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Independent in vitro regulation by the D-2 dopamine receptor of dopamine-stimulated efflux of cyclic AMP and K+-stimulated release of acetylcholine from rat neostriatum
Brain Research, 250 (1982) 263-270 Elsevier Biomedical Press
263
Independent In Vitro Regulation by the D-2 Dopamine Receptor of Dopamine-Stimulated Efflux of Cyclic AMP and K+-Stimulated Release of Acetylcholine from Rat Neostriatum J. C. STOOF* and J. W. KEBABIAN
Biochemical Neuropharmacology Section, Experimental Therapeutics Branch, N1NCDS, NIH, Bethesda, MD 20205 (U.S.A.) (Accepted April 8th, 1982)
Key words: cAMP efflux - - ACh release - - D-1 receptor - - D-2 receptor - - rat neostriatum
Two types of dopamine receptors whose stimulation affect cAMP efflux (and by inference formation) could be identified in rat neostriatum. One type of receptor, called D-I receptor, increased cAMP efflux whereas stimulation of a second type of dopamine receptor, called D-2 receptor, was followed by a reduction in cAMP efflux induced by stimulation with a D-1 receptor agonist. D-2 receptor agonists inhibited the effects olD-1 receptor agonists on cAMP efflux in a non-competitive way. These inhibiting effects of D-2 receptor agonists occurred also in the absence of Ca2+-ions which could imply that some of the D-2 receptors are located on cells possessing D-1 receptors. The dopamine receptor mediating inhibition of the release of radiolabeled acetylcholine (ACh) in the neostriatumappearedto have the same pharmacological characteristics as the D-2 dopamine receptor mediating the inhibition of the D-I receptor agonist induced cAMP efflux. Selective D-2 receptor agonists like LY 141865 and RU 24926 stimulated this receptor while the D-1 receptor agonist SKF 38393 was inactive. Effects of the selective D-2 receptor agonists could be antagonized by (--)-sulpiride, a selective D-2 receptor antagonist. Although the pharmacological characteristics of the dopamine receptors mediating inhibition of both ACh release and (D-1 dopamine receptor agonist induced) cAMP efflux appeared to be similar, drugs stimulating cAMP efflux did not affect ACh release or LY 141865 induced inhibition of ACh release from rat neostriatum. Therefore it is still questionable whether the dopamine receptor mediating inhibition of both ACh release and cAMP efflux is one and the same functional entity. INTRODUCTION A c c o r d i n g to one classification schema, there exist two categories o f d o p a m i n e r e c e p t o r designated as D-1 a n d D-2 receptors is. Several drugs discriminate between these two d o p a m i n e receptors. S K F 38393 is substantially m o r e p o t e n t t h a n d o p a m i n e as an a g o n i s t u p o n the D-1 r e c e p t o r 29, b u t is substantially less p o t e n t t h a n d o p a m i n e as an agonist u p o n the D2 r e c e p t o r 27. L Y 1418653a a n d R U 249267,8 selectively stimulate the D-2 d o p a m i n e receptor. T h e substituted b e n z a m i d e , ( - - ) - s u l p i r i d e , b l o c k s the stimulating actions o f drugs o n l y on the D-2 receptor18,22. B o t h types o f d o p a m i n e r e c e p t o r occur in the n e o s t r i a t u m o f the rat b r a i n a n d affect the synthesis
o f a d e n o s i n e 3',5' m o n o p h o s p h a t e ( c A M P ) 31. Stim u l a t i o n o f the D-1 r e c e p t o r increases c A M P form a t i o n 17 while s t i m u l a t i o n o f the D-2 d o p a m i n e r e c e p t o r decreases the c A M P f o r m a t i o n s t i m u l a t e d by D-1 agonists al. I n addition, a D-2 d o p a m i n e r e c e p t o r regulates the c o n t e n t o f neostriatal acetylcholine (ACh). The c o n t e n t o f neostriatal A C h is increased b y D-2 d o p a m i n e r e c e p t o r agonists a n d is decreased b y D-2 d o p a m i n e r e c e p t o r antagonists 7, 2s. T h e d o p a m i n e r g i c regulation o f neostriatal A C h levels correlates with the presence o f synaptic contacts between d o p a m i n e c o n t a i n i n g n e u r o n s with choline acetyltransferase-staining n e u r o n a l elements in the n e o s t r i a t u m 13. The present investigations were u n d e r t a k e n with two goals. T h e first goal was to further characterize
* On leave from the Department of Neurology, Free University, Amsterdam, The Netherlands. Address reprint requests to: J. C. Stoof, Dept. of Neurology, Free University, van der Boechorststr. 7, 1081 BT, Amsterdam, The Netherlands. 0006-8993/82/0000-0000/$02.75 © (1982) Elsevier Biomedical Press
264 the D-2 dopaminergic inhibition of the dopaminestimulated effiux of cAMP from neostriatal slices; the second goal was to characterize the pharmacology of the dopamine receptor inhibiting the depolarization-induced release of ACh from blocks of neostriatal tissue14, 2°,3°. Both of these endeavours utilize a previously described superfusion apparatus to collect either the cAMP or the ACh released from neostriatal tissueZL The availability of drugs discriminating between the D-1 and the D-2 dopamine receptors (in the classification schema of Kebabian and Calne) facilitated these investigations. MATERIALS A N D METHODS
Drugs used and suppliers were as follows: IBMX and 8Br cAMP (Sigma), LY 141865 (Eli Lilly), RU 24926 (Roussel) and SKF 38393 (Smith Kline and French).
Preparation of chopped neostriatal tissue Following decapitation of male Sprague-Dawley rats (weighing between 300 and 350 g), the brains were removed and placed in a brain holder which permitted two successive 2 mm thick coronal slices to be made between anterior planes A-7,000 and A-11,00019. The neostriatal tissue was dissected from these two slices and subsequently minced by passing it twice through a Mcllwain tissue chopper (micrometer setting 300 #m). Prior to the second pass, the surface was rotated 90°. The resulting blocks of neostriatal tissue (measuring 2 × 0.3 × 0.3 mm) were transferred to a balanced salt solution (BSS) containing 116.4 mM NaC1, 5.4 mM KC1, 26.2 mM NaHCOz, 1.0 mM NaH2PO4, 0.6 mM MgSO4, 5.6 mM D-glucose, 1.3 mM CaCI2 and 10 mg/1 phenol red. The tissue from all rats (12 for cAMP studies, 6 for ACh studies) was pooled. The BSS used for the dissection and subsequent experimental manipulations was kept under an atmosphere of 95 % 02 and 5 % COg.
Cyclic AMP efflux studies Aliquots of the pooled blocks of neostriatal tissue (containing approximately 20 mg wet weight of tissue) were placed in each of the 24 chambers of a previously described superfusion apparatus2L Superfusion at a rate of 0.1 ml/min with BSS fortified
with 3-isobutyl 1-methyl xanthine (1 mM) and bovine serum albumin (BSA, 2.5 mg/ml) began at superfusion time t = 0 min. Beginning at superfusion time t = 60 min, a 3.0 ml (30 min) aliquot (fraction 1) of the superfusion medium was collected from each chamber to permit estimation of the basal efflux of cAMP. At superfusion time t ::: 90 rain, drugs were added to the superfusion medium; beginning at superfusion time t ~ 105 rain, a 1.0 ml (10 min) aliquot (fraction 2) of the superfusion medium was collected. Previously, it has been shown that the maximal effiux of cAMP stimulated by a dopaminergic agonist occurred 15-25 min after exposure to dopaminergic agonists (see Fig. 1 of reference 31). The cAMP contents of duplicate 100 #1 aliquots of fractions 1 and 2 collected from each chamber were estimated by radioimmunoassay as described previouslylL The amount of cAMP in fraction 2 was expressed as a percentage of the amount of cAMP in fraction 1 (the basal efltux), thereby permitting each chamber to serve as its own control and correcting for differences in the amounts of tissue present in each of the superfusion chambers.
[3H]ACh release studies The pooled blocks of neostriatal tissue were incubated for 10 min at 37 °C in 10 ml of BSS. Subsequently, the BSS was removed and replaced with 10 ml of BSS containing [SH]choline (10/~Ci; spec. act. 80 Ci/mmol). The blocks of neostriatal tissue were incubated for an additional 15 min. At the end of the incubation period, the tissue was washed (twice with 2 ml of BSS) and aliquots of the tissue (containing approximately 10 mg wet weight of tissue) were placed in each of the 24 chambers of a previously described superfusion apparatus 25. Superfusion at a rate of 0.2 ml/min with BSS began at superfusion time t = 0 min. Beginning at superfusion time t = 30 min, 5 successive 4 ml (20 min) aliquots of the superfusion medium were collected from each chamber. During the first 5 min of the second (t = 50--70 min) and the fourth (t = 90-110 min) fractions, the concentrations of KC1 and NaCI in BSS were changed to 17 mM and 106 mM, respectively. Drugs, when present, were added to the superfusion medium beginning at superfusion time t = 70 rain. The first exposure to an elevated concentration of KC1 verified that the amount of [ZH]ACh
265 released from the tissue in each of the 24 superfusion chambers was equivalent (i.e. within 10~o of the mean); the second exposure to an elevated concentration of KCI tested the ability of a drug to inhibit the release of [aH]ACh. To conclude the experiment, the tissue was superfused for 20 min (beginning at t = 130 min) with 0.1 N HCI (fraction 6). The amount of radioactivity present in each of the fractions of the superfusion medium and the HC1 extract was estimated by liquid scintillation spectcoscopy (60 ~o counting efficiency for 3H). In order to correct for differences in the amount of tissue present in each of the superfusionchambers, data were expressed as a fractional rate (i.e. the amount of radioactivity released during each collection period was divided by the sum of the amount of radioactivity released in that collection period, subsequent collection periods and the HC1 extraction). In order to estimate the effect of different drugs, the K+-induced increase in the fractional rate of release of radioactivity during the fourth collection period (i.e. the difference between the fractional rate in the fourth collection period and the mean of the fractional rates in the third and fifth collection periods) is expressed as a percentage of the K+-induced increase in the fractional rate of release of control tissue. RESULTS
cAMP efflux studies In accord with previous demonstrations, SKF 38393, an agonist on the D-1 dopamine receptor, increased in a dose-dependent manner the effiux of cAMP from blocks of rat neostriatum 31. SKF 38393 was half maximally effective at a concentration of 2 × 10-7 M (Fig. 1). LY 141865, an agonist upon the D-2 dopamine receptor, diminished the magnitude of the SKF 38393-induced efflux of cAMP but did not change the concentration of SKF 38393 giving the half maximal effect. Similarly, dopamine increased the efflux of cAMP from neostriatal tissue. The dopamine-stimulated efflux of cAMP was half maxim al at a concentration of approximately 15/~M (Fig. 2). (--)-Sulpiride (50 #M) markedly potentiated the magnitude of the dopamine-stimulated etttux of cAMP but did not appreciably alter the potency of dopamine (Fig. 2). Omission of CaCl2 from the superfusion medium (during the entire superfusion proto-
col) did not abolish the ability either of SKF 38393 to induce an etttux of cAMP or of LY 141865 to diminish the SKF 38393-stimulated efflux of cAMP (Fig. 3). However, omission of CaC12 from the superfusion medium did increase the basal effiux of cAMP (see legend to Fig. 3).
[3H]ACh release studies Previously, the K+-evoked overflow of tritium from slices of rat neostriatum incubated in the presence of [3H]choline has been shown to reflect the release of [aH]ACh11,23,24. The experimental results obtained in a typical experiment are presented in Fig. 4A. Fig. 4B presents the data from Fig. 4A following calculation of the fractional rate of release. I
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Fig. 1. Inhibition by LY 141865 of the SKF 38393-stimulated
efflux of cAMP from blocks of rat neostriatum. The efflux of cAMP from neostriatal tissue stimulated with the indicated concentrations of SKF 38393 was estim,'tted in the absenc~ (open circles)and presence(filledsquares) of 5/~M LY 141865. The data arc expressed as the percentage of the basal efltux (for details see Materials and Methods). Data were colic(ted in two separate exl~criments;each exl~rimental condition was tested in 8 separate supcffusion chambers. Values reprcsent mean 4- S.E. * P < 0.005 vs SKF 38393 alone (Student's t-tes0. Basal efflux of cAMP in the two experiments was 18.4 4- 0.6 and 23.5 4- 0.7 fmol/min.
266 LY 141865 inhibited in a d o s e - d e p e n d e n t m a n n e r the release o f [3H]ACh from blocks o f rat neostriaturn (Fig. 4C). The m a x i m a l i n h i b i t o r y effect o f L Y 141865 was a 53 ~ reduction o f the fractional rate o f release o f [ZH]ACh. L Y 141865 was h a l f - m a x i m a l l y effective at a c o n c e n t r a t i o n o f 7 × 10 -8 M. R U 24926, an agonist on the D-2 receptor, also inhibited the release o f [ 3 H ] - A C h f r o m blocks o f rat neostriaturn (Fig. 4C). The m a x i m a l i n h i b i t o r y effect o f R U 24926 was a 52 ~ r e d u c t i o n o f the fractional rate o f release o f [3H]ACh; h a l f m a x i m a l inhibition was achieved at 7 × 10 -9 M. S K F 38393 d i d n o t inhibit (and at high c o n c e n t r a t i o n s slightly stimulated) the
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Fig. 3. Inhibition by LY 141865 of the SKF 38393-stimulated efflux of cAMP occurs in the presence and absence of CaC12 in the superfusion medium. The efflux of cAMP from neostriatal tissue was estimated in the absence of drugs (control) and in the presence of 2/~M LY 141865, 2/~M SKF 38393, or a combination of LY 141865 and SKF 38393 (each at 2 #M). The data are expressed as the percentage of the basal etttux (for details see Materials and Methods). Data were collected in two separate experiments; each experimental condition was tested in 6 separate superfusion chambers. Values represent mean & S.E. * P < 0.001 vs SKF 38393 (Student's t-test). Basal efflux of cAMP in the presence, of CaCle was 13.3 -L 0.8 and in the absence of CaC12 28.6 5:1.0 fmol/min.
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release o f [ a H ] A C h (Fig. 4C). ( - - ) - S u l p i r i d e reversed in a d o s e - d e p e n d e n t m a n n e r the inhibition o f [3H]ACh release i n d u c e d by either 1 F M L Y 141865 or 0.1 # M R U 24926 (Fig. 5). ( ÷ ) - S u l p i r i d e was less effective t h a n ( - - ) - s u l p i r i d e in a n t a g o n i z i n g the effects o f either D-2 agonist. Alone, ( - - ) - s u l p i r i d e slightly increased [3H]ACh release• A t r o p i n e d i d n o t reverse the effects o f either L Y 141865 or R U 24926
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Fig. 2. Potentiation by (--)-sulpiride of the dopamine-stimulated efflux of cAMP from blocks of rat neostriatum. The efflux of cAMP from ncostriatal tissue stimulated with the indicated concentrations of dopamine was estimated in the absence (filled squares) and presence (open circles) of 50 #M (--)-sulpiride. The data are express~ as the percentage of the basal ettlux (for details see Materials and Methods). Data were collected in two separate experiments; each experimental condition was tested in 6 separate superfusion chambers• Values represent mean -t- S.E. * P < 0.001 vs Dopamine + (--)-Sulpiride (Student's t-test). Basal efflux of cAMP in the two experiments was 16.6 ± 0.7 and 26.2 ± 2.1 fmol/min.
( d a t a n o t shown)• L-Isoproterenol, I B M X , cholera toxin a n d S K F 38393, agents increasing n e o s t r i a t a l c A M P content 9, 21,31, d i d n o t affect the release o f [aH]ACh (Table I, P a r t A). Similarly, 8Br c A M P , a c o m p o u n d mimicking the action o f c A M P u p o n m a n y tissues, was ineffective in altering the release o f [ a H ] A C h (Table I, P a r t A). Either in the presence or absence o f S K F 38393, cholera toxin, or 8Br c A M P , L Y 141865 inhibited the release o f [3H]ACh to a p p r o x i m a t e l y 50 % o f c o n t r o l (Table I, P a r t B). DISCUSSION Previously, we have p r o p o s e d t h a t in the neos t r i a t u m two d o p a m i n e receptors regulate the efflux
267 a direct interaction between the D-1 and the D-2 receptor. It is commonly accepted that in the absence of Ca ~+ ions, the release of neurotransmitters and hormones is markedly attenuatedz~. The observation that the inhibitory effect of LY 141865 upon the SKF 38393-induced efflux of cAMP occurs even when CaClz is omitted from the superfusion
(and by inference the formation) of cAMP. Stimulation of the D-1 dopamine receptor increases the formation of cyclic AMP. Stimulation of the D-2 dopamine receptor reduces the cAMP formation stimulated by D-1 agonists. Based on the present observations we hypothesize that the D-2 agonistinduced inhibition of cAMP formation results from
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Fig. 4. Inhibition by D-2 agonists of the release of [3H]ACh from blocks of rat neostriatum. A: inhibition by LY 141865 of the K +stimulated release of [aH]-ACh from blocks of rat neostriatal tissue. Results from a typical experiment. The amount of radioactivity (observed cpm) released into the superfusion medium during the experimental protocol described in Materials and Methods is plotted as a function of the superfusion time. The solid line across the top of each bar represents data (mean 4- S.E.) from control superfusions; the broken line across the top of each bar represents data (mean :E S.E.) from tissue exposed to LY 141865 (2/~M) besinning at superfusion time t = 70 rain. The amount of radioactivity released in the HC1 fraction (which is not shown in the figure) was 25039 4- 997 cpm in the absence and 26540 4- 1087 cpm in the presence of LY 141865. The symbol K + and the thickened abscissa represent the period of time when the concentration of NaC1 and KC1 in the supeffusion medium was altered as described in Materials and Methods. B: mathematical transformation of the data presented in A. Using the data presented in A, the fractional rate of release of [SH]ACh was calculated using the protocol described in Materials and Methods. Symbols are as in A. C: inhibition by LY 141865 and R U 24926 of the K+-stimulated release of [aH]ACh from blocks of rat neostriatal tissue. Dose--response relationship. Using the protocol described in Materials and Methods the effects of indicated concentrations of LY 141865 (open squares), R U 24926 (filled circles) or SKF 38393 (open triangles) were calculated. Data represent mean 4- S.E., n = 4 . Each drug was tested in a separate experiment.
268 TABLE I ,
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Drugs stimulating neostriatal cyclic A M P Jbrmation do not affect :'3H}ACh release or L Y 141865 induced inhibition o f 3H]ACh release from blocks o f rat neostriatum
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Data were obtained, fractional rate was calculated and the effect of a drug was calculated as shown in Fig. 4. Data represent mean ~ S.E. (n = 4) and were obtained in 2 separate experiments. Part A: because of the delay in the stimulation of cAMP formation by cholera toxin 15, this agent was added to the superfusion medium at time t 30 rain; all other drugs were added to the superfusion medium at time t 70 rain. Part B: cholera toxin, IBMX and 8Br cAMP were added to the superfusion medium at t = 30 rain; SKF 38393 or LY 141865 were added at t - 70 rain.
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Fig. 5. Reversal by (--)-sulpiride of either the LY 141865inhibited or the R U 24926-inhibited release or [SH]ACh from blocks of rat neostriatal tissue. Data were obtained and the fractional rate was calculated using protocols similar to those shown in Fig. 4A and B (see also Materials and Methods). The effect of a drug was calculated using a protocol similar to that shown in Fig. 4C (,see also Materials and Methods). Left: the release of [3H]ACh was determined in the presence of no drug (open square), (±)-sulpiride alone (open triangle) or R U 24926 (0.1/~M) in combination with either (--)-sulpiHde (open circles) or (+)-sulpiride (filled triangle). Data represent mean q- S.E. (n = 6) and were obtained in two separate experiments. Right: the release of [SH]ACh was determined in the presence of no drug (open square), (--)-sulpiride alone (open triangle), or LY 141865 (1.0 ~M) in combination with either (--)-sulpiride (open circles) or (+)-sulpiride (filled triangle). Data represent mean dz S.E. (n = 6) and were obtained in two separate experiments.
Part A L-Isoproterenol (1 #M) 1-Isobutyl 3-methylxanthine (IBMX; 1 raM) Cholera toxin (30 nM) 8-Br-cAMP (3 raM) SKF 38393 (1/~M) ± I B M X (1 raM)
medium supports the view that at least some of the D-2 dopamine receptors in the neostriatum occur on the cells possessing D-1 dopamine receptors. The existence upon a single cell of a receptor stimulating the formation of cAMP together with an additional receptor inhibiting the formation of cAMP has been previously demonstrated in simple peripheral tissues. For example, in the intermediate lobe of the rat pituitary gland, stimulation of a fl~-adrenoceptor increases the formation of cAMP while stimulation of a D-2 dopamine receptor inhibits the responsiveness of the flz-adrenoceptor 2,26. The D-2 agonists tested in this study decreased the maximal efflux of cAMP caused by stimulation of the D-1 receptor but do not affect the molar potency of the D-1 agonists. This contrasts with the effects of direct acting dopaminergic antagonists (e.g. fluphenazine) which decreased the molar potency of D-1 agonists but does not attenuate the maximal re-
sponse to the agonist6. The D-2 agonist-induced decrease in the responsiveness of the D-1 receptor resembles the non-competitive inhibition by D-2 agonists of the responsiveness of the fl2-adrenoceptor in the intermediate lobe of the rat pituitary gland. Recently, the biochemical events underlying the dopaminergic inhibition of adenylate cyclase activity in the intermediate lobe of the rat pituitary gland have been extensively characterizedL Previously, other investigators have demonstrated that selective D-2 dopamine receptor agonists cause an accumulation of ACh in the neostriatum of the rat brain 7,2s. The present results show again that in vivo accumulation of ACh may arise, in part, as a consequence of diminished release of ACh and suggest that this accumulation can be induced by selective D-2 agonists. Such an action of selective D2 agonists may be of theoretical interest for the treatment of Parkinson's disease/. A balance be-
Part B LY-141865 (1/~M) Cholera toxin (30 nM) ± LY-141865 (1/~M) 8-Br-cAMP (3 raM) + LY-141865 (1/~M) SKF 38393 (1/~M) d- I B M X (1 raM) -k LY-141865 (1 #M)
95.2 ± 3.6 100.0 ± 3.0 102.8 ± 1.4 96.0 ± 2.6 115.1 ± 1.5
48.8 ± 0.6 55.6 ± 0.3 44.1 ± 0.4 49.5 -b 1.1
269 tween the dopaminergic and the cholinergic system is thought to participate in the regulation of extrapyramidal function by the neostriatum. The deficiency of neostriatal dopamine occurring in Parkinsonism disrupts the balance between dopamine and ACh. Prior to the advent of L-DOPA therapy for Parkinsonism, cholinergic antagonists were widely used to alleviate the symptoms of Parkinsonism. The D-2 dopaminergic agonists used in the treatment of Parkinsonism (e.g. bromocriptine, lergotrile and lisuride) may achieve some of their therapeutic effect by interacting with the D-2 dopamine receptor on cholinergic interneurons and inhibiting the release of ACh. The inhibition of ACh release would tend to restore the balance between dopaminergic and cholinergic function in the neostriatum. However, the selective D-2 agonists may have additional effects entirely unrelated to cholinergic activity in the neostriatum (the inhibition of the efltux of cAMP stimulated by D-1 agonists is an example of such an additional effect of D-2 agonists). Other investigators have previously postulated that cAMP does not participate in the regulation of neostriatal cholinergic functionL The present observations also fail to provide any indication that cAMP participates in the release of ACh. Nonetheless, the pharmacology of the D-2 dopamine receptor inhibiting the release of [ZH]ACh resembles the
pharmacology of both the D-2 dopamine receptor inhibiting hormone release and cyclic A M P formation in the pituitary gland and the D-2 dopamine receptor decreasing the responsiveness of the D-1 dopamine receptor in the neostriatum. LY 141865 and R U 24926, selective D-2 dopamine receptor agonists, and (--)-sulpiride, a selective D-2 dopamine receptor antagonist, are active at each of these D-2 receptors. Given the complexity of the neostriatum, it is not surprising that little is known about the physiological mechanisms underlying the dopaminergic inhibition of ACh release. Therefore, it is of interest to examine the effects of dopamine upon the release of pituitary hormones. In the cases of prolactin release from mammotrophs and aMSH release from melanotrophs, some investigators have postulated that dopanfine regulates electrical activity that gates the movement of calcium ions across the plasma membrane and thereby regulates the release of hormone 4,5,32. However, additional evidence indicates that dopamine can also inhibit the capacity of either the mammotroph 10 or the melanotroph 27 to synthesize cAMP. The insight gained from the pituitary gland may facilitate the design of future experiments; however, ultimately it will be necessary to obtain a relatively pure population of neostriatal cholinergic interneurons before conclusive biochemical experiments can be performed.
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6 Dowling, J. E. and Watling, K. J., Dopaminergic mechanisms in the teleost retina, J. Neurochem., 36 (1981)569-579. 7 Euvrard, C., Premont, J., Oberlander, C., Boissier, J. R. and Bockaert, J., Is dopamine sensitive adenylate cyclase involved in regulating the activity of striatal cholinergic neurons, Naunyn-Schmiedeberg's Arch. PharmacoL, 309 (1979) 241-245. 8 Euvrard, C., Ferland, L., Dipaolo, T., Beaulieu,M., Labrie, F., Oberlander, C., Raynaud, J. P. and Boissier, J. R., Activity of two new potent dopaminergic agonists at the striatal and anterior pituitary levels, Neuropharmacology, 19 (1980) 379-386. 9 Forn, J., Krueger, B. K. and Greengard, P., Adenosine 3',5'-monophosphate content in rat caudate nucleus: demonstration of dopaminergic and adrenergic receptors, Science, 186 (1974) 1118-1120. 10 Gianattasio, G., Deferrari, M. E. and Spada, A., Dopamine-inhibited adenylate cyclase in female rat adenohypophysis, Life Sci., 28 (1981) 1605-1612. 11 Hadhazy, P. and Szerb, J. C., The effect of cholinergic drugs on [aH]acetylcholinerelease from slicesof rat hippo-
1 Calne, D. B., Parkinsonism, clinical and neuropharmacological aspects, Postgrad. Med., 64 (1978) 82-88. 2 Cote, T. E., Grewe, C. W. and Kebabian J. W., Stimulation of a D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland decreases the responsiveness of the fl-adrenoceptor: biochemicalmechanism, Endocrinology, 108 (1981)420-426. 3 Cote, T. E., Grewe, C. W., Tsuruta, K., Stoof, J. C., Eskay, R. L. and Kebabian, J. W., D-2 dopamine receptormediated inhibition of adeoylate cyclase activity in the intermediate lobe of the rat pituitary gland requires guanosine 5"-triphosphate, Endocrinology, 110 (1982) 812-819. 4 Douglas, W. W. and Taraskevich, P. S., Action potentials in gland cells of rat pituitary pars intermedia: inhibition by dopamine an inhibitor of MSH secretion, J. PhysioL (Lond.), 285 (1978) 171-184. 5 Douglas, W. W. and Taraskevich, P. S., Calcium component to action potentials in rat pars intermedia cells, J. Physiol. (Lond.), 309 (1980) 623-630.
270 campus, striatum and cortex, Brain Research, 123 (1977) 311-322. 12 Harper, J. F. and Brooker, G. J., Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by anhydride in aqueous solution, J. cyclic Nucleotide Res., 1 (1975) 207-218. 13 Hattori, T., Singh, V. K., McGeer, E. G. and McGeer, P. L., Immunohistochemical localization of cholineacetyltransferase containing neostriatal neurons and their relationship with dopaminergic synapses, Brain Research, 102 (1976) 164-173. 14 Hertting, G., Zumstein, A., Jackisch, R., Hoffmann, I. and Starke, K., Modulation by endogenous dopamine of the release of acetylcholine in the caudate nucleus of the rabbit, Naunyn-Schmiedeberg's Arch. PharmacoL, 315 (1980) 111117. 15 Holmgren, J., Actions of cholera toxin and the prevention and treatment of cholera, Nature (Lond.), 292 ( 1981) 413417. 16 Katz, B. and Miledi, R., The role of calcium in neuromuscular facilitation, J. Physiol. (Lond.), 195 (1968) 481492. 17 Kebabian, J. W., Petzold, G. L. and Greengard, P., Dopamine sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the dopamine receptor, Proc. nat. Acad. Sci. U.S.A., 69 (1972) 2145-2149. 18 Kebabian, J. W. and Calne, D. B., Multiple receptors for dopamine, Nature (Lond.), 277 (1979) 93-96. 19 K/Snig, J. F. R. and Knippel, R. A., The Rat Brain, A Stereotaxie Atlas, Krieger, New York, 1979. 20 Miller, J. C. and Friedhoff, A. J., Dopamine receptorcoupled modulation of the K+-depolarized overflow of 3H-acetylcholine from rat striatal slices: alteration after chronic haloperidol and alpha-methyl-p-tyrosine pretreatment, Life Sci., 25 (1979) 1249-1255. 21 Miller, R. J. and Kelly, P. H., Dopamine-like effects of choleratoxin in the central nervous system, Nature (Lond.), 255 (1975) 163-166. 22 Miller, R. J. and McDermed, J., Dopamine-sensitive adenylate cyclase. In A. S. Horn, J. Korf and B. H. C. Westerink (Eds.), Neurobiology Of Dopamine, Academic Press, London, 1979, pp. 159-178. 23 Molenaar, P. C., Nicholson, V. J. and Polak, R. C., Pre-
24
25
26
27
28
29
30
31
32
33
ferential release of newly synthesized eH-acetylcholine from rat cerebral cortex slices in vitro, Brit. J. Pharmacol., 47 (1973) 97-108. Mulder, A. H., Yamamura, M. 1., Kuhar, M. J. and Snyder, S. H., Release of acetylcholine from hippocampal slices by potassium depolarization: dependence on high affinity choline uptake, Brain Research, 70 (1974) 372-376. Mulder, A. H., van den Berg, W. B. and Stoof, J. C., Calcium dependent release of radiolabeled catecholamines and serotonin from rat brain synaptosomes in a superfusion system, Brain Research, 99 (1975) 419-424. Munemura, M., Eskay, R. L. and Kebabian, J. W., Release of a-melanocyte stimulating hormone from dispersed cells of the intermediate lobe of the rat pituitary gland: involvement of catecholamines and adenosine 3',5'-monophosphate, Endocrinology, 106 (1980) 1795-1803. Munemura, M., Cote, T. E., Tsuruta, K., Eskay, R. L. and Kebabian, J. W., The dopamine receptor in the intermediate lobe of the rat pituitary gland: pharmacological characterization, Endocrinology, 107 (1980) 1676-1683. Sethy, V. H., Regulation of striatal acetylcholine concentration by D-2 dopamine receptors, Europ. J. PharmacoL, 60 (1979) 397-398. Setler, P. E., Sarau, H. M., Zirkle, C. L. and Saunders, H. L., The central effects of a novel dopamine agonist, Europ. J. Pharmacol., 50 (1978)419-430. Stoof, J. C., Thieme, R. E., Vrijmoed-de Vries, M. C. and Mulder, A. H., In vitro acetylcholine release from rat caudate nucleus as a new model for testing drugs with dopamine receptor activity, Naunyn-Sehmiedeberg's Arch. Pharmacol., 309 (1979) 119-124. Stoof, J. C. and Kebabian, J. W., Opposing roles for the D-I and the D-2 dopamine receptors in efflux of cAMP from rat neostriatum, Nature (Lond.), 294 (1981) 366-368. Thorner, M. O., Hackett, J. T., Murad, F. and McLeod, R. M., Calcium rather than cyclic AMP as the physiological regulator of prolactin release, Neuroendocrinology, 31 (1980) 390-402. Tsuruta, K., Frey, E. A., Grewe, C. W., Cote, T. E., Eskay, R. L. and Kebabian, J. W., Evidence that LY-141865 specifically stimulates the D-2 dopamine receptor, Nature (Lond.), 292 (1981) 463-465.
263
Independent In Vitro Regulation by the D-2 Dopamine Receptor of Dopamine-Stimulated Efflux of Cyclic AMP and K+-Stimulated Release of Acetylcholine from Rat Neostriatum J. C. STOOF* and J. W. KEBABIAN
Biochemical Neuropharmacology Section, Experimental Therapeutics Branch, N1NCDS, NIH, Bethesda, MD 20205 (U.S.A.) (Accepted April 8th, 1982)
Key words: cAMP efflux - - ACh release - - D-1 receptor - - D-2 receptor - - rat neostriatum
Two types of dopamine receptors whose stimulation affect cAMP efflux (and by inference formation) could be identified in rat neostriatum. One type of receptor, called D-I receptor, increased cAMP efflux whereas stimulation of a second type of dopamine receptor, called D-2 receptor, was followed by a reduction in cAMP efflux induced by stimulation with a D-1 receptor agonist. D-2 receptor agonists inhibited the effects olD-1 receptor agonists on cAMP efflux in a non-competitive way. These inhibiting effects of D-2 receptor agonists occurred also in the absence of Ca2+-ions which could imply that some of the D-2 receptors are located on cells possessing D-1 receptors. The dopamine receptor mediating inhibition of the release of radiolabeled acetylcholine (ACh) in the neostriatumappearedto have the same pharmacological characteristics as the D-2 dopamine receptor mediating the inhibition of the D-I receptor agonist induced cAMP efflux. Selective D-2 receptor agonists like LY 141865 and RU 24926 stimulated this receptor while the D-1 receptor agonist SKF 38393 was inactive. Effects of the selective D-2 receptor agonists could be antagonized by (--)-sulpiride, a selective D-2 receptor antagonist. Although the pharmacological characteristics of the dopamine receptors mediating inhibition of both ACh release and (D-1 dopamine receptor agonist induced) cAMP efflux appeared to be similar, drugs stimulating cAMP efflux did not affect ACh release or LY 141865 induced inhibition of ACh release from rat neostriatum. Therefore it is still questionable whether the dopamine receptor mediating inhibition of both ACh release and cAMP efflux is one and the same functional entity. INTRODUCTION A c c o r d i n g to one classification schema, there exist two categories o f d o p a m i n e r e c e p t o r designated as D-1 a n d D-2 receptors is. Several drugs discriminate between these two d o p a m i n e receptors. S K F 38393 is substantially m o r e p o t e n t t h a n d o p a m i n e as an a g o n i s t u p o n the D-1 r e c e p t o r 29, b u t is substantially less p o t e n t t h a n d o p a m i n e as an agonist u p o n the D2 r e c e p t o r 27. L Y 1418653a a n d R U 249267,8 selectively stimulate the D-2 d o p a m i n e receptor. T h e substituted b e n z a m i d e , ( - - ) - s u l p i r i d e , b l o c k s the stimulating actions o f drugs o n l y on the D-2 receptor18,22. B o t h types o f d o p a m i n e r e c e p t o r occur in the n e o s t r i a t u m o f the rat b r a i n a n d affect the synthesis
o f a d e n o s i n e 3',5' m o n o p h o s p h a t e ( c A M P ) 31. Stim u l a t i o n o f the D-1 r e c e p t o r increases c A M P form a t i o n 17 while s t i m u l a t i o n o f the D-2 d o p a m i n e r e c e p t o r decreases the c A M P f o r m a t i o n s t i m u l a t e d by D-1 agonists al. I n addition, a D-2 d o p a m i n e r e c e p t o r regulates the c o n t e n t o f neostriatal acetylcholine (ACh). The c o n t e n t o f neostriatal A C h is increased b y D-2 d o p a m i n e r e c e p t o r agonists a n d is decreased b y D-2 d o p a m i n e r e c e p t o r antagonists 7, 2s. T h e d o p a m i n e r g i c regulation o f neostriatal A C h levels correlates with the presence o f synaptic contacts between d o p a m i n e c o n t a i n i n g n e u r o n s with choline acetyltransferase-staining n e u r o n a l elements in the n e o s t r i a t u m 13. The present investigations were u n d e r t a k e n with two goals. T h e first goal was to further characterize
* On leave from the Department of Neurology, Free University, Amsterdam, The Netherlands. Address reprint requests to: J. C. Stoof, Dept. of Neurology, Free University, van der Boechorststr. 7, 1081 BT, Amsterdam, The Netherlands. 0006-8993/82/0000-0000/$02.75 © (1982) Elsevier Biomedical Press
264 the D-2 dopaminergic inhibition of the dopaminestimulated effiux of cAMP from neostriatal slices; the second goal was to characterize the pharmacology of the dopamine receptor inhibiting the depolarization-induced release of ACh from blocks of neostriatal tissue14, 2°,3°. Both of these endeavours utilize a previously described superfusion apparatus to collect either the cAMP or the ACh released from neostriatal tissueZL The availability of drugs discriminating between the D-1 and the D-2 dopamine receptors (in the classification schema of Kebabian and Calne) facilitated these investigations. MATERIALS A N D METHODS
Drugs used and suppliers were as follows: IBMX and 8Br cAMP (Sigma), LY 141865 (Eli Lilly), RU 24926 (Roussel) and SKF 38393 (Smith Kline and French).
Preparation of chopped neostriatal tissue Following decapitation of male Sprague-Dawley rats (weighing between 300 and 350 g), the brains were removed and placed in a brain holder which permitted two successive 2 mm thick coronal slices to be made between anterior planes A-7,000 and A-11,00019. The neostriatal tissue was dissected from these two slices and subsequently minced by passing it twice through a Mcllwain tissue chopper (micrometer setting 300 #m). Prior to the second pass, the surface was rotated 90°. The resulting blocks of neostriatal tissue (measuring 2 × 0.3 × 0.3 mm) were transferred to a balanced salt solution (BSS) containing 116.4 mM NaC1, 5.4 mM KC1, 26.2 mM NaHCOz, 1.0 mM NaH2PO4, 0.6 mM MgSO4, 5.6 mM D-glucose, 1.3 mM CaCI2 and 10 mg/1 phenol red. The tissue from all rats (12 for cAMP studies, 6 for ACh studies) was pooled. The BSS used for the dissection and subsequent experimental manipulations was kept under an atmosphere of 95 % 02 and 5 % COg.
Cyclic AMP efflux studies Aliquots of the pooled blocks of neostriatal tissue (containing approximately 20 mg wet weight of tissue) were placed in each of the 24 chambers of a previously described superfusion apparatus2L Superfusion at a rate of 0.1 ml/min with BSS fortified
with 3-isobutyl 1-methyl xanthine (1 mM) and bovine serum albumin (BSA, 2.5 mg/ml) began at superfusion time t = 0 min. Beginning at superfusion time t = 60 min, a 3.0 ml (30 min) aliquot (fraction 1) of the superfusion medium was collected from each chamber to permit estimation of the basal efflux of cAMP. At superfusion time t ::: 90 rain, drugs were added to the superfusion medium; beginning at superfusion time t ~ 105 rain, a 1.0 ml (10 min) aliquot (fraction 2) of the superfusion medium was collected. Previously, it has been shown that the maximal effiux of cAMP stimulated by a dopaminergic agonist occurred 15-25 min after exposure to dopaminergic agonists (see Fig. 1 of reference 31). The cAMP contents of duplicate 100 #1 aliquots of fractions 1 and 2 collected from each chamber were estimated by radioimmunoassay as described previouslylL The amount of cAMP in fraction 2 was expressed as a percentage of the amount of cAMP in fraction 1 (the basal efltux), thereby permitting each chamber to serve as its own control and correcting for differences in the amounts of tissue present in each of the superfusion chambers.
[3H]ACh release studies The pooled blocks of neostriatal tissue were incubated for 10 min at 37 °C in 10 ml of BSS. Subsequently, the BSS was removed and replaced with 10 ml of BSS containing [SH]choline (10/~Ci; spec. act. 80 Ci/mmol). The blocks of neostriatal tissue were incubated for an additional 15 min. At the end of the incubation period, the tissue was washed (twice with 2 ml of BSS) and aliquots of the tissue (containing approximately 10 mg wet weight of tissue) were placed in each of the 24 chambers of a previously described superfusion apparatus 25. Superfusion at a rate of 0.2 ml/min with BSS began at superfusion time t = 0 min. Beginning at superfusion time t = 30 min, 5 successive 4 ml (20 min) aliquots of the superfusion medium were collected from each chamber. During the first 5 min of the second (t = 50--70 min) and the fourth (t = 90-110 min) fractions, the concentrations of KC1 and NaCI in BSS were changed to 17 mM and 106 mM, respectively. Drugs, when present, were added to the superfusion medium beginning at superfusion time t = 70 rain. The first exposure to an elevated concentration of KC1 verified that the amount of [ZH]ACh
265 released from the tissue in each of the 24 superfusion chambers was equivalent (i.e. within 10~o of the mean); the second exposure to an elevated concentration of KCI tested the ability of a drug to inhibit the release of [aH]ACh. To conclude the experiment, the tissue was superfused for 20 min (beginning at t = 130 min) with 0.1 N HCI (fraction 6). The amount of radioactivity present in each of the fractions of the superfusion medium and the HC1 extract was estimated by liquid scintillation spectcoscopy (60 ~o counting efficiency for 3H). In order to correct for differences in the amount of tissue present in each of the superfusionchambers, data were expressed as a fractional rate (i.e. the amount of radioactivity released during each collection period was divided by the sum of the amount of radioactivity released in that collection period, subsequent collection periods and the HC1 extraction). In order to estimate the effect of different drugs, the K+-induced increase in the fractional rate of release of radioactivity during the fourth collection period (i.e. the difference between the fractional rate in the fourth collection period and the mean of the fractional rates in the third and fifth collection periods) is expressed as a percentage of the K+-induced increase in the fractional rate of release of control tissue. RESULTS
cAMP efflux studies In accord with previous demonstrations, SKF 38393, an agonist on the D-1 dopamine receptor, increased in a dose-dependent manner the effiux of cAMP from blocks of rat neostriatum 31. SKF 38393 was half maximally effective at a concentration of 2 × 10-7 M (Fig. 1). LY 141865, an agonist upon the D-2 dopamine receptor, diminished the magnitude of the SKF 38393-induced efflux of cAMP but did not change the concentration of SKF 38393 giving the half maximal effect. Similarly, dopamine increased the efflux of cAMP from neostriatal tissue. The dopamine-stimulated efflux of cAMP was half maxim al at a concentration of approximately 15/~M (Fig. 2). (--)-Sulpiride (50 #M) markedly potentiated the magnitude of the dopamine-stimulated etttux of cAMP but did not appreciably alter the potency of dopamine (Fig. 2). Omission of CaCl2 from the superfusion medium (during the entire superfusion proto-
col) did not abolish the ability either of SKF 38393 to induce an etttux of cAMP or of LY 141865 to diminish the SKF 38393-stimulated efflux of cAMP (Fig. 3). However, omission of CaC12 from the superfusion medium did increase the basal effiux of cAMP (see legend to Fig. 3).
[3H]ACh release studies Previously, the K+-evoked overflow of tritium from slices of rat neostriatum incubated in the presence of [3H]choline has been shown to reflect the release of [aH]ACh11,23,24. The experimental results obtained in a typical experiment are presented in Fig. 4A. Fig. 4B presents the data from Fig. 4A following calculation of the fractional rate of release. I
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efflux of cAMP from blocks of rat neostriatum. The efflux of cAMP from neostriatal tissue stimulated with the indicated concentrations of SKF 38393 was estim,'tted in the absenc~ (open circles)and presence(filledsquares) of 5/~M LY 141865. The data arc expressed as the percentage of the basal efltux (for details see Materials and Methods). Data were colic(ted in two separate exl~criments;each exl~rimental condition was tested in 8 separate supcffusion chambers. Values reprcsent mean 4- S.E. * P < 0.005 vs SKF 38393 alone (Student's t-tes0. Basal efflux of cAMP in the two experiments was 18.4 4- 0.6 and 23.5 4- 0.7 fmol/min.
266 LY 141865 inhibited in a d o s e - d e p e n d e n t m a n n e r the release o f [3H]ACh from blocks o f rat neostriaturn (Fig. 4C). The m a x i m a l i n h i b i t o r y effect o f L Y 141865 was a 53 ~ reduction o f the fractional rate o f release o f [ZH]ACh. L Y 141865 was h a l f - m a x i m a l l y effective at a c o n c e n t r a t i o n o f 7 × 10 -8 M. R U 24926, an agonist on the D-2 receptor, also inhibited the release o f [ 3 H ] - A C h f r o m blocks o f rat neostriaturn (Fig. 4C). The m a x i m a l i n h i b i t o r y effect o f R U 24926 was a 52 ~ r e d u c t i o n o f the fractional rate o f release o f [3H]ACh; h a l f m a x i m a l inhibition was achieved at 7 × 10 -9 M. S K F 38393 d i d n o t inhibit (and at high c o n c e n t r a t i o n s slightly stimulated) the
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Fig. 3. Inhibition by LY 141865 of the SKF 38393-stimulated efflux of cAMP occurs in the presence and absence of CaC12 in the superfusion medium. The efflux of cAMP from neostriatal tissue was estimated in the absence of drugs (control) and in the presence of 2/~M LY 141865, 2/~M SKF 38393, or a combination of LY 141865 and SKF 38393 (each at 2 #M). The data are expressed as the percentage of the basal etttux (for details see Materials and Methods). Data were collected in two separate experiments; each experimental condition was tested in 6 separate superfusion chambers. Values represent mean & S.E. * P < 0.001 vs SKF 38393 (Student's t-test). Basal efflux of cAMP in the presence, of CaCle was 13.3 -L 0.8 and in the absence of CaC12 28.6 5:1.0 fmol/min.
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release o f [ a H ] A C h (Fig. 4C). ( - - ) - S u l p i r i d e reversed in a d o s e - d e p e n d e n t m a n n e r the inhibition o f [3H]ACh release i n d u c e d by either 1 F M L Y 141865 or 0.1 # M R U 24926 (Fig. 5). ( ÷ ) - S u l p i r i d e was less effective t h a n ( - - ) - s u l p i r i d e in a n t a g o n i z i n g the effects o f either D-2 agonist. Alone, ( - - ) - s u l p i r i d e slightly increased [3H]ACh release• A t r o p i n e d i d n o t reverse the effects o f either L Y 141865 or R U 24926
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Fig. 2. Potentiation by (--)-sulpiride of the dopamine-stimulated efflux of cAMP from blocks of rat neostriatum. The efflux of cAMP from ncostriatal tissue stimulated with the indicated concentrations of dopamine was estimated in the absence (filled squares) and presence (open circles) of 50 #M (--)-sulpiride. The data are express~ as the percentage of the basal ettlux (for details see Materials and Methods). Data were collected in two separate experiments; each experimental condition was tested in 6 separate superfusion chambers• Values represent mean -t- S.E. * P < 0.001 vs Dopamine + (--)-Sulpiride (Student's t-test). Basal efflux of cAMP in the two experiments was 16.6 ± 0.7 and 26.2 ± 2.1 fmol/min.
( d a t a n o t shown)• L-Isoproterenol, I B M X , cholera toxin a n d S K F 38393, agents increasing n e o s t r i a t a l c A M P content 9, 21,31, d i d n o t affect the release o f [aH]ACh (Table I, P a r t A). Similarly, 8Br c A M P , a c o m p o u n d mimicking the action o f c A M P u p o n m a n y tissues, was ineffective in altering the release o f [ a H ] A C h (Table I, P a r t A). Either in the presence or absence o f S K F 38393, cholera toxin, or 8Br c A M P , L Y 141865 inhibited the release o f [3H]ACh to a p p r o x i m a t e l y 50 % o f c o n t r o l (Table I, P a r t B). DISCUSSION Previously, we have p r o p o s e d t h a t in the neos t r i a t u m two d o p a m i n e receptors regulate the efflux
267 a direct interaction between the D-1 and the D-2 receptor. It is commonly accepted that in the absence of Ca ~+ ions, the release of neurotransmitters and hormones is markedly attenuatedz~. The observation that the inhibitory effect of LY 141865 upon the SKF 38393-induced efflux of cAMP occurs even when CaClz is omitted from the superfusion
(and by inference the formation) of cAMP. Stimulation of the D-1 dopamine receptor increases the formation of cyclic AMP. Stimulation of the D-2 dopamine receptor reduces the cAMP formation stimulated by D-1 agonists. Based on the present observations we hypothesize that the D-2 agonistinduced inhibition of cAMP formation results from
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Fig. 4. Inhibition by D-2 agonists of the release of [3H]ACh from blocks of rat neostriatum. A: inhibition by LY 141865 of the K +stimulated release of [aH]-ACh from blocks of rat neostriatal tissue. Results from a typical experiment. The amount of radioactivity (observed cpm) released into the superfusion medium during the experimental protocol described in Materials and Methods is plotted as a function of the superfusion time. The solid line across the top of each bar represents data (mean 4- S.E.) from control superfusions; the broken line across the top of each bar represents data (mean :E S.E.) from tissue exposed to LY 141865 (2/~M) besinning at superfusion time t = 70 rain. The amount of radioactivity released in the HC1 fraction (which is not shown in the figure) was 25039 4- 997 cpm in the absence and 26540 4- 1087 cpm in the presence of LY 141865. The symbol K + and the thickened abscissa represent the period of time when the concentration of NaC1 and KC1 in the supeffusion medium was altered as described in Materials and Methods. B: mathematical transformation of the data presented in A. Using the data presented in A, the fractional rate of release of [SH]ACh was calculated using the protocol described in Materials and Methods. Symbols are as in A. C: inhibition by LY 141865 and R U 24926 of the K+-stimulated release of [aH]ACh from blocks of rat neostriatal tissue. Dose--response relationship. Using the protocol described in Materials and Methods the effects of indicated concentrations of LY 141865 (open squares), R U 24926 (filled circles) or SKF 38393 (open triangles) were calculated. Data represent mean 4- S.E., n = 4 . Each drug was tested in a separate experiment.
268 TABLE I ,
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Drugs stimulating neostriatal cyclic A M P Jbrmation do not affect :'3H}ACh release or L Y 141865 induced inhibition o f 3H]ACh release from blocks o f rat neostriatum
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Data were obtained, fractional rate was calculated and the effect of a drug was calculated as shown in Fig. 4. Data represent mean ~ S.E. (n = 4) and were obtained in 2 separate experiments. Part A: because of the delay in the stimulation of cAMP formation by cholera toxin 15, this agent was added to the superfusion medium at time t 30 rain; all other drugs were added to the superfusion medium at time t 70 rain. Part B: cholera toxin, IBMX and 8Br cAMP were added to the superfusion medium at t = 30 rain; SKF 38393 or LY 141865 were added at t - 70 rain.
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Drug
' 3H / A Ch release (as % o f control)
Fig. 5. Reversal by (--)-sulpiride of either the LY 141865inhibited or the R U 24926-inhibited release or [SH]ACh from blocks of rat neostriatal tissue. Data were obtained and the fractional rate was calculated using protocols similar to those shown in Fig. 4A and B (see also Materials and Methods). The effect of a drug was calculated using a protocol similar to that shown in Fig. 4C (,see also Materials and Methods). Left: the release of [3H]ACh was determined in the presence of no drug (open square), (±)-sulpiride alone (open triangle) or R U 24926 (0.1/~M) in combination with either (--)-sulpiHde (open circles) or (+)-sulpiride (filled triangle). Data represent mean q- S.E. (n = 6) and were obtained in two separate experiments. Right: the release of [SH]ACh was determined in the presence of no drug (open square), (--)-sulpiride alone (open triangle), or LY 141865 (1.0 ~M) in combination with either (--)-sulpiride (open circles) or (+)-sulpiride (filled triangle). Data represent mean dz S.E. (n = 6) and were obtained in two separate experiments.
Part A L-Isoproterenol (1 #M) 1-Isobutyl 3-methylxanthine (IBMX; 1 raM) Cholera toxin (30 nM) 8-Br-cAMP (3 raM) SKF 38393 (1/~M) ± I B M X (1 raM)
medium supports the view that at least some of the D-2 dopamine receptors in the neostriatum occur on the cells possessing D-1 dopamine receptors. The existence upon a single cell of a receptor stimulating the formation of cAMP together with an additional receptor inhibiting the formation of cAMP has been previously demonstrated in simple peripheral tissues. For example, in the intermediate lobe of the rat pituitary gland, stimulation of a fl~-adrenoceptor increases the formation of cAMP while stimulation of a D-2 dopamine receptor inhibits the responsiveness of the flz-adrenoceptor 2,26. The D-2 agonists tested in this study decreased the maximal efflux of cAMP caused by stimulation of the D-1 receptor but do not affect the molar potency of the D-1 agonists. This contrasts with the effects of direct acting dopaminergic antagonists (e.g. fluphenazine) which decreased the molar potency of D-1 agonists but does not attenuate the maximal re-
sponse to the agonist6. The D-2 agonist-induced decrease in the responsiveness of the D-1 receptor resembles the non-competitive inhibition by D-2 agonists of the responsiveness of the fl2-adrenoceptor in the intermediate lobe of the rat pituitary gland. Recently, the biochemical events underlying the dopaminergic inhibition of adenylate cyclase activity in the intermediate lobe of the rat pituitary gland have been extensively characterizedL Previously, other investigators have demonstrated that selective D-2 dopamine receptor agonists cause an accumulation of ACh in the neostriatum of the rat brain 7,2s. The present results show again that in vivo accumulation of ACh may arise, in part, as a consequence of diminished release of ACh and suggest that this accumulation can be induced by selective D-2 agonists. Such an action of selective D2 agonists may be of theoretical interest for the treatment of Parkinson's disease/. A balance be-
Part B LY-141865 (1/~M) Cholera toxin (30 nM) ± LY-141865 (1/~M) 8-Br-cAMP (3 raM) + LY-141865 (1/~M) SKF 38393 (1/~M) d- I B M X (1 raM) -k LY-141865 (1 #M)
95.2 ± 3.6 100.0 ± 3.0 102.8 ± 1.4 96.0 ± 2.6 115.1 ± 1.5
48.8 ± 0.6 55.6 ± 0.3 44.1 ± 0.4 49.5 -b 1.1
269 tween the dopaminergic and the cholinergic system is thought to participate in the regulation of extrapyramidal function by the neostriatum. The deficiency of neostriatal dopamine occurring in Parkinsonism disrupts the balance between dopamine and ACh. Prior to the advent of L-DOPA therapy for Parkinsonism, cholinergic antagonists were widely used to alleviate the symptoms of Parkinsonism. The D-2 dopaminergic agonists used in the treatment of Parkinsonism (e.g. bromocriptine, lergotrile and lisuride) may achieve some of their therapeutic effect by interacting with the D-2 dopamine receptor on cholinergic interneurons and inhibiting the release of ACh. The inhibition of ACh release would tend to restore the balance between dopaminergic and cholinergic function in the neostriatum. However, the selective D-2 agonists may have additional effects entirely unrelated to cholinergic activity in the neostriatum (the inhibition of the efltux of cAMP stimulated by D-1 agonists is an example of such an additional effect of D-2 agonists). Other investigators have previously postulated that cAMP does not participate in the regulation of neostriatal cholinergic functionL The present observations also fail to provide any indication that cAMP participates in the release of ACh. Nonetheless, the pharmacology of the D-2 dopamine receptor inhibiting the release of [ZH]ACh resembles the
pharmacology of both the D-2 dopamine receptor inhibiting hormone release and cyclic A M P formation in the pituitary gland and the D-2 dopamine receptor decreasing the responsiveness of the D-1 dopamine receptor in the neostriatum. LY 141865 and R U 24926, selective D-2 dopamine receptor agonists, and (--)-sulpiride, a selective D-2 dopamine receptor antagonist, are active at each of these D-2 receptors. Given the complexity of the neostriatum, it is not surprising that little is known about the physiological mechanisms underlying the dopaminergic inhibition of ACh release. Therefore, it is of interest to examine the effects of dopamine upon the release of pituitary hormones. In the cases of prolactin release from mammotrophs and aMSH release from melanotrophs, some investigators have postulated that dopanfine regulates electrical activity that gates the movement of calcium ions across the plasma membrane and thereby regulates the release of hormone 4,5,32. However, additional evidence indicates that dopamine can also inhibit the capacity of either the mammotroph 10 or the melanotroph 27 to synthesize cAMP. The insight gained from the pituitary gland may facilitate the design of future experiments; however, ultimately it will be necessary to obtain a relatively pure population of neostriatal cholinergic interneurons before conclusive biochemical experiments can be performed.
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