European Journal of Pharmacology, 195 (1991) 295-300 0 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991002856
295
EfP 51791
fl=Adrenoceptor-sensitive enylate cyclaseisi Y ac receptors in rat striatal neurons Bernard J. Van Vliet, Sigrid R. Ruuls, Benjamin Drukarch ‘, Arie H. Mulder and Anton N.M. Schoffelmeer Departments of Pharmacology and ’ Neurology, Free University. Medical Faculty, Van der Boechorststraat 7, 1081 ET Amsterdam, The Netherlands Received 17 September 1990. revised MS received 8 January 1991, accepted 15 January 1991
The fi-adrenoceptor-sensitive adenylate cyclase in primary cultures of rat striatd neurons was inhibited by opioids, unlike that in rat striatal slices. Isoprenaline (1 PM)-stimulated cyclic AMP production was dose dependently inhibited by the p-opioid receptor agonist, [D-AlaZ,MePhe4,Gly-o15]enkephalin (DAGO, EC,, = 0.02 PM, 36% inhibition), and only slightly reduced by relatively high concentrations of the d-opioid receptor agonist, [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE, 1 PM). The highly selective and potenr S-opioid receptor agonist, [D-Ser’(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET). and the a-opioid receptor agonist, U50-488, were ineffective in concentrations up to 3 pM. Naloxone reversed equally well the inhibitory effects of DPDPE and of DAGO, indicating the involvement of functional F-opioid receptors. The isoprenaline (1 PM)-stimulated adenylate cyclase activity in cultured glial cells. which exceeded that in neurons about IO-fold. was not affected by opioids. Therefore, opioids were ineffective in rat brain slices probably due to the fact that cyclic AMP production induced by b-adrenoceptor activation occurs primarily in the glial cells, where it is not subject to inhibition by opioids. These data indicate for the first time the existence of an interaction between functional p-opioid receptors and P-adrenoceptors on striatal neurons of the rat. Opioid receptors; P-Adrenoceptors; Adenylate cyclase; Striatal neurons
1. Introduction It is now well established that opioids exert their effect by acting on receptors classified into at least three different types, i.e. CL-(morphine), 6- (enkephalin) and K- (dynorphin) opioid receptors (Martin, 1984; Goldstein, 1987; Simon, 1987). One major function of opioids in the brain appears to be the inhibition of monoaminergic neurotransmission processes. Thus, it has been shown that activation of opioid receptors results in presynaptic inhtbition of the electrically evoked release of dopamine (DA) and noradrenaline (NA) from slices of different regions of the brain (Chesselet, 1984; Jackisch et al., 1986a,b; Mulder et al., 1984; 1988; Schoffelmeer et al., 1988; Werling et al., 1988). The modulatory effect of opioid drugs at the postsynaptic level has been demonstrated in case of dopaminergic neurotransmission. No effect of opioid receptor activation on fi-adrenoceptor-sensitive adenylate cyclase was observed in rat striatal slices, where DA-sensitive
adenylate cyclase was found to be inhibited by activation of CL-and b-opioid receptors (Schoffelmeer et al.. 1986; 1987; 1988). Since, in rat brain, P-adrenoceptors occur primarily on astrocytes (Maderspach and Fajszi. 1983; Burgess et al., 1985), cyclic AMP production induced by /3-adrenoceptor activation may be expected to occur primarily in this type of cell. Moreover, little is known about the cellular distribution and possible colocalization of P-adrenoceptor and opioid receptors on neurons and glial cells. Therefore, we now used primary cultures of rat striatal neurons and glial cells (Van Vliet et al., 1990; Ebersolt et al., 1981) to investigate the possible modulation by opioids of the P-adrenoceptorstimulated cyclic AMP production in neurons. thought to be a major postsynaptic action of released NA in the brain.
2. Materials and methods 2.1. Preparation of striatal slices
Correspondence to: A.N.M. Schoffelmeer, Department of Pharmacology, Medical Faculty, Free University, Van der Boechorststraat 7. 1081 BT Amsterdam, The Netherlands.
Male Wistar rats (180-220 g body weight) were decapitated and the striatum was rapidly dissected from the brain. Slices (0.3 x 0.3 x 2 mm) were prepared using a McIlwain tissue chopper.
1 neuronal cultures were prepared as previibed (Van Vliet et al.. 1990). according to a reported by Bockaert et al. (1986) and Weiss et _ (1986) for the culture of mouse striatal neurons. efly. striata were dissected from 17-day-old Wistar rat embryos and were mechanically dissociated, using a fire-narrowed Pasteur pipet. in a serum-free medium. Cells were plated in 12-well Linbro culture dishes (6 X 1Q5 cells,/ml per well). previously coated with poly-Lornithine (1.5 pg/ml) and medium containing 10% sup~~erne~t~ calf serum. The culture medium was composed of a 1 : I mixture of Dulbecco’s modified Eagles medium and F-12 nutrient and contained glucose (0.6%) glutamirle (2 mM). sodium bicarbonate (3 mM), HEPES buffer (5 mM). streptomycin (100 pg/ml) and penicillin (100 Itl/ml). A defined hormone and salt mixture was added. consisting. of insulin (25 pg/ml). transferrin (100 rtg/mlf. progesterone (LO nM). putrescine (60 FM), /3-estradiol fl phiI) and selenium sodium salt (30 aM). Wnder our culture conditions. neurons developed an extensive matrix of dendrites and synapses, as described by Bockaert et al. (1986) and survived for more than 4 weeks. Since neurons were grown in a serum-free (hormone-supplemented) medium, only a few glial cells were observed (about only 7% of total cells. Bockaert et al.. 1986) after lo-13 days in vitro. when cultures were used for esperiments. 23 Preparation of primaty nrbes
of striatal ustrocy~es
Cultures of astrocytes were prepared as neuronal cultures. except that the cells were grown in medium containing 10% supplemented calf serum instead of the hormone and salt mixture (according to Ebersolt et al., 1981). The medium was changed twice a week. Glial cell cultures consisted of a nearly homogeneous population of type I astrocytes. The cells had reached confluency after 2 weeks. and almost no neuron survived in the cultures after 4 weeks, when cultures were used for experiments (Ebersolt et al., 1981).
were transferred to edch of the 24 chambers of a superfusion apparatus (about 3-6 mg tissue per chamber of 0.2 ml volume) and superfused (0.1 ml/min) in presence of 95% O,-5% CO, at 37*C. After 30 min. the slices were exposed to medium containing drugs and the inhibitor. 3-isobutyl-l-methyl-xanphosphodiesterase thine (IBMX. 1 mM). for 30 min. The reaction was stopped by superfusion of the slices with ice-cold TCA (5%) for 15 min. [“HICyclic AMP and [‘H]ATP present in the effluent of this extraction period were separated by sequential chromatography on Dowex and alumina columns (Salomon et al., 1974).
The cultures were washed with phosphate-buffered saline (PBS) containing (mM): 137 NaCl, 2.7 KCl, 8 Na,HPO,, 1.5 KH,PO,, 0.5 MgCl,, 1.2 CaCl, and 5 glucose (pH 7.3) before incubation with 2 &i [‘Hladenine at 37OC. After 2 h, the cultures were again washed with PBS and exposed for 5 min to a PBS solution containing drugs and 1 mM IBMX. The reaction was stopped by aspiration of the media and addition of 1 ml ice-cold trichloroacetic acid (TCA, 5%). [‘H]Cyclic AMP was separated from 13H]ATP through sequential chromatography on Dowex and alumina columns (Salomon et al., 1974). Cyclic AMP production was expressed as: Zconvrrsion =
{3H]cAhlP
[ ,HIATP
+ i,HlcAMP
X
100
This prelabelling technique, originally described by Shimizu et al. (1969), has yielded results completely consistent with those based on measurement of endogenous levels of cyclic AMP (Daly, 1977). and has been used in various studies with cell cultures (see Weiss et al., 1986, and references quoted therein). 2.5. Statistics The statistical significance of differences was determined by one-way analysis of variance (ANOVA) followed by Student’s t-test. 2.6. ~adioche~~i~a~sand drugs
Cyclic AMP production in slices and cell cultures was determined by measuring the conversion of 13Hj&enine nucleotide precursors (mainly [3H]ATP) to [ ’ HIcyclic AMP. 2.4. I. Sil%atal slict?s
Striatal slices were washed twice with Krebs-Ringerbicarbonate medium containing (mM): 121 NaCl, 1.9 KCl. 25 NaHCOx, 1.2 KH2P0,, 1.2 MgSO,, 1.2 CaCl, and 10 glucose (pH 7.3) before incubation with f3H]adenine (4 ,uCi/ml) for 1 h. Subsequently, they
The following drugs were obtained commercially: 3-isobutyl-1-methyl-xanthine (IBMX) from Aldrich: selenium sodium salt, from Merck; calf serum, from Hyclone; [ ‘Hladenine (15-25 Ci/mmol), from Amersham; poly-L-ornithine, streptomycin, penicillin, insulin, transferrin, progesterone, putrescine, P-estradiol, isoprenaline, propanolol and naloxone hydrochlo~de, from Sigma: Dulbecco’s modified Eagles medium and F-12 nutrient, from Gibco; and [D-Ala’,MePhe4,Gly-o15]enkephalin (DAGB) and [D-peni~illal~ne2,D-penicillamines]enkephalin (DPDPE), from Bachem. Trans-3.4dichloro-N-methyl-N-[2-(l-pyrrolidinyl)-cyclohexyl]-
297
80
60
9
- IOQ jsoprenalne cont. (M)
-
8
log DAGO
7 COIIC.
(M)
Fig. 1. Effect of isoprenaline on adenylate eyclase activity in cuhured striatal neurons and its in~bition by basal adenylate cyclase activity in the absence (0) and presence (0) of pmpranoid (1 PM). (b) Inhibition cyclase activity by DAGO. In the absence of drugs, the conversion of ATP to cyclic AMP amounted pM)-induced conversion of ATP to cyclic AMP amounted to 0.46 + 0.01% in the absence of opioids. The data nine observations obtained in three separate experiments.
benzeneacetamide methanesulfonate hydrate (U50-488) and [D-Ser’(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET) were generous gifts from Upjohn (Kalamazoo, MI. USA) and Dr. B.P. Roques (Universite Ren6 Descartes, Paris, France), respectively.
3. Results Activation of /3-adrenoceptors in rat striatal slices by isoprenaline (3 @vi) resulted in a 3.6-fold increase in adenylate cyclase activity, which was not affected by the :I receptor agonist DAGO (1 PM). the 6 receptor agonist DPDPE (1 PM) or the K receptor agonist U50488 {l FM), respectively {table 1). Activation of ~-adren~~ptors in cultured striatal neurons by isoprenaline (fig, la) resulted in a dose-de-
TABLE l Effect of opioid agonists on isoprenaline-stimulated adenylate cydase activity in rat striatal slices. fn the absence of drugs, conversion of ATP to cyclic AMP amounted to 0.37~0.02%. The isoprenaline (3 PM)-stimulated conversion of ATP in cyclic AMP amounted to 1.35 +0.06% in the absence of opioids. The data are neank S.E.M. values from 8-16 observations obtained in 4 separate experiments. NS: not significantly different from the value in the absence of opioid Adenylate cyclase activity (% of control)
Drugs lsoprenaline lsoprenaline lsoprenaline lsoprenaline
3 pM 3 pM + DAGO 1 PM 3 FM + DPDPE 1 I_LM 3 PM + U50-488 1 NM
lOOf 104+7NS 102&3 NS llSi-7 NS
DAGO. (a) Effect of isoprenahne on of i~prenaIine-stimui~ted adenylate to 0.13+0.01%. The isoprenahne (1 are mean f. S.E.M. (bars) values from
pendent increase in adenylate cyclase activity, which could be fully antagonized by 1 PM propranolol. Interestingly, the isoprenaline (1 PM)-stimulated cyclic AMP production was dose dependently inhibited by DAGO (EC, = 0.02 FM, 36% maximal in~bitio~ fig. lb), but only slightly reduced by relatively high concentrations of DPDPE (1 PM) (table 2). Moreaver. the selective 6 receptor agonist, DSTBULET. and K receptor agonist, U50-488, were not effective at concentrations up to 3 PM (table 2). In order to further investigate the inhibitory effects of DAGO and DPDPE, we examined the effect of the selective c receptor antagonist, naloxone. In neuronal cultures, naloxone reversed equally well the
TABLE 2 Inhibition by opioid agonists of isop~naIine-stimuiat~ adenylate cyclase activity in striatat neuronal cultures. In the absence of drugs. conversion of ATP to cyclic AMP amounted to 0.13 &0.01X. The isoprenaline (3 PM)-stimulated conversion of ATP in cyclic AMP amounted to 0.62 &0.03% in the absence of opioids. The data are mean +_S.E.M. values from six to nine observations obtained in three separate experiments. ND: not determined Drug cont. (PM)
0 0.03 0.1 0.3 1.0 3.0
lsoprenaline (1 PM)-stimulated adenylate cyclase activity (as I of control) DPDPE
DSTBULET
U50-488
100+3 ND 105+5 93*5 SO+2 a 83&3”
lOOzk2 99f4 101&l 88~3 87+2 Q2f4
100+3 ND ND 93+2 84*4 89+_3
” Significantly different from control (P < 0.01).
in neuronal cultures (4-fold stimulation). The stimulated adenylate cyclase activity in the glial cells, unlike that in neurons, was not affected by any of the opioid agonists used (table 3).
+‘8 0
-
7
log
naioxone
6
cont.
M
Fig. 2. Antagonism by naloxone of the inhibitory effect of submaximal& effective concentrations of opioid agonists on isoprenalinestimulated adenylate cyclase activity in striatal neuronal cultures. In the absence of drugs, conversion of ATP to cyclic AMP amounted to 0.15 50.01%. The isoprenaline (1 PM)-induced conversion of ATP to cyclic AMP amounted to 0.59&0.02% in the absence of opioids. Alone. naloxone did not affect ATP conversion. The effect of naloxone was measured in the presence of 0.01 pM DAGO ( DPDPE (0). The data are mean~S.EM. (bars) values of 13-15 observations obtained in five separate experiments. Values significantly different from those with maximally effective concentrations of naloxone are indicated * P c 0.001.
inhibitory effects of submaximally effective concentrations of DAGO (0.1 PM) and DPDPE (1 FM) (fig. 2). Activation of /3-adrenoceptors by isoprenaline (1 PM) caused a much greater increase in adenylate cyclase activity (43-fold stimulation) in cultured glial cells than
TABLE 3 Effect of opioid agonists on isoprenaline-stimulated adenylate cyclase activity in striataf @ial cell cultures. In the absence of drugs, conversion of ATP to cyclic AMP amounted to 0.20+0.02%. The isoprenahne (3 pM)-stimulated conversion of ATP in cyclic AMP amounted to 8.66 ?0.43% in the absence of opioids. The data are mean + S.E.M. values from 8-16 observations obtained in four separate experiments. NS: not significantly different from the value in the absence of opioids Adenylate cyclase activity (I of control) lsoprenaline Isoprenaline Isoprenaline koprenaline
1 FM 1 pM + DAGO 1 pM 1 pM+ DPDPE 1 pM 1 PM+ U50-488 1 pM
look3 104&4 NS 107+4 NS 117*7 NS
We have shown earlier that activation of CL-and b-opioid receptors results in inhibition of the cyclic AMP production induced by exogenous as well as endogenous DA (Schoffelmeer et al., 1988; He&a et al., 1989). However, regarding a possible postsynaptic modulation by opioids of noradrenergic neurotransmission, no effect of these peptides was found on P-adrenoceptor-stimulated adenylate cyclase activity (Schoffelmeer et al., 1987). Since one of the major postsynaptic effects of released NA in the brain is an increased cyclic AMP production in neurons with @-adrenoceptors, these observations could indicate that modulation of postsynaptic neurotransmission processes does not always occur, as far as NA is concerned. There are controversial data regarding the cellular localization of P-adrenoceptors in brain. However, in view of the fact that /3-adrenoceptors have been shown to be located on neurons (Strader et al., 1983) and also on gfial cells (Maderspach and Fajszi, 1983; Burgess et al., 1985) the postsynaptic effects of released NA on neurons at /.I-adrenoceptors could be overshadowed by cyclic AMP production in non-neuronal, i.e. glial cells. We have shown recently that the inhibitory coupling of opioid receptors to dopamine 0, receptor-sensitive adenylate cyclase can also be studied in primary cell cultures derived from fetal rat striatum (Van Vliet et al., 1990). This in vitro model, extensively described by Bockaert and coworkers (Bockaert et al., 1986; Pin et al., 1988; 1989), has the advantage that, depending on cu!ture conditions, an almost pure population of biologically active neurons or glial cells can be obtained. Obviously, since the striatum does not contain the cell bodies of dopaminergic and noradrenergic neurons, these cells will not be present in the cultures. The present study with primary cultures of rat striatal neurons shows for the first time that p receptor activation results in the inhibition of P-adrenoceptor-stimulated cyclic AMP production. Thus, DAGO, a highly selective agonist for p receptors (Goldstein and Naidu, 1989), was by far the most potent inhibitory opioid agonist (EC,, = 0.02 PM, 36% maximal inhibition of isoprenaline (1 PM)-stimulated cyclic AMP production). The selective K receptor agonist, U50-488 (Von Voigtlander et al., 1983) had no effect at a concentration of 3 PM, indicating that K receptors are not involved in opioid action. To investigate the possible involvement of 6 receptors, two selective agonists, DPDPE (Mosberg et al., 1983) and DSTBULET (Delay-Goyet et al., 1988),
299
were used. DPDPE slightly reduced (by almost 20%) the isoprenaline (1 PM)-stimulated cyclic AMP production, although at a relatively high concentration (1 FM), while DSTBULET had no effect. In order to further characterize the opioid receptors mediating the action of DPDPE, we used the p receptor antagonist, naloxone. Since naloxone displays a 20-fold selectivity for 11 binding sites (Paterson et al., 1983) and functional p receptors in rat brain (Schoffelmeer et al., 1986), it should be more potent to reduce the effect of DAGO than of DPDPE if the latter agonist exerted its inhibitory action through activation of 6 receptors. However, naloxone reduced equally well the inhibitory effects of DAGO and DPDPE, suggesting that only p receptors are involved in the inhibitory effects of the opioid agonist used in this study. Taken together, our observations leave little doubt that Cc-opioid receptors mediate the inhibition by opioids of isoprenaline-stimulated adeuylate cyclase activity in striatal neurons. When the possible existence of a functional interaction between opioid receptors and P-adrenoceptors in cultured glial cells was investigated, it was observed that /I-adrenoceptor activation caused a much greater increase in adenylate cyclase activity in these cells (43-fold stimulation; see also Hansson and Rbnnbsick, 1988; Rougon et al., 1983) than in neuronal cultures (Cfold stimulation). Furthermore, activation of opioid receptors by 1 I_IM DAGO, DPDPE or U50-488 did not inhibit isoprenaline-stimulated cyclic AMP production. Therefore, opioids were ineffective in rat striatal slices probably because cyclic AMP production induced by P-adrenoceptor activation occurs primarily in glial cells, where it is not subject to inhibition by opioids. Interestingly, in cultured astrocytes prepared from cerebral cortex of newborn rats, opioid receptor activation by morphine or [Met’]enkephalin strongly inhibited the NA-stimulated cyclic AMP production (Rougon et al., 1983). Thus, in contrast to that in the cerebral cortex, the interaction between opioid and P-adrenoceptors in the striatum seems to be exclusively localized on neuronal cells, suggesting a heterogeneous distribution of opioid receptors on cultured neurons and glial cells in the brain. Together, our previous (Van Vliet et al., 1990) and present results indicate that p receptors in striatal neurons inhibit adenylate cyclase stimulated by neurotransmitters such as DA and NA. An inbibitory effect of opioids on neurotransmitter-sensitive adenylate cyclase may not only play a role in the modulation of central neurotransmission by endogenovs opioid peptides but may also underlie the adaptive changes that occur in neurotransrmssion processes following long-term opioid treatment leading to tolerance, dependence and withdrawal phenomena. In this respect, it is worth noting that recent studies suggest that prolonged activation of receptors that inhibit adenylate cyclase may result in
a profound supersensitivity of this second messenger (Thomas and Hoffman, 1987).
Acknowledgements The ;L.rthorswish to thank Dr. Jot1 Bockaert, Dr. Jean-Philippe Pin and Dr. Michele Sebben (Centre CNRS INSERM de Phannacologie et E;rdocrinologie. Montpellier, France) for giving one of them (Bernard J. Van Vliet) the opportunity to gain experience with cultured central neurons and glial cells. This work was supported by a Senior Fellowship of the Royal Netherlands Academy of Sciences and Arts (K.N.A.W.) awarded to Anton N.M. Schoffehneer.
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EfP 51791
fl=Adrenoceptor-sensitive enylate cyclaseisi Y ac receptors in rat striatal neurons Bernard J. Van Vliet, Sigrid R. Ruuls, Benjamin Drukarch ‘, Arie H. Mulder and Anton N.M. Schoffelmeer Departments of Pharmacology and ’ Neurology, Free University. Medical Faculty, Van der Boechorststraat 7, 1081 ET Amsterdam, The Netherlands Received 17 September 1990. revised MS received 8 January 1991, accepted 15 January 1991
The fi-adrenoceptor-sensitive adenylate cyclase in primary cultures of rat striatd neurons was inhibited by opioids, unlike that in rat striatal slices. Isoprenaline (1 PM)-stimulated cyclic AMP production was dose dependently inhibited by the p-opioid receptor agonist, [D-AlaZ,MePhe4,Gly-o15]enkephalin (DAGO, EC,, = 0.02 PM, 36% inhibition), and only slightly reduced by relatively high concentrations of the d-opioid receptor agonist, [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE, 1 PM). The highly selective and potenr S-opioid receptor agonist, [D-Ser’(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET). and the a-opioid receptor agonist, U50-488, were ineffective in concentrations up to 3 pM. Naloxone reversed equally well the inhibitory effects of DPDPE and of DAGO, indicating the involvement of functional F-opioid receptors. The isoprenaline (1 PM)-stimulated adenylate cyclase activity in cultured glial cells. which exceeded that in neurons about IO-fold. was not affected by opioids. Therefore, opioids were ineffective in rat brain slices probably due to the fact that cyclic AMP production induced by b-adrenoceptor activation occurs primarily in the glial cells, where it is not subject to inhibition by opioids. These data indicate for the first time the existence of an interaction between functional p-opioid receptors and P-adrenoceptors on striatal neurons of the rat. Opioid receptors; P-Adrenoceptors; Adenylate cyclase; Striatal neurons
1. Introduction It is now well established that opioids exert their effect by acting on receptors classified into at least three different types, i.e. CL-(morphine), 6- (enkephalin) and K- (dynorphin) opioid receptors (Martin, 1984; Goldstein, 1987; Simon, 1987). One major function of opioids in the brain appears to be the inhibition of monoaminergic neurotransmission processes. Thus, it has been shown that activation of opioid receptors results in presynaptic inhtbition of the electrically evoked release of dopamine (DA) and noradrenaline (NA) from slices of different regions of the brain (Chesselet, 1984; Jackisch et al., 1986a,b; Mulder et al., 1984; 1988; Schoffelmeer et al., 1988; Werling et al., 1988). The modulatory effect of opioid drugs at the postsynaptic level has been demonstrated in case of dopaminergic neurotransmission. No effect of opioid receptor activation on fi-adrenoceptor-sensitive adenylate cyclase was observed in rat striatal slices, where DA-sensitive
adenylate cyclase was found to be inhibited by activation of CL-and b-opioid receptors (Schoffelmeer et al.. 1986; 1987; 1988). Since, in rat brain, P-adrenoceptors occur primarily on astrocytes (Maderspach and Fajszi. 1983; Burgess et al., 1985), cyclic AMP production induced by /3-adrenoceptor activation may be expected to occur primarily in this type of cell. Moreover, little is known about the cellular distribution and possible colocalization of P-adrenoceptor and opioid receptors on neurons and glial cells. Therefore, we now used primary cultures of rat striatal neurons and glial cells (Van Vliet et al., 1990; Ebersolt et al., 1981) to investigate the possible modulation by opioids of the P-adrenoceptorstimulated cyclic AMP production in neurons. thought to be a major postsynaptic action of released NA in the brain.
2. Materials and methods 2.1. Preparation of striatal slices
Correspondence to: A.N.M. Schoffelmeer, Department of Pharmacology, Medical Faculty, Free University, Van der Boechorststraat 7. 1081 BT Amsterdam, The Netherlands.
Male Wistar rats (180-220 g body weight) were decapitated and the striatum was rapidly dissected from the brain. Slices (0.3 x 0.3 x 2 mm) were prepared using a McIlwain tissue chopper.
1 neuronal cultures were prepared as previibed (Van Vliet et al.. 1990). according to a reported by Bockaert et al. (1986) and Weiss et _ (1986) for the culture of mouse striatal neurons. efly. striata were dissected from 17-day-old Wistar rat embryos and were mechanically dissociated, using a fire-narrowed Pasteur pipet. in a serum-free medium. Cells were plated in 12-well Linbro culture dishes (6 X 1Q5 cells,/ml per well). previously coated with poly-Lornithine (1.5 pg/ml) and medium containing 10% sup~~erne~t~ calf serum. The culture medium was composed of a 1 : I mixture of Dulbecco’s modified Eagles medium and F-12 nutrient and contained glucose (0.6%) glutamirle (2 mM). sodium bicarbonate (3 mM), HEPES buffer (5 mM). streptomycin (100 pg/ml) and penicillin (100 Itl/ml). A defined hormone and salt mixture was added. consisting. of insulin (25 pg/ml). transferrin (100 rtg/mlf. progesterone (LO nM). putrescine (60 FM), /3-estradiol fl phiI) and selenium sodium salt (30 aM). Wnder our culture conditions. neurons developed an extensive matrix of dendrites and synapses, as described by Bockaert et al. (1986) and survived for more than 4 weeks. Since neurons were grown in a serum-free (hormone-supplemented) medium, only a few glial cells were observed (about only 7% of total cells. Bockaert et al.. 1986) after lo-13 days in vitro. when cultures were used for esperiments. 23 Preparation of primaty nrbes
of striatal ustrocy~es
Cultures of astrocytes were prepared as neuronal cultures. except that the cells were grown in medium containing 10% supplemented calf serum instead of the hormone and salt mixture (according to Ebersolt et al., 1981). The medium was changed twice a week. Glial cell cultures consisted of a nearly homogeneous population of type I astrocytes. The cells had reached confluency after 2 weeks. and almost no neuron survived in the cultures after 4 weeks, when cultures were used for experiments (Ebersolt et al., 1981).
were transferred to edch of the 24 chambers of a superfusion apparatus (about 3-6 mg tissue per chamber of 0.2 ml volume) and superfused (0.1 ml/min) in presence of 95% O,-5% CO, at 37*C. After 30 min. the slices were exposed to medium containing drugs and the inhibitor. 3-isobutyl-l-methyl-xanphosphodiesterase thine (IBMX. 1 mM). for 30 min. The reaction was stopped by superfusion of the slices with ice-cold TCA (5%) for 15 min. [“HICyclic AMP and [‘H]ATP present in the effluent of this extraction period were separated by sequential chromatography on Dowex and alumina columns (Salomon et al., 1974).
The cultures were washed with phosphate-buffered saline (PBS) containing (mM): 137 NaCl, 2.7 KCl, 8 Na,HPO,, 1.5 KH,PO,, 0.5 MgCl,, 1.2 CaCl, and 5 glucose (pH 7.3) before incubation with 2 &i [‘Hladenine at 37OC. After 2 h, the cultures were again washed with PBS and exposed for 5 min to a PBS solution containing drugs and 1 mM IBMX. The reaction was stopped by aspiration of the media and addition of 1 ml ice-cold trichloroacetic acid (TCA, 5%). [‘H]Cyclic AMP was separated from 13H]ATP through sequential chromatography on Dowex and alumina columns (Salomon et al., 1974). Cyclic AMP production was expressed as: Zconvrrsion =
{3H]cAhlP
[ ,HIATP
+ i,HlcAMP
X
100
This prelabelling technique, originally described by Shimizu et al. (1969), has yielded results completely consistent with those based on measurement of endogenous levels of cyclic AMP (Daly, 1977). and has been used in various studies with cell cultures (see Weiss et al., 1986, and references quoted therein). 2.5. Statistics The statistical significance of differences was determined by one-way analysis of variance (ANOVA) followed by Student’s t-test. 2.6. ~adioche~~i~a~sand drugs
Cyclic AMP production in slices and cell cultures was determined by measuring the conversion of 13Hj&enine nucleotide precursors (mainly [3H]ATP) to [ ’ HIcyclic AMP. 2.4. I. Sil%atal slict?s
Striatal slices were washed twice with Krebs-Ringerbicarbonate medium containing (mM): 121 NaCl, 1.9 KCl. 25 NaHCOx, 1.2 KH2P0,, 1.2 MgSO,, 1.2 CaCl, and 10 glucose (pH 7.3) before incubation with f3H]adenine (4 ,uCi/ml) for 1 h. Subsequently, they
The following drugs were obtained commercially: 3-isobutyl-1-methyl-xanthine (IBMX) from Aldrich: selenium sodium salt, from Merck; calf serum, from Hyclone; [ ‘Hladenine (15-25 Ci/mmol), from Amersham; poly-L-ornithine, streptomycin, penicillin, insulin, transferrin, progesterone, putrescine, P-estradiol, isoprenaline, propanolol and naloxone hydrochlo~de, from Sigma: Dulbecco’s modified Eagles medium and F-12 nutrient, from Gibco; and [D-Ala’,MePhe4,Gly-o15]enkephalin (DAGB) and [D-peni~illal~ne2,D-penicillamines]enkephalin (DPDPE), from Bachem. Trans-3.4dichloro-N-methyl-N-[2-(l-pyrrolidinyl)-cyclohexyl]-
297
80
60
9
- IOQ jsoprenalne cont. (M)
-
8
log DAGO
7 COIIC.
(M)
Fig. 1. Effect of isoprenaline on adenylate eyclase activity in cuhured striatal neurons and its in~bition by basal adenylate cyclase activity in the absence (0) and presence (0) of pmpranoid (1 PM). (b) Inhibition cyclase activity by DAGO. In the absence of drugs, the conversion of ATP to cyclic AMP amounted pM)-induced conversion of ATP to cyclic AMP amounted to 0.46 + 0.01% in the absence of opioids. The data nine observations obtained in three separate experiments.
benzeneacetamide methanesulfonate hydrate (U50-488) and [D-Ser’(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET) were generous gifts from Upjohn (Kalamazoo, MI. USA) and Dr. B.P. Roques (Universite Ren6 Descartes, Paris, France), respectively.
3. Results Activation of /3-adrenoceptors in rat striatal slices by isoprenaline (3 @vi) resulted in a 3.6-fold increase in adenylate cyclase activity, which was not affected by the :I receptor agonist DAGO (1 PM). the 6 receptor agonist DPDPE (1 PM) or the K receptor agonist U50488 {l FM), respectively {table 1). Activation of ~-adren~~ptors in cultured striatal neurons by isoprenaline (fig, la) resulted in a dose-de-
TABLE l Effect of opioid agonists on isoprenaline-stimulated adenylate cydase activity in rat striatal slices. fn the absence of drugs, conversion of ATP to cyclic AMP amounted to 0.37~0.02%. The isoprenaline (3 PM)-stimulated conversion of ATP in cyclic AMP amounted to 1.35 +0.06% in the absence of opioids. The data are neank S.E.M. values from 8-16 observations obtained in 4 separate experiments. NS: not significantly different from the value in the absence of opioid Adenylate cyclase activity (% of control)
Drugs lsoprenaline lsoprenaline lsoprenaline lsoprenaline
3 pM 3 pM + DAGO 1 PM 3 FM + DPDPE 1 I_LM 3 PM + U50-488 1 NM
lOOf 104+7NS 102&3 NS llSi-7 NS
DAGO. (a) Effect of isoprenahne on of i~prenaIine-stimui~ted adenylate to 0.13+0.01%. The isoprenahne (1 are mean f. S.E.M. (bars) values from
pendent increase in adenylate cyclase activity, which could be fully antagonized by 1 PM propranolol. Interestingly, the isoprenaline (1 PM)-stimulated cyclic AMP production was dose dependently inhibited by DAGO (EC, = 0.02 FM, 36% maximal in~bitio~ fig. lb), but only slightly reduced by relatively high concentrations of DPDPE (1 PM) (table 2). Moreaver. the selective 6 receptor agonist, DSTBULET. and K receptor agonist, U50-488, were not effective at concentrations up to 3 PM (table 2). In order to further investigate the inhibitory effects of DAGO and DPDPE, we examined the effect of the selective c receptor antagonist, naloxone. In neuronal cultures, naloxone reversed equally well the
TABLE 2 Inhibition by opioid agonists of isop~naIine-stimuiat~ adenylate cyclase activity in striatat neuronal cultures. In the absence of drugs. conversion of ATP to cyclic AMP amounted to 0.13 &0.01X. The isoprenaline (3 PM)-stimulated conversion of ATP in cyclic AMP amounted to 0.62 &0.03% in the absence of opioids. The data are mean +_S.E.M. values from six to nine observations obtained in three separate experiments. ND: not determined Drug cont. (PM)
0 0.03 0.1 0.3 1.0 3.0
lsoprenaline (1 PM)-stimulated adenylate cyclase activity (as I of control) DPDPE
DSTBULET
U50-488
100+3 ND 105+5 93*5 SO+2 a 83&3”
lOOzk2 99f4 101&l 88~3 87+2 Q2f4
100+3 ND ND 93+2 84*4 89+_3
” Significantly different from control (P < 0.01).
in neuronal cultures (4-fold stimulation). The stimulated adenylate cyclase activity in the glial cells, unlike that in neurons, was not affected by any of the opioid agonists used (table 3).
+‘8 0
-
7
log
naioxone
6
cont.
M
Fig. 2. Antagonism by naloxone of the inhibitory effect of submaximal& effective concentrations of opioid agonists on isoprenalinestimulated adenylate cyclase activity in striatal neuronal cultures. In the absence of drugs, conversion of ATP to cyclic AMP amounted to 0.15 50.01%. The isoprenaline (1 PM)-induced conversion of ATP to cyclic AMP amounted to 0.59&0.02% in the absence of opioids. Alone. naloxone did not affect ATP conversion. The effect of naloxone was measured in the presence of 0.01 pM DAGO ( DPDPE (0). The data are mean~S.EM. (bars) values of 13-15 observations obtained in five separate experiments. Values significantly different from those with maximally effective concentrations of naloxone are indicated * P c 0.001.
inhibitory effects of submaximally effective concentrations of DAGO (0.1 PM) and DPDPE (1 FM) (fig. 2). Activation of /3-adrenoceptors by isoprenaline (1 PM) caused a much greater increase in adenylate cyclase activity (43-fold stimulation) in cultured glial cells than
TABLE 3 Effect of opioid agonists on isoprenaline-stimulated adenylate cyclase activity in striataf @ial cell cultures. In the absence of drugs, conversion of ATP to cyclic AMP amounted to 0.20+0.02%. The isoprenahne (3 pM)-stimulated conversion of ATP in cyclic AMP amounted to 8.66 ?0.43% in the absence of opioids. The data are mean + S.E.M. values from 8-16 observations obtained in four separate experiments. NS: not significantly different from the value in the absence of opioids Adenylate cyclase activity (I of control) lsoprenaline Isoprenaline Isoprenaline koprenaline
1 FM 1 pM + DAGO 1 pM 1 pM+ DPDPE 1 pM 1 PM+ U50-488 1 pM
look3 104&4 NS 107+4 NS 117*7 NS
We have shown earlier that activation of CL-and b-opioid receptors results in inhibition of the cyclic AMP production induced by exogenous as well as endogenous DA (Schoffelmeer et al., 1988; He&a et al., 1989). However, regarding a possible postsynaptic modulation by opioids of noradrenergic neurotransmission, no effect of these peptides was found on P-adrenoceptor-stimulated adenylate cyclase activity (Schoffelmeer et al., 1987). Since one of the major postsynaptic effects of released NA in the brain is an increased cyclic AMP production in neurons with @-adrenoceptors, these observations could indicate that modulation of postsynaptic neurotransmission processes does not always occur, as far as NA is concerned. There are controversial data regarding the cellular localization of P-adrenoceptors in brain. However, in view of the fact that /3-adrenoceptors have been shown to be located on neurons (Strader et al., 1983) and also on gfial cells (Maderspach and Fajszi, 1983; Burgess et al., 1985) the postsynaptic effects of released NA on neurons at /.I-adrenoceptors could be overshadowed by cyclic AMP production in non-neuronal, i.e. glial cells. We have shown recently that the inhibitory coupling of opioid receptors to dopamine 0, receptor-sensitive adenylate cyclase can also be studied in primary cell cultures derived from fetal rat striatum (Van Vliet et al., 1990). This in vitro model, extensively described by Bockaert and coworkers (Bockaert et al., 1986; Pin et al., 1988; 1989), has the advantage that, depending on cu!ture conditions, an almost pure population of biologically active neurons or glial cells can be obtained. Obviously, since the striatum does not contain the cell bodies of dopaminergic and noradrenergic neurons, these cells will not be present in the cultures. The present study with primary cultures of rat striatal neurons shows for the first time that p receptor activation results in the inhibition of P-adrenoceptor-stimulated cyclic AMP production. Thus, DAGO, a highly selective agonist for p receptors (Goldstein and Naidu, 1989), was by far the most potent inhibitory opioid agonist (EC,, = 0.02 PM, 36% maximal inhibition of isoprenaline (1 PM)-stimulated cyclic AMP production). The selective K receptor agonist, U50-488 (Von Voigtlander et al., 1983) had no effect at a concentration of 3 PM, indicating that K receptors are not involved in opioid action. To investigate the possible involvement of 6 receptors, two selective agonists, DPDPE (Mosberg et al., 1983) and DSTBULET (Delay-Goyet et al., 1988),
299
were used. DPDPE slightly reduced (by almost 20%) the isoprenaline (1 PM)-stimulated cyclic AMP production, although at a relatively high concentration (1 FM), while DSTBULET had no effect. In order to further characterize the opioid receptors mediating the action of DPDPE, we used the p receptor antagonist, naloxone. Since naloxone displays a 20-fold selectivity for 11 binding sites (Paterson et al., 1983) and functional p receptors in rat brain (Schoffelmeer et al., 1986), it should be more potent to reduce the effect of DAGO than of DPDPE if the latter agonist exerted its inhibitory action through activation of 6 receptors. However, naloxone reduced equally well the inhibitory effects of DAGO and DPDPE, suggesting that only p receptors are involved in the inhibitory effects of the opioid agonist used in this study. Taken together, our observations leave little doubt that Cc-opioid receptors mediate the inhibition by opioids of isoprenaline-stimulated adeuylate cyclase activity in striatal neurons. When the possible existence of a functional interaction between opioid receptors and P-adrenoceptors in cultured glial cells was investigated, it was observed that /I-adrenoceptor activation caused a much greater increase in adenylate cyclase activity in these cells (43-fold stimulation; see also Hansson and Rbnnbsick, 1988; Rougon et al., 1983) than in neuronal cultures (Cfold stimulation). Furthermore, activation of opioid receptors by 1 I_IM DAGO, DPDPE or U50-488 did not inhibit isoprenaline-stimulated cyclic AMP production. Therefore, opioids were ineffective in rat striatal slices probably because cyclic AMP production induced by P-adrenoceptor activation occurs primarily in glial cells, where it is not subject to inhibition by opioids. Interestingly, in cultured astrocytes prepared from cerebral cortex of newborn rats, opioid receptor activation by morphine or [Met’]enkephalin strongly inhibited the NA-stimulated cyclic AMP production (Rougon et al., 1983). Thus, in contrast to that in the cerebral cortex, the interaction between opioid and P-adrenoceptors in the striatum seems to be exclusively localized on neuronal cells, suggesting a heterogeneous distribution of opioid receptors on cultured neurons and glial cells in the brain. Together, our previous (Van Vliet et al., 1990) and present results indicate that p receptors in striatal neurons inhibit adenylate cyclase stimulated by neurotransmitters such as DA and NA. An inbibitory effect of opioids on neurotransmitter-sensitive adenylate cyclase may not only play a role in the modulation of central neurotransmission by endogenovs opioid peptides but may also underlie the adaptive changes that occur in neurotransrmssion processes following long-term opioid treatment leading to tolerance, dependence and withdrawal phenomena. In this respect, it is worth noting that recent studies suggest that prolonged activation of receptors that inhibit adenylate cyclase may result in
a profound supersensitivity of this second messenger (Thomas and Hoffman, 1987).
Acknowledgements The ;L.rthorswish to thank Dr. Jot1 Bockaert, Dr. Jean-Philippe Pin and Dr. Michele Sebben (Centre CNRS INSERM de Phannacologie et E;rdocrinologie. Montpellier, France) for giving one of them (Bernard J. Van Vliet) the opportunity to gain experience with cultured central neurons and glial cells. This work was supported by a Senior Fellowship of the Royal Netherlands Academy of Sciences and Arts (K.N.A.W.) awarded to Anton N.M. Schoffehneer.
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