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Transient down-regulation of neonatal rat brain μ-opioid receptors upon in utero exposure to buprenorphine
DEVELOPMENTAL BRAIN RESEARCH ELSEVIER
Developmental Brain Research 80 (1994) 158-162
...........
Research Report
Transient down-regulation of neonatal rat brain/ -opioid receptors upon in utero exposure to buprenorphine M a r i a n a M . B e l c h e v a , S a m u e l D a w n , J a c o b B a r g , R o b e r t J. M c H a l e , M a t t h e w T . H o , E l e n a I g n a t o v a , C a r m i n e J. C o s c i a * E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1400 South Grand Boulet,ard, St. Louis, MO 63104-1079, USA
Accepted 8 February 1994
Abstract
Gestational actions of the mixed agonist-antagonist buprenorphine on/~-and K1-opioid binding in neonatal and maternal rat brain were investigated. Upon exposure of pregnant rats to 0.5 mg/kg buprenorphine for 7 days prior to birth, postnatal day-one (P1) and P7 offspring brain /z-binding parameters ( K a and Bmax) were assessed with 3H-labeled [o-Ala2,Mephe4,Gly-ol 5] enkephalin (DAMGE). DAMGE binding was attenuated by 64% in P1 membranes, whereas P7 preparations showed no changes. The same buprenorphine regimen resulted in diminished DAMGE Bmax values in mothers' brains, 2 but not 7 days after cessation of drug administration. Receptor density changes were not accompanied by alteration of/~-binding affinities. Although the postnatal developmental profile of K1 opioid receptors in rat brain measured with [3H]U69593 revealed the presence of an ample number of sites for detection, their binding parameters in P1, P7 pups and mothers were unaffected by 0.5 mg/kg buprenorphine. In summary, buprenorphine administration to pregnant rats transiently down-regulates # opioid receptors in neonatal and maternal brain. Key words: Ontogenesis; Opioid receptor; Buprenorphine; Rat central nervous system; Opioid peptide; In utero drug exposure;
Down-regulation
1. Introduction
Maternal drug abuse and its treatment is placing offspring at considerable risk [8,21]. Buprenorphine, an oripavine alkaloid closely related to diprenorphine, is presently undergoing clinical evaluation for its ability to antagonize the reinforcing effects of both heroin and cocaine in humans [4,7,12,18]. This mixed agonistantagonist displays fewer adverse side effects and less potential for abuse liability than opioid agonists presently used in drug rehabilitation [2,3,16]. In a recent study we have found that in vivo administration of buprenorphine engendered a dose-dependent downregulation of forebrain ~ and up-regulation of K and one subtype of 6-opioid binding in neonatal and adult
rat brain membranes [1]. However, little is known about the in utero effects of buprenorphine or other mixed agonist-antagonists on opioid receptor adaptation. Such information would have ramifications on the adoption of buprenorphine for the treatment of maternal drug abuse. Moreover, opioid receptors in developing brain are particularly susceptible to opiate exposure [8,11,21]. Here we report on /x- and Kt-opioid binding in brain membrane preparations f r o m P1, P7 and maternal rats after gestational buprenorphine treatment of the mothers.
2. Materials and methods 2.1. Chemicals
* Corresponding author. Fax: (1) (314) 577-8156. E-mail: [email protected]. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00027-W
DAMGE was obtained from Multiple Peptide Systems (San Diego, CA); buprenorphine and U69593 were from NIDA Drug Supply (Research Triangle, NC). All other chemicalswere purchased from Sigma Chemical Co. (St. Louis, MO).
159
M.M. Belcheva et al. / Developmental Brain Research 80 (1994) 158-162 2.2. Buprenorphine treatment of rats Timed-pregnant S p r a g u e - D a w l e y rats were obtained 1 - 2 weeks before parturition and were injected with buprenorphine intraperitoneally once daily for 7 days (0.5-1.0 m g / k g body weight) beginning on gestation day 14. The dams were not injected with drug on the day of delivery or during their lactation period. Precautions were taken during injections to avoid damaging the placenta. Treated mothers were killed after 2 or 7 days postpartum and forebrains (without cerebella) were harvested. Control mothers were either naive or injected with saline alone for 14 days prior to birth and killed 7 days postpartum. No differences in opioid binding properties were found between saline-treated and naive animals (data not shown). Birth-dated (random sex) pups were killed either 12-24 h (P1) or 156-168 h (P7) after birth and brains (with cerebella) were removed. A single P7 brain provided sufficient protein to generate one binding curve. For P1 assays, two brains from littermates were pooled. In some cases, the P1 and P7 pups tested were littermates. For some of the binding assays, separate experiments were performed using neonates from independent litters. Animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Brain specimens were immediately frozen in dry ice and stored at - 70°C until binding assays were performed.
7 days and [3H]DAMGE binding was measured in the brains of their P1 and P7 offspring. In all experiments brain membrane preparations were washed 5 times with 50 mM Tris buffer to facilitate residual drug removal. A significant (64%) down-regulation of /zopioid binding in P1 membranes was induced by the in utero buprenorphine treatment (Fig. 1). In contrast, the same drug regimen had no effect on the density of /z-opioid receptors in P7 rat brain membranes. Buprenorphine exposure did not affect DAMGE binding affinity in brains of pups of both ages. Brains of P1 and P7 rats from mothers treated with 0.5 mg/kg buprenorphine for 7 days prior to delivery were also analyzed for Krbinding parameters (Bmax and K a) with [3H]U69593 (Fig. 1). Although
DAMGE 100
5
2.3. Washing procedure to remove residual buprenorphine
%M e m b r a n e preparations from neonatal or adult rat brains were washed at least 5 times with 50 m M Tris-HCI, pH 7.4. At each washing step the suspension was centrifuged at 20,000 g for 10 min. This procedure proved to be sufficient to remove most of the residual buprenorphine. This was previously established in control experiments wherein tritiated buprenorphine was administered to P1 and P7 rats and remaining radioactivity was estimated [1].
SL
2.4. Opioid receptor binding assays
Control
1oo Neonatal and adult rat brain m e m b r a n e preparations were assayed for opioid binding activity as described [1]. Briefly, m e m b r a n e preparations ( 3 0 0 - 8 0 0 / ~ g protein) were incubated in duplicate with 1 nM of each [3H]DAMGE, (35 C i / m m o l , Multiple Peptide Systems) or [3H]U69593 (57 C i / m m o l , A m e r s h a m , Arlington Heights, IL) at 25°C for 1 h. Bmax and K,j values were estimated from homologous competition binding assays performed in the presence of 12 different concentrations of the corresponding unlabelled ligand. W h e n levels of opioid binding sites are relatively low as in rat pup brains, we found that the homologous competition approach was superior to saturation plots [17]. Although Bmax values determined by homologous competition analysis may not represent absolute amounts, the relative changes observed between control and treated animals reflect actual differences in maximal binding. Protein concentrations were determined by the m e t h o d of Lowry et al. [10] with bovine serum albumin as standard. Statistical analyses of data were performed using Student's t-test. Binding parameters ( K d and Bmax) were estimated using the L I G A N D program [13].
3. Results 3.1. Effect of in utero buprenorphine treatment on txand Kl-opioid binding in P1 and P7 rat brain membranes
Beginning on gestation day 14, rats were treated with buprenorphine (0.5 mg/kg body weight) daily for
U69,fi93
77"A Buprenorphine
2:
om m o
r
5o
P1
i
P7
Fig. 1. [3H]DAMGE (/J.) and [3H]U69593 (K 1) binding densities in P1 and P7 rat brains after buprenorphine treatment of their mothers. Pregnant rats were treated with buprenorphine (0.5 m g / k g ) for 7 days prior to delivery as described in section 2. p.- and Kl-opioid binding parameters (Bmax and K a) were estimated in m e m b r a n e s from P1 and P7 brains. K d values for D A M G E binding were 0.90+0.06 and 0.92+0.21 nM (for P1 controls and treated, respectively); 1.07+0.06 and 0.89+0.00 nM (for P7 controls and treated, respectively). K d values for U69593 binding were 2.4+0.3 and 2.3 +0.5 nM (for P1 controls and treated, respectively); 3.1 -+0.7 and 4.5-+0.4 nM (for P7 controls and treated, respectively). Maximum radioactivity bound per assay was for ,u., 840-2170 dpm and for KI, 530-1290 dpm. * m e a n s significantly different from control pups, P < 0.05, n = 3-6, except for D A M G E binding to m e m b r a n e s from buprenorphine-treated P7 pups, where n = 2.
160
M.M. Belchet,a et al. / Decelopmental Brain Research 80 (1994) 1.58-102
[3H]U69593 is a K1 specific ligand, its ontogenic profile in rat brain (Fig. 2) did not differ appreciably from that of the total K population [5,6,14,15]. Noteworthy is the fact that this receptor subtype is present at birth in rat brain at concentrations comparable to those of the adult. Consistent with previous studies on adult male rats [1], administration of the same amount of buprenorphine (0.5 mg/kg) had no effect on either K~-binding affinity or density (Fig. 1). Although higher levels of buprenorphine (1.0 and 2.5 mg/kg) were found to up-regulate adult male K1 receptors, the 1.0 mg/kg dose proved lethal to all three pregnant females treated at this level for 4-6 days. There was no incidence of mortality in all saline-treated control and 0.5 mg/kg buprenorphine-treated pregnant animals (n = 10).
DAMGE •~
1oo
__T
~
5o
z O
U69,593 o
I
40
~--~ CONTROL 2 DAYS 7 DAYS
3.2. Buprenorphine effects on t.L- and K~-opioid binding in maternal brain membranes [3H]DAMGE and [3H]U69593 binding parameters were also determined in brain membrane preparations from the mothers that were on the above-described
80
~
60
~
40
S
] Y
2O
m
2O
Fig. 3. Effects of buprenorphine on I~- and rl-opioid binding in maternal rat brains. Mothers. treated with buprenorphine (0.5 m g / k g ) for 7 days prior to delivery as described in section 2. were killed 2 or 7 days after the last injection. # ([3H]DAMGE) and r x ([3H]U69593) binding parameters (Bmax and K d) were estimated. K d values for D A M G E binding to control and buprenorphine-treated m e m b r a n e s were 1.4_+0.03. 1.5 _+0.03 (2 days) and 1.5 + 0.2 n M (7 days), respectively. K o values for U69593 binding to control and buprenorphine-treated m e m b r a n e s were 2.7 +_0.2. 3.1 + 0.8 (2 days) and 3.9 + 0.7 nM (7 days), respectively. Maximum radioactivity bound per assay was for /z, t590-2830 d p m and for Kl, 490--1160 dpm. * M e a n s significantly different from control mothers, P < 0.05. n = 3.
0 O3
r
)
I
/ / /
I
buprenorphine regimen and were killed 2 or 7 days after treatment (Fig. 3). A down-regulation (18%) of #- opioid binding was detected in the brains collected 2 days after the buprenorphine treatment and no change in t~ receptor density was found for brains obtained 7 days after drug exposure. This level of buprenorphine neither affected Kl-opioid binding in mothers' brains in both paradigms nor binding affinities of [3H]DAMGE and [3H]U69593.
~.0 I
t~
4
// I
Pl
P7
I
P14
1
P21
/ /
I
ADULT
Fig. 2. Developmental profile of K~-opioid receptors in rat brain. 3H-U69593 binding parameters (Bm~ and K d) were m e a s u r e d in brain m e m b r a n e preparations from rats of different ages. From P1 to adult, K d values were 2.4+0.3, 3.1+0.7, 2.6_+0.2, 3.6_+0.3 and 3.3 _+0.6 nM. n = 3.
4. Discussion
Our experiments have focused on the in utero effects of a mixed agonist-antagonist such as buprenorphine on rat brain /z- and Kl-opioid receptor binding. 6-Opioid receptors, which are up,regulated by buprenorphine in adult and P7 rat brain [1], were not
M.M. Belcheva et al. / Developmental Brain Research 80 (1994) 158-162
studied because they are not detectable in the perinatal period of rat brain ontogeny [9,14,15,17,24]. The density of/z opioid receptors was decreased in P1 pups of mothers exposed to buprenorphine (0.5 mg/kg), whereas no changes were found in P7 pup brains. Similarly, buprenorphine down-regulated /z-opioid binding in maternal brains 2 days after the treatment but not 7 days later. More than likely, the down-regulation has subsided by P7 as buprenorphine turns over in the brain [1]. Buprenorphine-induced /z receptor density changes in maternal and P1 pup brain represent additional proof for the agonist behavior of this drug at Iz opioid receptors. Moreover, density abatement was unaccompanied by changes in K d values suggesting that authentic receptor down-regulation occurs. An apparent loss in sites due to receptor blockade by residual buprenorphine was ruled out by several lines of evidence in a previous study [1]. The use of formerly established binding conditions, known to prevent masking of receptor sites by buprenorphine and the lack of affinity changes of the radiolabeled ligands observed here, verify the occurrence of /z opioid receptor down-regulation. In utero exposure to opioid agonists often results in increased nociceptive thresholds [8,11,21] and transient changes in opioid receptor binding [19,20,23]. Tempel et al. found that implantation of a 75 mg morphine pellet into rats on gestation day 16 induced Iz receptor down-regulation in their offspring which was detected at birth and appeared to persist until P5, dissipating by the second week postnatal [19,20]. Similarly, daily subcutaneous injection of rat pups with 5-10 mg/kg morphine beginning on P1 resulted in /z receptor downregulation of P4, but not later (P8, P21 and P28). The authors explained their findings as being due to the existence of unique plasticity of the immature opioid receptor system that may be lost with maturation. Implicit in this line of reasoning is the possibility of the occurrence of /z opioid receptor subtype variations during development. The fact that prenatal exposure to buprenorphine has no effect on P7 ~-binding in our study could also be explained by the loss of this hypothetical plasticity. However, a 50% down-regulation of P7 brain opioid receptors occurred when 0.5 mg/kg buprenorphine was administered daily for 6 days [1]. Neonates displayed a greater sensitivity to lower doses of buprenorphine than the adult in these and our previous experiments [1]. The results on maternal brain opioid binding are consistent with our observations of a dose-dependent /~ opioid receptor down-regulation in adult male brain upon buprenorphine administration. At 0.75 mg/kg, buprenorphine caused a 25% reduction on ~z opioid receptor density in adult male brain. In contrast to ~z opioid receptor down-regulation, buprenorphine did not alter Kl-binding in maternal, P1
161
and P7 rat brain membrane preparations in this study. Nevertheless, we documented that rat brain K1 sites are present at least as early as P1. Previously, we found a dose dependent up-regulation of U69593 binding in P7 and adult rat brain after buprenorphine treatment [1]. Up-regulation was observed at buprenorphine concentrations >_ 1.0 mg/kg in adults. Neither male nor female adult brain K1 receptors were affected by 0.5 mg/kg buprenorphine. P7 neonates were more sensitive to 0.5 mg/kg buprenorphine and displayed a 1.9fold increase in K~-binding [1]. The fact that the K i of buprenorphine for [3H]U69593 binding in rat brain is almost two orders of magnitude greater than that for [3H]DAMGE [1], indicates that higher concentrations may be necessary to elicit in utero effects on K1 receptors. However, higher levels of buprenorphine (1 mg/kg) proved lethal when administered during gestation, consistent with earlier findings [22]. There were no mortalities in saline-treated control and 0.5 mg/kg buprenorphine-treated pregnant animals. Moreover, of 140 rat pups either saline- (n = 37) or buprenorphinetreated postnatally for 20 h (n = 10) or 7 days (n = 93), only seven died, of which all were chronically exposed [1]. Of the latter seven, all but one were administered > 0.5 mg/kg buprenorphine. The /z opioid receptor down-regulation upon in utero exposure of mothers to buprenorphine observed here cannot be due to drug-elicited changes on enkephalin levels. Tiong and Olley found that buprenorphine (1 and 2 mg/kg, subcutaneously administered) did not affect met- and leu-enkephalin levels in developing rat brain after mothers were treated with the drug [22]. In contrast, methadone (8 mg/kg) reduced enkephalin levels in striatum using the same drug administration regimen. In summary, the present investigation indicates that buprenorphine administered to pregnant females transiently affects maternal and fetal /x opioid receptor densities. The observed receptor down-regulation appeared to be reversible and was curtailed at least 7 days (P7) if not earlier after the last drug treatment. Furthermore, this level of buprenorphine shows selectivity for prenatal /z receptor, as it has no apparent effect on Kropioid binding under the conditions of these experiments. A typical daily sublingual buprenorphine dose used in clinical studies is 0.11 mg/kg [18]. In addition to down-regulation, a number of neurochemical and behavioral correlates accompany opioid agonist actions [8,11,21]. For example, we have found that buprenorphine inhibits DNA synthesis in fetal brain cell aggregates (J. Barg and C.J. Coscia, unpublished observations). There is also extensive documentation of the pathological manifestations of prenatal opioid exposure including physiological and psychological delays [21]. Confounding variables in clinical experiments complicate the determination of the role of the
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M.M. Belcheva et al. / Developmental Brain Research 80 (1994) 158-162
various pharmacological correlates in the etiology of these manifestations. Since buprenorphine is being evaluated in clinical trials for the treatment of dual cocaine and heroin abuse [18], the in utero actions of the drug have ramifications for its use in maternal drug abuse and its effects should be assessed in developing animal model systems.
Acknowledgements This research was supported in part by NIDA Grant DA 05412.
References [1] Belcheva, M.M., Barg, J., McHale, R.J., Dawn, S. Ho, M., Ignatova, E., Coscia, C.J., Differential down- and up-regulation of rat brain opioid receptor types and subtypes by buprenorphine, Mol. Pharmacol., 44 (1993) 173-179. [2] Cowan, A., Lewis, J.W. and MacFarlane, I.R., Agonist and antagonist properties of buprenorphine, a new antinociceptive agent, Br. J. Pharmacol., 60 (1977) 537-545. [3] Jasinski, D.R., Pevnick, J.S. and Griffith, D., Human pharmacology and abuse potential of the analgesic buprenorphine, Arch. Gen. Psychiatry, 35 (1978) 601-616. [4] Johnson, R.E., Jaffe, J.H. and Fudala, P.J., A controlled trial of buprenorphine treatment for opioid dependence, JAMA, 267 (1992) 2750-2755. [5] Kitchen, I., Kelly, M. and Viveros, M.P., Ontogenesis of K-opioid receptors in rat brain using [3H]U-69593 as a binding ligand, Eur. J. Pharmacol. 175 (1990) 93-96. [6] Kornblum, H.I., Hurlbut, D.E. and Leslie, F.M., Postnatal development of multiple opioid receptors in rat brain, Dev. Brain Res., 37 (1987) 21-41. [7] Kosten, T.R., Kleber, H.D. and Morgan, C., Treatment of cocaine abuse with buprenorphine, Biol. Psychiatry, 26 (1989) 637-639. [8] Kuhn, C.M., Windh, R.T. and Little, P.J., Effects of perinatal opiate addiction on neurochemical development of the brain, In M.W. Miller (Ed.), Development of the Central Nervous System, Wiley-Liss, New York, 1992, pp. 341-361. [9] Leslie, F.M., Tso, S. and Hurlbut, D.E., Differential appearance
of opiate receptor subtypes in neonatal rat b~ain, lnti' Scr. ~1 (1982) 1393-1396. [10l Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.. Protein measurement with the Folin phenol reagent. J Biol. Chem., 193 (1951) 265-275. [11] McDowell, J., Kitchen, I., Development of opioid systems: peptides, receptors and pharmacology, Brain Res. Re~.. t2 (1987) 307-421. [12] Mello, N.K. and Mendelson, J.H., Buprenorphme suppresses heroin use by heroin addicts, Science, 207 (1980) 657-650. [13] Munson, PJ. and Rodbard, D., Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem., 11)7(1980) 220-239, [14] Petrillo, P., Tavani, A., Verotta, D., Robsom L.E. and Kosterlitz, H.W., Differential postnatal development of ~-, 8- and K-opioid binding sites in rat brain, Dev. Brain Res, 3t (Iq87) 53-58. [15] Spain, J.W,, Roth, B.L. and Coscia, C.J., Differential ontogeny of multiple opioid receptors (/x, 8 and K), J. Neurosci., 5 (19851 584-588. [16] Strain, E.C., Preston, K.L., Liebson, I.A. and Bigelow, G.E., Acute effects of buprenorphine, hydromorphone and naloxone in methadone-maintained volunteers, J. Pharmacol. Eff~. Ther., 261 (1992) 985-993. [17] Sziics, M. and Coscia, C.J., Evidence for 6 opioid binding and GTP regulatory proteins in 5 day-old rat brain membranes, J. Neurochem., 54 (1990) 1419-1425. [18] Teoh, S.K., Mendelson, J.H., Mello, N.K., Kuehnle, J., Sintavanarong, P. and Rhoades, E.M., Acute interactions of buprenorphine with intravenous cocaine and morphine: an investigational new drug phase I safety evaluation, J. Clin. Psychopharm;, 13 (1993) 87-99. [19] Tempel, A., Habas, J., Paredes, W. and Barr, G.A., Morphineinduced down regulation of #-opioid receptors in neonatal rat brain, Dev. Brain Res., 41 (1988) 129-133. [20] Tempel, A., Visualization of Iz opiate receptor down regulation following morphine treatment in neonatal rat brain, Dec. Brain Res., 64 (1991) 19-26. [21] Tempel, A., Regulation of the opioid system by exogenous drug administration, In M.W. Miller (Ed.), Development of the Central Nervous System, Wiley-Liss, New York, 1992, pp. 3t9-339. [22] Tiong, G.K.L. and Olley, J.E., Effects of exposure in utero to methadone and buprenorphine on enkephalin levels in the developing rat brain, Neurosci. Lett.. 93 (1988) 101-106. [23] Tsang, D. and Ng, S.C., Effect of antenatal exposure to opiates on the development of opiate receptors in rat brain; Brain Res.. 188 (1980) 199-206. [24] Wohltmann, M., Roth, B.L and Coscia, C.J., Differential postnatal development of p. and 6 receptors, Dev. Brain Res.. 3 (1982) 679-684.
Developmental Brain Research 80 (1994) 158-162
...........
Research Report
Transient down-regulation of neonatal rat brain/ -opioid receptors upon in utero exposure to buprenorphine M a r i a n a M . B e l c h e v a , S a m u e l D a w n , J a c o b B a r g , R o b e r t J. M c H a l e , M a t t h e w T . H o , E l e n a I g n a t o v a , C a r m i n e J. C o s c i a * E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1400 South Grand Boulet,ard, St. Louis, MO 63104-1079, USA
Accepted 8 February 1994
Abstract
Gestational actions of the mixed agonist-antagonist buprenorphine on/~-and K1-opioid binding in neonatal and maternal rat brain were investigated. Upon exposure of pregnant rats to 0.5 mg/kg buprenorphine for 7 days prior to birth, postnatal day-one (P1) and P7 offspring brain /z-binding parameters ( K a and Bmax) were assessed with 3H-labeled [o-Ala2,Mephe4,Gly-ol 5] enkephalin (DAMGE). DAMGE binding was attenuated by 64% in P1 membranes, whereas P7 preparations showed no changes. The same buprenorphine regimen resulted in diminished DAMGE Bmax values in mothers' brains, 2 but not 7 days after cessation of drug administration. Receptor density changes were not accompanied by alteration of/~-binding affinities. Although the postnatal developmental profile of K1 opioid receptors in rat brain measured with [3H]U69593 revealed the presence of an ample number of sites for detection, their binding parameters in P1, P7 pups and mothers were unaffected by 0.5 mg/kg buprenorphine. In summary, buprenorphine administration to pregnant rats transiently down-regulates # opioid receptors in neonatal and maternal brain. Key words: Ontogenesis; Opioid receptor; Buprenorphine; Rat central nervous system; Opioid peptide; In utero drug exposure;
Down-regulation
1. Introduction
Maternal drug abuse and its treatment is placing offspring at considerable risk [8,21]. Buprenorphine, an oripavine alkaloid closely related to diprenorphine, is presently undergoing clinical evaluation for its ability to antagonize the reinforcing effects of both heroin and cocaine in humans [4,7,12,18]. This mixed agonistantagonist displays fewer adverse side effects and less potential for abuse liability than opioid agonists presently used in drug rehabilitation [2,3,16]. In a recent study we have found that in vivo administration of buprenorphine engendered a dose-dependent downregulation of forebrain ~ and up-regulation of K and one subtype of 6-opioid binding in neonatal and adult
rat brain membranes [1]. However, little is known about the in utero effects of buprenorphine or other mixed agonist-antagonists on opioid receptor adaptation. Such information would have ramifications on the adoption of buprenorphine for the treatment of maternal drug abuse. Moreover, opioid receptors in developing brain are particularly susceptible to opiate exposure [8,11,21]. Here we report on /x- and Kt-opioid binding in brain membrane preparations f r o m P1, P7 and maternal rats after gestational buprenorphine treatment of the mothers.
2. Materials and methods 2.1. Chemicals
* Corresponding author. Fax: (1) (314) 577-8156. E-mail: [email protected]. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00027-W
DAMGE was obtained from Multiple Peptide Systems (San Diego, CA); buprenorphine and U69593 were from NIDA Drug Supply (Research Triangle, NC). All other chemicalswere purchased from Sigma Chemical Co. (St. Louis, MO).
159
M.M. Belcheva et al. / Developmental Brain Research 80 (1994) 158-162 2.2. Buprenorphine treatment of rats Timed-pregnant S p r a g u e - D a w l e y rats were obtained 1 - 2 weeks before parturition and were injected with buprenorphine intraperitoneally once daily for 7 days (0.5-1.0 m g / k g body weight) beginning on gestation day 14. The dams were not injected with drug on the day of delivery or during their lactation period. Precautions were taken during injections to avoid damaging the placenta. Treated mothers were killed after 2 or 7 days postpartum and forebrains (without cerebella) were harvested. Control mothers were either naive or injected with saline alone for 14 days prior to birth and killed 7 days postpartum. No differences in opioid binding properties were found between saline-treated and naive animals (data not shown). Birth-dated (random sex) pups were killed either 12-24 h (P1) or 156-168 h (P7) after birth and brains (with cerebella) were removed. A single P7 brain provided sufficient protein to generate one binding curve. For P1 assays, two brains from littermates were pooled. In some cases, the P1 and P7 pups tested were littermates. For some of the binding assays, separate experiments were performed using neonates from independent litters. Animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Brain specimens were immediately frozen in dry ice and stored at - 70°C until binding assays were performed.
7 days and [3H]DAMGE binding was measured in the brains of their P1 and P7 offspring. In all experiments brain membrane preparations were washed 5 times with 50 mM Tris buffer to facilitate residual drug removal. A significant (64%) down-regulation of /zopioid binding in P1 membranes was induced by the in utero buprenorphine treatment (Fig. 1). In contrast, the same drug regimen had no effect on the density of /z-opioid receptors in P7 rat brain membranes. Buprenorphine exposure did not affect DAMGE binding affinity in brains of pups of both ages. Brains of P1 and P7 rats from mothers treated with 0.5 mg/kg buprenorphine for 7 days prior to delivery were also analyzed for Krbinding parameters (Bmax and K a) with [3H]U69593 (Fig. 1). Although
DAMGE 100
5
2.3. Washing procedure to remove residual buprenorphine
%M e m b r a n e preparations from neonatal or adult rat brains were washed at least 5 times with 50 m M Tris-HCI, pH 7.4. At each washing step the suspension was centrifuged at 20,000 g for 10 min. This procedure proved to be sufficient to remove most of the residual buprenorphine. This was previously established in control experiments wherein tritiated buprenorphine was administered to P1 and P7 rats and remaining radioactivity was estimated [1].
SL
2.4. Opioid receptor binding assays
Control
1oo Neonatal and adult rat brain m e m b r a n e preparations were assayed for opioid binding activity as described [1]. Briefly, m e m b r a n e preparations ( 3 0 0 - 8 0 0 / ~ g protein) were incubated in duplicate with 1 nM of each [3H]DAMGE, (35 C i / m m o l , Multiple Peptide Systems) or [3H]U69593 (57 C i / m m o l , A m e r s h a m , Arlington Heights, IL) at 25°C for 1 h. Bmax and K,j values were estimated from homologous competition binding assays performed in the presence of 12 different concentrations of the corresponding unlabelled ligand. W h e n levels of opioid binding sites are relatively low as in rat pup brains, we found that the homologous competition approach was superior to saturation plots [17]. Although Bmax values determined by homologous competition analysis may not represent absolute amounts, the relative changes observed between control and treated animals reflect actual differences in maximal binding. Protein concentrations were determined by the m e t h o d of Lowry et al. [10] with bovine serum albumin as standard. Statistical analyses of data were performed using Student's t-test. Binding parameters ( K d and Bmax) were estimated using the L I G A N D program [13].
3. Results 3.1. Effect of in utero buprenorphine treatment on txand Kl-opioid binding in P1 and P7 rat brain membranes
Beginning on gestation day 14, rats were treated with buprenorphine (0.5 mg/kg body weight) daily for
U69,fi93
77"A Buprenorphine
2:
om m o
r
5o
P1
i
P7
Fig. 1. [3H]DAMGE (/J.) and [3H]U69593 (K 1) binding densities in P1 and P7 rat brains after buprenorphine treatment of their mothers. Pregnant rats were treated with buprenorphine (0.5 m g / k g ) for 7 days prior to delivery as described in section 2. p.- and Kl-opioid binding parameters (Bmax and K a) were estimated in m e m b r a n e s from P1 and P7 brains. K d values for D A M G E binding were 0.90+0.06 and 0.92+0.21 nM (for P1 controls and treated, respectively); 1.07+0.06 and 0.89+0.00 nM (for P7 controls and treated, respectively). K d values for U69593 binding were 2.4+0.3 and 2.3 +0.5 nM (for P1 controls and treated, respectively); 3.1 -+0.7 and 4.5-+0.4 nM (for P7 controls and treated, respectively). Maximum radioactivity bound per assay was for ,u., 840-2170 dpm and for KI, 530-1290 dpm. * m e a n s significantly different from control pups, P < 0.05, n = 3-6, except for D A M G E binding to m e m b r a n e s from buprenorphine-treated P7 pups, where n = 2.
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[3H]U69593 is a K1 specific ligand, its ontogenic profile in rat brain (Fig. 2) did not differ appreciably from that of the total K population [5,6,14,15]. Noteworthy is the fact that this receptor subtype is present at birth in rat brain at concentrations comparable to those of the adult. Consistent with previous studies on adult male rats [1], administration of the same amount of buprenorphine (0.5 mg/kg) had no effect on either K~-binding affinity or density (Fig. 1). Although higher levels of buprenorphine (1.0 and 2.5 mg/kg) were found to up-regulate adult male K1 receptors, the 1.0 mg/kg dose proved lethal to all three pregnant females treated at this level for 4-6 days. There was no incidence of mortality in all saline-treated control and 0.5 mg/kg buprenorphine-treated pregnant animals (n = 10).
DAMGE •~
1oo
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~
5o
z O
U69,593 o
I
40
~--~ CONTROL 2 DAYS 7 DAYS
3.2. Buprenorphine effects on t.L- and K~-opioid binding in maternal brain membranes [3H]DAMGE and [3H]U69593 binding parameters were also determined in brain membrane preparations from the mothers that were on the above-described
80
~
60
~
40
S
] Y
2O
m
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Fig. 3. Effects of buprenorphine on I~- and rl-opioid binding in maternal rat brains. Mothers. treated with buprenorphine (0.5 m g / k g ) for 7 days prior to delivery as described in section 2. were killed 2 or 7 days after the last injection. # ([3H]DAMGE) and r x ([3H]U69593) binding parameters (Bmax and K d) were estimated. K d values for D A M G E binding to control and buprenorphine-treated m e m b r a n e s were 1.4_+0.03. 1.5 _+0.03 (2 days) and 1.5 + 0.2 n M (7 days), respectively. K o values for U69593 binding to control and buprenorphine-treated m e m b r a n e s were 2.7 +_0.2. 3.1 + 0.8 (2 days) and 3.9 + 0.7 nM (7 days), respectively. Maximum radioactivity bound per assay was for /z, t590-2830 d p m and for Kl, 490--1160 dpm. * M e a n s significantly different from control mothers, P < 0.05. n = 3.
0 O3
r
)
I
/ / /
I
buprenorphine regimen and were killed 2 or 7 days after treatment (Fig. 3). A down-regulation (18%) of #- opioid binding was detected in the brains collected 2 days after the buprenorphine treatment and no change in t~ receptor density was found for brains obtained 7 days after drug exposure. This level of buprenorphine neither affected Kl-opioid binding in mothers' brains in both paradigms nor binding affinities of [3H]DAMGE and [3H]U69593.
~.0 I
t~
4
// I
Pl
P7
I
P14
1
P21
/ /
I
ADULT
Fig. 2. Developmental profile of K~-opioid receptors in rat brain. 3H-U69593 binding parameters (Bm~ and K d) were m e a s u r e d in brain m e m b r a n e preparations from rats of different ages. From P1 to adult, K d values were 2.4+0.3, 3.1+0.7, 2.6_+0.2, 3.6_+0.3 and 3.3 _+0.6 nM. n = 3.
4. Discussion
Our experiments have focused on the in utero effects of a mixed agonist-antagonist such as buprenorphine on rat brain /z- and Kl-opioid receptor binding. 6-Opioid receptors, which are up,regulated by buprenorphine in adult and P7 rat brain [1], were not
M.M. Belcheva et al. / Developmental Brain Research 80 (1994) 158-162
studied because they are not detectable in the perinatal period of rat brain ontogeny [9,14,15,17,24]. The density of/z opioid receptors was decreased in P1 pups of mothers exposed to buprenorphine (0.5 mg/kg), whereas no changes were found in P7 pup brains. Similarly, buprenorphine down-regulated /z-opioid binding in maternal brains 2 days after the treatment but not 7 days later. More than likely, the down-regulation has subsided by P7 as buprenorphine turns over in the brain [1]. Buprenorphine-induced /z receptor density changes in maternal and P1 pup brain represent additional proof for the agonist behavior of this drug at Iz opioid receptors. Moreover, density abatement was unaccompanied by changes in K d values suggesting that authentic receptor down-regulation occurs. An apparent loss in sites due to receptor blockade by residual buprenorphine was ruled out by several lines of evidence in a previous study [1]. The use of formerly established binding conditions, known to prevent masking of receptor sites by buprenorphine and the lack of affinity changes of the radiolabeled ligands observed here, verify the occurrence of /z opioid receptor down-regulation. In utero exposure to opioid agonists often results in increased nociceptive thresholds [8,11,21] and transient changes in opioid receptor binding [19,20,23]. Tempel et al. found that implantation of a 75 mg morphine pellet into rats on gestation day 16 induced Iz receptor down-regulation in their offspring which was detected at birth and appeared to persist until P5, dissipating by the second week postnatal [19,20]. Similarly, daily subcutaneous injection of rat pups with 5-10 mg/kg morphine beginning on P1 resulted in /z receptor downregulation of P4, but not later (P8, P21 and P28). The authors explained their findings as being due to the existence of unique plasticity of the immature opioid receptor system that may be lost with maturation. Implicit in this line of reasoning is the possibility of the occurrence of /z opioid receptor subtype variations during development. The fact that prenatal exposure to buprenorphine has no effect on P7 ~-binding in our study could also be explained by the loss of this hypothetical plasticity. However, a 50% down-regulation of P7 brain opioid receptors occurred when 0.5 mg/kg buprenorphine was administered daily for 6 days [1]. Neonates displayed a greater sensitivity to lower doses of buprenorphine than the adult in these and our previous experiments [1]. The results on maternal brain opioid binding are consistent with our observations of a dose-dependent /~ opioid receptor down-regulation in adult male brain upon buprenorphine administration. At 0.75 mg/kg, buprenorphine caused a 25% reduction on ~z opioid receptor density in adult male brain. In contrast to ~z opioid receptor down-regulation, buprenorphine did not alter Kl-binding in maternal, P1
161
and P7 rat brain membrane preparations in this study. Nevertheless, we documented that rat brain K1 sites are present at least as early as P1. Previously, we found a dose dependent up-regulation of U69593 binding in P7 and adult rat brain after buprenorphine treatment [1]. Up-regulation was observed at buprenorphine concentrations >_ 1.0 mg/kg in adults. Neither male nor female adult brain K1 receptors were affected by 0.5 mg/kg buprenorphine. P7 neonates were more sensitive to 0.5 mg/kg buprenorphine and displayed a 1.9fold increase in K~-binding [1]. The fact that the K i of buprenorphine for [3H]U69593 binding in rat brain is almost two orders of magnitude greater than that for [3H]DAMGE [1], indicates that higher concentrations may be necessary to elicit in utero effects on K1 receptors. However, higher levels of buprenorphine (1 mg/kg) proved lethal when administered during gestation, consistent with earlier findings [22]. There were no mortalities in saline-treated control and 0.5 mg/kg buprenorphine-treated pregnant animals. Moreover, of 140 rat pups either saline- (n = 37) or buprenorphinetreated postnatally for 20 h (n = 10) or 7 days (n = 93), only seven died, of which all were chronically exposed [1]. Of the latter seven, all but one were administered > 0.5 mg/kg buprenorphine. The /z opioid receptor down-regulation upon in utero exposure of mothers to buprenorphine observed here cannot be due to drug-elicited changes on enkephalin levels. Tiong and Olley found that buprenorphine (1 and 2 mg/kg, subcutaneously administered) did not affect met- and leu-enkephalin levels in developing rat brain after mothers were treated with the drug [22]. In contrast, methadone (8 mg/kg) reduced enkephalin levels in striatum using the same drug administration regimen. In summary, the present investigation indicates that buprenorphine administered to pregnant females transiently affects maternal and fetal /x opioid receptor densities. The observed receptor down-regulation appeared to be reversible and was curtailed at least 7 days (P7) if not earlier after the last drug treatment. Furthermore, this level of buprenorphine shows selectivity for prenatal /z receptor, as it has no apparent effect on Kropioid binding under the conditions of these experiments. A typical daily sublingual buprenorphine dose used in clinical studies is 0.11 mg/kg [18]. In addition to down-regulation, a number of neurochemical and behavioral correlates accompany opioid agonist actions [8,11,21]. For example, we have found that buprenorphine inhibits DNA synthesis in fetal brain cell aggregates (J. Barg and C.J. Coscia, unpublished observations). There is also extensive documentation of the pathological manifestations of prenatal opioid exposure including physiological and psychological delays [21]. Confounding variables in clinical experiments complicate the determination of the role of the
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various pharmacological correlates in the etiology of these manifestations. Since buprenorphine is being evaluated in clinical trials for the treatment of dual cocaine and heroin abuse [18], the in utero actions of the drug have ramifications for its use in maternal drug abuse and its effects should be assessed in developing animal model systems.
Acknowledgements This research was supported in part by NIDA Grant DA 05412.
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