Pharmacological actions of oximino-propofol analogues at GABA(B) autoreceptors.

1. GABA(B) autoreceptors are a subclass of GABA(B) receptors that inhibit the release of [(3) H]GABA from GABAergic nerve terminals. Baclofen is an agonist that reduces [(3)H]GABA, whilst the antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911) enhances [(3)H]GABA release in electrically-stimulated rat neocortical brain slices preloaded with [(3)H]GABA. Here, the pharmacological actions of a series of compounds derived from the positive allosteric modulator, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930), were examined on GABA(B) autoreceptors. 2. The compound, 3-(3,5-ditbutyl-4-hydroxyphenyl)-2,2-dimethyl-1-oximinopropane (compound 2), at 10 µmol/L had little effect on the stimulation-induced overflow of [(3)H]GABA when superfused alone, but when superfused in the presence of baclofen (2 µmol/L) inhibited the overflow of [(3)H]GABA. These effects were reversed by Sch 50911 (10 µmol/L). Although compounds 1-(4-chlorophenyl)-3-(4-hydroxy-3,5-diisopropylphenyl)-2-methyl-1-oximinopropane (compound 1), 1-[(3,5-ditbutyl-4-hydroxyphenyl)methyl]-1-oximinomethylcyclohexane (compound 3), 3-(3,5-ditbutyl-4-hydroxyphenyl)-1,2-diphenyl-1-oximinopropane (compound 4) and 4-(3,5-ditbutyl-4-hydroxyphenyl)-3-methyl-2-oximinobutane (compound 5) (each at 10 µmol/L) tended to reduce the stimulation-induced overflow in the presence of baclofen, an effect reversed by Sch 50911, their status as modulators is not confirmed in the present study. 3. Another derivative, 3-(3,5-ditbutyl-4-hydroxyphenyl)-1-(4-chlorophenyl)-2-methyl-1-oximinopropane (compound 6) (10 µmol/L), acted as an agonist as it inhibited the release of [(3)H]GABA by 32% (EC(50) of 3.3 µmol/L), an effect reversed by Sch 50911 (10 µmol/L). The other compounds, 1-[(3,5-ditbutyl-4-hydroxyphenyl)methyl]-1-methyl-2-oximinocyclohexane (compound 7), 4-(3,5-ditbutyl-4-hydroxyphenyl)-3,3-dimethyl-2-oximinobutane (compound 8) and 4-(4-hydroxy-3,5-diisopropylphenyl)-3,3-dimethyl-2-oximinobutane (compound 9) (each at 10 µmol/L), were inactive. 4. These findings indicate that this series of compounds show different modes of activity at GABA(B) autoreceptors.

The CGP7930 analogue 2,6-di-tert-butyl-4-(3-hydroxy-2-spiropentylpropyl)-phenol (BSPP) potentiates baclofen action at GABA(B) autoreceptors.

The pharmacological actions of 2,6-di-tert-butyl-4-(3-hydroxy-2-spiropentylpropyl)-phenol (BSPP), a putative presynaptic GABA(B) receptor modulator, were examined in electrically stimulated rat neocortical brain slices preloaded with [3H]-GABA or [3H]-glutamic acid. At 10 mmol/L, BSPP inhibited the release of [3H]-GABA in the presence of baclofen, but not that of [3H]-glutamic acid. This effect was sensitive to the GABA(B) receptor antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911). Alone, BSPP had no effect on the release of [3H]-GABA or [3H]-glutamic acid. It is concluded that BSPP selectively potentiates the action of baclofen at GABA(B) autoreceptors, but not heteroreceptors and may be a useful ligand to discriminate between presynaptic GABA(B) receptor subtypes.

Uptake and release of [(3)H]GABA in human dental pulp.

The purpose of this study was to determine whether (a) an uptake system for gamma-aminobutyric acid (GABA) exists in human dental pulp, (b) GABA can be released from nerves in this tissue, and (c) GABA(B) autoreceptors modulate release of this transmitter. Segments of vital pulp were incubated in [(3)H]GABA (0.1-10 microM) for up to 120 min, washed, and the retained [(3)H] extracted and assayed. Some tissues were treated with GABA uptake inhibitors (nipecotic acid or NO-711) prior to incubation. At concentrations of 0.1 and 1.0 microM the uptake of [(3)H]GABA was saturated after 90 min of incubation. At 10 microM, at least two uptake compartments were apparent, and the amount of [(3)H]GABA retained was five-fold greater than 0.1 microM. The uptake inhibitors reduced [(3)H]GABA accumulation by more than 80%. In the release study, pulp was incubated in [(3)H]GABA (0.5 microM) for 90 min, and superfused with Krebs solution containing NO-711 (5 microM). Electrical stimulation increased the overflow of [(3)H]; a GABA(B) autoreceptor agonist (baclofen) inhibited, whilst an antagonist, Sch 50911, enhanced this release. The effects of baclofen were reversed by Sch 50911. These results imply that GABA can be taken up and bound firmly in compartments within human dental pulp, GABA can be released from isolated pulp segments by electrical stimulation, and this release is modulated by GABA(B) autoreceptors.

3-Chloro,4-methoxyfendiline is a potent GABA(B) receptor potentiator in rat neocortical slices.

Using grease-gap recording from rat neocortical slices, the GABA(B) receptor agonist baclofen elicited reversible and concentration-dependent hyperpolarizing responses (EC50=18+/-2.3 microM). The hyperpolarizations were antagonised by the GABA(B) receptor antagonist Sch 50911 [(+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid). (+)-N-1-(3-chloro-4-methoxyphenyl)ethyl-3,3-diphenylpropylamine (3-chloro,4-methoxyfendiline; 3-Cl,4-MeO-fendiline) reversibly potentiated baclofen-induced hyperpolarizing responses, which were reduced by Sch 50911, producing leftward shifts of the baclofen concentration-response curves, with a marked increase in the maximal hyperpolarization (EC50=2+/-0.5 microM). In slices preincubated with either [3H]GABA or [3H]glutamic acid, 3-Cl,4-MeO-fendiline (1 microM) potentiated the inhibitory effect of baclofen (2 microM) on the electrically evoked release of [3H]GABA and had a similar effect on the release of [3H]glutamic acid at a concentration of 0.5 microM, without affecting the basal release. These effects were blocked by Sch 50911 (10 microM). Our findings suggest that 3-Cl,4-MeO-fendiline is a potent potentiator of pre- and postsynaptic GABA(B) receptor-mediated functions.

Gabapentin activates presynaptic GABAB heteroreceptors in rat cortical slices.

In electrically stimulated rat neocortical brain slices preloaded with [3H]gamma-aminobutyric acid (GABA) or [3H]glutamic acid, the pharmacological actions of 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin, Gp) were compared with the GABAB receptor agonists baclofen (Bac) and (3-amino-2-(S)-hydroxypropyl)-methylphosphinic acid (CGP 44532). Gabapentin, baclofen and CGP 44532 all reduced the electrically stimulated release of [3H]glutamic acid (IC50=20 microM, 0.8 microM and 2 microM, respectively). These effects were sensitive to the GABAB receptor antagonists (+)-(S)-5,5 dimethylmorpholinyl-2-acetic acid (Sch 50911) or N-3-[[1-(S)-(3,4-dichlorophenyl)ethyl]amino]-2-(S)-hydroxypropyl-P-(cyclo-hexylmethyl)-phosphinic acid (CGP 54626). By contrast, gabapentin was without effect on the release of [3H]GABA, whilst baclofen (IC50=8 microM) and CGP 44532 (IC50=1 microM) inhibited [3H]GABA release. It is concluded that gabapentin selectively activates presynaptic GABAB heteroreceptors, but not GABAB autoreceptors, and may be a useful ligand to discriminate between presynaptic GABAB receptor subtypes.