MIXED ANTAGONISTIC EFFECTS OF BILOBALIDE AT  1 GABA C RECEPTOR S. H. HUANG, a R. K. DUKE, a,b * M. CHEBIB, b K. SASAKI, c K. WADA c AND G. A. R. JOHNSTON a a Adrien Albert Laboratory of Medicinal Chemistry, Department of Phar- macology D06, Faculty of Medicine, The University of Sydney, NSW 2006, Australia b Pharmaceutical Chemistry, Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia c Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Kanazawa 1757, Ishikari- Tobetsu, Hokkaido 061-0293, Japan Abstract—Bilobalide was found to be a moderately potent antagonist with a weak use-dependent effect at recombinant human  1 GABA C receptors expressed in Xenopus oocytes using two-electrode voltage clamp methodology. Antago- nism of bilobalide at homomeric  1 GABA C receptors ap- peared to be mixed. At low concentration, bilobalide (3 M) caused a parallel right shift and surmountable GABA maxi- mal response of the GABA dose-response curve character- istic of a competitive antagonist. At high concentrations, bilobalide (10 –100 M) caused nonparallel right shifts and reduced maximal GABA responses of GABA dose-response curves characteristic of a noncompetitive antagonist. The potency of bilobalide appears to be dependent on the con- centrations of GABA and was more potent at lower GABA concentrations. The mechanism of action of bilobalide at  1 GABA C receptors appears to be similar to that of the chloride channel blocker picrotoxinin. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: picrotoxinin, bilobalide, mixed antagonist, use- dependent action, GABA C receptors, two-electrode voltage clamp. GABA, the most ubiquitously distributed inhibitory neuro- transmitter in the mammalian CNS, mediates fast neuro- transmission via ionotropic GABA receptors, termed GABA A and GABA C receptors. GABA C receptors were identified from their insensitivity to the classical GABA A competitive antag- onist bicuculline, as well as the GABA B agonist baclofen (Drew et al., 1984). GABA C receptors also differ from GABA A receptors in that they are relatively insensitive to classical GABA A modulators such as benzodiazepines, barbiturates and neurosteroids (Johnston, 1996; Qian and Ripps, 2001) and are competitively inhibited by (1,2,5,6- tetrahydropyridine-4-yl)-methyl-phosphinic acid (TPMPA) (Muratra et al., 1996; Ragozzino et al., 1996; Duke et al., 2000; Johnston, 2002). GABA C receptors are generally made up of  subunits (Kusama et al., 1993a; Zhang et al., 1995) forming func- tional homomeric receptors with  1 or  2 subunits (Kusama et al., 1993a,b), or heteromeric receptors with a mixture of  1 and  2 subunits (Enz and Cutting, 1999; Zhang and Pan, 2001; Johnston, 2002). More recent evidence indi- cates that the GABA C  1 subunit can also co-assemble with GABA A  1 and  2 subunits (Qian and Ripps, 1999; Pan et al., 2000; Milligan et al., 2004). Furthermore, het- eromeric receptors coexpressed by the  1 and the  2 subunits in Xenopus oocytes have been shown to display response properties very similar to those of the native GABA C receptors on retinal bipolar cells (Qian and Ripps, 1999). GABA C receptors are distributed throughout the CNS and are enriched mainly on neurons of the vertebrate retina (Qian and Ripps, 2001) and less abundant in cere- bellum (Drew et al., 1984), spinal cord (Johnston et al., 1975) and hippocampus (Strata and Cherubini, 1994). A range of CNS functions including vision, sleep, cognition and memory is believed to involve GABA C receptors (Johnston et al., 2003). Like the GABA A receptor, the GABA C receptor is con- sidered to be made up of five protein subunits arranged around a central channel axis that is directly gated by GABA. The channel of both GABA A and GABA C receptors is blocked by the plant convulsant picrotoxinin (Fig. 1, Macdonald and Olsen, 1994; Zhorov and Bregestovski, 2000). Picrotoxinin is a potent noncompetitive antagonist of GABA A receptors (Newland and Cull-Candy, 1992; Ca- sida, 1993; Yoon et al., 1993), and a mixed antagonist of lobster muscle GABA receptors (Constanti, 1978; Smart and Constanti, 1986) and GABA C receptors (Woodward et al., 1992; Qian and Dowling, 1994; Wang et al., 1995; Zhang et al., 1995; Qian et al., 2005). Picrotoxinin exhib- ited characteristic features of noncompetitive and (appar- ent) competitive antagonism at GABA C receptors. Picro- toxinin competitive antagonism at GABA C receptors is con- sidered to be allosteric (Qian and Dowling, 1994; Qian et al., 2005) as its site of action is distinct from the agonist binding site of GABA C receptors (Wang et al., 1995; Sedelnikova et al., 2005). Bilobalide (Fig. 1) is one of the active constituents of the Ginkgo biloba leaf extract. The ginkgo leaves have long been used in traditional Chinese remedies and the extracts of its leaves are currently being utilized widely for *Correspondence to: R. Duke, Department of Pharmacology D06, The University of Sydney, NSW 2006, Australia. Tel: +61-2-9351-6204; fax: +61-2-9351-3868. E-mail address: rujeek@pharmacol.usyd.edu.au (R. Duke). Abbreviations: DMSO, dimethyl sulfoxide; EC n , effective doses that evoked n% of maximal or control current generated by GABA for each individual cell; I, peak amplitude of current at a given dose of agonist or agonist/antagonist; IC 50 , effective doses that inhibited 50% of maximal or control current generated by GABA for each individual cell; I max , maximal or control current generated by GABA for each individual cell; n H , Hill coefficient; S.E.M., standard error of the mean; TPMPA, (1,2,5,6-tetrahy- dropyridine-4-yl)-methyl-phosphinic acid. Neuroscience 137 (2006) 607– 617 0306-4522/06$30.00+0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.08.071 607