, 1300, 1330, 1400, and 1500 h. At 0815 h, subjects received a small standardized

454

H. Ehrenreichet al. : hCRF, ACTH, and Opioid ReceptorBlockade

breakfast (200 kcal) and then remained fasting, except for water, and supine throughout the study session. Respiratory rate, pulse, and blood pressure were measured in 10- to 15-min intervals throughout the study session. Heart rate and rhythms were monitored continuously using an electrocardiogram. Subjective and objective symptoms were protocolled. Occasion 1 (six subjects): At 1030 h, synthetic h C R F (Bissendorf Peptide, FRG, 2 gg/kg body weight in 5 ml 0.9% saline solution) was given as an intravenous bolus injection over 30 s. Occasion 2 (six subjects): At 1000 h, an intravenous bolus injection of naloxone (1.6 mg) was given, followed by an infusion of naloxone at a rate of 1.2 mg/h in 0.9% saline solution; at 1030 h, h C R F was injected as described for occasion 1. Occasion 3 (three subjects) Subjects received naloxone as described for occasion 2, but no hCRF. Occasion 4 (three subjects): Subjects received only an infusion of 0.9% saline solution, with bolus injections of saline at 1000 and 1030 h.

Radioimmunoassays ( RIA ) ACTH was measured by RIA using N-terminal specific antibodies after silicic acid plasma extraction, as described earlier [12]. Synthetic human 1-39 ACTH (Ciba-Geigy, Basel, Switzerland) served as standard (pg/ml). Blood specimens for ACTH were collected on ice, immediately centrifuged and deep frozen ( - 4 0 ° C) until analyzed. Cortisol was measured by RIA without prior extraction, as described previously [12]. The intraassay coefficient of variation for ACTH was less than 10% and for cortisol less than 5%, the interassay variation was less than 18% for ACTH and less than 11% for cortisol. Statistical evaluation of the data was performed using the nonparametric Wilcoxon matched-pairs signed-ranks test.

Results

hnmediately after the injection (p.i.) of h C R F (2 pg/kg b.w.), flushing of face and neck appeared in all six subjects accompanied by an intense feeling of facial warmth, lasting 30-90 min. Three of the six men experienced a transitory (5-10 min) sensation of mild dyspnea (tightness of the chest, sense of shortage of air) although there was no apparent change in respiratory rate in any of the subjects tested. Other short-lasting subjective symptoms (described by one person each) were coolness in mouth and throat, slight numbness,

and crawling sensation in the fingers. All six subjects manifested a slight, transient (5-15 rain) increase in heart rate (15%-25%) occurring 2-3 min p.i. hCRF. There was no change in blood pressure or EKG in response to hCRF. The side-effects of h C R F seemed milder during simultaneous treatment with naloxone. Naloxone alone did not cause any of these symptoms. In all subjects, plasma ACTH significantly rose in response to hCRF, displaying its peak level at individually variable intervals between 10 and 30 rain p.i. hCRF, and gradually returning to baseline at about 90 min p.i. (Fig. 1). Subsequently, h C R F provoked an increase in plasma cortisol commencing at 10-20 min p.i. with peak values to be noted at about 40 min p.i. and a return to baseline at 90-120 rain p.i. (Fig. 1). Simultaneous naloxone treatment clearly potentiated the hCRF-induced elevation of plasma ACTH, with ACTH values being significantly higher immediately before and at 10, 20, and 30 min p.i. h C R F as compared to treatment with h C R F alone. The peak level of ACTH following naloxone/hCRF occurred at 10 min p.i. h C R F in all subjects and was 2 to 3 times that after h C R F alone (Fig. 1). Natoxone also enhanced the response of plasma cortisol to hCRF. The augmentation of the hCRF-induced rise of cortisol by opioid receptor blockade, however, seemed milder and somewhat delayed as compared to the ACTH curve. Cortisol elevation became significant as early as 30 min p.i. naloxone, i.e., immediately before the injection of hCRF, then gradually climbed up to achieve its peak at about 50 min p.i. of h C R F and very slowly declined thereafter to reach baseline at the end of the study session. During this delayed return to baseline, cortisol values were significantly higher at all points as compared to the curve following h C R F alone (Fig. 1). Following naloxone alone, plasma ACTH increased 20 to 40 min after the bolus injection of the opioid antagonist, achieved its highest values, which almost reached those lbund after pure h C R F stimulation, at between 30 and 80 min p.i. and tended to remain elevated above baseline levels throughout the study session (Fig. 2). Plasma cortisol began to rise at 20 to 30 min p.i. of naloxone, displayed a peak, also comparable in size to that following pure h C R F stimulation, at about 60 to 70 min p.i. and remained elevated above baseline up to the end of the study period (Fig. 2). During saline infusion alone, ACTH and cortisol levels decreased from the higher morning to lower day values that were consistently to be measured throughout the study period (Fig. 2).

START OF NALOXONE TREATHENT !

160

c~ B( LU$

i

120

E w-, v

-i-

i,

80

1--

,~0

!

!

,1100

'

,=i00

'

tL00

'

I

•

!

I

l&

'

i~00 TIME (h I

3O "ID

-.... 20

I-OC

o

10

~ B.O0

,.. ~

~,

9.~

\

I

11iO0

I0.00

"~ ....

I.

!

12O0

13.~

~,

~

I~,~

:

15.~ TIME ( h )

Fig, 1. Plasma A C T H and cortisol responses in six normal subjects to a bolus injection of h C R F (2 ~tg/kg b.w. at 1030 h) with ( o - - - o - - - o ) or without (o - o - - o ) simultaneous naloxone administration (1.6 mg i.v. bolus at 1000 h, followed by an i.v, infusion at a rate of 1.2 mg/h until 1500 h). Mean_+ S,E.M.; *, significant difference using Wilcoxon matched-pairs signed-ranks test (n = 6 ; P < 0.05)

START OF NALOXONE

I00I

TREATMENT 1

"

~° t 20

0

8.00

9.00

10.00

11.00

12.00

131)0

1/,,.00

15.00

TIME ( h )

} "-~

,c.~..-,/"~':~.,.,, / 4

u~-" OrY

~

o

8. 'oo

'

9 ;oo

'

40.00

•

j .'~-l~%

"

'

1, Ioo

=

'

" ~2.oo

...-I .,~

~ .~__. "-...

",,,. -" - - 4

'

~Lo o

.~

'

"

' ~.oo

'

' ,~,oo

TIME (h) Fig. 2. Individual plasma ACTH and cortisol responses in three normal subjects to natoxone administration (1.6 mg i.v. bolus at 1000 h, followed by an i.v. infusion at a rate of 1.2 mg/h until 1500 h) ( e - - - e - - - e ) and to an i.v. control infusion of 0.9% saline solution (o-- - - e - - - e )

456

It. Ehrenreich et al. : hCRF, ACTH, and Opioid Receptor Blockade

Discussion The administration of h C R F to six normal men produced a rise in plasma A C T H , followed by an increase in circulating cortisol. This is in line with previous work, attributing to C R F the role of a physiological stimulatory factor of the hypophyseal corticotrophs [3, 5, 12, 13, 17]. C R F is k n o w n to induce the concomitant release of P O M C - d e rived peptides, e.g., A C T H and fl-endorphin [8, 10]. The functions o f coreleased fl-endorphin, however, are still obscure. Peripheral effects have been postulated, but n o t yet clearly demonstrated. The existence of at least three different opioid systems, giving rise to multiple endogenous opioid peptides, makes it difficult to attribute certain opioid effects to one specific opioid peptide [2, 7]. The finding that fl-endorphin given to normal subjects suppresses A C T H and cortisol levels has led to the conclusion that the site o f its action could be at h y p o t h a l a m i c (modulation of CRF-release) as well as at pituitary level [19]. In our study, treatment with the opioid antagonist naloxone, concurrent with the administration of h C R F , d e a r l y potentiated the h C R F - i n d u c e d rise in A C T H a n d enhanced the cortisol response to h C R F . It is therefore suggested that naloxone acts by antagonizing an inhibitory ultra-short-loop feedback effect of coreleased fl-endorphin on pituitary corticotrophs, thereby amplifying the net effect o f h C R F , i.e., the release of A C T H . The involvement o f other opioids, however, or the participation of other neuroendocrine substances, cooperating with opioids, c a n n o t be completely excluded f r o m this study. The elevation of plasma A C T H and cortisol induced by naloxone alone, a p h e n o m e n o n t h a t has previously been described by others [4, 11, 21], points to the existence o f a tonic inhibitory opioidergic control function on the secretion of P O M C related peptides. A m a x i m u m response to physiologically released a m o u n t s of C R F would thus, according to our hypothesis, be prevented by an inhibitory feedback action of fl-endorphin on pituitary corticotrophs - an effect, u n m a s k e d by opioid receptor blockade. Similar mechanisms have been postulated for several other sites of the central nervous system, e.g., the neurohypophysis, where metenkephalin, coexisting with oxytocin in the same nerve terminals, is t h o u g h t to exert an autoregulatory ultra-short-loop feedback effect on the secretion of oxytocin [1, 9]. An enzymatic m o d u l a t i o n of the local availability of fl-endorphin or transient modifications of the opioid receptor intrinsic activity by the actual metabolic and endocrine environment, for instance, could represent possible regula-

tory principles of such a mechanism on anterior pituitary level. The ability of the h y p o t h a l a m u s pituitary-adrenal axis to react to certain C R F - m e diated stimuli m a y thus quantitatively depend on the actual efficacy o f the fl-endorphinergic feedback system controlling the release o f A C T H and, subsequently, o f cortisol.

Acknowledgements: The authors would like to thank Dr. M.A. Schreiber for his kind help in the statistical analysis of the data and Dr. C. Pittius, Mrs. B. R6ttger, Mrs. K. Schedo, Mrs. M. Scheidter, and Mrs. J. Stalla for their excellent technical assistance.

References 1. Ehrcnreich H, Riisse M, Schams D, Hammerl J, Herz A (1985) An opioid antagonist stimulates myometrial activity in early postpartum cows. Theriogenology 23 : 309-324 2. Ehrenreich H, Goebel F-D (1986) The role of opioids in the endocrine function of the pancreas. Diabetes Res 3 : 59-66 3. Grossman A, Nieuwenhuijzen Kruseman AC, Perry L, Tomlin S, Schally AV, Coy DH, Rees LH, Comaru-Schally A-M, Besser GM (1982) New hypothalamic hormone, corticotropin-releasing factor, specifically stimulates the release of adrenocorticotropic hormone and cortisol in man. Lancet II : 921-922 4. Grossman A, Gaillard RC, McCartney P, Rees LH, Besser GM (1982) Opiate modulation of the pituitary-adrenal axis: effects of stress and circadian rhythm. Clin Endocrinol 17: 279-286 5. Hermus ARMM, Pieters GFFM, Pesman GJ, Buys WCAM, Smals AGH, Benraad TJ, Kloppenborg PWC (1984) Differential effects of ovine and human corticotropin-releasing factor in human subjects. Clin Endocrinol 21 : 589-595 6. H6kfelt T, Fahrenkrug J, Tatemoto K, Mutt V, Werner S, Hulting A-L, Terenius L, Chang KJ (1983) The PHI (PHI-27)/corticotropin-releasing factor/enkephalin immunoreactive hypothalamic neuron: possible morphological basis for integrated control of prolactin, corticotropin, and growth hormone secretion. Proc Nat Acad Sci USA 80:895-898 7. Hrllt V (1983) Multiple endogenous opioid peptides. Trends Neurosci 6: 24-26 8. Jackson RV, De Cherney GS, Debold CR, Sheldon WR, Alexander AN, Rivier J, Vale W, Orth DN (t984) Synthetic ovine corticotropin-releasing hormone: simultaneous release of proopiolipomelanocortin peptides in man. J Clin EndocrinoI Metab 58 : 740--743 9. Martin R, Voigt KH (1981) Enkephalins co-exist with oxytocin and vasopressin in nerve terminals of rat neurohypophysis. Nature 289:502-504 10. McLoughlin L, Tomlin S, Grossman A, Lytras N, Schally AV, Coy D, Besser GM, Rees LH (1984) CRF-41 stimulates the release of fl-lipotrophin and fl-endorphin in normal human subjects. Neuroendocrinology 38:282--284 l l. Morley JE, Baranetsky NG, Wingert TD, Carlson HE, Hershman JM, Melmed S, Levin SR, Jamison KR, Weitzman R, Chang RJ, Varner AA (1980) Endocrine effects of naloxone-induced opiate receptor blockade. J Clin Endocrinol Metab 50:251-257 12. Mfiller OA, D6rr ttG, Hagen B, Stalla GK, yon Werder K (1982) Corticotropin releasing factor (CRF)-stimulation

H. Ehrenreich et al. : hCRF, ACTH, and Opioid Receptor Blockade test in normal controls and patients with disturbances of the hypothalamo-pituitary-adrenal axis. Klin Wochenschr 60:1485-1491 13. Mfiller OA, Stalla GK, v. Werder K (1983) Corticotropin releasing factor: a new tool for the differential diagnosis of Cushing's syndrome. J Clin Endocrinol Metab 57:227229 14. Racz K, Glaz E, Kiss R, Lada G, Varga I, Vida S, DiGleria K, Medzihradszky K, Lichtwald K, Vecsei P (1980) Adrenal cortex - a newly recognized peripheral site of action of enkephalins. Biochem Biophys Res Commun 97:134(~i 353 15. Roth KA, Weber E, Barchas JD, Chang D, Chang J-K (1983) Immunoreactive dynorphin (1-8) and corticotropinreleasing factor in subpopulation of hypothalamic neurons. Science 219:189-191 16. Shibahara S, Morimoto Y, Futurani Y, Notake M, Takahashi H, Shimizu S, Horikawa S, Numa S (1983) Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. EMBO J 2:775-779 17. Stalla GK, Hartwimmer J, Schopohl J, v. Werder K, Mtiller OA (1986) Intravenous application of ovine and human corticotropin releasing factor (CRF): ACTH, cortisol and CRF levels. Neuroendocrinology 42:1-5 18. Stubbs WA, Delitala G, Jones A, Jeff?oate WJ, Edwards CRW, Ratter SJ, Besser GM, Bloom SR, Alberti KGMM

457 (1978) Hormonal and metabolic responses to an enkephalin analogue in normal man. Lancet II:1225--1227 19. Taylor T, Dluhy RG, Williams GH (1983) fl-Endorphin suppresses adrenocorticotropin and cortisot levels in normal human subjects. J Clin Endocrinol Metab 57: 592-596 20. Vale W, Spiess J, Rivier C, Rivier J (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and fl-endorphin. Science 213 : 1394-1397 21. Volavka J, Cho D, Mallya A, Bauman J (1979) Naloxone increases ACTH and cortisol levels in man. N Engl J Med 300:1056-1057

Received: November 18, 1986 Returned for revision: December 16, 1986 Accepted: December 30, 1986

Prof. Dr. F.-D. Goebel Medizinische Poliktinik Universit~it Mfinchen Pettenkoferstr. 8a D-8000 Miinchen 2