European Journal of Pharmacology, 175 (1990) 93-96

93

Elsevier EJP 20258 Short communication

Ontogenesis of x-opioid receptors in rat brain using [3H]U-69593 as a binding ligand I a n K i t c h e n , M a r y K e l l y a n d M. P a z Viveros 1 Department of Biochemistry, Molecular Toxicology and Pharmacology Group, University of Surrey, Guildford, Surrey GU2 5XH, U.K.

Received 31 October 1989, accepted 7 November 1989

The ontogenesis of x-opioid receptors has been studied in the postnatal period from day 5 to day 30 using the highly selective K-site ligand [3H]U-69593 in binding studies. Analyses of saturation curves revealed a marked increase in the binding capacities between day 5 and day 10 with no further increment in the number of sites up to adult ages confirming a distinct ontogenetic profile from /~- and g-sites. When expressed per mg protein the number of sites declined from day 10 to adult. At all postnatal ages there was little change in receptor affinity. This ontogenetic profile is broadly in agreement with studies using non-selective ~-ligands but the number of sites labelled by [3H]U-69593 is markedly lower in both the neonate and the adult. U-69593; Opioid ontogeny; x Opioid receptor binding

1. Introduction The ontogeny of opioid receptors has been studied using both ligand binding to brain homogenates and autoradiography (for review see McDowell and Kitchen, 1987). Binding studies with ligands highly selective for/~- and 8-opioid receptors have demonstrated a distinct postnatal development of the sites in rate brain (Spain et al. 1985; McDowell and Kitchen 1986). The ontogenesis of x-opioid receptors has mostly been followed using radiolabelled ligands such as ethylketocyclazocine or bremazocine which exhibit poor selectivity for the x-site (Petrillo et al., 1987; Kornblum et al., 1987), and in these studies binding to/~- and &sites has been removed by the use

1 Present address: Departamento de Biologia animal II, Universidad Complutense, Madrid, Spain. Correspondence to: I. Kitchen, Department of Biochemistry, Molecular Toxicologyand PharmacologyGroup, University of Surrey, Guildford, Surrey GU2 5XH, U.K.

of cold suppressor ligands. With the availability of the radiolabelled x-compound U-69593 which exhibits high selectivity at this receptor (Lahti et al., 1985) the opportunity to provide a clearer picture of the development of x-opioid receptors now exists. To this end we have studied the ontogenesis of x-opioid binding sites in rat brain using [3H]U69593 for comparison with the previously reported ontogeny of the receptor using far less selective ligands.

2. Materials and methods Wistar albino rats (University of Surrey strain) of mixed sexes were used in all experiments. Animals were equilibrated in a quiet laboratory 24 h before experimentation and experimental procedures began between 08:30 and 09:30 h. Preweanling pups were removed from the maternal cage i m m e d i a t e l y before killing. M e m b r a n e homogenates (whole brain minus cerebellum) were freshly prepared for each binding assay (Mc-

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94 TABLE 1 Dissociation constants, binding capacities and specific binding ratio of [3H]U-69593 in developing rat brain. Values for K D and Bmax are the means +_S.E. of 3-6 Scatchard plots. Age (days)

Dissociation constant (KD, nM)

Binding capacity (Bmax) pmol/brain fmol/mg wet wt.

fmol/mg prot.

Specific binding ratio (mean specific/total bound)

5 10 15 20 25 30 Adult

1.71 +_0.69 1.54 ± 0.40 2.45 +_0.32 1.51 +_0.37 1.87 +_0.77 1.98 +_0.48 1.66 +_0.10

0.11 +_0.014 0.47 ± 0.076 0.74 +_0.17 0.66 ± 0.15 0.50 +_0.13 0.62 ± 0.15 0.63 +_0.066

6.9 +_0.32 15.5 +_3.3 13.7 ± 4.5 10.5 ± 2.4 7.9 ± 2.2 8.0 +_2.4 7.3 +_0.91

0.63 0.77 0.64 0.72 0.66 0.66 0.60

0.20 ± 0.03 0.54 _+0.1 0.63 ± 0.17 0.54 _+0.1 0.38 +_0.1 0.44 ± 0.1 0.40 +_0.044

Dowell a n d Kitchen, 1986). F o r 5 a n d 10 day old n e o n a t e s pooled tissue from 8-11 b r a i n s were used; for 15-30 day olds, 5-6 brains were used a n d for adults ( > 60 days), m e m b r a n e s were prepared from 4 b r a i n s for each s a t u r a t i o n experiment. [3H]U69593 (New E n g l a n d N u c l e a r Corporation, 40 C i / m m o l ) was purified b y reverse phase H P L C using a linear acetronitrile gradient (20-90% over 20 m i n at 1 m l / m i n in 6.25% v / v a m m o n i a , p H 3.2). S a t u r a t i o n b i n d i n g studies were carried out in triplicate, in 50 m M Tris HC1, p H 7.4 at 25 ° C for 75 min. T e n c o n c e n t r a t i o n s of ligand (0.1-7 n M ) were used in each assay a n d non-specific b i n d i n g was d e t e r m i n e d using 1/~M diprenorphine. Assays were t e r m i n a t e d b y filtration over W h a t m a n G F / B filters presoaked for 2 h in 0 . 1 % polyethylene±mine a n d washed 3 times with ice-cold 50 m M Tris buffer. Scatchard analysis was used to determine the e q u i l i b r i u m dissociation c o n s t a n t ( K D ) a n d maximal b i n d i n g capacity (Bmax). Values for Bma x w e r e calculated per g wet weight of tissue, per b r a i n a n d per mg protein after d e t e r m i n a t i o n of p r o t e i n c o n c e n t r a t i o n s in b r a i n h o m o g e n a t e s (McDowell a n d Kitchen, 1986).

3. Results [3H]U-69593 b i n d i n g p r o d u c e d linear Scatchard plots at all ages studied. Table 1 shows the b i n d ing capacities a n d affinities for [3H]U-69593 dur-

ing p o s t n a t a l development. [3H]U-69593 b i n d i n g was detectable at day 5 a n d there was a rapid increase in b i n d i n g b e t w e e n day 5 a n d 10 representing a 7.3-fold increase when expressed per whole brain, a n d 2.5- a n d 2.1-fold w h e n expressed per mg wet weight tissue or per mg protein, respectively. A t day 10 the n u m b e r of sites (expressed per mg wet weight or per b r a i n ) was equivalent to that observed in adults. W h e n expressed per mg protein, a progressive fall in sites was observed from day 10 to day 25 w h e n the adult level was reached. D u e to the low n u m b e r of sites it was n o t always possible to achieve reproducible b i n d i n g at 5 days of age a n d accordingly earlier time points were n o t studied. T h r o u g h o u t the p o s t n a t a l period the b i n d i n g affinities for [3H]U-69593 were n o t significantly different ( A N O V A ; P > 0.05). TABLE 2 Brain wet weights and brain protein concentrations during postnatal development. Values are the means+_S.E, of 3-6 observations. Age (days)

Brain weight (g)

Protein concentration (mg/g brain tissue)

5 10 15 20 25 30 Adult

0.52 _+0.05 0.91 _+0.02 1.18+_0.06 1.22 +_0.03 1.30 +_0.02 1.40 +_0.04 1.62 +_0.04

29 +-2.8 35 +-1.0 49+_3.6 51 +_7.2 90 +_2.9 50 ± 4.3 55 ± 2.0

95 Table 2 shows the brain weights and protein concentrations throughout development. F r o m day 5 to 15 there was a marked increase in protein levels relative to brain mass but thereafter protein concentrations and brain weight increased in parallel to adulthood.

4. Discussion [3H]U-69593 labels a single population of receptors at all ages as evidenced by the Scatchard analyses and in c o m m o n with all opioid receptor studies the affinities of the receptors are unaltered during development (McDowell and Kitchen, 1987). The characteristics of the development of K-opioid receptors using [3H]U-69593 as a binding ligand are broadly speaking similar to those reported for [3H]bremazocine in the presence of cold ligands to suppress/~- and g-binding (Petrillo et al., 1987). Using [3H]bremazocine a 2-fold increase in sites (expressed per mg brain) was observed between day 3 and day 14 with no further increments by adult ages. However, some studies with [3H]ethylketocyclazocine suggest that K-sites continue to develop at later ages up to day 35 (Barr et al., 1986; Hill et al., 1984; Spain et al., 1985). Clearly this is not observed when using a highly selective x ligand. Further, this study confirms a distinct ontogenetic profile for x-opioid receptors from the previously reported development of tt- and 8-opioid receptors (Spain et al., 1985; McDowell and Kitchen, 1986). The apparent fall in site n u m b e r when expressed per mg protein accords with the recent findings of Allerton et al. (1989) who demonstrated a 6-fold fall in U-69593 sites in rat spinal cord between 9-16 day old and adult animals. Similarly others using non-selective K-ligands (Pertrillo et al., 1987; Spain et al., 1985) have observed this in brain tissue. It is unlikely that this developmental decrement in site n u m b e r is an actual loss of receptors as it is generally recognised that ontogenetic increases in binding capacities are lower when expressed per mg protein due to the general increase in protein concentration within the brain. However, of the opioid receptors x-sites are the only ones to show decreases after day 10

confirming the assertion that the full development of this site is observed by this age. The n u m b e r of sites labelled by U-69593 is clearly less than is seen with non-selective K-ligands in agreement with adult studies by others (Lahti et al., 1985; N o c k et al., 1988) and this has raised the question as to whether U-69593 labels a x-receptor subtype. If, as has been suggested, U-69593 labels a x 1-receptor which is p r o p o s e d to be in low concentration in rat brain (Zukin et al., 1988) then it is this site which is fully developed by day 10. A ligand with high selectivity for the proposed x2-site would help demonstrate if there are x-receptors which show delayed development. In conclusion this study is the first report of K-opioid receptor o n t o g e n y using a ligand highly selective for this receptor. It shows the presence of K-receptors in the early postnatal period and that the major development of these sites labelled with [3H]U-69593 occurs in the first 10 days after birth. The rapid development of these sites is at odds with some studies using ethylketocyclazocine.

References Allerton, C.A., P.R. Boden, R.G. Hill, J. Hughes, J.C. Hunter and J.A.M. Smith, 1989, Correlation of [3H]U-69593 binding sites with antinociceptive activity in the 9-16 day old rat spinal cord, J. Physiol. 409, 38P. Barr, G.A., W. Paredes, K.L. Erickson and R.S. Zukin, 1986, ~¢-Opioid receptor mediated analgesia in the developing rat, Dev. Brain Res. 29, 145. Hill, H.F., Y. Watanabe and T. Shibuya, 1984, Differential postnatal ontogeny of opiate and benzodiazepine receptor subtypes in rat cerebral cortex: binding characteristics of trifluadom and brotizolam, Jap. J. Pharmacol. 36, 15. Kornblum, H.I., D.E. Hurlbut and F.M. Leslie, 1987, Postnatal development of multiple opioid receptors in rat brain, Dev. Brain Res. 37, 121. Lahti, R.A., M.M. Mickelson, J.M. McCall and P.F. Von Voigtlander, 1985, [3H]U-469593, a highly selective ligand for the x-opioid receptor, European J. Pharmacol. 109, 281. McDowell, J. and I. Kitchen, 1986, Ontogenesis of 8-opioid receptors in rat brain using [3H][D-Pen2,D-PenS]enkephalin as a binding ligand, European J. Pharmacol. 128, 287. McDowell, J. and I. Kitchen, 1987, Development of opioid systems: peptides, receptors and pharmacology, Br. Rev. Res. 12, 397. Nock, B., A. Rajpara, L.H. O'Connor and T.J. Cicero, 1988, [3H]U-69593 labels a subtype of kappa opiate receptor with characteristics different from that labelled by [3H]ethylketocyclazocine, Life Sci. 42, 2403.

96 Petrillo, P., A. Tavani, D. Verotta, L.E. Robson and H.W. Kosterlitz, 1987, Differential postnatal development of/~-, 8- and ~-opioid binding sites in rat brain, Dev. Brain Res. 31, 53. Spain, J.W., B.L. Roth and C.J. Coscia, 1985, Differential ontogeny of multiple opioid receptors (/x, 8 and x), J. Neurosci. 5, 584.

Zukin, R.S., M. Eghbali, D. Olive, E.M. Unterwald and A. Tempel, 1988, Characterization and visualization of rat and guinea pig brain x opioid receptors; evidence for xl and x2 opioid receptors, Proc. Natl. Acad. Sci. 85, 4061.

Ontogenesis of kappa-opioid receptors in rat brain using [3H]U-69593 as a binding ligand.

The ontogenesis of kappa-opioid receptors has been studied in the postnatal period from day 5 to day 30 using the highly selective kappa-site ligand [...
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