The existence of a receptor for γ-aminobutyric acid (GABA) on neurones of the mammalian central nervous system (CNS) is now firmly established1–3. It is generally accepted that bicuculline (and its methohalide salts) is an antagonist of the actions of GABA4,5, although resistance to bicuculline has been described6,7. The view that bicuculline prevents GABA from interacting with a membrane recognition site is supported by results obtained in radiolabelled ligand binding studies8,9. Bicuculline-sensitive GABA receptors are not confined to neurones in the CNS but are also present on neurones and axons of the peripheral nervous system10,11. Their existence on neurones in sympathetic ganglia made us consider the possibility that they are also present on the terminals of such neurones. This has recently been tested12,13 by assuming that if GABA depolarises the terminals in a manner similar to the cell bodies, evoked transmitter output might be decreased (see ref. 14). GABA (ED50, 4µM) clearly decreased the evoked release of accumulated 3H-noradrenaline from rat atria in vitro and 3H-acetylcholine from preganglionic terminals in the rat superior cervical ganglion in vitro without affecting the basal release of tritium. In neither system was the effect of GABA antagonised by bicuculline methobromide, even though the ganglion terminal depolarisation was13. This suggested that the two phenomena, depolarisation and inhibition of transmitter release, were separate. The decrease in transmitter release not surprisingly leads to a decrease in the postsynaptic response15. Again, the decrease in response was not prevented by bicuculline or other GABA antagonists. We believe that these results indicate the presence of a novel GABA receptor on nerve terminals, a theory supported by results obtained with a variety of GABA analogues. For example, 3-aminopropane sulphonic acid (3-APS) which is at least as active as GABA at bicuculline sensitive sites10,16,17 is inactive at the terminal receptor. By contrast, the analogue baclofen (β-chlorophenyl GABA) is inactive at bicuculline-sensitive sites18,19 but is as active as GABA in reducing evoked transmitter output15. This effect of baclofen is stereospecific, the (−) isomer being > 100-fold more active than the (+) isomer15. We now report the presence of the novel GABA receptor within the mammalian CNS.

Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor

The presence of a novel receptor for the neurotransmitter gamma-aminobutyric acid (GABA) on peripheral autonomic nerve terminals and in mammalian brain slices has been described recently. This receptor differs from the classical GABA site as it is unaffected by recognized GABA antagonists such as bicuculline and is not sensitive to the majority of accepted GABA-mimetics such as 3-aminopropanesulphonic acid (3-APS) or isoguvacine. We propose to designate the classical site as the GABA A and the novel site as the GABA B receptor. The beta-p-chlorophenyl derivative of GABA, baclofen, is stereospecifically active at the GABA B site whereas it is devoid of activity at the classical GABA A3 site. We now report that high-affinity saturable binding of 3H-baclofen and 3H-GABA to the GABA B site can be detected in fragments of crude synaptic membranes prepared from rat brain. The results support the concept of a novel GABA receptor within the mammalian brain and show that GABA and baclofen can compete for the same recognition site.

3 H-baclofen and 3 H-GABA bind to bicuculline-insensitive GABA B sites in rat brain

I. Introduction 1485II. Scope of This Review 1485III. Transformations of Cyclobutane Derivatives inOrganic Syntheses1486A. Ring-Opening Reactions 1486B. Ring-Contraction Reactions 1493C. Ring-Expansion Reactions 14951. Five-Membered Rings 14952. Six-Membered Rings 15093. Seven-Membered Rings 15174. Eight-Membered Rings 15215. Nine-Membered Rings 1523IV. Transformations of Cyclobutane Derivatives inNatural Product Syntheses1524A. Ring-Opening Reactions 1524B. Ring-Expansion Reactions 15261. Five-Membered Rings 15262. Six-Membered Rings 15293. Seven-Membered Rings 15324. Eight-Membered Rings 1533V. Conclusion 1534VI. Acknowledgments 1534VII. References 1534

The application of cyclobutane derivatives in organic synthesis.

Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals.

1 Saturable binding of (+/-)-[3H]-baclofen and [3H]-gamma- aminobutyric acid ([3H]-GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 2 The binding of [3H]-baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+ -dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. 3 The saturable portion of bound [3H]-baclofen formed 20.3 +/- 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 microM), (-)-baclofen (0.04 microM) and to a much lesser extent by (+)-baclofen (33 microM). Isoguvacine, piperidine-4-sulphonic acid and bicuculline methobromide were inactive (up to 100 microM) and muscimol was only weakly active (IC50 = 12.3 microM). 4 Saturable binding of [3H]-GABA increased on adding CaCl2 or MgSO4 (up to 2.5 mM and 5.0 mM respectively) to the Tris-HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline-sensitive binding of GABA (GABAA site binding) and could be completely displaced by (-)-baclofen (IC50 = 0.13 microM). 5 Increasing the Ca2+ concentration (0 to 2.5 mM) increased the binding capacity of the membranes without changing their affinity for the ligand. 6 The binding of [3H]-GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non-ionic detergent Triton-X-100 (0.05% v/v) reduced the binding capacity by 50%. 7 The pharmacological profile of displacers of [3H]-GABA from GABAB sites correlated well with that for [3H]-baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. 8 It is concluded that [3H]-baclofen or [3H]-GABA are both ligands for the same bicuculline-insensitive, divalent cation-dependent binding sites in the rat brain.

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Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes.

THERE is considerable evidence that Na+-independent C3H]GABA binding to synaptic membranes of mammalian brain involves interaction with GABA receptor sites (ZUKIN et al., 1974; ENNA & SNYDER, 1975; ENNA et al., 1977; HITZEMANN & LOH, 1978). Thus, measurement of Na+independent C3H]GABA binding provides a useful biochemical technique to ascertain directly in virro interaction of GABA analogues with the synaptic GABA receptor. In the present study, we have evaluated the relative potencies of a series of 8-substituted GABA derivatives in inhibiting C3H]GABA binding in synaptic membranes of rat brain. This research was undertaken to define the requirements of the substituents of the PC atom of the GABA molecule to retain affinity for the receptor sites and to achieve additional information on the mechanism of action of some GABA derivatives with central inhibitory activity, like P-phenyl-GABA (Phenygam) and fi-(p-chloropheny1)-GABA (Baclofen).

Inhibition of Na+-independent [3H]GABA binding to synaptic membranes of rat brain by beta-substituted GABA derivatives.

The thermal isomerisation of isoxazol-5-ylhydrazines (I) led to 1-aminopyrazolin-5-ones (II), 4-aminopyrazolin-5-ones (III), and 1,2,4-triazin-6-ones (IV), in ratios depending on the nature of the substituents and the solvents used. A reaction mechanism involving a bicyclic intermediate, is suggested on the basis of the behaviour of isoxazolyl hydrazines methylated on the hydrazine moiety towards heating. Heating the methylated isoxazolyl-hydrazines (IXg and h) afforded the corresponding azirinecarbohydrazides (XII).

Thermally Induced isomerisation of Isoxazol-5-ylhydrazines

1-Aminopyrazolin-5-ones: Synthesis and Reactivity

Hydrogenolysis of 1-aminopyrazoles: Synthesis of primary and secondary alkylamines †

The behaviour of 4-hydroxy-4-methylcyclopent-2-enone and related 4-substituted cyclopent-2-enones towards 1,3-dipolar cycloaddition with nitrile oxides is studied. The reactions are always completely regioselective while the diastereofacial selectivity depends on the nature of the substituents. Remarkably, the reaction of 4-acetoxycyclopent-2-enone shows complete regio- and diastereo-facial selectivity.

Giorgio Adembri

Papers

Inhibition of Na+-independent [3H]GABA binding to synaptic membranes of rat brain by beta-substituted GABA derivatives.

Thermally Induced isomerisation of Isoxazol-5-ylhydrazines

1-Aminopyrazolin-5-ones: Synthesis and Reactivity

Hydrogenolysis of 1-aminopyrazoles: Synthesis of primary and secondary alkylamines †

Regio- and diastereo-selectivity in 1,3-dipolar cycloadditions of nitrile oxides to 4-substituted cyclopent-2-enones