Colleen A. Drew

Bicuculline-insensitive GABA receptors: Studies on the binding of (−)-baclofen to rat cerebellar membranes

The binding of [3H](-)-baclofen to synaptic membranes prepared from rat cerebellum was studied. Consistent with pharmacological data that (-)-baclofen is the more active stereoisomer, studies on the binding of [3H](-)-baclofen showed increased specific binding, a higher affinity Kd and a lower Bmax than equivalent studies using [3H](+/-)-baclofen. Divalent metal ions (mercury, lead, calcium and zinc) inhibited the binding of [3H](-)-baclofen. The effects of some conformationally restricted analogues of gamma-aminobutyric acid (GABA) on [3H](-)-baclofen binding indicated that GABA interacts with (-)-baclofen-sensitive binding sites (GABAB) in extended rather than folded conformations, and that folded analogues of GABA may interact with a class of binding site (GABAc?) insensitive to (-)-baclofen and bicuculline.

Bicuculline‐ and Baclofen‐Insensitive γ‐Aminobutyric Acid Binding to Rat Cerebellar Membranes

Abstract: Up to 60% of γ‐[3H]aminobutyric acid ([3H]GABA) bound specifically to rat cerebellar membranes in the absence of Ca2+ was insensitive to the GABAA antagonist bicuculline and to the GABAB agonist baclofen. This indicates that a significant component of specifically bound [3H]GABA is associated with non‐GABAA, non‐GABAB binding sites. The presence of this binding component appeared seasonal, peaking in the month of September (early spring) each year over a 4‐year period. The calcium independence and bicuculline and baclofen insensitivity of the binding indicate that this binding is not to the classical GABAA and GABAB binding sites. High concentrations of muscimol and isoguvacine inhibited non‐GABAA, non‐GABAB binding. Scatchard analysis of the non‐GABAA, non‐GABAB binding sites indicated two kinetic components: KDI= 42 nM and KD2= 9.2 μM; Bmaxi= 1.6 pmol/mg of protein and Bmax2= 28 pmol/mg of protein.

A new synthesis resolution and in vitro activities of (R)- and (S)-β-Phenyl-Gaba

Abstract β-Phenyl-GABA (2) was resolved by separation by crystallization and/or h.p.l.c. of the diastereoisomeric (R)-(-)-pantolactone esters of the N-phthalimido protected β-phenyl-GABA. The absolute stereochemical assignments obtained from chiroptical studies of the enantiomers(8a) and(8b) and an X-ray crystallographic study of the diastereoisomer(7a) were supported by the activities of the enantiomers(8a) and(8b) in binding and electrophysiological studies. Details of synthesis, binding, electrophysiological, chiroptical and X-ray crystallographic studies are reported.

Inhibition of high affinity l-glutamic acid uptake into rat cortical synaptosomes by the conformationally restricted analogue of glutamic acid, cis-1-aminocyclobutane-1,3-dicarboxylic acid

The action of two cyclobutane derivatives of L-glutamic acid on the high affinity uptake of L-glutamic acid was investigated using a preparation of synaptosomes from rat cerebral cortex. cis-1-Aminocyclobutane-1,3-dicarboxylic acid (also known as trans-2,4-methanoglutamic acid) potently inhibited L-glutamic acid uptake (IC50 30 microM), whereas trans-1-aminocyclobutane-1,3-dicarboxylic acid (also known as cis-2,4-methanoglutamic acid), a potent N-methyl-D-aspartate (NMDA) agonist, was inactive. Analysis of the kinetics of L-glutamic acid uptake in the presence and absence of cis-1-aminocyclobutane-1,3-dicarboxylic acid (CACB) suggests that it may act as a competitive inhibitor (Ki 8 microM). CACB may be substrate for the L-glutamic acid high-affinity uptake carrier since preincubation of CACB with the synaptosomal preparation increased its potency in inhibiting L-glutamic acid uptake. The conformationally restricted structure of CACB may be indicative of the conformations of L-glutamic acid that interact with the high affinity uptake carrier.

Inhibition of baclofen binding to rat cerebellar membranes by phaclofen, saclofen, 3-aminopropylphosphonic acid and related GABAB receptor antagonists.

The inhibition of the binding of the GABAB agonist [3H](-)-baclofen to rat cerebellar membranes by some sulfonic and phosphonic acid analogues of GABA has been studied. These analogues have been shown to act as GABAB antagonists in the rat cortical wedge and the guinea-pig isolated ileum preparations. The order of potency of phaclofen (IC50 118 microM), 2-hydroxysaclofen (IC50 5.1 microM) and saclofen (IC50 7.8 microM) as inhibitors of [3H](-)-baclofen binding was similar to the order of potency of these compounds as GABAB antagonists, whereas 3-aminopropylphosphonic acid (IC50 1.5 microM) and 4-aminobutyl-phosphonic acid (IC50 3.9 microM) were much more potent than anticipated from their relatively weak GABAB antagonist actions. These results indicate that inhibition of [3H](-)-baclofen binding to rat cerebellar membranes does not reflect antagonist activity at GABAB receptors seen in the rat cortical wedge preparation or the guinea-pig isolated ileum preparation. This may indicate a heterogeneity of GABAB binding and receptor sites.

Acute effects of lead at central synapses in vitro

The acute effects of lead in the rat CNS in vitro were studied on synaptic transmission in the isolated hemisected spinal cord from newborn rats and on the transport of exogenous GABA, acetylcholine and cis-3-aminocyclohexane carboxylic acid (ACHC) from slices of cerebral cortex from adult rats. Lead had quite variable effects on monosynaptic reflexes and synaptic potentials. When it occurred, the depression of synaptic transmission by lead (typically at 18.5 mumol/liters of added lead acetate) was reversible provided exposure times were less than 15 min; furthermore, depression could be antagonised by increasing the external calcium concentration. Lead had no effect on the postsynaptic responses of motoneurons to the putative transmitters L-glutamate, GABA and glycine or to eledoisin-related peptide. The effects of lead on uptake and release of exogenous GABA and ACHC were dependent on the perfusion buffer employed: minimal effects were seen in solutions buffered with either phosphate or carbonate. When Tris HCl was used as buffer, lead inhibited the uptake of GABA and potentiated the spontaneous release of GABA with an EC50 = 50 mumol/liters as added lead acetate. In Tris HCl buffer, lead acetate (100 mumol/liters) produced a two-fold enhancement in the spontaneous release of acetylcholine under conditions where choline and acetylcholine re-uptake was blocked by hemicholinium. The availability of free lead cations in solution is highly dependent on the concentrations of other ions (particularly phosphates) and the pH. Under the appropriate conditions, lead can inhibit CNS synaptic function acutely in a manner consistent with lead competing with calcium ions in transmitter release processes as has been established for acetylcholine release at peripheral synapses.

Effects of lead salts on the uptake, release, and binding of γ-aminobutyric acid: the importance of buffer composition

Abstract: The effects of lead on the uptake and release of γ‐[3H]aminobutyric acid ([3H]GABA) from rat brain slices were examined in solutions buffered with Tris‐HCl, sodium phosphate, and sodium bicarbonate. Lead acetate (10–250 μM) inhibited uptake and potassium‐stimulated release and facilitated spontaneous efflux only in solutions buffered with Tris‐HCl. Calcium‐independent binding of [3H]GABA was unaffected by lead acetate (1–100 μM) in Tris‐citrate buffer but was significantly inhibited by 3 μM lead acetate in Tris‐HCl solution. At the rat soleus neuromuscular junction, lead caused a dose‐dependent reduction of end‐plate potential amplitude at concentrations of 10–100 μM lead acetate in HEPES‐buffered solution but had no effect at these concentrations in phosphate‐buffered solution. Stability constants of lead complexes indicate that buffers containing carbonate and phosphate are unlikely to contain a significant concentration of Pb2+, as complexing by these anions would reduce the availability of free Pb2+. This study indicates that the choice of buffer is important when investigating the effects of lead on biological systems and that negative findings may result from the use of inappropriate buffers. It also has important clinical implications suggesting that some effects of lead poisoning may result from its ability to affect neurotransmitter systems directly and that local changes in pH and complexing anion concentrations in the CNS may influence its biological availability and, hence, variable biological responses.

Thioether analogues of baclofen, phaclofen and saclofen

Analogues of baclofen, phaclofen and saclofen, incorporating a sulfur atom within the methylene chain, have been tested against responses induced by baclofen for activity at gamma-aminobutyric acid-B (GABAB) receptor sites, using a number of preparations including the guinea-pig isolated ileum and vas deferens, rat brain cortical slices and displacement of (-)-[3H]baclofen in rat cerebellar membranes. Results indicate that 2-([2-amino-1-(4-chlorophenyl)ethyl]thio)ethanephosphonic acid 2d is the most active of the new compounds. 2d is some 2-5 times weaker than phaclofen as a GABAB antagonist and approximately half as potent as phaclofen as an inhibitor of GABAB binding.

Effect of chronic exposure to lead on GABA binding in developing rat brain

The effect of chronic treatment with lead on the growth and the development of calcium-dependent GABA(B) (forebrain and cerebellum) and calcium-independent GABA(A) binding (forebrain), at 10, 21 and 84 days post-natally (PN), was studied in rats whose dams received either lead acetate (7 gl(?1), 18.5 mM) or sodium acetate (controls) in their drinking water from conception to weaning and normal animals given only water. The body and brain weight of lead treated pups was significantly less than that of normal and control pups. Chronic lead treatment produced receptor specific effects on binding at different times during development. In the forebrain GABA(A) binding was reduced at PN 10 (decreased affinity and density) but showed increased receptor density at PN 84. GABA(B) binding, while reduced during the developmental period (increased affinity and decreased density), appeared to have recovered at maturity. The development of cerebellar calcium-dependent binding was more variable in showing an increase in affinity in adult animals and decreased density at PN 10 and 84. There was no difference in normal and control binding except at PN 10 for both GABA(A) and GABA(B) binding. Chronic lead treatment affects calcium-dependent and calcium-independent binding differently suggesting that within the period of greatest vulnerability to the effects of lead, development in different receptor systems may be affected in quite different ways according to the role of calcium in their normal function.

GABA receptors and related binding sites

GABA (4-aminobutanoic acid, γ-aminobutyric acid) may be the most important inhibitory synaptic transmitter in mammalian brain. Although it possesses a relatively simple molecular structure, GABA appears to selectively activate a number of structurally specific receptors which are linked to a variety of sites that interact with certain classes of centrally active drugs such as barbiturates and benzodiazepines. GABA receptors may be part of complex domains consisting of specific binding sites for transmitters and modulators, channels for specific ions and associated gating apparatus, and enzyme complexes. The behaviour of GABA receptors is likely to be highly dependent on the local environment and this may explain many of the apparent differences between GABA receptor properties studied in situ by electrophysiological means and those studied in vitro by ligand binding techniques.