Lisuride hydrogen maleate: An ergoline with β-adrenergic antagonist activity

European Journal of Pharmacology, 59 (1979) 303--306 © Elsevier/North-Holland Biomedical Press

303

Short c o m m u n i c a t i o n LISURIDE HYDROGEN MALEATE: AN ERGOLINE WITH ~-ADRENERGIC ANTAGONIST ACTIVITY THOMAS COTE, MASAHIDE MUNEMURA and JOHN KEBABIAN Experimental Therapeutics Branch, National Institute of Neurological and Communicative Disorders and Stroke, Building 10, Room 61916, National Institutes of Health, Bethesda, Maryland, 20205, U.S.A.

Received 17 September 1979, accepted 20 September 1979

T. COTE, M. MUNEMURA and J. KEBABIAN, Lisuride hydrogen maleate: an ergoline with ~J-adrenergic antagonist activity, European J. Pharmacol. 59 (1979) 303--306. Lisuride hydrogen maleate is identified as a potent ~-adrenergic antagonist using a hormone-sensitive adenylate cyclase system and [3H]dihydroalprenolol binding in cell free homogenates of rabbit cerebellum. Lisuride and two other ergolines, lergotrile and bromocriptine, and the phenothiazine, fluphenazine, all interact with spiroperidol binding sites (dopamine receptors) in the anterior pituitary; however, among these compounds lisuride is unique in its ability to antagonize the ~-adrenoceptor.

Lisuride

~-Adrenoceptor

Adenylate cyclase

1. Introduction The ergolines, a class of semisynthetic ergot derivatives, interact with receptors for a number of monoamines (for a review see Berde and Schild, 1978}. Lisuride hydrogen maleate ( N - [ 6-methyl -8-(9-ero len yl) ] -N', N'-diethyl urea) is an ergoline which interacts with receptors for serotonin, dopamine, and noradrenaline (Berde and Schild, 1978; Pieri et al., 1978a, b; Kehr, 1978). The present communication demonstrates that lisuride is a potent, competitive antagonist of the fl-adrenoceptor in the rabbit cerebellum.

2. Materials and methods Drugs were obtained from the following sources: [3H]-dihydroalprenolol ([3H]-DHA), specific activity 46 Ci/mmol) and [3H]-spiroperidol {specific activity 23.6 Ci/mmol), New England Nuclear Corp; ATP, GTP, 1-isoproterenol bitartrate and d,l-propranolol, Sigma Chemical Co.. lergotrile methane sulfonate,

Dihydroalprenolol

Spiroperidol

Eli Lilly; fluphenazine hydrocl~loride and iisuride hydrogen maleate, Schering; bromocriptine, Sandoz. Other chemicals were obtained from commercial sources. Female New Zealand white rabbits were obtained from Dutchland Laboratory Animals. The rabbits were killed by injecting 60 ml of air into the ear vein; the brain was removed and the cerebellum was rapidly dissected. Homogenates of the cerebellar cortex were prepared; both adenylate cyclase activity and the binding of [3H]-DHA were assayed with previously described procedures (Cote and Kebabian, 1978). The procedure of Caron et al. {1978) was used to prepare membrane from frozen bovine anterior pituitary obtained from either a local abbatoir or Pel Freeze Biologicals. The binding assays were performed in a final volume of 500 pl containing Tri.'s-HC1, pH 7.4, 2 5 m M ; MgC12, 2 m M ; [3H]-spiroperidol, 1 nM; 1.5--2 mg of freshly prepared membrane protein, and the indicated compounds. The incubation was initiated with the addition of the pituitary membranes, and was conducted

304

for 30 min at 25°C with gentle shaking in metabolic incubator. 'Nonspecific' binding was estimated by including 2 pM fluphenazine in the assay system. The incubation was terminated with the addition of 0.1 ml of a suspension of Norit SGX charcoal (10 g/100 ml) and antifoam A concentrate (2 ml/100 ml) in a solution of 25 mM Tris HC1, pH 7.4, and 2 mM MgC12. The assay tubes were kept on ice for 10 min and subsequently centrifuged {1000 × g, 15 min, 4°C). Aliquots (300 pl) of the supernatant were added to 5 ml of Aquasol (New England Nuclear) and counted in a Packard PRIAS Liquid Scintillation Counter. The values shown represent the mean of triplicate observations, the S.E.M. was less than 5% of the mean, The amount of [aH]spiroperidol bound to the membranes ranged from 13% to 24% of the total radioactivity added to the assay system; nonspecific binding represented less than 10% of the radioactive material bound to the membranes. 'Specific' binding represents the difference between total and non-specific binding.

T. COTE ET AL. -T-I~

>_-E <

i

,

[

,

A ~-adrenoceptor regulates adenylate cyclase in rabbit cerebellum (Cote and Kebabian, 1978). Lisuride produces a concentration-dependent blockade of the isoproterenol~stimulated increase in cerebellar adenylate cyclase activity (fig. lA); a half-maximal decrease of the effect of 1-isoproterenol (10 pM) occurs in the presence of 0.7 pM lisuride. In the absence of isoproterenol, the ergoline has no effect upon enzyme activity. In the presence of lisuride (1 pM), higher concentrations of isoproterenol are required to stimulate enzyme activity (fig. 1B); this inhibitory effect of lisuride is overcome by concentrations 1-isoproterenol greater than 10 ttM. The results in fig. 1B are consistent with a kinetic competition between lisuride and isoproterenol for the /3-adrenoceptor regulating cerebellar adenylate cyclase. Using previously described methodology (Cote and

• 1~

,

25

c_

0-,'

W~15

j.. -~

D'

i

i

A

i

i

-8

-7

-6

-5

4

LOG LISLIRIDE (MI

~ r - 4~---

~,,p -¢o

L

A

x

-7

-6

-5

LOG ISOPROTERENOL

-4 (M)

.....

OE

~

25

.J

, 3. Results

[

3o

-

.oo

-8

-7

-6

LOG LISURIDE ( M I

Fig. 1. Blockade o f the ~-adrenoceptor in rabbit cerebellum by lisuride. Panel A. Adenylate cyclase activity was measured in the presence of the indicated c o n c e n t r a t i o n of lisuride in the absence (open circles) and presence (filled circles) of 10/.IM 1-isoproterenol. Panel B. Adenylate cyclase activity was measured in presence of the indicated c o n c e n t r a t i o n s of l-isoproterenol in the absence (open circles) and presence (filled circles) of I #M lisuride. The affinity (Ki) of the 13-adrenoceptor for lisuride was calculated from the relationship, Ki = i / ( K ~ / K M -- 1) where i is the concentration of lisuride (1 #M), and K h and K M are the concentrations of l-isoproterenol causing half maximal stimulation of e n z y m e activity in the presence and absence, respectively, o f lisuride. Panel C. Competition b e t w e e n lisuride and 3H-DHA for specific binding sites. The c o n c e n t r a t i o n of 3H-DHA was 3.0 nM, the c o n c e n t r a t i o n s of lisuride are indicated. Data points represent the m e a n of triplicate observations; the standard error of the mean was less than 8%. The affinity of lisuride for the specific 3H-DHA binding sites, 21 nM, is calculated from the relationship given in the caption of table 1 (the affinity of [ 3 H ] - D N A for the specific binding sites is 1.3 nM).

LISURIDE BLOCKS ~-ADRENOCEPTORS

305

TABLE 1 Effects o f dopaminergic drugs at spiroperidol binding sites and a ~-adrenergic receptor. The assays of 3H-spiroperidol binding and isoproterenol-stimulated adenylate cyclase were performed as indicated in Methods. The affinity of the drugs for each category of receptor was calculated from the relationship K! = (ICs0)(1 + S/KM) -l ; where IC50 is the concentration of drug producing a 50% decrease in the measured response, S is the concentration of ligand (1 nM 3H-spiroperidol, 10/~M isoproterenol) and Km is the affinity of ligand for the receptor (0.45 nM for spiroperidol, 0.4 pM for isoproterenol). Bromocriptine, fluphenazine, and lergotrile were inactive upon the ~-adrenoceptor at concentrations as great as (30 pM).

Drug

Affinity of drugs for a H-spiroperidol binding sites

ICs0 Lisuride Bromocriptine Fluphenazine Lergotrile

10 80 80 2000

~-Adrenoceptor regulatingcerebellar adenylate cyclase

Ki nM nM nM nM

3 25 25 621

nM nM nM nM

Kebabian, 1978); the affinity of the fl-adrenoceptor for lisuride can be calculated to be 45 nM. The ~-adrenoceptor found in the rabbit cerebellum can be identified with [aH]DHA (Cote and Kebabian, 1978). Like other drugs active upon the fl-adrenoceptor, lisuride competes effectively with [3H]-DHA for these specific binding sites {fig. 1C). The affinity of these specific binding sites for lisuride is 21 nM. Lisuride, like other ergolines and phenothiazines, is active upon dopamine receptors. In the anterior pituitary, specific binding sites for [3H]-spiroperidol have been identified as dopamine receptors (Creese et al., 1977). Table 1 shows that lisuride has a high affinity for specific [3H]-spiroperidol binding sites in anterior pituitary; two other ergolines, bromocriptine, and lergotrile, and the phenothiazine, fluphenazine, also display a high affinity for this binding site. In contrast, lisuride is unique among these four compounds in its ability to interact with the fl-receptor regulating adenylate cyclase; both of the other ergolines and the phenothiazine are inactive.

ICs0

Ki

300 nM Inactive Inactive Inactive

28 nM ----

4. Discussion The present study shows that lisuride is an antagonist of a ~-adrenoceptor in brain. The interaction between lisuride and the ~-adrenoceptor displays an affinity ranging from 21 nM to 45 nM in the different experimental protocols used in our studies. Thus, lisuride is approximately one tenth as potent as the ~-adrenergic antagonists, alprenolol or propranolol. The two other ergots tested in the present study were devoid of activity as antagonists of the ~-adrenoceptor. However, at least one other ergot, LSD, antagonizes the ~-adrenoceptor (Dolphin et al., 1978). Other pharmacological similarities between lisuride and LSD are: (a) their ability to inhibit the spontaneous firing of identified serotonergic neurons (Rogawski and Aghajanian, 1979); and (b) the ability of the two drugs to antagonize the dopamine receptor which stimulates adenylate cyclase (Da Prada et al., 1975; Pieri et al., 1978a). Lisuride interacts with a-adrenoceptors, dopamine receptors {both D-1 and D-2)and serotonin receptors, in addition to blocking

306

fi-adrenoceptors. The ability of lisuride to stimulate receptors for either dopamine or serotonin has been proposed to account for certain behavioral and biochemical effects of the drug (Pieri et al., 1978b). Similarly, the ability of lisuride to act as a 'blocker of noradrenaline receptors' {which may be -adrenoceptors) has been proposed to account for the lisuride-stimulated increase in 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (Pieri et al., 1978a). At present, there is no well
References Berde, B. and H.O. Schild, 1978, Ergot Alkaloids and Related Compounds (Springer, Heidelberg). Caron, M.G., M. Eaulieu, V. Raymond, B. Gagne, J. Drouin, R.J. Lefkowitz and F. Labrie, 1978, Dopaminergic receptors in the anterior pituitary, J. Biol. Chem. 253, 2244. Cote, T.E. and J.W. Kebabian, 1978, Beta-adrenergic

T. COTE ET AL. receptor in the brain: comparison of 3H-dihydroalperenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in ceUfree homogenates, Life Sci. 23, 1703. Creese, I., R. Schneider and S.H. Snyder, 1977, 3Hspiroperidol labels dopamine receptors in pituitary and brain, European J. Pharmacol. 46, 377. Da Prada, M., A. Saner, W.P. Burkard, G. Bartholini and A. Pletscher, 1975, Lysergic acid diethylamide: evidence for stimulation of cerebral dopamine receptors, Brain Res. 94, 67. Dolphin, A., A. Enjalbert, J-P. Tassin, M. Lucas and J. Bockaert, 1978, Direct interaction of LSD with central beta-adrenergic receptors, Life Sci. 22, 345. Kehr, W., 1978, Effect of lisuride and other ergot derivatives on monoaminergic mechanisms in rat brain, European J. Pharmacol. 41,261. Pieri, L., H.H. Keller, W. Burkard and M. Da Prada, 1978a, Effects of lisuride and LSD on cerebral monoamine systems and hallucinosis, Nature 272, 278. Pieri, M., R. Schaffner, L. Pieri, M. Da Prada and W. Haefely, 1978b, Turning in MFB-lesioned rats and antagonism of neuroleptic-induced catalepsy after lisuride and LSD, Life Sci. 22, 1615. Rogawski, M.A. and G.K. Aghajanian, 1979, Response of central monoaminergic neurons to lisuride: comparison with LSD, Life Sci. 24, 1289.