(+)-Hydrastine, a Potent Competitive Antagonist at Mammalian GABAA Receptors
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,'-. 27-30© Pes Lt, 99 Macmillan Press Ltd, 1990 Br. Br.J.Phamaol.(190,J. Pharmacol. (1990), ", 727-730 (+)-Hydrastine, a potent competitive antagonist at mammalian GABAA receptors 'Jun-Hua Huang & 2Graham A.R. Johnston Department of Pharmacology, The University of Sydney, NSW, 2006, Australia 1 (+)-Hydrastine is a phthalide isoquinoline alkaloid, isolated from Corydalis stricta. It has the same 1S,9R configuration as the competitive GABAA receptor antagonist bicuculline and is the enantiomer of the commercially available (- )hydrastine. 2 (+)-Hydrastine (CD50 0.16mgkg-', i.v.) was twice as potent as bicuculline (CD50 0.32mgkg 1, i.v.) as a convulsant in mice. This action was stereoselective in that (+)-hydrastine was 180 times as potent as (-)-hydrastine. 3 (+)-Hydrastine was a selective antagonist at bicuculline-sensitive GABAA receptors in the guinea-pig isolated ileum. It did not influence phaclofen-sensitive GABAB receptors or acetylcholine receptors in this tissue. (+)-Hydrastine was a competitive antagonist of GABAA responses (pA2 6.5) more potent than bicuculline (pA2 6.1). 4 When tested against the binding of [3H]-muscimol to high affinity GABAA binding sites in rat brain membranes, (+)-hydrastine (IC50 2.37 yM) was 8 times more potent than bicuculline (IC5o 19.7 pM). 5 As an antagonist of the activation of low affinity GABAA receptors as measured by the stimulation by GABA of [3H]-diazepam binding to rat brain membranes, (+)-hydrastine (IC5o 0.4 gM) was more potent than bicuculline (IC50 2.3 pM). 6 (+)-Hydrastine, 10 nm to 1 mM, did not inhibit the binding of [3H]-(-)-baclofen to GABAB binding sites in rat brain membranes. Introduction Methods The discovery of bicuculline as an antagonist of certain inhibi- Convulsant potencies tory actions of 4-aminobutanoic acid (GABA) on central neu- rones provided vital pharmacological evidence for the role of Convulsant potencies were assessed on injection of aqueous neurotransmitter in the CNS (Curtis solutions of the alkaloids as hydrochlorides into the tail veins GABA as an inhibitory value et al., 1970). Mammalian GABA receptors are currently classi- of male mice weighing between 18 and 25g. The CD50 fied into at least two pharmacological classes: GABAA recep- was defined as the dose causing convulsive seizures in 50% of tors are antagonized by bicuculline and insensitive to mice within 1 min of injection. baclofen, whereas GABAB receptors are activated by baclofen, antagonized by phaclofen and insensitive to bicuculline (Hill Bowery, 1981; Johnston, 1986; Kerr et al., 1987). & 0 2N CH30 Bicuculline is a phthalide isoquinoline alkaloid first isolated 4 and subsequently from Dicentra cucullaria (Manske, 1932) 0 CH30 111CH3 from a variety of species of Corydalis, Dicentra and Adlumia HHHCH H H was (Kametani, 1969). Its convulsant action reported by 3. io0 Welch & Henderson (1934). Many isoquinoline alkaloids 6'3 produce convulsions on systemic administration to mammals, antagonism appears to be restricted to the phtha- 120 0 but GABA 0 0olmn (S R lide isoquinoline alkaloids that have the lS,9R configuration, i.e. bicuculline and corlumine (Figure 1, Curtis & Johnston, 1974). Thus (-)-bicuculline (1R,9S), (-)-hydrastine (1R,9S), Bicuculline (iS, 9R) Corlumine (iS, 9R) (-)-adlumine (lR,9R) and (+)-adlumine (lS,9S) are inactive as GABA antagonists (Curtis & Johnston, 1974). Bicuculline is more potent than the closely structurally 0 " related corlumine (Figure 1) as a convulsant and as a selective GABA antagonist (Rice, 1938; Johnston et al., 1972). The H H present study concerns (+)-hydrastine, a 1S,9R, configured 0 phthalide isoquinoline alkaloid structurally related to bicuculline and corlumine, which has been isolated from 0 Corydalis stricta (Fang et al., 1981) and is the enantiomer of CH30 ICH3 the commercially available (-)-hydrastine (Figure 1). (+)- CH30 OCH3 be more potent than bicuculline in 4 Hydrastine appears to (1R, 9S) test systems using 3 animal species. (+)-Hydrastine (1S, 9R) (-)-Hydrastine Figure 1 Structures of the phthalide isoquinoline alkaloids and showing 1 On leave from Department of Pharmacology, Institute of Materia bicuculline, corlumine, (+)-hydrastine (-)-hydrastine, China. the numbering system and indicating the absolute configurations Medica, Chinese Academy of Medical Sciences, 100050 Beijing, 1 2 Author for correspondence. about carbons and 9. 728 J.-H. HUANG & G.A.R. JOHNSTON Guinea-pig isolated ileum preparation a competitive antagonist for its receptor) were calculated from Clark plots (see Krantis & Kerr, 1981) and CD5o values (dose Guinea-pigs of either sex, weighing between 200-400g, were producing convulsions in 50% of animals) from log-probit stunned by a blow on the head, segments of the distal ileum analyses as described by Tallarida & Murray (1981). Values 3-4cm in length were quickly removed, emptied of their con- are expressed as means + standard error (s.e.). tents and mounted vertically in a 20 ml organ bath containing modified Krebs solution of following composition (mM): Na' 151.0, K+ 4.6, Mg2+ 0.6, Ca2+ 2.8, Cl- 134.9, HCO- 24.9, Results H2PO4 1.3, S02- 0.6, glucose 7.7 (pH 7.4 at 370C). The Krebs solution was continuously gassed with a mixture of 95% 02 and 5% CO2. The tissues were initially placed under a resting Convulsant potencies tension of 1 g and were allowed to equilibrate for 60 min in the bath. Isometric contractions of the longitudinal muscle were Bicuculline and (+)-hydrastine produced similar convulsions measured with a Grass FT03 force transducer and recorded in mice on intravenous injection. (+}Hydrastine was more on a Grass polygraph. Antagonists were added at least 2min potent than bicuculline on the basis of relative CD5o values: before agonists were tested at 8-15min intervals, depending 0.16 + 0.01mgkg-' and 0.32 + 0.01mgkg-1 respectively on the recovery of the tissue responses to base line level. (means + s.e., n = 10). The action of (+)-hydrastine was Added drug volumes never exceeded 2% of the bath volume stereoselective in that (-)-hydrastine was some 180 times less (Krantis & Kerr, 1981; Ong et al., 1988). potent (CD50 29.8 mg kg- 1) than (+ )-hydrastine. Ligand binding studies [3H]-muscimol, [3H]-diazepam, and [3H]-(-)-baclofen Guinea-pig ileal responses to GABA and acetylcholine Whole rat brain was homogenized in 1:10 (w/v) volumes of When GABA was applied to the guinea-pig isolated ileum, cold 0.32 M sucrose. The homogenate was centrifuged at it produced either contractions or biphasic responses 1000g for 10min. The sucrose supernatant was centrifuged at (transient contractions followed immediately by relaxation). 12,000 g for 20 min to yield a crude mitochondrial pellet which The GABA-induced contractions, which are known to be was subsequently processed in different ways for the different mediated by bicuculline-sensitive GABAA receptors, were ligand binding assays. antagonized in a dose-dependent manner by (+)-hydrastine, [3H]-diazepam binding was studied by a filtration assay which had no effect upon tissue activity when added to the essentially as described by Skerritt et al. (1982a): the well- bath alone. (+)-Hydrastine at doses of 1 and 5juM elicited a washed crude mitochondrial pellet was incubated for 20min parallel shift to the right in the dose-response curve for at 4°C in 1 ml containing 530 ug membrane protein, 50mM GABA-induced contractions (Figure 2). Clark analysis indi- Tris HCl buffer, 560pM [3H]-diazepam and test compounds. cated the pA2 value for (+)-hydrastine was 6.5 + 0.2 (0.31 gM) Nonspecific binding was determined by the addition of 100pM while the pA2 value for bicuculline was 6.12 + 0.14 (0.75 jM) unlabelled diazepam. (Krantis & Kerr, 1981). The pA2values showed that (+)- [3H]-muscimol binding was studied in a similar manner hydrastine is 2.4 times stronger than bicuculline in inhibiting using membranes extracted with 0.05% Triton X-100 Tris GABA-induced contractile responses in guinea-pig ileum. citrate buffer at 37°C for 30min. After thorough washing the (+)-Hydrastine at 1 gM did not show any effect on Triton treated membranes were incubated for 60 min at 4°C in acetylcholine-induced contractile responses in guinea-pig a volume of 1 ml containing 690 jg membrane protein, 50mM ileum or on relaxations mediated by GABAB receptors. Tris HCl buffer (pH 7.4), 1.47 nm [3H]-muscimol and test compounds. Nonspecific binding was determined by the addi- tion of 100 M unlabelled GABA. Muscimol binding to high affinity GABAA binding sites [3H]-(-)-baclofen binding was studied in a centrifugation on rat brain membranes assay as described by Drew et al. (1984). Protein for all of the receptor binding assays was measured Equilibrium binding assays revealed that (+)-hydrastine and by the method of Lowry et al. (1951). bicuculline inhibited [3H]-muscimol binding to rat brain membranes, whereas (-)-hydrastine was inactive (Figure 3). Materials The following IC50 values were determined by computer analyses: (+)-hydrastine 2.37 + 0.45 gM; bicuculline 19.7 (+)-Hydrastine, isolated from Corydalis stricta, was the gift of + 5.2juM. Scatchard analysis of the results of [3H]-muscimol Professor Fang Qi-Cheng in the Institute of Materia Medica, binding studies indicated binding to a single population of Chinese Academy of Medical Sciences, Beijing. Bicuculline high affinity sites with a Kd of 2.91 + 0.86nM and a B,,, of methochloride and phaclofen were synthesized in the Depart- 1.09 + 0.18 nmol mg- ' protein. ment of Pharmacology at the University of Sydney by Ms Christine Apostopoulos and