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Home , Apomorphine, Ergoline, Lergotrile, Pergolide

Proc. Natl. Acad. Sci. USA Vol. 77, No. 6, pp. 3725-3728, June 1980 Neurobiology

Interaction of with central receptors (/adenylate cyclase) MENEK GOLDSTEIN*, ABRAHAM LIEBERMAN*, Jow Y. LEW*, TAKU ASANO*, MYRNA R. ROSENFELDt, AND MAYNARD H. MAKMANt *New York University Medical Center, Departments of Psychiatry and Neurology, 560 First Avenue, New York, New York 10016; and tAlbert Einstein College of , Departments of Biochemistry and Molecular Pharmacology, Bronx, New York 10461 Communicated by Michael Heidelberger, February 12,1980

ABSTRACT The activity of pergolide, an N-propylergoline MATERIALS AND METHODS derivative, has been tested for stimulation of central dopa- minergic receptors. Binding to receptors shows that Materials. [3H]Dopamine (8.4 Ci/mmol), [3H]Spiroperidol pergolide acts as an with respect to these receptors. GTP ([3H]Spi) (23 Ci/mmol), N-n-[3H] decreases the potencies of dopamine and of pergolide, ([3H]NPA) (75 Ci/nmol) were purchased from New England but not of , to displace [3HJspiroperidol {43HSpi) Nuclear (1 Ci = 3.7 X 1010 becquerels). Pergolide was a gift from striatal membrane sites. The GTP-sensitive site labeled from Eli Lilly, and bromocriptine from Sandoz Pharmaceu- by [3HJSpi seems to be localized on intrastriatal dopamine re- tical. ceptors. The potency of dopamine agonists and of pergolide to Binding Assay. Preparation of bovine or rat membranes and displace [3HJSpi from striatal sites is reduced in membranes exposed to higher temperatures. Pergolide, but not the ensuing binding assay were carried out as described (13). hitherto-tested dopaminergic , stimulates do amine- For routine assay each tube contained 1.8 ml. of membrane sensitive adenylate cyclase in striatal homogenates. Thus, per- suspension (5 mg of wet tissue), 0.1 ml of radioactive (2.5 golide, unlike other dopaminergic ergots, acts as an agonist on nM [3H]dopamine, 0.5 nM [3H]Spi, or 0.5 nM [3H]NPA), and, GTP-sensitive components of [3HJSpi binding and stimulates when indicated, various concentrations of drugs dissolved in dopamine receptors linked to dopamine-sensitive adenylate 0.1% ascorbic acid. compounds were dissolved in ap- cyclase. The drug also induces turning behavior in rats with proximately 25 ,l of glacial acetic acid and brought up to 1 mM 6-OH-dopamine lesions and relieves in monkeys with with a solution containing 50% (vol/vol) ethanol in 0.01 M HCG ventromedial tegmental lesions for a longer time at a lower dose The in were incubated for 10 min than other tested dopaminergic ergots. Other studies have tubes, triplicate, at 37°C shown that it is effective in the treatment of patients with ad- when [3H]dopamine was used, or 15 min with [3H]Spi or vanced parkinsonism. [3H]NPA, then cooled to 0WC, and filtered. Radioactivity was measured in the filters. Saturable and specific binding of It is now established that the major abnormality in parkinsonism [3H]dopamine, [3H]NPA, and [3H]Spi was measured as the is the degeneration of the nigrostriatal 3,4-dihydroxyphenyl- excess over "blanks" measured in the presence of 1 1uM apo- ethylamine (dopamine) neurons. The effectiveness of 3,4- (for [3H]dopamine or [3H]NPA) or of 0.1 ,uM (+)- dihydroxyphenylalanine (L-dopa) in parklnsonism is dependent (for [3H]Spi). on the capacity of the remaining nigrostriatal dopamine neu- Values for the mean inhibitory concentration, ICso, were rons to from It has derived by log probit analysis. Values for the dissociation con- synthesize dopamine administered L-dopa. stant, Kd, for each radioactive ligand were determined from become apparent that therapeutic response diminishes after the corresponding Scatchard plots, and values for the inhibitor prolonged treatment with L-dopa (1, 2). This decrease might constant, Ki, were determined according to the equation Ki = be due to progressive degeneration of the nigrostriatal dopa- IC50/(I + C/Kd), in which C represents the concentration of mine neurons or to a decreased sensitivity of dopamine recep- the radioactive ligand. tors in the striatum. It was therefore of interest to investigate Dopamine-Sensitive Adenylate Cyclase. Male Sprague- the effectiveness of drugs that directly stimulate dopamine Dawley rats were sacrificed by decapitation, and striata were receptors in the . Among various dopamine agonists tested, rapidly dissected. The activity of adenylate cyclase was de- certain ergot were found to stimulate dopamine re- termined in triplicate for each assay as described (14). The final ceptors (3, 4), and their therapeutic effectiveness in parkin- dilution of tissue was 1:300, 3',5'-cyclic AMP (cAMP) was sonism was investigated (5, 6). measured by a protein-binding assay (15), and protein was We now describe properties of a semi- measured by the method of Lowry et al. (16). synthetic derivative, pergolide (8fl-[8-(methylthio)- Surgical Lesions. Unilateral 6-OH-dopamine lesions were methyl]-6-propylergoline), tested both in vitro and in two an- induced in and decortication was performed on male imal models that simulate certain features of parkinsonism. The Sprague-Dawley rats weighing 150-200 g (17, 18). Green potency of pergolide was compared with that of bromocriptine monkeys (Cercopithecus sabeus) weighing 3-4 kg were used (2-bromo-a-ergocriptine), another ergot derivative currently for placing unilateral radiofrequency ventromedial tegmental used in the treatment of parkinsonism (7, 8). The long duration (VMT) lesions in the brain stem (19). The animals developed of action of pergolide as an agonist (9, 10) prompted us to in- hypokinesia and resting tremor (4-6 cycles per sec) 1-2 weeks vestigate its therapeutic effectiveness in parkinsonism patients. after surgery (20). Preliminary reports on this study have been presented (9, 11, Turning Behavior. The turning behavior in rats was mea- 12). sured in a transparent plastic cage and the number of 3600 turns was recorded for 3 min every 15 min (17). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: Spi, spiroperidol; NPA, N-n-propylnorapomorphine; vertisement" in accordance with 18 U. S. C. §1734 solely to indicate cAMP, 3',5'-cyclic AMP; AIM, abnormal involuntary movements; this fact. VMT, ventromedial tegmental. 3725 Downloaded by guest on October 1, 2021 3726 Neurobiology: Goldstein et al. Proc. Natl. Acad. Sci. USA 77 (1980)

Table 1. Affinities of pergolide and of other drugs at bovine Table 3. Effect of unilateral decortication on binding of [3H]Spi striatal binding sites to rat striatal membrane sites in the presence and absence of GTP Ki, nM Receptor binding, Drug [3H]dopamine [3H]NPA [3H]Spi pmol/g tissue Conc., Control side Lesion side Pergolide 12.8 I 0.8 41.1 ± 5.5 34.2 ± 2.5 Drug OM -GTP +GTP -GTP +GTP Bromocriptine 49.0 3.5 54.7 I 7.0 1.8 + 0.2 Dopamine 12.5 ± 1.0 50.9 ± 8.0 D-Butaclamol 0.1 19.6 17.4 12.2 10.9 6.6 ± 0.4 3.5 ± 0.5 42.8 ± 5.5 Bromocriptine 0.05 13.9 12.9 8.5 7.6 Spi 6.5 1.0 0.3 + 0.05 Pergolide 0.05 6.6 4.6 3.6 0.4 135 ± 18.0 32.8 ± 4.5 5.5 ± 0.9 Binding to receptor was measured 2-3 weeks after decortication Binding was assayed in presence of four to six concentrations of in intact and decorticated striatum. The specific binding (excess over each compound in triplicate. The values are the mean : SEM from blanks in the presence of the drug) was determined in the absence and at least three experiments. The Kd and maximal binding, B,, were presence of 50 ,uM GTP. The results are the mean of two experiments; obtained from Scatchard analysis of the saturation data. Kd (nM): SEM was +3-10%o. [3H]Spi = 0.41 : 0.03, [3H]dopamine =S14.80 + 0.8, [3H]NPA = 0.35 ± 0.05 and 1.30 I 0.20. B... (pmol/g oftissue): [3H]Spi = 27.0 ± 1.2, cyclase with high affinity for agonists are localized on in- [3H]dopamine = 22.3 + 1.1, [3H]NPA = 12.4 I 0.8 and 27.0 1 1.5. The trastriatal neurons (18). To determine whether removal of two values for [3H]NPA indicate high- and low-affinity binding sites, corticostriatal dopamine receptors alters receptor binding af- respectively. finity of pergolide or of bromocriptine we have measured their capacity to displace [3H]Spi in decorticated rat striatum. Tremor and Abnormal Involuntary Movements (AIM). In agreement with previously reported findings (18), Tremor was recorded on an electroencephalograph and AIM decortication results in a decrease of [3H]Spi binding dis- were observed visually (20). placeable by D-butaclamol in striatum (Table 3). Pergolide- and bromocriptine-displaceable [3H]Spi binding is also decreased RESULTS on the decorticated side of the striatum. GTP inhibits more Dopamine receptor binding studies effectively the displacement of [3H]Spi by pergolide in the decorticated striatum than in the intact striatum, indicating that Affinities for Agonist and Antagonist Sites. The capacity GTP-sensitive sites labeled by [3H]Spi are localized on the in- DA and antago- of Pergolide to displace radiolabeled agonists trastriatal neurons and that Pergolide interacts with these do- nists from bovine striatal membrane sites was investigated (Table 1). Pergolide effectively displaces [3H]dopamine, pamine receptors. [3H]NPA, and [3H]Spi from striatal membrane sites and has a Thermal Exposure of Striatal Membranes. Thermal ex- higher affinity for [3H]dopamine binding sites and a lower posure of striatal membranes decreases the number of binding affinity for [3H]Spi binding sites than does bromocriptine. sites for dopamine agonists, but not for antagonists (23). We [3H]NPA appears to label with approximately equal affinity have therefore determined the effects of thermal exposure of agonist and antagonist receptor sites. The affinities of pergolide, bovine striatal membranes on the binding of mixed dopamine dopamine, and bromocriptine for [3H]NPA sites are about the agonists and antagonists such as pergolide and bromocriptine. same (Table 1). The capacity of pergolide to displace [3H]Spi like that of do- Inhibition of GTP. Because the binding of dopamine ago- pamine agonists is reduced in thermally exposed membranes nists, but not of antagonists, is inhibited by GTP (21, 22), we from ICWo = 83.2 + 4.9 in control membranes to IC50 = 355.0 have determined the effects of this nucleotide on the binding i 21.0. Displacement by bromocriptine is not altered (ICso = affinity of pergolide and of bromocriptine. Table 2 shows that 3.2 + 0.2 for control membranes, 2.8 + 0.2 for thermal exposed GTP decreases the capacity of Pergolide, but not of bromo- membrane). Pergolide therefore interacts with thermolabile criptine, to displace [3H]Spi from bovine striatal membrane sites receptors that have high affinity for dopamine agonists (23). and causes a significant increase in the inhibition constant for pergolide and apomorphine, but not for bromocriptine or Spi. Table 4. Stimulation of rat striatal adenylate cyclase These results indicate that [3H]Spi binds to multiple striatal pergolide membrane sites and one of them is sensitive to regulation by activity by guanine nucleotides. Pergolide, but not bromocriptine, interacts Increment over basal activity with the GTP-sensitive site. due to pergolide, Effect of Decortication. Evidence has been presented that Pergolide, pmol cAMP/mg protein dopamine receptors with high affinity for antagonists are lo- AM -GTP +GTP

calized on corticostriatal neurons and those linked to adenylate 0.3 41 ± 10 (3) 62 10 (3) 1.0 110± 43 (4) 181 + 53 (4) Table 2. Effect of GTP on the affinity of pergolide and 3.0 122 10 (4) 152 + 4 (4) bromocriptine for bovine striatal [3H]Spi binding sites 6.0 114 11 (2) 164 + 29 (2) 10.0 75 20 (4) 89 + 72 (4) K;, nM 100.0 (Inhibitory) (4) 32 (1) Drug Control +GTP Values represent mean increments in adenylate cyclase activity Pergolide 34.2 ± 2.5 170.0 ± 22.0 above basal activity measured without pergolide. Numbers ofseparate Bromocriptine 1.8 ± 0.2 2.1 I 0.3 experiments carried out on different days are given in parentheses. Apomorphine 41.0 ± 3.8 168.0 ± 25.0 Triplicate determinations were carried out. Basal activities in the Spi 0.30 0.05 0.36 ± 0.05 absence of GTP and in the presence of 1 ,M GTP were, respectively, 269 + 16 and 230 + 23 pmol of cAMP per mg of protein (n = 4). In- Various concentrations of each drug in the presence and absence crement over basal value without GTP due to 10)M dopamine (a of 50MM GTP were added to tubes containing [3H]Spi (0.5 nM). The maximally effective concentration) was 370 + 81 pmol of cAMP per values are the mean + SEM from three experiments. mg of protein (n = 4). Downloaded by guest on October 1, 2021 Neurobiology: Goldstein et al. Proc. Natl. Acad. Sci. USA 77 (1980) 3727

Table 5. Effect of pergolide on turning behavior in rats with (26), but it remains to be determined whether this is a direct unilateral 6-OH-dopamine lesion of the nigrostriatal dopamine effect. neurons and on relief of tremor in monkeys with VMT lesions Evidence has been presented that GTP regulates the coupling Dose, Rotation Relief of between receptors and adenylate cyclase. mg/kg time,* hr tremor,t hr Thus, GTP in the presence of pergolide may interact with the coupling subunit and activate dopamine-sensitive adenylate 0.05 4-8 cyclase activity in cell-free preparations. Indeed, the results of 0.1 8-12 3-5 our study show that the stimulation of adenylate cyclase by 0.2 >24 5-8 pergolide is enhanced in the presence of GTP. 0.5 12-24 The most striking features of pergolide are its potency and 1.0 >72 its long-lasting action. Pergolide relieves tremor in monkeys * Twelve rats were tested in four separate groups. Pergolide elicited with VMT lesions and induces turning behavior in rats with rotation in all rats (28-36 turns per 3 min per rat). 6-OH-dopamine lesions of the nigrostriatal dopamine neurons t Three monkeys with VMT lesions were used. Recordings of for a longer time, at doses 1/10th to 1/20th of those of other were obtained on an electroencephalograph with a transducer, and dopaminergic ergot compounds. Furthermore, a single-dose the tremograms were quantitatively analyzed by integration of the administration of pergolide in humans results in an inhibition amplitudes per unit time and determination of the frequency. of secretion that persists for more than 24 hr (10). The long-lasting action of pergolide indicates that the drug is tightly Adenylate Cyclase Activity. Pergolide was found to stim- bound to dopamine receptors or that the concentration in the ulate directly the adenylate cyclase activity of rat striatal ho- vicinity of the receptors is maintained. mogenates (Table 4). In the absence of GTP it produced a rel- Because the ultimate test for the utility of a dopamine agonist atively small but significant increase in activity, with maximal is its anti-parkinsonism efficacy in humans, it was of consid- stimulation at 1 MM. At higher concentrations this stimulatory erable interest to evaluate this drug in parkinsonism. In pre- effect was reversed, and at 100MM pergolide actually inhibited liminary clinical trials, pergolide was found to be effective in enzyme activity. In the presence of 1 ,M GTP, a concentration patients with advanced parkinsonism (27). Particularly, per- that did not significantly change basal cyclase activity or do- golide was effective in attenuating the L-dopa-induced "on- pamine-sensitive cyclase activity, stimulation by pergolide was off" phenomena (oscillation in performance) by increasing the enhanced, with a maximal increase of 79% obtained at 1 MM. time in which the patient could function actively. The stimulatory effect of 3 MM pergolide was completely an- It is assumed that decreased buffer capacity produced by tagonized by an equimolar concentration of the dopamine progressive loss of nigrostriatal dopamine neurons in parkin- antagonist haloperidol, but not by , an antagonist at sonism contributes to the "on-off" effect during treatment with sertonergic receptors. This implies that pergolide acts in a L-dopa. These "on-off" phenomena are one of the major ad- specific manner, and that this interaction is most likely with a verse effects occurring during L-dopa treatment. Thus, a potent dopaminergic receptor. dopamine agonist, such as pergolide, that has long duration of Dopamine agonist activity in vivo action and that does not require intact nigrostriatal dopamine neurons for its action should produce minimal fluctuation in Dopamine agonist activity of pergolide was evaluated in two performance. At present, therefore, pergolide seems to be a animal models that simulate parkinsonism. The results are promising anti-parkinsonism agent. summarized in Table 5. In rats with unilateral 6-OH-dopamine After long-term treatment of rats (34 weeks) with pergolide, lesions of the nigrostriatal dopamine pathway, intraperitoneal there is a significant decrease in the maximum number of administration of pergolide produces dose-dependent and [3H]Spi binding sites (9). These findings raise the question of long-lasting rotation contralateral to the lesion. At a dose of 0.05 the long-range efficacy of pergolide in parkinsonism. The mg/kg, turning behavior persists up to 8 hr and at a dose of 0.2 biochemical studies reported here indicate that pergolide has mg/kg up to at least 24 hr. Administration of haloperidol de- a spectrum of action different from other dopaminergic ergots. creases the induced turning behavior in a dose-dependent It will therefore be of interest to determine which of these manner. characteristics are responsible for its therapeutic efficacy and In monkeys with VMT lesions, the intraperitoneal adminis- its propensity to develop undesirable side effects, such as AIM tration of pergolide results in a relief of tremor. Immediately or mental changes (27). after administration tremor disappears for several hours, and at a higher dose (0.2 mg/kg) longer than 24 hr. The relief of A portion of this work was carried out by M.R.R. in partial fulfill- tremor is concomitant with the appearance of AIM, which ment of the requirements for Doctor of Philosophy degree. This work persist for almost the entire tremor-free period. was supported by National Institute of Mental Health Grants 02717 and 31773 and by National Institute of Neurological Diseases and DISCUSSION Stroke Grants 06801 and 09649. The findings that GTP inhibits more effectively the displace- 1. Ludin, H. P. & Bass-Verrey, F. (1976) J. Neural Transm. 38, ment of [3H]Spi by pergolide in decorticated striatal membrane 249-258. suggests that pergolide interacts with intrastriatal dopamine 2. Marsden, C. D. & Parkes, J. C. (1977) Lancet i, 345-349. receptors that are linked to dopamine-sensitive adenylate cy- 3. Fuxe, K., Fredholm, B. B., Ogren, S. O., Agnati, L. F., Hokfelt, clase. This idea is further corroborated with our findings that T. & Gustafsson, J. A. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. pergolide directly stimulates dopamine-sensitive adenylate 37,2181-91. cyclase in cell-free preparations. Thus, pergolide acts differently 4. Goldstein, M., Lew, J. Y., Nakamura, S., Battista, A. F., Lieber- man, A. & Fuxe, K. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. from other dopaminergic ergot compounds such as bromo- 37,78-82. criptine, , or (24, 25), which have been found 5. Lieberman, A., Miyamoto, T., Battista, A. F. & Goldstein, M. to be ineffective in stimulating adenylate cyclase in striatal (1975) Neurology 25, 459-462. homogenates. More recently, it was reported that bromocriptine 6. Calne, D. B., Teychenne, P. F. & Leigh, P. N. (1974) Lancet ii, stimulates dopamine-sensitive adenylate cyclase in striatal slices 1355-1356. Downloaded by guest on October 1, 2021 3728 Neurobiology: Goldstein et al. Proc. Natl. Acad. Sci. USA 77 (1980)

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