Repeated administration of opioids alters characteristics of membrane-bound phorbol ester binding in rat brain

ejp

ELSEVIER

European Journal of Pharmacology 271 (1994) 547-550

Short communication

Repeated administration of opioids alters characteristics of membrane-bound phorbol ester binding in rat brain Minoru Narita, Yangzheng Feng, Mizue Makimura, Beth Hoskins, I.K. Ho

*

Department of Pharmacology and Tox'icology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA Received 18 October 1994; accepted 21 October 1994

Abstract

Scatchard analysis of saturation binding data indicated that dissociation c o n s t a n t ( g D) of [3H]phorbol 12,13-dibutyrate (PDB) binding to the membrane-bound protein kinase C of rat cortex and midbrain was significantly decreased following systemic repeated administration of morphine (/x-opioid receptor agonist) and butorphanol (/x/~/K-mixed opioid receptor agonist). No change in the receptor density (Bmax) of [3H]PDB binding was found following repeated treatment with morphine and butorphanol. On the other hand, neither the Bmax n o r K D values in pons/medulla were altered in these rats. These results suggest that systemic repeated opioid treatment, such as morphine and butorphanol leads to an increased affinity for phorbol ester binding to the membrane-bound protein kinase C in rat cortex and midbrain.

Keywords: Chronic opioid treatment; Protein kinase C; Phorbol ester binding; Morphine; Butorphanol

1. Introduction

Pharmacological studies have defined at least three classes of opioid receptors, termed /x, t~ and K. Complementary DNAs encoding three opioid receptors have been isolated and characterized (Reisine and Bell, 1993). Repeated or sustained exposure to opioids results in a reduction in their potencies in evoking pharmacological effects. It has been reported that alterations in intracellular messenger proteins could be part of an overall mechanism underlying opioid addiction (Nestler et al., 1993). Protein kinase C is an important third messenger in the regulation of neuronal excitability and signal transduction, and protein kinase C exists in both cytosolic and membrane fractions (Kikkawa et al., 1982; Nishizuka, 1988). Multiple forms of protein kinase C have been cloned, with the brain known to contain at least seven species of the enzyme, and the protein kinase C family can be classified into two distinct classes; the Ca2+-dependent and -independent protein kinase C subspecies (Nishizuka, 1988; Akita et al.,

* Corresponding author. Tel. (601) 984-1600, fax (601) 984-1637. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 6 4 8 - 2

1990). Additionally, the activation of protein kinase C by second messengers is believed to be associated with its translocation from the cytosolic fraction to the membrane fraction (Akita et al., 1990). In recent studies, we found that systemic repeated treatment of rats with morphine led to an enhancement of cytosolic, but not membrane, protein kinase C activity in rat brain, when high concentrations of calcium chelators were present in the assay medium (Narita et al., 1994). These findings indicate that systemic repeated morphine treatment may activate the CaZ+-independent cytosolic protein kinase C subspecies, but not Ca 2+independent membrane-bound protein kinase C in rat brain. With this in mind, we examined whether the CaZ+-dependent membrane-bound protein kinase C could be influenced by systemic repeated opioid treatment. Phorbol 12,13-dibutyrate (PDB) is a phorbol ester with high affinity (nM) for various protein kinase C isoenzymes and is known to be a selective ligand and a potent activator of the regulatory subunit of these isoenzymes (Bazzi and Nelsestuen, 1989). The tritiated PDB binding activity co-chromatographs with enzymatic activity of protein kinase C. Since PDB binding is less affected by experimental conditions than catalytic activity of the enzyme, it represents a more convenient

548

M. Narita et al. / European Journal of Pharmacology 271 (1994) 547-550

measure for detecting the distribution and characteristics of protein kinase C (Bazzi and Nelsestuen, 1989). In the present study, we investigated whether systemic repeated morphine treatment can change the characteristics of PDB binding to protein kinase C in the membrane fractions of rat brain in the absence of Ca 2÷ chelators. Butorphanol (17-cyclobutylmethyl-3,14-dihydroxymorphinan) is a potent mixed agonist/antagonist opioid analgesic which has multiple actions on the opioid receptor system including /.~-, 8- and K-opioid receptors (Horan and Ho, 1989). Butorphanol exhibits some pharmacological and biochemical differences as well as similarities to the prototype/z-opioid receptor agonist, morphine (Horan and Ho, 1989). Therefore, our second purpose was to compare the effects of systemic repeated treatment with morphine and butorphanol on PDB binding.

2. Materials and methods 2.1. Animals

Seven- to eight-week old male Sprague-Dawley rats (Charles River, Wilmington, MA, USA), weighing 225250 g, were used. Animals were kept in a room with an ambient temperature of 21 + 2°C and a 12 h light-dark cycle with free access to food and water, for a week prior to experiments. 2.2. Membrane preparation

For the PDB binding experiment, rats were divided into three treatment groups (n = 10-15) designated as repeated administration of morphine (20 mg/kg per day, i.p.), butorphanol (20 mg/kg per day, i.p.), and saline. Drug/saline administration was performed between 11:00-13:00 h each day for 7 consecutive days. After the appropriate treatments, rats were killed by decapitation 24 h after the last injection and their brains were quickly excised on an ice-cold petri-dish. The brain regions (cortex, midbrain and pons/medulla) were dissected. Each brain region of two or three rats was pooled as one sample. Brain regions were homogenized in 10 volumes of ice-cold 0.32 M sucrose using the Potter-Elvehjem tissue grinder with teflon pestle (PGC Scientifics Corp., Gaithersburg, MD, USA). The homogenate was centrifuged at 900 x g for 10 min and the supernatant was centrifuged at 11500 x g for 20 min. The resultant pellet was disrupted by suspending it in 10 volumes of 50 mM Tris-HC1 buffer (pH 7.4) and the suspension was centrifuged at 11 500 × g for 20 min. The resulting pellet was resuspended in 10 volumes of 50 mM Tris-HC1 buffer (pH 7.4), and then stored at - 70°C until use. On the day of binding assay,

the frozen membrane fractions were thawed and suspended in 50 mM Tris-HC1 buffer (pH 7.4), and centrifuged at 11500 x g for 20 min. The pellets (P2 fraction) obtained were then resuspended in the 50 mM Tris-HCl buffer (pH 7.4) for binding assay. 2.3. Binding assay

The suspensions of P2 fraction were incubated with [20-3H (N)]-PDB (18.6-20.7 Ci/mmol; New England Nuclear, Boston, MA, USA) at 25°C for 60 min. The reaction was terminated by filtering through Whatman G F / B glass filters that had been immersed in 0.1% polyethylenimine using a cell harvester (Model M-24, Brandel, MD, USA). The filters were washed twice with 50/zM Tris-HCl buffer (pH 7.4). Filters were put into individual vials filled with 6 ml of scintillation cocktail (Packard, Downers Grove, IL, USA). After shaking, the radioactivity trapped on the filters was counted in a liquid scintillation counter using a Packard liquid scintillation analyzer (Model 2,200 CA). The differences in the amounts of [3H]PDB bound (0.5, 1.0, 2.0, 5.0, 10, 20 and 50 nM) in the presence and absence of 10 /zM of PDB (Research Biochemicals International, Natick, MA, USA) were designated as specific binding. All reactions were run in triplicate. Comparable results were obtained from four to five independent sets of experiments. 2.4. Statistical analysis

The binding data for the determination of the density and affinity of binding sites were evaluated by computer-assisted non-linear regression analysis with the computerized curve fitting programs, EBDA and LIGAND (Biosoft, Cambridge, UK). The data are presented as the mean + S.E.M. Newman-Keuls multiple comparison test was used for the statistical analysis of the data.

3. Results

The amount of non-specific binding was < 5% of the total binding under these conditions. To distinguish between the dissociation constants (K D) and maximum numbers of binding sites (Bmax), Scatchard analysis of [3H]PDB binding saturation experiments were performed. Table 1 shows the effect of chronic opioid treatment on the characteristics of [3H]PDB binding in the rat brain regions. Scatchard plots were linear under all experimental conditions, indicating the presence of a single class of high-affinity binding sites. In cortices from animals receiving repeated treatment with morphine and butorphanol, the apparent K D was significantly decreased when compared to chronic saline-

M. Narita et al. / European Journal of Pharmacology 271 (1994) 547-550

Table 1 Effects of repeated opioid treatment on dissociation c o n s t a n t (K D) and receptor density (Bma~) for [3H]PDB binding to the membrane fractions of rat brains

KD (nM)

Bmax (pmol/mg of protein)

7.71 + 0.71 5.17 + 0.47 a 4.96 + 0.50 b

29.13 + 1.08 25.83 + 1.13 26.12 _+1.41

6.57 + 0.54 4.22 + 0.21 a 4.21 + 0.37

26.97 + 1.68 24.74 + 1.77 23.74 + 2.07

4.66 + 0.39 4.60 + 0.38 4.12 +_0.36

9.69 + 0.31 9.88 + 0.29 9.69 + 0.32

Cortex

Chronic Saline Chronic Morphine Chronic Butorphanol Midbrain

Chronic Saline Chronic Morphine Chronic Butorphanol

a

P o n s / medulla

Chronic Saline Chronic Morphine Chronic Butorphanol

Drug administration was performed once each day for 7 consecutive days (saline, morphine 20 mg/kg per day, butorphanol 20 mg/kg per day). Rats were killed 24 h after the last injection, and PDB binding was performed. Data are expressed as means + S.E.M. from four to five independent experiments performed in tripricate, a p < 0.01, b p < 0.05, significantlydifferent from the saline group. treated rats, whereas the B m a x w a s not changed. The percent decrease in K D values were 32.9% and 35.7% in the morphine- and butorphanol-treated groups, respectively. In the midbrain, the binding data clearly showed a significant decrease in K D with no change in Bmax. There were 35.8% and 35.9% decrease in K D values in rats repeatedly treated with morphine and butorphanol, respectively. However, there were no differences in the characteristics of [3H]PDB binding in the p o n s / m e d u l l a region between saline-, morphineand butorphanol-treated groups.

4. Discussion We report here, for the first time, that systemic repeated treatment of rats with morphine and butorphanol resulted in an increased affinity for [3H]PDB binding in m e m b r a n e s of the cortex and midbrain, but not p o n s / m e d u l l a . These data provide further evidence for changes in the characteristics of m e m b r a n e bound protein kinase C in rat cortex and midbrain following repeated opioid treatment. The protein kinase C family can be classified into sub-groups, depending on whether the enzymes require Ca 2+ for activation or not (Nishizuka, 1988; Akita et al., 1990). U n d e r Ca2+-chelating conditions, we found that repeated treatment of rats with morphine enhanced the cytosolic protein kinase C activity in rat p o n s / m e d u l l a , while having no effect on the m e m b r a n e protein kinase C activity (Narita et al., 1994). Recently, it has been reported that in the presence of added Ca 2÷ the

549

[3H]PDB binding sites in the m e m b r a n e fraction of rat brain exhibit a higher affinity with no change in the n u m b e r of binding sites (Spieler et al., 1993), Others have reported that chronic morphine treatment is associated with increased synaptosomal Ca 2+ levels and upregulation of dihydropyridine binding sites (L-type Ca 2÷ channel) in brain (Garcia and Harlan, 1993). Our findings therefore suggest that repeated systemic opioid treatment causes an increase in intracellular Ca 2+ levels which, in turn, enhances Ca2+-dependent m e m brane-bound protein kinase C activity in rat cortex and midbrain. The pharmacology of butorphanol is complex due to its apparent multiplicity of actions on the opioid receptor system. In in vitro radioligand displacement studies, butorphanol was shown to have a high affinity not only for /x-opioid receptors but also for 6- and K-opioid receptors (Horan and Ho, 1989). In the present studies, repeated systemic treatment with butorphanol resuited in [3H]PDB binding parameters which were similar to those found for repeated morphine treatment. In a preliminary study, we found that repeated systemic treatment with a selective K-opioid receptor agonist produced an increased affinity for m e m b r a n e bound [3H]PDB binding sites without the change in the receptor density (unpublished data). It is still unclear which opioid receptor type(s) contribute to an enhancement of [3H]PDB binding induced by repeated administration of butorphanol. In conclusion, we have demonstrated that repeated systemic treatment with morphine and butorphanol leads to an increased affinity for [3H]PDB binding in m e m b r a n e s of rat cortex and midbrain, but not p o n s / m e d u l l a . These alterations of the m e m b r a n e bound phorbol ester binding in rat brain, most probably associated with activation of protein kinase C, can be dissociated by Ca 2÷ chelation. Therefore, we hypothesize that some chronic opioid-induced phenomena are modulated by upregulation of protein kinase C system.

Acknowledgements We wish to thank Dr. Takehiko Ito, Mr. James G. Bennett and Miss Kathia Lawson for their helpful guidance and technical assistance. This research was supported by G r a n t D A 05828 from the National Institute on Drug Abuse.

References Akita, Y., S. Ohno, Y. Konno, A. Yano and K. Suzuki, 1990, Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C, J. Biol. Chem. 265, 354.

550

M. Narita et al. /European Journal of Pharmacology 271 (1994) 547-550

Bazzi, M.D. and G.L. Nelsestuen, 1989, Properties of protein kinase C-phorbol-ester interaction, Biochemistry 28, 3577. Garcia, M.M. and R.E. Harlan, 1993, Stimulus-transcription coupling at the/.~ opiate receptor, in: The Neurobiology of Opiates, ed. R.P. Hammer (CRC Press, Inc., Boca Raton, Florida) p. 257. Horan, P. and I.K. Ho, 1989, Comparative pharmacological and biochemical studies between butorphanol and morphine, Pharmacol. Biochem. Behav. 34, 847. Kikkawa, U., Y. Takai, R. Minakuchi, S. Inohara and Y. Nishizuka, 1982, Ca2+-activated, phospholipid-dependent protein kinase from rat brain: subcellular distribution, purification, and properties, J. Biol. Chem. 257, 13348. Narita, M., M. Makimura, Y.Z. Feng, B. Hoskins and I.K. Ho, 1994,

Influence of chronic morphine treatment on protein kinase C activity: comparison with butorphanol and implication for opioid tolerance, Brain Res. 650, 175. Nestler, E.J., B.T. Hope and K.L. Widnell, 1993, Drug addiction: a model for the molecular basis of neural plsticity, Neuron 11, 995. Nishizuka, Y., 1988, The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature 334, 661. Resisine, T. and G.I. Bell, 1993, Molecular biology of opioid receptors, Trends Neurosci. 16, 506. Spieler, K., P. Schoch, J.R. Martin and W. Haefely, 1993, Environmental stimulation promotes changes in the distribution of phorbol ester receptors, Pharmacol. Biochem. Behav. 46, 553.