0028-3908/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc
Neuropharmacology Vol. 29, No. 9, pp. 799-804, 1990 Printed in Great Britain. All rights reserved
OPIATE
RECEPTORS IN NEURONAL CULTURES
PRIMARY
P. S. ERIKSSON,’ELISABETHHANSSON’ and L. R~NNB;~CK’Q~ Institute of Neurobiology and *Department of Neurology, University of Giiteborg, GBteborg, Sweden (Accepred 30 April 1990) Summary-The occurrence and characteristics of p-, 6- and a-receptors were studied as effects of the respective agonists on forskolin-stimulated accumulation of CAMP in neuronal enriched primary cultures
from the cerebral cortex of foetal rats. Morphine or [o-Ala*,N-Me-Phe4,GIy5-ol]-enkephalin(DAGO) were used as h-receptor agonists. [o-Ala*@Let?]-Enkephalin (DADLE) or [D-Pen*,D-Pen’]-enkephahn (DPDPE) were used as &receptor agonists and dynorphin 1-13 (Dyn) or U-50,488H were used as K-receptor agonists. In the presence of lO-*-1O-5 M morphine or lO-*-1O-s M DAGO, there was a dose-related inhibition of the 1W5 M forskolin-stimulated accumulation of CAMP. The inhibitory action of morphine or DAGO was reversed by naloxone. In the presence of 10-9-10-6M DADLE or 10-9-10-6 M DPDPE, there was also a dose-related inhibition of the forskolin-stimulated accumulation of CAMP and a similar result was obtained in the presence of 1O-p-lO-s M Dyn or 10-q-10-5M U-50,488 H. These findings indicate that neurones from the cerebral cortex in culture express n-. 6- and rc-receptors, that inhibit the forskolin-stimulated accumulation of CAMP. Administration of 10TsM
morphine and low6M DADLE or 1O-6M DPDPE together, resulted in a non-additive inhibition of the forskolin-stimulated accumulation of CAMP, indicating the presence of both p- and &receptors on the same population of cells. Key words--morphine, DAGO, DADLE, DPDPE, dynorphin, neurones, primary culture, cerebral cortex, p-receptor, 6-receptor, k-receptor, CAMP.
Within the last two decades an extensive research effort has been undertaken to determine the molecular properties and functional roles of opioid receptors (Casy, 1989). As the methods for the analysis of receptors have become more refined, the complexities of opioid pharmacology have increased. This, in turn, has resulted in the need for more refined model systems, among which primary cultures of neuronal cells are one. It is well established that the b-receptor is coupled negatively to adenylate cyclase, as has been demonstrated, particularly on the neuroblastoma x glioma cell line NG 108- 15 (Sharma, Klee and Nirenberg, 1975: Law, Wu, Koehler and Loh, 1981; Law, Horn and Loh, 1985). There are also studies indicating a negative coupling of h- and K-receptors with adenylate cyclase (Schoffelmeer, Hogenboom and Mulder, 1987; Attali, Saya and Vogel, 1989b). Primary cultures of neurones have been used in the study of p- and 6-receptors from the striatum of the rat (Chneiweiss, Glowinski and Premont, 1988). ICReceptors have been studied in primary cocultures of spinal cord and dorsal root ganglia (Attali, Saya, Nah and Vogel, 1989a: Attali et al., 1989b). In these culture studies, the opiate receptors were indicated to be negatively coupled to adenylate cyclase. The cerebral cortex of the rat is, beside the striatum, a region rich in 11. and 6-receptors (Quirion, Zajac, Morgat and Roques, 1983; McLean, Rothman and Herkenham, 1986), while the K-receptor population is less prominent (Mansour, Khachaturian, Lewis, Akil and
Watson, 1987). The distribution of the different opiate receptors has been shown to be differentiated in the cortex of the brain of the rat, with p-receptors predominantly in layers I and IV, 6-receptors in layers II, III, V and VI, ic-receptors in layers II, III, V and VI (Mansour et al., 1987). The physiological significance of several subclasses of opioid-receptors, with apparently similar effects, is still somewhat puzzling, especially since more than one subtype of receptor has been shown to be located on the same neurone (Egan and North, 1981; Chneiweiss et al., 1988). The present study was undertaken to elucidate the appearance and localization of p-, 6 - and Kreceptors on neurones in primary culture from the cerebral cortex of the brain of foetal rats. The effects of selective opiate agonists on forskolin-stimulated accumulation of 3’,5’cyclic adenosine monophosphate (cyclic AMP) were used to detect opioid ligand-receptor interactions. METHODS
Cell cultures
The cultures were prepared from 15-l ‘f-day-old rat foetuses (Sprague-Dawley strain, A-lab, Stockholm, Sweden). The cerebral cortex was dissected according to Sijderblck, Hansson, Tottmar and Riinnblick (1989). The cultures were grown for 10 days on poly-t.-lysine (Pettmann, Louis and Sensenbrenner, 1979) (Sigma Chemical Company, St Louis, Missouri,
P. S. ERKSSON er al
800 U.S.A.) Roskilde,
coated
multidishes
Denmark),
in
(4 15 mm; a humidified
Nunc
A/S,
atmosphere,
5% CO, in air. The cells were grown in Eagle’s minimum essential medium (MEM, Flow Laboratories Ltd, Irvine, Scotland), supplemented to make up the following final composition: double concentrations of amino acids, 2 mM glutamine, 30 mM glucose, quadruple concentrations of vitamins, 5 pg insulin/ml (Sigma), 250,000 IU penicillin/liter, 1% streptomycin and 20% (v/v) foetal calf serum (Gibco Bio Cult. Lab. Ltd, London, U.K.), all at pH 7.3. The medium was changed after one day of cultivation and then on the 6th day, the amount of foetal calf-serum being changed from 20% to 10%. On the 6th day, the cultures were treated with 1O-5 M cytosine-I-P-Darabinofuranoside (c-ARA; Sigma) to suppress the growth of dividing cells. with
QuantiJication
were used as k-receptor agonists and (-)-2(3-furylmethyl)-noretazocine = (-)-(lR,5R,9R)-5,9diethyl - 2 - (3 - furylmethyl) - 2 - hydroxy - 6,7 - benzo morphan (Mr 2266; Boehringer Ingelheim, Ingelheim, F.R.G.) as a K--receptor antagonist. Forskolin (Sigma) was used as an adenylate cyclase activator. RESULTS
Morphine, DAGO, DPDPE, DADLE, Dyn or U-50,488H did not stimulate the accumulation of CAMP in primary cultures from the cerebral cortex, nor did their respective antagonists. An increase in accumulation of CAMP, compared with controls, was seen after stimulation with forskolin, this stimulatory effects being dose-related in the range 10-6-10m4 M (Fig. 1). In the presence of
of CAMP
The incubation medium was changed to serum-free Eagle’s MEM 1 hr before the experiments. The cultures were exposed to drugs in different combinations including the adenylate cyclase activator, 10m5 M forskolin. Receptor antagonists, when used, were always added prior to the receptor agonists. The exposure time was 10 min and the incubations were made in the presence of the phosphodiesterase inhibitor 10 -’ M iso-butyl-I-methylxantine (IBMX; Sigma). After the incubation, the medium was decanted and the cultures were immediatly exposed to 2 M ice-cold perchloric acid. The cells were scraped off, glass/glass homogenized and centrifuged at 2000 g for 20 min, all at 4°C. Protein determinations were done according to Lowry, Rosebrough, Farr and Randall (1951), with bovine serum albumin as standard. The supernatants were run on anion exchange resins, Ag I-X8, 200400 mesh (BioRad Laboratories, Richmond, California, U.S.A.) at pH 6.95-7.05. The CAMP was eluted using 8 ml of 2 M formic acid. After freeze-drying and dissolving in 2.0 ml, 0.05 M sodium acetate at pH 6.2, CAMP was determined using radioimmunoassay kits (Steiner, Parker and Kipnis, 1972; Steiner, Pagliara, Chase and Kipnis, 1972) (NEN, Boston, Massachusetts, U.S.A.).
Forskolin-stimulated
14r
accumulation
of
CAMP
of neurona 1 cultures
, 7
I
I
I
I
6
5
4
3
-log
[Forskolinl
M
Fig. 1. Accumulation of CAMP in the presence of lO-6-1O-4 M forskolin. Incubations were performed for 10 min in the presence of IO-’ M IBMX. The results are expressed as accumulation of CAMP in “times control”. Each point represents the mean of 6 dishes, obtained in 2 experiments. SEM values are given.
Effects r-
stimulated
of
morphine
on forskolln-
accumulation
of
CAMP
Drugs Morphine (Apoteksbolaget AB, Stockholm, Sweden) and [D-Ala*,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO; Sigma) were used as p-receptor agonists and naloxone (Sigma) as an antagonist. [D-Ala*,DLeu’]-enkephalinamide (DADLE; Sigma) and [DPen*@-Pen’]-enkephalin (DPDPE; Sigma) were used as a-receptor agonists and NJ-diallyl-tyr-Aib-AibPhe-Leu (ICI 174,864; Cambridge Research Biochemicals Ltd, Cambridge, England) was used as an antagonist. Dynorphin 1-13 (Dyn; Cambridge Research Biochemicals) and tram-( -t )-3,4-dichloroN-methyl- N-[2-( 1-pyrrolidinyl)cyclohexyl]-benzeneacetamide methane sulfonate (U-50,488H; The Upjohn Company, Kalamazoo, Michigan, U.S.A.)
-log
[Morphmel
M
Fig. 2. Effects of 1O-8-lO-5 M morphine on the accumulation of CAMP, stimulated with 10m5M forskolin. The inhibitory effects were more prominent at larger doses of morphine. The hatched area shows the effect of lO-‘M forskolin. The bar labelled naloxone shows the effect of 1O-5 M of naloxone, in the presence of lo-’ M morphine and 10m5 M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of three dishes. Statistical analysis according to Student’s t-test. SEM values are given. **P < 0.01, ***P < 0.001.
Opioid receptors on cortex neurones Effects of DAGO on forskolin stimulated accumulation of CAMP
801 Effects of DPDPE on forskolinstimulated accumulation of CAMP
,,
9
6
7
-log
5
6
CDAGOI
,
_I IO
9
M
7
6
-log
CDPDPEI
6 M
Fig. 3. Effects of lO-8-1O-5 M DAGO on the accumulation of CAMP, stimulated with 10e5M forskolin. The hatched area shows the effect of IO-’ M forskolin. The bar labelled naloxone shows the effect of 10e5M of naloxone, in the presence of 10e5 M DAGO and 10e5 M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of three dishes. Statistical analysis according to Student’s r-test. SEM values are given *P < 0.05, **p< 0.01.
Fig. 5. Effects of 10-9-10-6 M DPDPE on the accumulation of CAMP, stimulated with 10m5M forskolin. The hatched area shows the effect of 10m5M forskolin. The bar labelled ICI 174,864 shows the effect of 10e5 M ICI 174,864, in the presence of 10m6M DPDPE and 10m5M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of 6 cultures from 2 experiments. Statistical analysis according to Student’s f-test. SEM values are given. *P < 0.05, ***Pi 0.001.
1O-8-lO-5 M morphine, there was a dose-related inhibition of the forskolin-stimulated accumulation of CAMP (Fig. 2). The inhibitory action of 10-j M morphine on 10-j M forskolin-stimulated accumulation of CAMP was reversed with 10-6-10-4 M naloxone in a dose-related manner. The 10-j M forskolin-stimulated accumulation of CAMP was inhibited by DAGO in a dose-related manner. Naloxone lO_‘M antagonized the effect of 10d5 M DAGO (Fig. 3). In a concentration of 10-9-10-6 M, DADLE was shown to inhibit the forskolin-stimulated accumulation of CAMP in a dose-related manner and 10-j M ICI 174,864 reversed the inhibitory effect of 10m6 M DADLE (Fig. 4). The drug DPDPE was shown to be inhibitory, in the range 10e910m6 M, of the forskolin-stimulated accumulation of
CAMP. The inhibitory action of 10m6 M DPDPE was antagonized with 10-j M ICI 174,864 (Fig. 5). Dynorphin elicited a dose-related inhibitory action on the forskolin-stimulated accumulation of CAMP (Fig. 6). In the presence of U-50,488H, in increasing doses, there was also a dose-related inhibition of the forskolin-stimulated accumulation of CAMP (Fig. 7). The inhibitory actions of the two ~-receptor agonists were not completely blocked with the K-receptor antagonist, Mr 2266. When added together, the inhibitory effects of 10m6 M Dyn was additive with the inhibitory effects of p- or d-receptor stimulation. On the other hand, no additivity of the inhibitory effects on forskolin-stimulate accumulation of CAMP of 10-j M morphine upon administration together with 10e6M DADLE, was seen (Fig. 8). Similar results were obtained with 10m5 M morphine and 10e6M DPDPE.
Effect of DADLE on forskotinstlmuloted accumulation of CAMP
12r
0-
10
9
6
-log
C DADLEI
6
7
M
Fig. 4. Effects of 10~9~10-6 M DADLE on forskolin-stimulated accumulation of CAMP. The hatched area shows the effect of 10e5M forskolin. The bar labelled ICI 174,864 shows the effect of IO-’ M ICI 174,864, in the presence of 10-6M DADLE and 10m5M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of 9 cultures from 3 experiments. Statistical analysis according to Student’s r-test. SEM values are given. 'P < 0.05, ***p< 0.001.
Effects of Dyn on forskollnstimutoted accumulation of CAMP
2 OlO
I 9
I
I
I
I
6
7
6
5
-tog
tDyn1
1
4
M
Fig. 6. Effects of 10-9-10-5 M Dyn on the accumulation of CAMP, stimulated with 10m5M forskolin. The hatched area shows the effect of 10m5M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of 6 cultures from 2 experiments. Statistical analysis according to Student’s r-test. SEM values are given. *,*p < 0.01, ***p< 0.001.
802
P. S. ERK~~ON et al. Effects of stimulated
U 50,468H on forskolinaccumulation of CAMP
6I
10
I 9
I 6 -log
I 7
I 6
CU-50,488H1
I 5 M
Fig. 7. Effects of 10~‘-10~5 M U-50,488H on the accumulation of CAMP. stimulated with 10m5 M forskolin. The hatched area shows the effects of 1O-5 M forskolin. Accumulation of CAMP is expressed as “time control”. Each point represents the mean of 3 cultures. Statistical analysis according to Student’s l-test. SEM values are given. **p < 0.01, ***p < 0.001.
Neuron01
oplote
receptor
interactlow
IOr 6
3
-6
FO
Fo+Mo
6
Fo + DADLE
4
Fo+ MO+ Dyn
2
Fo + MO + DADLE + Dyn
8
Fo+ Dyn Fo + MO+ DADLE
E i=
Fo + DADLEtDyn
-
0:
Fig. 8. Effects of 10m5 M morphine, 10e6 M DADLE or 10m6 M Dyn on IO-’ M forskolin-stimulated accumulation of CAMP. An inhibition of the forskolin-induced accumulation of CAMP is seen. When 10m5 M morphine and 10m6 M DADLE was administered together, the effects were not additive, suggesting the localization of p- and b-receptors together on the same population of cells. Upon the simultaneous administration of morphine and Dyn, DADLE and Dyn or morphine, DADLE and Dyn, there was an additive inhibition of the forskolin-induced accumulation of CAMP. This possibly indicates that the K-receptors are localized on different populations of cells, compared with the cells exhibiting p- and d-receptors. Fo = Forskolin, MO = morphine, SEM values are given.
DISCUSSION
Since one type of cell can be enriched, primary cultures comprise an interesting model system when effects of the stimulation of receptors are studied on accumulation of second messengers. In the present study, neuronal cultures, derived from the cerebral cortex of foetal rats were used. The cultures, previously characterized in this laboratory (SGderbick, et al., 1989), contain predominantly cells staining with antibodies against the neuronal specific enolase (NSE) and are thus considered to be neurones. These cells are morphologically differentiated, forming a
network of processes. Neurones with different morphological appearance exist within one culture. Small neurones (cellbody 6 pm in diameter), mainly forming aggregates, and oval neurones (cellbody 10 pm), with two polar or more processes, were identified between the aggregates. Some larger neurones (cellbody 20pm), with networks of processes, were also observed (Saderbdck er al., 1989). A very small number of cells stained with antisera against glial fibrillary acidic protein (GFAp) or SIOO and were considered to be astrocytes. The majority of the non-neuronal population of cells are morphologically undifferentiated cells, not stained. Endothelial cells (lo-20 cells per dish) and some macrophages were present. The existence of synapses in the primary cultures is at present unclear. Opioid receptors have been classified into at least three main types (p, 6 and K) (Martin, 1984). Recent reports indicate that subclasses also may exist (Callahan and Pasternak, 1987; Castanas, Bourhim, Giraud, Boudouresque, Cantau and Oliver, 1985; Casy, 1989). Analysis of the properties and function of opioid receptors is complicated by the finding that multiple types of receptor many exist together within a single cell (Egan and North, 1981; Chneiweiss et al., 1988). In the present study, DAGO or morphine were used as p-receptor agonists. The drug DAGO has previously been shown to possess a relative affinity of 0.994 for the p-receptor and 0.006 for the &-receptor in the brain of the guinea pig (Corbett, Gillan, Kosterlitz, McKnight, Paterson and Robson, 1984). Naloxone was used as a p-receptor antagonist. The relative affinity of naloxone for the three opioid sites has been shown to be p :0.85, 6 :06, and 0.09 for the ICsite (Paterson, Robson and Kosterlitz, 1983); DPDPE is considered to be one of the most selective 6 ligands (James and Goldstein, 1984), with a relative affinity of 0.996 for the a-receptor and 0.004 for the p site (Corbett er al., 1984) and DADLE has been shown to have 0.87 in relative affinity for the 6 site and 0.13 in relative affinity for the p site (Corbett et al., 1984). Also, ICI 174,864 has been shown to be 6-selective (Cotton, Giles, Miller, Shaw and Timms, 1984), with a relative affinity of 0.99 for the 6 site and 0.01 for the p site. Dyn has been shown to be potent at the ~-site McKnight, Magnan and Paterson, (Corbett, Kosterlitz, 1982), as has U-50,488H (James and Goldstein, 1984) and Mr 2266 has mainly a K-receptor affinity but it also has some affinity for the p site (Paterson et al., 1983). In order to study the inhibitory effect of opiates on the accumulation of CAMP, the formation of CAMP was stimulated with forskolin. This compound is thought to inhibit the association of the c( and By subunits of the G, protein, thus generating an x subunit stimulatory influence on adenylate cyclase (Barber and Goka, 1985; Bourne, 1989). However, it should be kept in mind that both CIand pi, subunits can induce effects in other effector systems, beside adenylate cyclase
Opioid receptors
(Neer and Clapham, 1988; Boume, 1989). The present results indicate that neurones in primary culture from cerebral cortex of the rat expressed JL-, 6- and K-receptors. Upon s~m~atiou, all three receptors responded inhibition of forwith the skolin-induced accumulation of CAMP. This is in line with previous work indicating the negative coupling of opioid-receptors to adenylate cyclase (~~hoffelmeer et lai., 1987; Attali et af,, 1989b) through Ci- or C&-proteins (Wang, ~moliou-Mason and Barnard, 1989). stimulation of &-receptors was antagonized with naloxone. Furthermore, the action of DADZE or DPDPE was antagonized with the selective 6-receptor antagonist, ICI 174,864, The action of 1V6 M Dyn was not compIetely reversed with the antagonist 10S5 M Mr 2266, neither was the effect of 10e6 M U-50,488H. However, both Dyn and U-50,488H are considers to be selective E-receptor agonists and Mr 2266 is a less selective antagonist (Paterson et al., 1983). Therefore the effects of Dyn and U-50,488H are ascribed to the K-receptor. Chneiweiss eb a!. (1988~ were unable to detect n- or &receptors on neurones in primary culture from the cerebral cortex. This could be due to differences in culture conditions, though the expression of receptors in primary cultures is sensitive to composition of the medium. In that study, effects of opioids on basai activity of adenylate cyclase were studied. Since the basal activity is lower than in the striatum, this could also explain the negative results obtained. Indications about the cellular localization of different types of receptors, coupled with negatively to adenylate cyciase, can be obtained by the dete~ination of whether inhibitory effects of the ~o~pondin8 agonists are additive or not. Addi~vity of the effects of two distinct agonists is compatible both with a localization of the corresponding receptors on separate cellular populations or on the same cell (Premont, Da8uet-deMontety, Herbet, ~lowinski, Bockaert and Procbiantz, 1983). Non-additivity d~rno~st~tes a localization of the different types of receptor on the same cell. The inhibitory efIet~s of p- and &receptor stimulation were not additive, indicating a localization of these receptors together on a subpopulation of cells. These receptors have also been demonstrated to be localized on neurones from the striatum of the mouse, in ~rirnar~ culture ~~bneiweiss et aE., 1988). The functional roles of pand &receptors localized together are still unclear. There were no detectable differences in the effects of ,ut- and &-receptors, as studied on the adenylate cyclase system. There are studies indicating diflerences in ion channel elects between these two receptors (McFadzean, 1988; North, 1986). Such differences could be aimed at the fine tuning of membrane excitability. Metabolic events, beyond the second messengers, can possibly also be differentially regulated, eventually involving second messen~rs other than CAMP. NP 2W-ii
on
cortex ~uron~
803
Acknowledgements-The
study was supported with grants from the Swedish Medical Research Council (project No.
25X-06005), from Central Ex~rirne~~l Animal Co~tte (Grant No. L-89-16), from the Mcdii Faculty of G~te~rg~ from Torsten and Ragnar Sitderbergs Fotmdations and from Goteborgs Ku&. Vetenskaps och Vitterhets-Samh%lle. We are grateful for the gift of U50,488H from Upjon AB Sweden and for the gift of Mr 2266 from Boehringer Ingelheim AB Sweden.
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Neuropharmacology Vol. 29, No. 9, pp. 799-804, 1990 Printed in Great Britain. All rights reserved
OPIATE
RECEPTORS IN NEURONAL CULTURES
PRIMARY
P. S. ERIKSSON,’ELISABETHHANSSON’ and L. R~NNB;~CK’Q~ Institute of Neurobiology and *Department of Neurology, University of Giiteborg, GBteborg, Sweden (Accepred 30 April 1990) Summary-The occurrence and characteristics of p-, 6- and a-receptors were studied as effects of the respective agonists on forskolin-stimulated accumulation of CAMP in neuronal enriched primary cultures
from the cerebral cortex of foetal rats. Morphine or [o-Ala*,N-Me-Phe4,GIy5-ol]-enkephalin(DAGO) were used as h-receptor agonists. [o-Ala*@Let?]-Enkephalin (DADLE) or [D-Pen*,D-Pen’]-enkephahn (DPDPE) were used as &receptor agonists and dynorphin 1-13 (Dyn) or U-50,488H were used as K-receptor agonists. In the presence of lO-*-1O-5 M morphine or lO-*-1O-s M DAGO, there was a dose-related inhibition of the 1W5 M forskolin-stimulated accumulation of CAMP. The inhibitory action of morphine or DAGO was reversed by naloxone. In the presence of 10-9-10-6M DADLE or 10-9-10-6 M DPDPE, there was also a dose-related inhibition of the forskolin-stimulated accumulation of CAMP and a similar result was obtained in the presence of 1O-p-lO-s M Dyn or 10-q-10-5M U-50,488 H. These findings indicate that neurones from the cerebral cortex in culture express n-. 6- and rc-receptors, that inhibit the forskolin-stimulated accumulation of CAMP. Administration of 10TsM
morphine and low6M DADLE or 1O-6M DPDPE together, resulted in a non-additive inhibition of the forskolin-stimulated accumulation of CAMP, indicating the presence of both p- and &receptors on the same population of cells. Key words--morphine, DAGO, DADLE, DPDPE, dynorphin, neurones, primary culture, cerebral cortex, p-receptor, 6-receptor, k-receptor, CAMP.
Within the last two decades an extensive research effort has been undertaken to determine the molecular properties and functional roles of opioid receptors (Casy, 1989). As the methods for the analysis of receptors have become more refined, the complexities of opioid pharmacology have increased. This, in turn, has resulted in the need for more refined model systems, among which primary cultures of neuronal cells are one. It is well established that the b-receptor is coupled negatively to adenylate cyclase, as has been demonstrated, particularly on the neuroblastoma x glioma cell line NG 108- 15 (Sharma, Klee and Nirenberg, 1975: Law, Wu, Koehler and Loh, 1981; Law, Horn and Loh, 1985). There are also studies indicating a negative coupling of h- and K-receptors with adenylate cyclase (Schoffelmeer, Hogenboom and Mulder, 1987; Attali, Saya and Vogel, 1989b). Primary cultures of neurones have been used in the study of p- and 6-receptors from the striatum of the rat (Chneiweiss, Glowinski and Premont, 1988). ICReceptors have been studied in primary cocultures of spinal cord and dorsal root ganglia (Attali, Saya, Nah and Vogel, 1989a: Attali et al., 1989b). In these culture studies, the opiate receptors were indicated to be negatively coupled to adenylate cyclase. The cerebral cortex of the rat is, beside the striatum, a region rich in 11. and 6-receptors (Quirion, Zajac, Morgat and Roques, 1983; McLean, Rothman and Herkenham, 1986), while the K-receptor population is less prominent (Mansour, Khachaturian, Lewis, Akil and
Watson, 1987). The distribution of the different opiate receptors has been shown to be differentiated in the cortex of the brain of the rat, with p-receptors predominantly in layers I and IV, 6-receptors in layers II, III, V and VI, ic-receptors in layers II, III, V and VI (Mansour et al., 1987). The physiological significance of several subclasses of opioid-receptors, with apparently similar effects, is still somewhat puzzling, especially since more than one subtype of receptor has been shown to be located on the same neurone (Egan and North, 1981; Chneiweiss et al., 1988). The present study was undertaken to elucidate the appearance and localization of p-, 6 - and Kreceptors on neurones in primary culture from the cerebral cortex of the brain of foetal rats. The effects of selective opiate agonists on forskolin-stimulated accumulation of 3’,5’cyclic adenosine monophosphate (cyclic AMP) were used to detect opioid ligand-receptor interactions. METHODS
Cell cultures
The cultures were prepared from 15-l ‘f-day-old rat foetuses (Sprague-Dawley strain, A-lab, Stockholm, Sweden). The cerebral cortex was dissected according to Sijderblck, Hansson, Tottmar and Riinnblick (1989). The cultures were grown for 10 days on poly-t.-lysine (Pettmann, Louis and Sensenbrenner, 1979) (Sigma Chemical Company, St Louis, Missouri,
P. S. ERKSSON er al
800 U.S.A.) Roskilde,
coated
multidishes
Denmark),
in
(4 15 mm; a humidified
Nunc
A/S,
atmosphere,
5% CO, in air. The cells were grown in Eagle’s minimum essential medium (MEM, Flow Laboratories Ltd, Irvine, Scotland), supplemented to make up the following final composition: double concentrations of amino acids, 2 mM glutamine, 30 mM glucose, quadruple concentrations of vitamins, 5 pg insulin/ml (Sigma), 250,000 IU penicillin/liter, 1% streptomycin and 20% (v/v) foetal calf serum (Gibco Bio Cult. Lab. Ltd, London, U.K.), all at pH 7.3. The medium was changed after one day of cultivation and then on the 6th day, the amount of foetal calf-serum being changed from 20% to 10%. On the 6th day, the cultures were treated with 1O-5 M cytosine-I-P-Darabinofuranoside (c-ARA; Sigma) to suppress the growth of dividing cells. with
QuantiJication
were used as k-receptor agonists and (-)-2(3-furylmethyl)-noretazocine = (-)-(lR,5R,9R)-5,9diethyl - 2 - (3 - furylmethyl) - 2 - hydroxy - 6,7 - benzo morphan (Mr 2266; Boehringer Ingelheim, Ingelheim, F.R.G.) as a K--receptor antagonist. Forskolin (Sigma) was used as an adenylate cyclase activator. RESULTS
Morphine, DAGO, DPDPE, DADLE, Dyn or U-50,488H did not stimulate the accumulation of CAMP in primary cultures from the cerebral cortex, nor did their respective antagonists. An increase in accumulation of CAMP, compared with controls, was seen after stimulation with forskolin, this stimulatory effects being dose-related in the range 10-6-10m4 M (Fig. 1). In the presence of
of CAMP
The incubation medium was changed to serum-free Eagle’s MEM 1 hr before the experiments. The cultures were exposed to drugs in different combinations including the adenylate cyclase activator, 10m5 M forskolin. Receptor antagonists, when used, were always added prior to the receptor agonists. The exposure time was 10 min and the incubations were made in the presence of the phosphodiesterase inhibitor 10 -’ M iso-butyl-I-methylxantine (IBMX; Sigma). After the incubation, the medium was decanted and the cultures were immediatly exposed to 2 M ice-cold perchloric acid. The cells were scraped off, glass/glass homogenized and centrifuged at 2000 g for 20 min, all at 4°C. Protein determinations were done according to Lowry, Rosebrough, Farr and Randall (1951), with bovine serum albumin as standard. The supernatants were run on anion exchange resins, Ag I-X8, 200400 mesh (BioRad Laboratories, Richmond, California, U.S.A.) at pH 6.95-7.05. The CAMP was eluted using 8 ml of 2 M formic acid. After freeze-drying and dissolving in 2.0 ml, 0.05 M sodium acetate at pH 6.2, CAMP was determined using radioimmunoassay kits (Steiner, Parker and Kipnis, 1972; Steiner, Pagliara, Chase and Kipnis, 1972) (NEN, Boston, Massachusetts, U.S.A.).
Forskolin-stimulated
14r
accumulation
of
CAMP
of neurona 1 cultures
, 7
I
I
I
I
6
5
4
3
-log
[Forskolinl
M
Fig. 1. Accumulation of CAMP in the presence of lO-6-1O-4 M forskolin. Incubations were performed for 10 min in the presence of IO-’ M IBMX. The results are expressed as accumulation of CAMP in “times control”. Each point represents the mean of 6 dishes, obtained in 2 experiments. SEM values are given.
Effects r-
stimulated
of
morphine
on forskolln-
accumulation
of
CAMP
Drugs Morphine (Apoteksbolaget AB, Stockholm, Sweden) and [D-Ala*,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO; Sigma) were used as p-receptor agonists and naloxone (Sigma) as an antagonist. [D-Ala*,DLeu’]-enkephalinamide (DADLE; Sigma) and [DPen*@-Pen’]-enkephalin (DPDPE; Sigma) were used as a-receptor agonists and NJ-diallyl-tyr-Aib-AibPhe-Leu (ICI 174,864; Cambridge Research Biochemicals Ltd, Cambridge, England) was used as an antagonist. Dynorphin 1-13 (Dyn; Cambridge Research Biochemicals) and tram-( -t )-3,4-dichloroN-methyl- N-[2-( 1-pyrrolidinyl)cyclohexyl]-benzeneacetamide methane sulfonate (U-50,488H; The Upjohn Company, Kalamazoo, Michigan, U.S.A.)
-log
[Morphmel
M
Fig. 2. Effects of 1O-8-lO-5 M morphine on the accumulation of CAMP, stimulated with 10m5M forskolin. The inhibitory effects were more prominent at larger doses of morphine. The hatched area shows the effect of lO-‘M forskolin. The bar labelled naloxone shows the effect of 1O-5 M of naloxone, in the presence of lo-’ M morphine and 10m5 M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of three dishes. Statistical analysis according to Student’s t-test. SEM values are given. **P < 0.01, ***P < 0.001.
Opioid receptors on cortex neurones Effects of DAGO on forskolin stimulated accumulation of CAMP
801 Effects of DPDPE on forskolinstimulated accumulation of CAMP
,,
9
6
7
-log
5
6
CDAGOI
,
_I IO
9
M
7
6
-log
CDPDPEI
6 M
Fig. 3. Effects of lO-8-1O-5 M DAGO on the accumulation of CAMP, stimulated with 10e5M forskolin. The hatched area shows the effect of IO-’ M forskolin. The bar labelled naloxone shows the effect of 10e5M of naloxone, in the presence of 10e5 M DAGO and 10e5 M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of three dishes. Statistical analysis according to Student’s r-test. SEM values are given *P < 0.05, **p< 0.01.
Fig. 5. Effects of 10-9-10-6 M DPDPE on the accumulation of CAMP, stimulated with 10m5M forskolin. The hatched area shows the effect of 10m5M forskolin. The bar labelled ICI 174,864 shows the effect of 10e5 M ICI 174,864, in the presence of 10m6M DPDPE and 10m5M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of 6 cultures from 2 experiments. Statistical analysis according to Student’s f-test. SEM values are given. *P < 0.05, ***Pi 0.001.
1O-8-lO-5 M morphine, there was a dose-related inhibition of the forskolin-stimulated accumulation of CAMP (Fig. 2). The inhibitory action of 10-j M morphine on 10-j M forskolin-stimulated accumulation of CAMP was reversed with 10-6-10-4 M naloxone in a dose-related manner. The 10-j M forskolin-stimulated accumulation of CAMP was inhibited by DAGO in a dose-related manner. Naloxone lO_‘M antagonized the effect of 10d5 M DAGO (Fig. 3). In a concentration of 10-9-10-6 M, DADLE was shown to inhibit the forskolin-stimulated accumulation of CAMP in a dose-related manner and 10-j M ICI 174,864 reversed the inhibitory effect of 10m6 M DADLE (Fig. 4). The drug DPDPE was shown to be inhibitory, in the range 10e910m6 M, of the forskolin-stimulated accumulation of
CAMP. The inhibitory action of 10m6 M DPDPE was antagonized with 10-j M ICI 174,864 (Fig. 5). Dynorphin elicited a dose-related inhibitory action on the forskolin-stimulated accumulation of CAMP (Fig. 6). In the presence of U-50,488H, in increasing doses, there was also a dose-related inhibition of the forskolin-stimulated accumulation of CAMP (Fig. 7). The inhibitory actions of the two ~-receptor agonists were not completely blocked with the K-receptor antagonist, Mr 2266. When added together, the inhibitory effects of 10m6 M Dyn was additive with the inhibitory effects of p- or d-receptor stimulation. On the other hand, no additivity of the inhibitory effects on forskolin-stimulate accumulation of CAMP of 10-j M morphine upon administration together with 10e6M DADLE, was seen (Fig. 8). Similar results were obtained with 10m5 M morphine and 10e6M DPDPE.
Effect of DADLE on forskotinstlmuloted accumulation of CAMP
12r
0-
10
9
6
-log
C DADLEI
6
7
M
Fig. 4. Effects of 10~9~10-6 M DADLE on forskolin-stimulated accumulation of CAMP. The hatched area shows the effect of 10e5M forskolin. The bar labelled ICI 174,864 shows the effect of IO-’ M ICI 174,864, in the presence of 10-6M DADLE and 10m5M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of 9 cultures from 3 experiments. Statistical analysis according to Student’s r-test. SEM values are given. 'P < 0.05, ***p< 0.001.
Effects of Dyn on forskollnstimutoted accumulation of CAMP
2 OlO
I 9
I
I
I
I
6
7
6
5
-tog
tDyn1
1
4
M
Fig. 6. Effects of 10-9-10-5 M Dyn on the accumulation of CAMP, stimulated with 10m5M forskolin. The hatched area shows the effect of 10m5M forskolin. Accumulation of CAMP is expressed as “times control”. Each point represents the mean of 6 cultures from 2 experiments. Statistical analysis according to Student’s r-test. SEM values are given. *,*p < 0.01, ***p< 0.001.
802
P. S. ERK~~ON et al. Effects of stimulated
U 50,468H on forskolinaccumulation of CAMP
6I
10
I 9
I 6 -log
I 7
I 6
CU-50,488H1
I 5 M
Fig. 7. Effects of 10~‘-10~5 M U-50,488H on the accumulation of CAMP. stimulated with 10m5 M forskolin. The hatched area shows the effects of 1O-5 M forskolin. Accumulation of CAMP is expressed as “time control”. Each point represents the mean of 3 cultures. Statistical analysis according to Student’s l-test. SEM values are given. **p < 0.01, ***p < 0.001.
Neuron01
oplote
receptor
interactlow
IOr 6
3
-6
FO
Fo+Mo
6
Fo + DADLE
4
Fo+ MO+ Dyn
2
Fo + MO + DADLE + Dyn
8
Fo+ Dyn Fo + MO+ DADLE
E i=
Fo + DADLEtDyn
-
0:
Fig. 8. Effects of 10m5 M morphine, 10e6 M DADLE or 10m6 M Dyn on IO-’ M forskolin-stimulated accumulation of CAMP. An inhibition of the forskolin-induced accumulation of CAMP is seen. When 10m5 M morphine and 10m6 M DADLE was administered together, the effects were not additive, suggesting the localization of p- and b-receptors together on the same population of cells. Upon the simultaneous administration of morphine and Dyn, DADLE and Dyn or morphine, DADLE and Dyn, there was an additive inhibition of the forskolin-induced accumulation of CAMP. This possibly indicates that the K-receptors are localized on different populations of cells, compared with the cells exhibiting p- and d-receptors. Fo = Forskolin, MO = morphine, SEM values are given.
DISCUSSION
Since one type of cell can be enriched, primary cultures comprise an interesting model system when effects of the stimulation of receptors are studied on accumulation of second messengers. In the present study, neuronal cultures, derived from the cerebral cortex of foetal rats were used. The cultures, previously characterized in this laboratory (SGderbick, et al., 1989), contain predominantly cells staining with antibodies against the neuronal specific enolase (NSE) and are thus considered to be neurones. These cells are morphologically differentiated, forming a
network of processes. Neurones with different morphological appearance exist within one culture. Small neurones (cellbody 6 pm in diameter), mainly forming aggregates, and oval neurones (cellbody 10 pm), with two polar or more processes, were identified between the aggregates. Some larger neurones (cellbody 20pm), with networks of processes, were also observed (Saderbdck er al., 1989). A very small number of cells stained with antisera against glial fibrillary acidic protein (GFAp) or SIOO and were considered to be astrocytes. The majority of the non-neuronal population of cells are morphologically undifferentiated cells, not stained. Endothelial cells (lo-20 cells per dish) and some macrophages were present. The existence of synapses in the primary cultures is at present unclear. Opioid receptors have been classified into at least three main types (p, 6 and K) (Martin, 1984). Recent reports indicate that subclasses also may exist (Callahan and Pasternak, 1987; Castanas, Bourhim, Giraud, Boudouresque, Cantau and Oliver, 1985; Casy, 1989). Analysis of the properties and function of opioid receptors is complicated by the finding that multiple types of receptor many exist together within a single cell (Egan and North, 1981; Chneiweiss et al., 1988). In the present study, DAGO or morphine were used as p-receptor agonists. The drug DAGO has previously been shown to possess a relative affinity of 0.994 for the p-receptor and 0.006 for the &-receptor in the brain of the guinea pig (Corbett, Gillan, Kosterlitz, McKnight, Paterson and Robson, 1984). Naloxone was used as a p-receptor antagonist. The relative affinity of naloxone for the three opioid sites has been shown to be p :0.85, 6 :06, and 0.09 for the ICsite (Paterson, Robson and Kosterlitz, 1983); DPDPE is considered to be one of the most selective 6 ligands (James and Goldstein, 1984), with a relative affinity of 0.996 for the a-receptor and 0.004 for the p site (Corbett er al., 1984) and DADLE has been shown to have 0.87 in relative affinity for the 6 site and 0.13 in relative affinity for the p site (Corbett et al., 1984). Also, ICI 174,864 has been shown to be 6-selective (Cotton, Giles, Miller, Shaw and Timms, 1984), with a relative affinity of 0.99 for the 6 site and 0.01 for the p site. Dyn has been shown to be potent at the ~-site McKnight, Magnan and Paterson, (Corbett, Kosterlitz, 1982), as has U-50,488H (James and Goldstein, 1984) and Mr 2266 has mainly a K-receptor affinity but it also has some affinity for the p site (Paterson et al., 1983). In order to study the inhibitory effect of opiates on the accumulation of CAMP, the formation of CAMP was stimulated with forskolin. This compound is thought to inhibit the association of the c( and By subunits of the G, protein, thus generating an x subunit stimulatory influence on adenylate cyclase (Barber and Goka, 1985; Bourne, 1989). However, it should be kept in mind that both CIand pi, subunits can induce effects in other effector systems, beside adenylate cyclase
Opioid receptors
(Neer and Clapham, 1988; Boume, 1989). The present results indicate that neurones in primary culture from cerebral cortex of the rat expressed JL-, 6- and K-receptors. Upon s~m~atiou, all three receptors responded inhibition of forwith the skolin-induced accumulation of CAMP. This is in line with previous work indicating the negative coupling of opioid-receptors to adenylate cyclase (~~hoffelmeer et lai., 1987; Attali et af,, 1989b) through Ci- or C&-proteins (Wang, ~moliou-Mason and Barnard, 1989). stimulation of &-receptors was antagonized with naloxone. Furthermore, the action of DADZE or DPDPE was antagonized with the selective 6-receptor antagonist, ICI 174,864, The action of 1V6 M Dyn was not compIetely reversed with the antagonist 10S5 M Mr 2266, neither was the effect of 10e6 M U-50,488H. However, both Dyn and U-50,488H are considers to be selective E-receptor agonists and Mr 2266 is a less selective antagonist (Paterson et al., 1983). Therefore the effects of Dyn and U-50,488H are ascribed to the K-receptor. Chneiweiss eb a!. (1988~ were unable to detect n- or &receptors on neurones in primary culture from the cerebral cortex. This could be due to differences in culture conditions, though the expression of receptors in primary cultures is sensitive to composition of the medium. In that study, effects of opioids on basai activity of adenylate cyclase were studied. Since the basal activity is lower than in the striatum, this could also explain the negative results obtained. Indications about the cellular localization of different types of receptors, coupled with negatively to adenylate cyciase, can be obtained by the dete~ination of whether inhibitory effects of the ~o~pondin8 agonists are additive or not. Addi~vity of the effects of two distinct agonists is compatible both with a localization of the corresponding receptors on separate cellular populations or on the same cell (Premont, Da8uet-deMontety, Herbet, ~lowinski, Bockaert and Procbiantz, 1983). Non-additivity d~rno~st~tes a localization of the different types of receptor on the same cell. The inhibitory efIet~s of p- and &receptor stimulation were not additive, indicating a localization of these receptors together on a subpopulation of cells. These receptors have also been demonstrated to be localized on neurones from the striatum of the mouse, in ~rirnar~ culture ~~bneiweiss et aE., 1988). The functional roles of pand &receptors localized together are still unclear. There were no detectable differences in the effects of ,ut- and &-receptors, as studied on the adenylate cyclase system. There are studies indicating diflerences in ion channel elects between these two receptors (McFadzean, 1988; North, 1986). Such differences could be aimed at the fine tuning of membrane excitability. Metabolic events, beyond the second messengers, can possibly also be differentially regulated, eventually involving second messen~rs other than CAMP. NP 2W-ii
on
cortex ~uron~
803
Acknowledgements-The
study was supported with grants from the Swedish Medical Research Council (project No.
25X-06005), from Central Ex~rirne~~l Animal Co~tte (Grant No. L-89-16), from the Mcdii Faculty of G~te~rg~ from Torsten and Ragnar Sitderbergs Fotmdations and from Goteborgs Ku&. Vetenskaps och Vitterhets-Samh%lle. We are grateful for the gift of U50,488H from Upjon AB Sweden and for the gift of Mr 2266 from Boehringer Ingelheim AB Sweden.
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