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β-endorphin levels in plasma, pituitary and brain of 10-day old rats
Life Sciences, Vol. Printed in the USA
52, pp. 1417-1424
Pergamon Press
EFFECTS OF HYPNORM R (FENTANYL) ON ACTH/B-ENDORPHIN LEVELS IN PLASMA, PITUITARY AND BRAIN OF 10-DAY OLD RATS Istv~in Barna 1, Zsuzsanna Acs 1 and James I. Koenig 2 1Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest P.O.BOX 67. (Hungary), 2Department of Physiology and Biophysics, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, D.C. 20007 (USA). (Received in final form February 12, 1993)
Summary. Administration of Hypnorm R, an anaesthetic containing the known /z-opiate receptor agonist fentanyl, elicited dose- and time-related elevation of plasma ACTH, B-endorphin and corticosterone levels in 10-day old rat pups. Pretreatment with specific antibodies (raised against CRH, AVP and ACTH resp.) revealed that Hypnorm R administration activated the ACTHcorticosterone system in the 10-day old rat and its effect is mediated by CRH and/or AVP. Hypnorm R anaesthesia was associated with significant decrease in the ACTH and B-endorphin levels in the pituitary lobes as well as in 6endorphin content of the hypothalamus and medulla oblongata. Latter results may indicate that the B-endorphinergic system in the brain of the 10-day old rat is activated by Hypnorm R, an effect most probably elicited by the opiate agonist fentanyl.
Hypothalamic regulation of pituitary hormone secretion in the postnatal period differs to some extent from that seen in the adult rat. This involves reduced reactivity of the hypothalamo-pituitary-adrenal system (HPA) to certain stressful stimuli during the first two weeks of postnatal life (for review see Ref. 1). During this period called stress hyporesponsive period (SHRP), very small plasma corticotropin (ACTH) and corticosterone (CS) response was observed after aether or electro-shock (2). In more recent studies a variety of stressors including endotoxin (3), hypoglycaemia (4), histamine and cold (5) elicited similar ACTH release in the neonatal and in the adult rats. On the other hand, after cold- or aether-stress serum B-endorphin (6E) concentration was unchanged in 10-day old but stimulated in older rats (6). These findings suggest that in the developing rat the responsiveness of the HPA is age and stressor specific and that there might be differences in the maturation of its components. Opiate agonists and antagonists are known to influence HPA activity (7). The fentanyl-fluanisone combination (Hypnorm R) is a widely used anaesthetic in the laboratory practice, furthermore fentanyl is commonly used in pediatric surgery. The
Sendcorrespondenceto:IsW~mBarna, lnstituteofExperimental Medicine, Hungarmn A~demyofSciences, H-1450 BudapestP.O.BOX..67. Hunga~. 0024-3205/93 Copyright
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1418
Fentanyl and ACTH/endorphin in Rat Pups
Vol. 52, No. 17, 1993
time- and dose-related increase in plasma immunoreactive t~E concentration following administration of the opiate agonist fentanyl and of Hypnorm R in adult rats has been reported earlier (8). In humans fentanyl was shown either to inhibit the ACTH response to surgery (9) or to potentiate the inhibitory effect of corticosteroids (9, 10), whereas in one study (11) fentanyl stimulated ACTH secretion in adult rats. In this work we studied the effect of the /z-opiate agonist containing Hypnorm R on the HPA in 10-day old rat pups measuring immunoreactive ACTH (ir-ACTH), BE (ir-I~E) and CS (ir-CS) levels in the plasma as well as ir-ACTH and/or ir-BE contents in the anterior and neurointermediate pituitary lobes, in the hypothalamus and in the medulla oblongata. Materials and methods Animals and treatments: Ten-day old (20-25 g) Wistar rats of our inbred colony of either sexes were used. The morning on which the pups were found in the cage of the mother was considered the first postnatal day, when litter size was reduced to 10. Pups were weighed and labelled the day before the experiment. Hypnorm R (a formulation containing 140 /zg/mL fentanyl and 7 ng/mL fluanisone, Jansen Pharm., Belgium) or physiological saline (controls) were injected subcutaneously (s.c.) using a 10 or 50 /ZL Hamilton syringe as appropriate. The pups were returned to the dam and left undisturbed until sacrifice. The "short-term effect" (0-40 min) and the "long-term effect" (0-180 min) of Hypnorm R was studied in two separate experiments. Antibodies, antiACTH (a-ACTH) raised in rabbits (Institute of Experimental Medicine, Budapest, Hungary), or the mixture of anti-corticotropin releasing hormone (a-CRH) raised in sheep (#4654/26/84 kindly provided by W.Vale,) and anti-arginine vasopressin (a-AVP) raised in goat (gift from T. Jangtky, Szeged, Hungary) were injected intraperitoneally (i.p.) 120 min before the Hypnorm R administration. In a pilot immunoneutralization experiment increasing volume of the antisera were tested for their effect on corticosterone response to Hypnorm R and the dose of 2 /ZL/g undiluted antibody was found to be effective. Control rats were given normal rabbit serum or a mixture of sheep and goat serum in corresponding dose. Sample collection and hormone measurements: Pups were sacrificed by decapitation and trunk blood was collected on ice into Eppendorf tubes containing 50 ~L of 20% K2-EDTA. Blood was centrifuged and the plasma was stored at -20°C until assayed. After decapitation the brain was quickly removed and dissected; tissue pieces were collected on ice in Eppendorf tubes containing 100/ZL 0.2 N HC1, and stored at -20 ° C until extraction. Plasma ir-ACTH and ir-BE concentrations were determined without extraction (12). Ir-ACrH/ir-f~E content of tissue samples were measured after acidic extraction and heat denaturation as described previously (12). Ir-CS was measured in plasma without extraction using a highly specific antibody which shows weak cross-reaction with deoxycorticosterone (1.5%) and progesterone (2.3 %), but no significant cross-reactions ( < 0 . 0 1 % ) with cortisol and cortisone and with further 12 naturally occurring steroids (13). Protein content of the tissue extracts were measured according to Lowry et al. (14). Statistical evaluation: Immunoreactive peptide levels (plasma: fmol/mL; pituitary: pmol/lobe; brain tissue: fmol/mg protein), ir-CS levels (plasma: pmol/mL) are presented as mean ± S.E.M. with the number of observations (number of pups). Data on plasma hormone levels approximated better the log normal than the normal distribution thus they were transformed to logarithms before statistical evaluation (15). Analysis of variance was followed by Dunn's or Dunnett's test (16,17) as appropriate.
VOI. 52, No. 17, 1993
Fentanyl and ACTH/endorphin in Rat Pups
1419
Results H y p n o r m R administration to 10-day old rats resulted in anaesthesia within 5 rain lasting for more than 120 min. A dose dependent increase in plasma ir-ACTH, ir-BE, and ir-CS concentration was seen 40 min after Hypnorm R administration (Fig. 1). FIG. 1. 300
2O0 8
8
E Ia
J
E
m
200
W "r h0 ,<
Plasma ACTH, t3E and CS levels 40 min after various doses of HypnormR s.c. in 10-day old rats. Values are geometric means with retransformed S.E.M. (n=6-7/group). a: significantly (p<0.01) different from saline injected group.
8
100
s
"'it
ACTH
[]
BE
E a
1 O0
Q. ,,.J
E
0
E
OS
o
[]0.2 [ ] 0.5 [ ] 1.0 pl./g H y p n o r m s.c.
saline
Plasma i r - A C T H / i r - a E levels were elevated as early as 5 min after 1 ~zL/g Hypnorm R s.c. (Fig. 2, Panel A) reaching maximal value 20 and 180 min with a nadir at 120 min (Fig. 2, Panel A and B). Neither A C T H nor f~E levels were changed in the anterior and in the neurointermediate pituitary, in the hypothalamus or in the medulla oblongata for 40 min after Hypnorm R injection ("short-term experiment"; not shown).
g E
400
3
n .
ij
'Ill A C T H
300
A ~
•
200
w"
"1I-
o
o
~
F--
100
o
o
..~ 0
-6 s 5
10
20
40
oo
400
e
200 100
0 0
minutes
B
[] ^erE [ ] BE
20
60
120
180
minutes
FIG. 2. Plasma ACTH and BE levels at various time after 1/~L/g HypnormR s.c. in 10-day old rats. A: "short-term" experiment; B: "long-term" experiment. Values are geometric means with retransformed S.E.M. (n=6-12/group). a: p<0.01; b: p<0.05 compared to control (0 min) group. Significantly reduced ir-ACTH/ir-f~E levels were found in the anterior pituitary (AL), in the neurointermediate pituitary (NIL), in the hypothalamus, and in the medulla oblongata at various time in the "long-term experiment" i.e. 60-180 min after the
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Fentanyl and ACTH/endorphin in Rat Pups
Vol. 52, No. 17, 1993
HypnormR administration (Table I and II). The ir-CS concentration in the plasma began to rise at 20 min and was still elevated at 120 min after HypnormR injection (Fig. 3). FIG. 3. 200
iF~
i~'~
t
E
Plasma CS levels at various time after 1 /aL/g Hypnorm R s.c. in 10-day old rats; effect of ACTH-antibody pretreatment on CS levels at 0, 20 and 60 min after Hypnorm R s.c. Values are geometric means with retransformed S.E.M. (n=4-6/group). a: p<0.01 relative to control (0 min); c: p<0.01 relative to the corresponding group not given ACTH-antibody.
a
no pretreatment a-ACTH pretreatment
"ll
I
8
.,J
E
O
100
O
E o.
[-7 0
5
10
20
40
60
120
minutes
Immunoneutralization with a-ACTH antibody blocked the HypnormR-induced elevation of plasma ir-CS levels as measured at 0, 20 and 60 min after HypnormR injection (Fig. 3). Pretreatment with the combined a-CRH/a-AVP antibody prevented the HypnormR elicited increase of plasma ir-ACTH but not of if-BE concentration (Fig.
4). Discussion Administration of HypnormR (containing fentanyl) to 10-day old rats resulted in the activation of the HPA system reflected by the elevated plasma ir-ACTH, Jr-BE and irCS levels. Opioids can both stimulate and inhibit the hypothalamic CRH release in adult rats (18). Elevation of plasma CS level by both /z- and k-opiate agonists has been described in neonatal rats indicating opiate stimulation of the HPA during the SHRP (19). Our results (Fig. 3) are consistent with these data and they revealed that HypnormR containing the ~z-opiate agonist fentanyl increased ir-ACTH/ir-BE plasma levels as well (Fig. 1 and 2). The increase of plasma ir-BE after HypnormR (Fig 1.) is consistent with the increase seen in adult rats (8). Since stressful stimuli (cold or ether) are without effect on plasma ir-BE levels in 10-day old rats (6) one might postulate that the effect of HypnormR containing the ~-opiate receptor agonist fentanyl (20) is mediated by specific opiate receptors rather than being a non-specific stress. On the other hand the biphasic effect of HypnormR on the plasma ir-ACTH/ir-BE levels i.e. an elevation of ir-ACTH/irBE plasma levels at 180 min (Fig. 2) seems to be due to the distress experienced by the pups after awakening from the anaesthesia. Secretion of ACTH and BE from the pituitary lobes are under different control and their plasma concentration is an indicator of the activity of the HPA. The anterior pituitary produces both ir-ACTH and Jr-BE (B-LPH and BE), the intermediate pituitary secretes a-MSH and ir-BE (N-acetylated and/or C-terminally shortened forms of BE). Secretion of both peptides from the anterior pituitary lobe is under the stimulatory control of the hypothalamic CRH and AVP both originating from the paraventricular nucleus, since the CRH containing neurons of the paraventricular nucleus also contain
Vol. 52, No. 17, 1993
Fentanyl and ACTH/endorphin in Rat Pups
1421
FIG. 4. 300
~:~ACTH
Hypnorm
I
E --I
SE
q,
~1,
a
200
E .,,.. IU 3: I-0 ,< 'B
Plasma ACTH and BE levels 20 min after 1 pL/g Hypnorm R s.c. in CRH+AVP antibody pretreated 10day old rats. Values are geometric means with retransformed S.E.M. (n-8-11/group). a: p<0.01 relative to control; c: p<0.01 relative to the ACTH level in normal serum treated group.
~ / ~ ii
100
untrented control
normal serum
a-CRH+ a-AVP
pretreatment
AVP in the same neurosecretory granule (21, 22; 23). The major inhibitory input for the anterior pituitary is the adrenocortical CS (24,25,26,) whereas for the intermediate lobe mainly the central dopaminergic system (27). TABLE I. ACTH Content of the Anterior Pituitary, the Hypothalamus and the Medulla Oblongata at Various Time after Hypnorm R s.c. in 10-day Old Rats. Values are mean_+S.E.M, with number of determinations in parentheses. time after Hypnorm R s.c. 0 min
120 min
180 min
26.0_+2.34 17.7_+2.81b (7) (7)
17.2_+1.36b (6)
18.6+1.85 b (6)
hypothalamus & 18.9_+2.03 (11)
17.6_+0.81 (7)
17.5_+1.21 (7)
18.5_+1.06 (7)
20.3_+1.18 (7)
medulla oblongata &
4.5_+0.36 (6)
4.1_+0.18 (7)
3.4_+0.09 (6)
4.3_+0.81 (7)
anterior pituitary lobe#
26.4+1.77 (9)
4.2+0.27 (10)
20 min
60 min
#: pmol ACTH/gland; &: fmol ACTH/mg protein b: p < 0.05 relative to 0 rain value Specific antibody pretreatments revealed that Hypnorm R administration activated not only ACTH but also CRF and/or AVP secretion; a-ACTH antibody pretreatment attenuated t h e CS release (Fig. 3) and combined administration of a-CRH/a-AVP antisera reduced the elevation in plasma ACTH levels (Fig. 4). Pretreatment with a-CRH only slightly reduces morphine administration induced increase of plasma ACTH levels in adult rats (18) therefore Nikolarakis et al. (18) postulated a CRH dependent and a CRH independent mechanism for the/z-receptor mediated ACTH release. Since combined aCRH/a-AVP pretreatment completely blocked the elevation of plasma ACTH levels
1422
Fentanyl
and ACTH/endorphin
in Rat Pups
Vol.
52, No. 17, 1993
(Fig. 4) the/~-opiate receptor agonist fentanyl seem to elicit ACTH release by activating either CRH or AVP path or both in 10-day old rats. TABLE II. B-Endorphin Content of the Anterior Pituitary, the Neurointermediate Lobe, the Hypothalamus and the Medulla Oblongata at Various time after HypnormR s.c. in 10-day Old Rats. Values are mean_+S.E.M, with number of determinations in parentheses. time after HypnormR s.c. 0 min
20 min
60 min
120 min
180 min
anterior pituitary lobe #
29.7_+1.59 (9)
36.0+1.48 (7)
26.8_+3.18 (7)
21.2+1.88b (6)
26.1+2.09 (7)
neurointermediate lobe #
19.5_+1.34 15.3_+1.09 (9) (7)
13.4+1.30a (7)
18.2+1.21 (6)
13.3+0.75a (6)
hypothalamus& 183.3_+17.93 148.9_+8.31 134.2+10.72 b 121.9+5.89a (7) (7) (11) (7)
125.7_+6.29b (7)
medulla oblongata&
16.7_+0.87 (10)
18.5_+1.79 (6)
11.0+0.37 a
(7)
11.7+0.49a (7)
11.5+1.36a (7)
#: pmol BE/gland; &: fmol BE/mg protein b: p < 0.05; a: p < 0.01 relative to 0 min value Under stressful conditions ir-BE concentration shows concomitant changes with plasma ir-ACTH levels in the plasma of adult rats (28) furthermore, immunoneutralization of CRH results in parallel changes in the circadian patterns of plasma ir-ACTH and ir-BE levels (29). Therefore our finding that a-CRH/a-AVP pretreatment failed to affect HypnormR-induced elevation in plasma ir-BE levels (Fig. 4) suggest that the enhanced plasma ir-aE content after HypnormR in the 10-day old rats may originate predominantly from the intermediate pituitary controlled primarily by the central dopaminergic system. This assumption is supported by the finding that ir-gE content in the NIL was diminished 60 and 180 min after HypnormR administration (Table I and II) indicating the stimulation of the melanotroph cells in the intermediate pituitary. Furthermore, the restored Jr-BE content of the NIL but not in the AL at 120 min (Table I and II) may indicate that under our experimental conditions the rate of BErelated peptide synthesis was higher in melanotrophs than in corticotrophs. The reduced ir-ACTH/ir-BE content in the AL 60-180 rain after HypnormR administration (Table I and II) could be the result of the persistent ACTH/BLPH/BE release from the corticotroph cells for at least 120 minutes (Fig. 2). ACTH and BE are also synthesized in extrapituitary tissues as hypothalamus and medulla oblongata (30). The hypothalamic BE system seems to be an important physiological regulator of the HPA (31,32). The decrease in the hypotbalamic Jr-BE but not ir-ACTH concentration after HypnormR (Table I and II) might be the consequence of the continuous release of ir-aE from hypothalamic BE/ACTH producing cells being involved in the hypophysiotropic function of the hypothalamus. On the other hand hypothalamic ACTH does not seem to play significant role in the HypnormR induced
Vol.
52, No. 17, 1993
Fentanyl
and A C T H / e n d o r p h i n
in Rat Pups
1423
changes in the activity of the HPA. Since the respiratory and cardiovascular effects of BE administered into the medulla and of centrally administered #-agonists are well known (33, 34, 35), the significantly reduced ir-BE concentration in the medulla oblongata 60, 120 and 180 min after HypnormR administration (Table II) might be related to the respiratory and cardiovascular response to fentanyl. We conclude that the brain BE-ergic system of the 10-day old rat is capable to react to the HPA-activating as well as to the respiratory and cardiovascular effects of HypnormR (fentanyl) anaesthesia. Our results that HypnormR administration activated CRH and/or AVP and the ACTH-corticosterone system during the "stress hyporesponsive" period in the rat seem to indicate that the/~-opiate receptor control of the HPA functions during the SHRP. Acknowled~,ements We thank Ms. M. Temesv~iri for her expert technical help and the NIDDK for the generous gift of the h-ACTH1_39. This work was supported by the grant OTKA 2922. References 1. R.M. SAPOLSKY and M.J. MEANEY, Brain Res. Rev. 11 65-76 (1986). 2. C.D. WALKER, M PERRIN, W VALE and C. RIVIER, Endocrinology 118 14451451 (1986). 3. L. WlTEK-JANUSEK, Am. J. Physiol. 255 E525-E530 (1988). 4. M. ARAI and E.P. WIDMAYER, Endocrinology 129 1505-1512 (1991). 5. A. KJOER, U. KNIGGE, F.W. BACH and J. WARBERG, Neuroendocrinology 56 419-428 (1992). 6. P. ANGELOGIANNI and C. GIANOULAKIS, Neuroendocrinology 50 372-381 (1989). 7. D. DE WlED, J.M. VAN REE and W. DE JONG, Narcotics and the Hypothalamus, E. Zimmermann and R. George (eds.), 251-266, Raven Press, New York (1974). 8. M.D. RAMIREZ-GONZALEZ, I. BARNA, V.M. WlEGANT and W. DE JONG, Life Sciences 48 1371-1377 (1991). 9. H. RAFF, A.J. NORTON, R.J. FLEMMA and J.W. FINDLING, J. clin. Endocrin. Metab. 65 295-298 (1987). 10. H. RAFF, R.J. FLEMMA and J.W. FINDLING, J. clin. Endocrin. Metab. 67 11461148 (1988). 11. T. KUDO, M. KUDO, A. MATSUKI and T. OYAMA, Masui 38 901-907 (1989). [abstract from MEDLINE 1989]. 12. I. BARNA and J.I. KOENIG, Brain Res. 593 69-76 (1992). 13. J.K. KOV,/~CS and P. PI~CZELY, Gen. Comp. Endocrinol. 84 192-198 (1991). 14. O.H. LOWRY, N.J. ROSEBOROUGH, A.L. FARR and R.J. RANDALL, J. biol. Chem. 265-275 (1951). 15. L. KRULICH, E. HEFCO, P. ILLNER and C.B. READ, Neuroendocrinology 16 293311 (1974). 16. O.J. DUNN, J. Am. Stat. Assoc. 56 52-64 (1961). 17. C.W. DUNNET-F, J. Am. Stat. Assoc. 50 1096-1121 (1955). 18. K. NIKOLARAKIS, A. PFEIFFER, G.K. STALLA and A. HERZ, Brain Res. 421 373-376 (1987). 19. A.T. ADAMSON, R.T. WlNDH, S. BLACKFORD and C.M. KUHN, Endocrinology 129 959-964 (1991).
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Fentanyl and ACTH/endorphin
in Rat Pups
Vol. 52, No. 17, 1993
20. J.W. VILLIGER, L.J. RAY and K.M. TAYLOR, Neuropharmacology 22 447-452 (1983). 21. G.B. MAKARA, E. STARK, M. KARTESZI, M. PALKOVITS and GY. RAPPAY, Am. J. Physiol. 240 E441-E446 (1981). 22. P.E. SAWCHENKO, L.W. SWANSON and W.W. VALE, Proc. Natl. Acad. Sci. 81 1883-1887 (1984). 23. F.A. ANTONI, Endocr. Rev. 7 351-378 (1989). 24. C. GRAMSCH, G. KLEBER, V. HOLLT, A. PASI, P. MEHRAEIN and A. HERZ, Brain Res. 192 109-119(1980). 25. G.E. GILLIES, E.A. LINTON and P.J. LOWRY, Nature 299 355-357 (1982). 26. M.E. KELLER-WOOD and M.F. DALLMAN, Endocr. Rev., 5 1-24 (1984). 27. C.G.J. SWEEP and V.M. WlEGANT, J. Neuroendocrinol. 2 531-537 (1990). 28. R. GUILLEMIN, T. VARGO, J. ROSSIER, S. MINICK, N. LING, C. RIVIER, W. VALE and F. BLOOM, Science 197 1367-1369 (1977). 29. Gy. BAGDY, G.P. CHROUSOS and A.E. CALOGERO, Neuroendocrinology 53 573-578 (1991). 30. H. KHACHATURIAN, M.E. LEWIS, K. TSOU and S.J. WATSON, Handbook of Chemical Neuroanatomy Vol 4, A. Bj6rklund and T.H6kfelt (eds.) 216-272, Elsevier, Amsterdam, (1985). 31. J.L. HARACZ, A.S. BLOOM, R.I.H. WANG, and L.F. TSENG, Neuropharmacology 33 170,175 (1981). 32. J.C. BUCKINGHAM, Neuropharmacology 42 148-152 (1986). 33. M.A. PETFY and W. DE JONG, Eur. J. Pharm. 81 449-457 (1982). 34. J. FLOREZ and M.A. HURLE and A. MEDIAVILLA, Life Sci. 31 2189-2192 (1982). 35. S. LAURENT and H. SCHMITF, Eur. J. Pharm. 96 165-169 (1983).
52, pp. 1417-1424
Pergamon Press
EFFECTS OF HYPNORM R (FENTANYL) ON ACTH/B-ENDORPHIN LEVELS IN PLASMA, PITUITARY AND BRAIN OF 10-DAY OLD RATS Istv~in Barna 1, Zsuzsanna Acs 1 and James I. Koenig 2 1Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest P.O.BOX 67. (Hungary), 2Department of Physiology and Biophysics, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, D.C. 20007 (USA). (Received in final form February 12, 1993)
Summary. Administration of Hypnorm R, an anaesthetic containing the known /z-opiate receptor agonist fentanyl, elicited dose- and time-related elevation of plasma ACTH, B-endorphin and corticosterone levels in 10-day old rat pups. Pretreatment with specific antibodies (raised against CRH, AVP and ACTH resp.) revealed that Hypnorm R administration activated the ACTHcorticosterone system in the 10-day old rat and its effect is mediated by CRH and/or AVP. Hypnorm R anaesthesia was associated with significant decrease in the ACTH and B-endorphin levels in the pituitary lobes as well as in 6endorphin content of the hypothalamus and medulla oblongata. Latter results may indicate that the B-endorphinergic system in the brain of the 10-day old rat is activated by Hypnorm R, an effect most probably elicited by the opiate agonist fentanyl.
Hypothalamic regulation of pituitary hormone secretion in the postnatal period differs to some extent from that seen in the adult rat. This involves reduced reactivity of the hypothalamo-pituitary-adrenal system (HPA) to certain stressful stimuli during the first two weeks of postnatal life (for review see Ref. 1). During this period called stress hyporesponsive period (SHRP), very small plasma corticotropin (ACTH) and corticosterone (CS) response was observed after aether or electro-shock (2). In more recent studies a variety of stressors including endotoxin (3), hypoglycaemia (4), histamine and cold (5) elicited similar ACTH release in the neonatal and in the adult rats. On the other hand, after cold- or aether-stress serum B-endorphin (6E) concentration was unchanged in 10-day old but stimulated in older rats (6). These findings suggest that in the developing rat the responsiveness of the HPA is age and stressor specific and that there might be differences in the maturation of its components. Opiate agonists and antagonists are known to influence HPA activity (7). The fentanyl-fluanisone combination (Hypnorm R) is a widely used anaesthetic in the laboratory practice, furthermore fentanyl is commonly used in pediatric surgery. The
Sendcorrespondenceto:IsW~mBarna, lnstituteofExperimental Medicine, Hungarmn A~demyofSciences, H-1450 BudapestP.O.BOX..67. Hunga~. 0024-3205/93 Copyright
© 1993 P e r g a m o n
$6.00 + .00
Press
Ltd
All
rights
reserved.
1418
Fentanyl and ACTH/endorphin in Rat Pups
Vol. 52, No. 17, 1993
time- and dose-related increase in plasma immunoreactive t~E concentration following administration of the opiate agonist fentanyl and of Hypnorm R in adult rats has been reported earlier (8). In humans fentanyl was shown either to inhibit the ACTH response to surgery (9) or to potentiate the inhibitory effect of corticosteroids (9, 10), whereas in one study (11) fentanyl stimulated ACTH secretion in adult rats. In this work we studied the effect of the /z-opiate agonist containing Hypnorm R on the HPA in 10-day old rat pups measuring immunoreactive ACTH (ir-ACTH), BE (ir-I~E) and CS (ir-CS) levels in the plasma as well as ir-ACTH and/or ir-BE contents in the anterior and neurointermediate pituitary lobes, in the hypothalamus and in the medulla oblongata. Materials and methods Animals and treatments: Ten-day old (20-25 g) Wistar rats of our inbred colony of either sexes were used. The morning on which the pups were found in the cage of the mother was considered the first postnatal day, when litter size was reduced to 10. Pups were weighed and labelled the day before the experiment. Hypnorm R (a formulation containing 140 /zg/mL fentanyl and 7 ng/mL fluanisone, Jansen Pharm., Belgium) or physiological saline (controls) were injected subcutaneously (s.c.) using a 10 or 50 /ZL Hamilton syringe as appropriate. The pups were returned to the dam and left undisturbed until sacrifice. The "short-term effect" (0-40 min) and the "long-term effect" (0-180 min) of Hypnorm R was studied in two separate experiments. Antibodies, antiACTH (a-ACTH) raised in rabbits (Institute of Experimental Medicine, Budapest, Hungary), or the mixture of anti-corticotropin releasing hormone (a-CRH) raised in sheep (#4654/26/84 kindly provided by W.Vale,) and anti-arginine vasopressin (a-AVP) raised in goat (gift from T. Jangtky, Szeged, Hungary) were injected intraperitoneally (i.p.) 120 min before the Hypnorm R administration. In a pilot immunoneutralization experiment increasing volume of the antisera were tested for their effect on corticosterone response to Hypnorm R and the dose of 2 /ZL/g undiluted antibody was found to be effective. Control rats were given normal rabbit serum or a mixture of sheep and goat serum in corresponding dose. Sample collection and hormone measurements: Pups were sacrificed by decapitation and trunk blood was collected on ice into Eppendorf tubes containing 50 ~L of 20% K2-EDTA. Blood was centrifuged and the plasma was stored at -20°C until assayed. After decapitation the brain was quickly removed and dissected; tissue pieces were collected on ice in Eppendorf tubes containing 100/ZL 0.2 N HC1, and stored at -20 ° C until extraction. Plasma ir-ACTH and ir-BE concentrations were determined without extraction (12). Ir-ACrH/ir-f~E content of tissue samples were measured after acidic extraction and heat denaturation as described previously (12). Ir-CS was measured in plasma without extraction using a highly specific antibody which shows weak cross-reaction with deoxycorticosterone (1.5%) and progesterone (2.3 %), but no significant cross-reactions ( < 0 . 0 1 % ) with cortisol and cortisone and with further 12 naturally occurring steroids (13). Protein content of the tissue extracts were measured according to Lowry et al. (14). Statistical evaluation: Immunoreactive peptide levels (plasma: fmol/mL; pituitary: pmol/lobe; brain tissue: fmol/mg protein), ir-CS levels (plasma: pmol/mL) are presented as mean ± S.E.M. with the number of observations (number of pups). Data on plasma hormone levels approximated better the log normal than the normal distribution thus they were transformed to logarithms before statistical evaluation (15). Analysis of variance was followed by Dunn's or Dunnett's test (16,17) as appropriate.
VOI. 52, No. 17, 1993
Fentanyl and ACTH/endorphin in Rat Pups
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Results H y p n o r m R administration to 10-day old rats resulted in anaesthesia within 5 rain lasting for more than 120 min. A dose dependent increase in plasma ir-ACTH, ir-BE, and ir-CS concentration was seen 40 min after Hypnorm R administration (Fig. 1). FIG. 1. 300
2O0 8
8
E Ia
J
E
m
200
W "r h0 ,<
Plasma ACTH, t3E and CS levels 40 min after various doses of HypnormR s.c. in 10-day old rats. Values are geometric means with retransformed S.E.M. (n=6-7/group). a: significantly (p<0.01) different from saline injected group.
8
100
s
"'it
ACTH
[]
BE
E a
1 O0
Q. ,,.J
E
0
E
OS
o
[]0.2 [ ] 0.5 [ ] 1.0 pl./g H y p n o r m s.c.
saline
Plasma i r - A C T H / i r - a E levels were elevated as early as 5 min after 1 ~zL/g Hypnorm R s.c. (Fig. 2, Panel A) reaching maximal value 20 and 180 min with a nadir at 120 min (Fig. 2, Panel A and B). Neither A C T H nor f~E levels were changed in the anterior and in the neurointermediate pituitary, in the hypothalamus or in the medulla oblongata for 40 min after Hypnorm R injection ("short-term experiment"; not shown).
g E
400
3
n .
ij
'Ill A C T H
300
A ~
•
200
w"
"1I-
o
o
~
F--
100
o
o
..~ 0
-6 s 5
10
20
40
oo
400
e
200 100
0 0
minutes
B
[] ^erE [ ] BE
20
60
120
180
minutes
FIG. 2. Plasma ACTH and BE levels at various time after 1/~L/g HypnormR s.c. in 10-day old rats. A: "short-term" experiment; B: "long-term" experiment. Values are geometric means with retransformed S.E.M. (n=6-12/group). a: p<0.01; b: p<0.05 compared to control (0 min) group. Significantly reduced ir-ACTH/ir-f~E levels were found in the anterior pituitary (AL), in the neurointermediate pituitary (NIL), in the hypothalamus, and in the medulla oblongata at various time in the "long-term experiment" i.e. 60-180 min after the
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Fentanyl and ACTH/endorphin in Rat Pups
Vol. 52, No. 17, 1993
HypnormR administration (Table I and II). The ir-CS concentration in the plasma began to rise at 20 min and was still elevated at 120 min after HypnormR injection (Fig. 3). FIG. 3. 200
iF~
i~'~
t
E
Plasma CS levels at various time after 1 /aL/g Hypnorm R s.c. in 10-day old rats; effect of ACTH-antibody pretreatment on CS levels at 0, 20 and 60 min after Hypnorm R s.c. Values are geometric means with retransformed S.E.M. (n=4-6/group). a: p<0.01 relative to control (0 min); c: p<0.01 relative to the corresponding group not given ACTH-antibody.
a
no pretreatment a-ACTH pretreatment
"ll
I
8
.,J
E
O
100
O
E o.
[-7 0
5
10
20
40
60
120
minutes
Immunoneutralization with a-ACTH antibody blocked the HypnormR-induced elevation of plasma ir-CS levels as measured at 0, 20 and 60 min after HypnormR injection (Fig. 3). Pretreatment with the combined a-CRH/a-AVP antibody prevented the HypnormR elicited increase of plasma ir-ACTH but not of if-BE concentration (Fig.
4). Discussion Administration of HypnormR (containing fentanyl) to 10-day old rats resulted in the activation of the HPA system reflected by the elevated plasma ir-ACTH, Jr-BE and irCS levels. Opioids can both stimulate and inhibit the hypothalamic CRH release in adult rats (18). Elevation of plasma CS level by both /z- and k-opiate agonists has been described in neonatal rats indicating opiate stimulation of the HPA during the SHRP (19). Our results (Fig. 3) are consistent with these data and they revealed that HypnormR containing the ~z-opiate agonist fentanyl increased ir-ACTH/ir-BE plasma levels as well (Fig. 1 and 2). The increase of plasma ir-BE after HypnormR (Fig 1.) is consistent with the increase seen in adult rats (8). Since stressful stimuli (cold or ether) are without effect on plasma ir-BE levels in 10-day old rats (6) one might postulate that the effect of HypnormR containing the ~-opiate receptor agonist fentanyl (20) is mediated by specific opiate receptors rather than being a non-specific stress. On the other hand the biphasic effect of HypnormR on the plasma ir-ACTH/ir-BE levels i.e. an elevation of ir-ACTH/irBE plasma levels at 180 min (Fig. 2) seems to be due to the distress experienced by the pups after awakening from the anaesthesia. Secretion of ACTH and BE from the pituitary lobes are under different control and their plasma concentration is an indicator of the activity of the HPA. The anterior pituitary produces both ir-ACTH and Jr-BE (B-LPH and BE), the intermediate pituitary secretes a-MSH and ir-BE (N-acetylated and/or C-terminally shortened forms of BE). Secretion of both peptides from the anterior pituitary lobe is under the stimulatory control of the hypothalamic CRH and AVP both originating from the paraventricular nucleus, since the CRH containing neurons of the paraventricular nucleus also contain
Vol. 52, No. 17, 1993
Fentanyl and ACTH/endorphin in Rat Pups
1421
FIG. 4. 300
~:~ACTH
Hypnorm
I
E --I
SE
q,
~1,
a
200
E .,,.. IU 3: I-0 ,< 'B
Plasma ACTH and BE levels 20 min after 1 pL/g Hypnorm R s.c. in CRH+AVP antibody pretreated 10day old rats. Values are geometric means with retransformed S.E.M. (n-8-11/group). a: p<0.01 relative to control; c: p<0.01 relative to the ACTH level in normal serum treated group.
~ / ~ ii
100
untrented control
normal serum
a-CRH+ a-AVP
pretreatment
AVP in the same neurosecretory granule (21, 22; 23). The major inhibitory input for the anterior pituitary is the adrenocortical CS (24,25,26,) whereas for the intermediate lobe mainly the central dopaminergic system (27). TABLE I. ACTH Content of the Anterior Pituitary, the Hypothalamus and the Medulla Oblongata at Various Time after Hypnorm R s.c. in 10-day Old Rats. Values are mean_+S.E.M, with number of determinations in parentheses. time after Hypnorm R s.c. 0 min
120 min
180 min
26.0_+2.34 17.7_+2.81b (7) (7)
17.2_+1.36b (6)
18.6+1.85 b (6)
hypothalamus & 18.9_+2.03 (11)
17.6_+0.81 (7)
17.5_+1.21 (7)
18.5_+1.06 (7)
20.3_+1.18 (7)
medulla oblongata &
4.5_+0.36 (6)
4.1_+0.18 (7)
3.4_+0.09 (6)
4.3_+0.81 (7)
anterior pituitary lobe#
26.4+1.77 (9)
4.2+0.27 (10)
20 min
60 min
#: pmol ACTH/gland; &: fmol ACTH/mg protein b: p < 0.05 relative to 0 rain value Specific antibody pretreatments revealed that Hypnorm R administration activated not only ACTH but also CRF and/or AVP secretion; a-ACTH antibody pretreatment attenuated t h e CS release (Fig. 3) and combined administration of a-CRH/a-AVP antisera reduced the elevation in plasma ACTH levels (Fig. 4). Pretreatment with a-CRH only slightly reduces morphine administration induced increase of plasma ACTH levels in adult rats (18) therefore Nikolarakis et al. (18) postulated a CRH dependent and a CRH independent mechanism for the/z-receptor mediated ACTH release. Since combined aCRH/a-AVP pretreatment completely blocked the elevation of plasma ACTH levels
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Fentanyl
and ACTH/endorphin
in Rat Pups
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(Fig. 4) the/~-opiate receptor agonist fentanyl seem to elicit ACTH release by activating either CRH or AVP path or both in 10-day old rats. TABLE II. B-Endorphin Content of the Anterior Pituitary, the Neurointermediate Lobe, the Hypothalamus and the Medulla Oblongata at Various time after HypnormR s.c. in 10-day Old Rats. Values are mean_+S.E.M, with number of determinations in parentheses. time after HypnormR s.c. 0 min
20 min
60 min
120 min
180 min
anterior pituitary lobe #
29.7_+1.59 (9)
36.0+1.48 (7)
26.8_+3.18 (7)
21.2+1.88b (6)
26.1+2.09 (7)
neurointermediate lobe #
19.5_+1.34 15.3_+1.09 (9) (7)
13.4+1.30a (7)
18.2+1.21 (6)
13.3+0.75a (6)
hypothalamus& 183.3_+17.93 148.9_+8.31 134.2+10.72 b 121.9+5.89a (7) (7) (11) (7)
125.7_+6.29b (7)
medulla oblongata&
16.7_+0.87 (10)
18.5_+1.79 (6)
11.0+0.37 a
(7)
11.7+0.49a (7)
11.5+1.36a (7)
#: pmol BE/gland; &: fmol BE/mg protein b: p < 0.05; a: p < 0.01 relative to 0 min value Under stressful conditions ir-BE concentration shows concomitant changes with plasma ir-ACTH levels in the plasma of adult rats (28) furthermore, immunoneutralization of CRH results in parallel changes in the circadian patterns of plasma ir-ACTH and ir-BE levels (29). Therefore our finding that a-CRH/a-AVP pretreatment failed to affect HypnormR-induced elevation in plasma ir-BE levels (Fig. 4) suggest that the enhanced plasma ir-aE content after HypnormR in the 10-day old rats may originate predominantly from the intermediate pituitary controlled primarily by the central dopaminergic system. This assumption is supported by the finding that ir-gE content in the NIL was diminished 60 and 180 min after HypnormR administration (Table I and II) indicating the stimulation of the melanotroph cells in the intermediate pituitary. Furthermore, the restored Jr-BE content of the NIL but not in the AL at 120 min (Table I and II) may indicate that under our experimental conditions the rate of BErelated peptide synthesis was higher in melanotrophs than in corticotrophs. The reduced ir-ACTH/ir-BE content in the AL 60-180 rain after HypnormR administration (Table I and II) could be the result of the persistent ACTH/BLPH/BE release from the corticotroph cells for at least 120 minutes (Fig. 2). ACTH and BE are also synthesized in extrapituitary tissues as hypothalamus and medulla oblongata (30). The hypothalamic BE system seems to be an important physiological regulator of the HPA (31,32). The decrease in the hypotbalamic Jr-BE but not ir-ACTH concentration after HypnormR (Table I and II) might be the consequence of the continuous release of ir-aE from hypothalamic BE/ACTH producing cells being involved in the hypophysiotropic function of the hypothalamus. On the other hand hypothalamic ACTH does not seem to play significant role in the HypnormR induced
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52, No. 17, 1993
Fentanyl
and A C T H / e n d o r p h i n
in Rat Pups
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changes in the activity of the HPA. Since the respiratory and cardiovascular effects of BE administered into the medulla and of centrally administered #-agonists are well known (33, 34, 35), the significantly reduced ir-BE concentration in the medulla oblongata 60, 120 and 180 min after HypnormR administration (Table II) might be related to the respiratory and cardiovascular response to fentanyl. We conclude that the brain BE-ergic system of the 10-day old rat is capable to react to the HPA-activating as well as to the respiratory and cardiovascular effects of HypnormR (fentanyl) anaesthesia. Our results that HypnormR administration activated CRH and/or AVP and the ACTH-corticosterone system during the "stress hyporesponsive" period in the rat seem to indicate that the/~-opiate receptor control of the HPA functions during the SHRP. Acknowled~,ements We thank Ms. M. Temesv~iri for her expert technical help and the NIDDK for the generous gift of the h-ACTH1_39. This work was supported by the grant OTKA 2922. References 1. R.M. SAPOLSKY and M.J. MEANEY, Brain Res. Rev. 11 65-76 (1986). 2. C.D. WALKER, M PERRIN, W VALE and C. RIVIER, Endocrinology 118 14451451 (1986). 3. L. WlTEK-JANUSEK, Am. J. Physiol. 255 E525-E530 (1988). 4. M. ARAI and E.P. WIDMAYER, Endocrinology 129 1505-1512 (1991). 5. A. KJOER, U. KNIGGE, F.W. BACH and J. WARBERG, Neuroendocrinology 56 419-428 (1992). 6. P. ANGELOGIANNI and C. GIANOULAKIS, Neuroendocrinology 50 372-381 (1989). 7. D. DE WlED, J.M. VAN REE and W. DE JONG, Narcotics and the Hypothalamus, E. Zimmermann and R. George (eds.), 251-266, Raven Press, New York (1974). 8. M.D. RAMIREZ-GONZALEZ, I. BARNA, V.M. WlEGANT and W. DE JONG, Life Sciences 48 1371-1377 (1991). 9. H. RAFF, A.J. NORTON, R.J. FLEMMA and J.W. FINDLING, J. clin. Endocrin. Metab. 65 295-298 (1987). 10. H. RAFF, R.J. FLEMMA and J.W. FINDLING, J. clin. Endocrin. Metab. 67 11461148 (1988). 11. T. KUDO, M. KUDO, A. MATSUKI and T. OYAMA, Masui 38 901-907 (1989). [abstract from MEDLINE 1989]. 12. I. BARNA and J.I. KOENIG, Brain Res. 593 69-76 (1992). 13. J.K. KOV,/~CS and P. PI~CZELY, Gen. Comp. Endocrinol. 84 192-198 (1991). 14. O.H. LOWRY, N.J. ROSEBOROUGH, A.L. FARR and R.J. RANDALL, J. biol. Chem. 265-275 (1951). 15. L. KRULICH, E. HEFCO, P. ILLNER and C.B. READ, Neuroendocrinology 16 293311 (1974). 16. O.J. DUNN, J. Am. Stat. Assoc. 56 52-64 (1961). 17. C.W. DUNNET-F, J. Am. Stat. Assoc. 50 1096-1121 (1955). 18. K. NIKOLARAKIS, A. PFEIFFER, G.K. STALLA and A. HERZ, Brain Res. 421 373-376 (1987). 19. A.T. ADAMSON, R.T. WlNDH, S. BLACKFORD and C.M. KUHN, Endocrinology 129 959-964 (1991).
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20. J.W. VILLIGER, L.J. RAY and K.M. TAYLOR, Neuropharmacology 22 447-452 (1983). 21. G.B. MAKARA, E. STARK, M. KARTESZI, M. PALKOVITS and GY. RAPPAY, Am. J. Physiol. 240 E441-E446 (1981). 22. P.E. SAWCHENKO, L.W. SWANSON and W.W. VALE, Proc. Natl. Acad. Sci. 81 1883-1887 (1984). 23. F.A. ANTONI, Endocr. Rev. 7 351-378 (1989). 24. C. GRAMSCH, G. KLEBER, V. HOLLT, A. PASI, P. MEHRAEIN and A. HERZ, Brain Res. 192 109-119(1980). 25. G.E. GILLIES, E.A. LINTON and P.J. LOWRY, Nature 299 355-357 (1982). 26. M.E. KELLER-WOOD and M.F. DALLMAN, Endocr. Rev., 5 1-24 (1984). 27. C.G.J. SWEEP and V.M. WlEGANT, J. Neuroendocrinol. 2 531-537 (1990). 28. R. GUILLEMIN, T. VARGO, J. ROSSIER, S. MINICK, N. LING, C. RIVIER, W. VALE and F. BLOOM, Science 197 1367-1369 (1977). 29. Gy. BAGDY, G.P. CHROUSOS and A.E. CALOGERO, Neuroendocrinology 53 573-578 (1991). 30. H. KHACHATURIAN, M.E. LEWIS, K. TSOU and S.J. WATSON, Handbook of Chemical Neuroanatomy Vol 4, A. Bj6rklund and T.H6kfelt (eds.) 216-272, Elsevier, Amsterdam, (1985). 31. J.L. HARACZ, A.S. BLOOM, R.I.H. WANG, and L.F. TSENG, Neuropharmacology 33 170,175 (1981). 32. J.C. BUCKINGHAM, Neuropharmacology 42 148-152 (1986). 33. M.A. PETFY and W. DE JONG, Eur. J. Pharm. 81 449-457 (1982). 34. J. FLOREZ and M.A. HURLE and A. MEDIAVILLA, Life Sci. 31 2189-2192 (1982). 35. S. LAURENT and H. SCHMITF, Eur. J. Pharm. 96 165-169 (1983).