Journal ofNeurochernistry Raven Press, New York 0 1986 International Society for Neurochemistry

Specific Inhibition of Dopamine D- 1 -Mediated Cyclic AMP Formation by Dopamine D-2, Muscarinic Cholinergic, and

Opiate Receptor Stimulation in Rat Striatal Slices

Eamonn Kelly and Stefan R. Nahorski

Department of Pharmacology and Therapeutics, University of Leicester, Leicester, England

Abstract: The ability of different receptors to mediate inhi- bition of cyclic AMP accumulation due to a variety of ago- nists was examined in rat striatal slices. In the presence of 1 mM 3-isobutyl- I -methylxanthine, dopamine D-2, musca- rinic cholinergic, and opiate receptor stimulation by RU 24926, carbachol, and morphine (all at 10-8-10-5 M ) , re- spectively, inhibited the increase in cyclic AMP accumula- tion in slices of rat striatum due to dopamine D- 1 receptor stimulation by 1 pM SKF 38393. In contrast, these inhibi- tory agents were unable to reduce the ability of a number of other agonists, including isoprenaline, prostaglandin El , 2- chloroadenosine, vasoactive intestinal polypeptide, and cholera toxin, to increase cyclic AMP levels in stnatal slices.

It is now known that in a number of cells, certain neurotransmitter receptors mediate inhibition of the enzyme adenylate cyclase and thus modify cellular function. For example, in the melanotrophs of rat pi- tuitary gland, 3,4-dihydroxyphenylethylamine (dopa- mine) D-2 receptor stimulation inhibits the ability of P-adrenoceptors to enhance intracellular cyclic AMP accumulation and, consequently, a-melanocyte-stim- ulating hormone secretion (Munemura et al., 1980; Cote et al., 1982). In the rat striatum, dopamine stim- ulates adenylate cyclase activity via D- 1 receptors (Kebabian and Calne, 1979), and, more recently, it has been shown that D-2 receptors, also present in rat striatum, are linked in an inhibitory fashion to this enzyme (Stoof and Kebabian, 1981; Battaglia et al., 1985; Onali et al., 1985; Weiss et al., 1985). Such in- teractions in this region of the brain may be impor- tant, because, in terms of motor behaviour, functional interplay between D- 1 and D-2 receptors has recently

Received March 7, 1986; revised May 2 1, 1986; accepted May 22, 1986.

Address correspondence and reprint requests to Prof. S. R. Na- horski at Department of Pharmacology and Therapeutics, Medical Sciences Building, University of Leicester, University Road, Leices- ter LEI 7RH, U.K.

These results suggest that in rat striatum either (a) dopamine D-2, muscarink cholinergic, and opiate receptors are only functionally linked to dopamine 1)-1 receptors or that (b) the D- 1 and D-2 receptors linked to adenylate cyclase lie on the cells, distinct from other receptors capable of elevating striatal cyclic AMP levels. Key Words: Cyclic AMP-Rat striatum-Dopamine D- 1 receptor-Dopamine D-2 recep- tor-Muscarinic cholinergic receptor-Opiate receptor. Kelly E. and Nahorski S. R. Specific inhibition of dopamine D- I-mediated cyclic AMP formation by dopamine D-2, muscarinic cholinergic, and opiate receptor stimulation in rat striatal slices. J. Neurochem. 47, 1 5 12- 15 16 ( 1986).

been demonstrated (Meller et al., 1985; Molloy and Waddington, 1985). Furthermore, both muscarink cholinergic and opiate receptors have been shown to inhibit dopamine-stimulated adenylate cyclase activ- ity (Olianas et al., 1983) and dopamine-stimulated cy- clic AMP accumulation (Minneman, 1977) or efflux (Schoffelmeer et al., 1985), respectively. In the present study, we have investigated whether,

in slices of rat striatum, dopamine D-2, muscarink cholinergic, and opiate receptors are able to inhibit D- 1 -stimulated cyclic AMP accumulation. We have also investigated whether these agents can reduce the cy- clic AMP accumulation produced by a number of other agonists and cholera toxin, all of which are known to stimulate striatal adenylate cyclase activity. We report that, in this preparation, dopamine D-2, muscarinic cholinergic, and opiate receptors appear to mediate a selective inhibition of D- 1 -stimulated cy- clic AMP accumulation.

Abbreviations used: dopamine, 3,4-dihydroxyphenylethylarnine; IBMX, 3-isobutyl- I-methylxanthine; N, and N,, GTP-binding reg- ulatory (inhibitory and stimulatory, respectively) protein; =El, prostaglandin El ; VIP, vasoactive intestinal polypeptide.

1512

INHIBITION OF CYCLIC AMP FORMATION 1513

MATERIALS AND METHODS

Incubation technique The incubation technique was adapted from a previously

described study (Lazareno et al., 1985). Cross-chopped stri- atal slices (0.35 x 0.35 mm) from male Sprague-Dawley rats (body weight, 150-350 g) were incubated in a shaking water bath for 90 rnin at 37'C in Krebs buffer (NaCl, 118 mM; KCI, 4.7 mM; CaC12, 1.3 mM; KH2P04, 1.2 mM; MgS04, 1.2 mM; NaHC03, 25 mM; glucose, 1 1.7 mM) containing 1 mM 3-isobutyl- 1 -methylxanthine (IBMX) and saturated with 95% 02/5% C02, with replacement of buffer every 20 min. Slices (20 pl) were then added to test tubes using an Eppendorf repeating pipette and preincubated for 5 rnin with antagonists or other drugs where indicated, followed by incubation for 20 rnin with agonists in a final volume of 300 pl, with continuous gentle vortex-mixing at 37'C in a heating block (Buchler Instruments). Tubes were gassed with 95% 02/5% C02 and capped for the 20-min incubation period. At the end of this period, 20 pl of concentrated HCl was added to each tube to elute cyclic AMP from the tissue, and the tubes were placed on ice. After >20 min, the sam- ples were neutralised with 200 pl of 1 M NaOH, and the samples were centrifuged at 1,000 g for I0 rnin at 4'C. Cyclic AMP was measured in duplicate 50-pl aliquots of superna- tant using a protein binding assay (Brown et al., 1971). Some aliquots of tissue were retained for protein assay, and following sonication the protein content was measured by the method of Lowry et al. (195 1).

Intrastriatal injection of cholera toxin Rats were anaesthetised with 1.5% halothane and immo-

bilised in a David Kopf stereotaxic frame. Intrastriatal injec- tions of 4 pg of cholera toxin were given by manual injection of 1 pg in 1 p1 of vehicle at four different sites (A 8.5 and 9.5, L 2.5, V 5.0 and 4.0; Konig and Klippel, 1963) via a 5-pl Hamilton syringe with Luer needle and at a rate of 0.5 pl/ min. The vehicle consisted of0.05 MTris-HC1,0.2 MNaC1, 0.003 M NaN3, and 0.001 M Na2EDTA, pH 7.5. Vehicle containing 4 pg of bovine serum albumin was injected at corresponding coordinates into the contralateral hemi- sphere. After each injection, the needle was Ieft in place a further 1 rnin before being slowly withdrawn. Following completion of injections, the skin overlying the skull was sealed with silk sutures and Superglue, and anaesthesia was discontinued. Animals recovered consciousness within 5 min.

Design, analysis, and statistics For every experiment, each treatment involved the use of

three to five tubes containing striatal slices. The concentra- tion-inhibition curves were constructed from experiments conducted on at least four different occasions, with the data from these being fitted to a four-parameter logistic model using the computer program ALLFIT (De Lean et al., 1978). Statistical analysis was performed using an unpaired Student's t test.

Drugs SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy- l-phenyl-

Iff-3-benzazepine; Smith Kline and French), SCH 23390 (7-chloro-2,3,4,5 - tetrahydro- 3 -methyl- 5 -phenyl- 1 H - 3 - benzazepine-7-01; Schering), (-)-sulpiride (Ravizza), RU 24926 [N-n-propyl di-p (3-hydroxypheny1)ethylamine; Roussel], LY 17 1555 (quinpirole hydrochloride; Lilly Re- search Laboratories), morphine (Mallinckrodt), and nalox-

one (Endo Laboratories) were kind gifts. [3H]Cyclic AMP (50-60 Ci/mmol) was purchased from Amersham Interna- tional. All other drugs, vasoactive intestinal polypeptide (VIP), and cholera toxin, were purchased from Sigma.

RESULTS In striatal slices incubated with 1 mM IBMX, basal

levels of cyclic AMP ranged between 8 and 15 pmol/ mg of protein. Inclusion of the D-1 agonist SKF 38393 (1 pM) in the incubation medium significantly enhanced intracellular cyclic AMP accumulation by 70-loo%, with this increase in turn being unaffected by the selective D-2 antagonist (-)-sulpiride (30 p M ) but being totally abolished by the selective D- 1 antag- onist SCH 23390 ( 1 pM; data not shown; see also La- zareno et al., 1985).

The D-2 agonist RU 24926, carbachol, and mor- phine all inhibited cyclic AMP accumulation due to SKF 38393 (1 pM), with ICso values of 0.05 k 0.02, 0.29 f 0.10, and 0.32 f 0.15 pM, respectively (Fig. 1). Inclusion of (-)-sulpiride (10 pM), atropine ( 1 pM), or naloxone ( 1 pM) antagonised the inhibitory effects of RU 24926, carbachol, and morphine, respectively, except at the highest doses of RU 24926 and mor- phine used (both 10 pM; Fig. 1). Atropine, naloxone, or yohimbine (each at 1 p M ) did not affect the ability of RU 24926 ( 1 p M ) to inhibit SKF 38393 (1 p M ) enhancement of cyclic AMP accumulation (data not shown).

Isoprenaline (1 0 pM), prostaglandin El (PGE, ; 1 pM), 2-chloroadenosine (100 pM; in the absence of IBMX), VIP (0.25 pM), and prior intrastriatal injec- tion of cholera toxin (4 pg) all enhanced cyclic AMP accumulation in striatal slices (Table 1). In no case was the effect of these agents significantly inhibited by RU 24926, carbachol, or morphine (each at 1 &I; Ta- ble 1). Even higher doses of these agonists remained ineffective (10 pM; data not shown). Also, RU 24926, carbachol, and morphine (each 1 pM) did not signifi- cantly inhibit basal accumulation of cyclic AMP in striatal slices (Table 1). Furthermore, the inability to inhibit stimulated cyclic AMP accumulation was in- dependent of incubation time, because following in- cubation for 5 or 10 rnin with VIP (0.25 pM), carba- chol and another selective D-2 agonist, LY 1 7 1 5 5 5, remained unable to reduce cyclic AMP accumulation due to the peptide (data not shown).

DISCUSSION

The results of this study indicate the selectivity of inhibition of striatal dopamine-stimulated cyclic AMP accumulation by different receptors. Stoof and Kebabian (1 98 1) originally demonstrated that striatal D-2 receptors are linked in an inhibitory fashion to D- 1 stimulation of cyclic AMP formation. It was ob- served that the D-Zselective antagonist (-)-sulpiride increased the ability of dopamine to enhance cyclic AMP efflux from striatal slices. Previous results from

J. Neurochem., Val. 47, No. 5, 1986

1514 E. KELLY AND S.

C 120-

100-

8P - 60-

40-

20-

R . NAHORSKI

""1 40

I 201 I - 0 -9 -8 -7 -6 -5 m

g 0 1 / 1

log [RU24926]

- 60 J n 401

2 20 r

I I -8 -7 -6 -5

ap O W / I 0

log [carbachol]

1

I I I -8 -7 -8 -5

0-1 I 0

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FIG. 1. Effect of RU 24926 (a)% carbachol (b), and morphine (c) on the SKF 38393-induced increase in cyclic AMP accumulation in striatal slices in the presence (0) or absence (0) of 10 jLM sulpiride (a), 1 j d A atropine (b), or 1 j d A naloxone (c), respectively. Slices were preincubated for 5 min in the presence or absence of each agonist or antagonist before addition of 1 pM SKF 38393 for a further 20 min. Each graph represents data from at least four ex- periments performed on different occasions, with each being ex- pressed as a percentage of the increase in cyclic AMP level in- duced by SKF 38393 alone (defined as 100°/o). Over 13 experi- ments, basal levels of cyclic AMP were 12.83 f 0.73 pmollmg of protein, and those stimulated by 1 jLM SKF 38393 were 22.83 f 1.08 pmol/mg of protein. In each experiment, the D-1 agonist significantly elevated cyclic AMP levels over basal values. Data are mean f SEM (bars) values. 'p -= 0.05 for values lower than in the presence of the antagonist, but at the same dose of agonist, by Student's t test.

this laboratory have confirmed and extended these findings by using a variety of selective dopamine an- tagonists and have demonstrated that such interac- tions reflect intracellular cyclic AMP generation rather than effects on the efflux mechanism (Lazareno et al., 1985). In the present study, we have elected to examine predominantly intracellular cyclic nucleo- tide generation, because the extracellular cyclic AMP level does not always reflect tissue levels (Lazareno et al., 1985) and can be influenced indirectly by certain drugs (Barber and Butcher, 1983).

The selective D-1 agonist SKF 38393 (Setler et al., 1978) enhanced cyclic AMP accumulation in striatal slices, and, as would be predicted, the ability of this agonist to elevate striatal cyclic AMP levels was un- affected by (-)-sulpiride but was abolished by the D- 1 selective antagonist SCH 23390 (Hyttel, 1983; Iorio et al., 1983). The D-Zselective dopamine agonist RU 24926 (Euvrard et al., 1979) potently inhibited D-l- stimulated cyclic AMP accumulation. The inhibition was almost certainly mediated by D-2 receptors, be- cause atropine, naloxone, and the qadrenergic an- tagonist yohimbine did not block the effect of RU 24926. On the other hand, 10 pM (-)-sulpiride dra- matically shifted the dose-response curve to the right, a result indicative of a competitive interaction with the receptor.

Activation of muscarinic cholinergic and opiate re- ceptors has been reported to inhibit striatal dopa- mine-stimulated adenylate cyclase (Olianas et al., 1983) and cyclic AMP accumulation (Minneman, 1977) or efflux (Schoffelmeer et al., 1985), respec- tively. The present study supports these findings, be- cause carbachol and morphine both inhibited SKF 38393-mediated cyclic AMP accumulation, and, in each case, inhibition was reversed by specific antago- nists. Thus, in rat striatum, cyclic AMP accumulation due to D-1 receptor activation is inhibited by concur- rent stimulation of D-2, muscarinic cholinergic, or opiate receptors.

Several other nondopaminergic agonists, including the P-adrenoceptor agonist isoprenaline (Minneman et al., 1978), PGEl (Minneman, 1977), VIP (Quik et al., 1978), and 2-chloroadenosine (Wilkening and Makman, 1975), are known to elevate striatal cyclic AMP accumulation. However, concurrent stimula- tion of D-2, muscarinic cholinergic, or opiate recep- tors did not attenuate the increase in cyclic AMP for- mation produced by each of these agents. This indi- cates that, in rat striatal slices, D-2, muscarinic cholinergic, and opiate receptors appear only to be linked in an inhibitory fashion to the pool of adenyl- ate cyclase that is activated by stimulation of D- 1 re- ceptors. Alternatively, those nondopaminergic recep- tors mediating enhanced levels of cyclic AMP in stria- tum exist on cells other than those containing D-1 receptors. In this respect, it is of interest that whereas dopamine D- 1 receptors are almost certainly located on striatal kainate-sensitive neuronal cell bodies (Coyle and Schwarcz, 1976), p-adrenoceptors, at least, predominantly survive such lesions, a result indicat- ing a glial cell localisation (Nahorski et al., 1979). On the other hand, in primary culture of striatal neu- rones, a D-Zmediated inhibition of VIP-stimulated cyclic AMP accumulation has been reported (Weiss et al., 1985). This difference between slice and cell cul- ture may relate to the presence of different cell types in the former or to the possibility that development of receptor-effector coupling in neurones in culture does not parallel that in intact brain.

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INHIBITION OF CYCLIC AMP FORMATION 1515

TABLE 1. Effect of RU 24926, carbachol, and morphine on enhancement of cyclic AMP accumulation in striatal slices by various agonists and cholera toxin

Enhancement of cyclic AMP level

Response in presence of other d r u c

Treatment over basal (%) RU 24926 (I pM) Carbachol(1 pA4) Morphine (1 p M )

SKF 38393 (1 p M ) 70 39 * 116 17%7b 35 % lSb Isoprenaline (1 0 pM) 450 108 t 16 1 1 1 % 10 121 + 8

220 110+ 19 124+ 13 1 1 3 t 2 5 2-Chloroadenosine ( 100 pM)’ 490 105 t 22 96+ 10 91 + 9 PGEi (1

VIP (0.25 p M ) 540 1 1 6 t 17 124 + 20 109 + 15 Basal - 95 t 12 97+ 10 96+ 10 Cholera toxin (4 pg) 220 89 k 6 101 + 11 98 + 14

Striatal slices were preincubated for 5 min in the presence or absence of RU 24926, carbachol, or morphine before addition of agonist for a further 20 min. In the case of cholera toxin, animals were injected intrastriatally 24 h previously, as described in Materials and Methods. The effect of RU 24926, carbachol, and morphine on basal accumulation was assessed using tissue from the vehicleinjected striaturn. Basal levels of cyclic AMP in these experiments ranged between 8.2 and 14.8 pmol/mg ofprotein. Data are mean + SEM values from three to five separate incubations and represent values in the presence of RU 24926, carbachol, or morphine expressed as a percentage of the mean response in the presence of agonist or cholera toxin alone (defined as 100%).

Expressed as a percent of the mean response to the cyclic AMP elevating treatment alone. p < 0.05 for cyclic AMP values lower than those values in the presence of the cyclic AMP stimulating agent alone by Student’s t test. The experiment with 2-chloroadenosine was performed in the absence of IBMX, which would act as an adenosine receptor antagonist at

I mM.

Cholera toxin causes a persistent increase in adenyl- ate cyclase activity by ADP-ribosylating the GTP- binding regulatory (stimulatory) protein N, (Johnson et al., 1978), which mediates receptor-linked stimula- tion of adenylate cyclase. As has previously been found following focal injection into the rat nucleus accumbens (Miller and Kelly, 1975), intrastriatal cholera toxin locally increased adenylate cyclase ac- tivity with a consequent elevation of intracellular cy- clic AMP in the presence of a phosphodiesterase in- hibitor. However, RU 24926, carbachol, and mor- phine remained unable to inhibit the increase in cyclic AMP level due to the toxin. This is in contrast to the pituitary gland, in which enhanced adenylate cyclase activity following preincubation with cholera toxin can be potently inhibited by dopamine D-2 receptor activation (Cote et al., 1982). The likely reason for this difference is that in striatum the pool of N, activated by cholera toxin includes that linked to all receptors capable of enhancing adenylate cyclase activity. Thus, any inhibition of that portion of N, linked to D- 1 re- ceptors would be difficult to detect.

The mechanism by which D-2, muscarinic, and opiate receptors inhibit D- 1 -stimulated cyclic AMP accumulation is almost certainly via the GTP-binding regulatory (inhibitory) protein Ni (Rodbell, 1980). Thus, in striatal membranes the D-2 inhibition ofade- nylate cyclase activity is dependent on the presence of GTP (Onali et al., 1985). Furthermore, pertussis toxin, which inactivates Ni by ADP-ribosylating its a subunit (Murayama and Ui, 1983), can also block D- 2 inhibition of cyclic AMP accumulation (Weiss et al., 1985) and muscarinic inhibition of D-I-stimulated cyclic AMP accumulation (Kelly et al., 1985) in the striatum.

In conclusion, the results of the present study indi- cate that the inhibition of striatal cyclic AMP accu- mulation by dopamine D-2, muscarinic cholinergic, and opiate receptor stimulation is probably specific for D-1 receptors, an observation suggesting the im- portance of these interactions in striatal function. It will be important in the future to ascertain whether inhibition of adenylate cyclase mediates some or all of the physiological actions of cerebral D-2 receptor stimulation.

Acknowledgment: The authors would like to thank the Wellcome Trust for financial support and Jenny Bell for manuscript preparation.

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