Michael A. Simmonds

Modulation of the GABA receptor complex by a steroid anaesthetic.

The interactions of a steroid anaesthetic, alphaxalone, with the GABA receptor-ionophore complex were investigated by two different experimental approaches. In the rat cuneate nucleus slice, alphaxalone (0.1-10 microM) potentiated depolarizing responses to superfused GABA and muscimol, but not those to glycine. The potentiating effect of alphaxalone was unaltered by the benzodiazepine antagonist Ro 15-1788. Alphaxalone (0.1-30 microM) also enhanced [3H]muscimol binding to rat brain membranes in the presence of Cl-ions; the enhancing effect on [3H]muscimol binding was abolished by Triton X-100. Analysis of binding curves for [3H]muscimol indicated that the steroid anaesthetic increases the affinity for [3H]muscimol of low affinity binding sites; this property is shared by pentobarbitone. The physiologically inactive beta-hydroxy isomer of the steroid was without activity in either of the experimental situations at 30 microM. It is suggested that alphaxalone and pentobarbitone share a common mode of action on the GABA system, which may be relevant to the mechanisms by which these drugs produce anaesthesia.

Effects of membrane cholesterol on the sensitivity of the GABAA receptor to GABA in acutely dissociated rat hippocampal neurones

The effects of membrane cholesterol on the GABA(A) receptor were investigated in acutely dissociated rat hippocampal neurones, using the whole-cell patch clamp technique. Neuronal cholesterol was manipulated within the range 56-250% control by incubation with methyl-beta-cyclodextrin for depletion and a complex of cholesterol and methyl-beta-cyclodextrin for enrichment. Manipulation over a narrower range was achieved with cholesterol + phosphatidylcholine liposomes. A complex of epicholesterol and methyl-beta-cyclodextrin was used to insert epicholesterol. Cholesterol enrichment reduced the potency of GABA, as did cholesterol depletion, with increases in EC(50) of up to 4-fold. Cholesterol enrichment reduced the potency of the competitive antagonist bicuculline but did not affect that of the non-competitive antagonist picrotoxinin. Cholesterol depletion did not affect the potencies of either antagonist. Epicholesterol substituted functionally for cholesterol with respect to the effects of enrichment. In cholesterol-depleted neurones, however, only incubation with cholesterol was able to restore GABA potency to normal. These results suggest a specific requirement for cholesterol at control levels to maintain optimal GABA potency, which may involve specific binding of cholesterol to the GABA(A) receptor. The reduction in GABA potency by enrichment with cholesterol or epicholesterol is more likely to be due to reduced plasma membrane fluidity.

A comparative study of some convulsant substances as γ‐aminobutyric acid antagonists in the feline cerebral cortex

1 By the use of microiontophoretic techniques, quantitative estimates were obtained of the depressant effects of γ‐aminobutyric acid (GABA) on single feline cortical neurones. 2 Picrotoxin, bicuculline, strychnine, (+)‐tubocurarine, penicillin and leptazol were also applied microiontophoretically to single neurones. Sequential GABA applications were made before, during and after the microiontophoresis of these substances and any effects on the time course of the GABA depression were measured as an estimate of antagonism or potentiation of GABA. 3 (+)‐Tubocurarine was found to be a potent GABA antagonist. Picrotoxin and bicuculline were rather less potent and strychnine and penicillin only weakly active as GABA antagonists. Leptazol appeared to be inactive against GABA depressions. 4 In addition, bicuculline and strychnine were found to be capable of potentiating the depressant action of GABA. This property was not shared by the other substances studied. 5 All the substances studied produced changes in neuronal firing rate that did not correlate with GABA antagonism. 6 In conclusion, several potent convulsants have been shown to be capable of GABA antagonism. It is not yet clear that this effect, rather than a direct effect on neuronal excitability, is the prime mechanism behind their convulsant properties.

PRESYNAPTIC ACTIONS OF β‐AMINOBUTYRIC ACID AND SOME ANTAGONISTS IN A SLICE PREPARATION OF CUNEATE NUCLEUS

1 A slice preparation of the rat cuneate nucleus is described which is suitable for electrophysiological studies on the presynaptic action of drugs. 2 Superfusion of a slice with γ‐aminobutyric acid (GABA) depolarized the afferent nerves in a concentration‐related manner. The responses were CI “‐dependent. Depolarizations to high concentrations of GABA often faded. Glycine and l‐glutamate had little effect. 3 (‐h)‐Bicuculline antagonized GABA in an apparently competitive manner (pA2 = 5.35) at low response levels. Strychnine was 10 times less potent. Responses to high concentrations of GABA were sometimes potentiated by (+)‐biculline and strychnine. 4 Bemegride and leptazol both antagonized GABA, but with low potency and in a manner which was clearly not competitive.

Differential antagonism by epipregnanolone of alphaxalone and pregnanolone potentiation of [3H]flunitrazepam binding suggests more than one class of binding site for steroids at GABAA receptors

In rat brain membranes, the 3 alpha-hydroxy pregnane steroids, pregnanolone, allopregnanolone, alphaxalone and 5 beta-alphaxalone potentiated 1 nM [3H]flunitrazepam binding at the GABAA receptor, with maximal potentiations of 140-150% of control. The potencies of the 5 alpha isomers were greater than the 5 beta and the presence of an 11-keto group conferred lower potency. The potentiation produced by these steroids was antagonised by the 3 beta-OH isomers epipregnanolone, isopregnanolone and betaxalone (60 microM). The dose-effect curves for pregnanolone and allopregnanolone were shifted to the right, with no reduction in the maximal potentiation. In contrast, the maximal effect of alphaxalone and 5 beta-alphaxalone was reduced with no change in EC50. Alphaxalone (1 microM) caused an increase in the binding of [3H]flunitrazepam in the presence of maximal concentrations of pregnanolone or allopregnanolone. These results suggest multiple sites of action for neurosteroids in the brain.

Multiple GABA receptors and associated regulatory sites

Abstract γ-Aminobutyric acid (GABA) functions as an inhibitory transmitter in a wide range of species 13 . Considerable information has accumulated on the pharmacological properties of the receptors for GABA and the ion channels controlled by those receptors in crustacea, molluscs and vertebrates. It is now apparent that the GABA receptor and ion channel complexes in the invertebrates differ from those in the vertebrates.

Spontaneous paroxysmal activity induced by zero magnesium and bicuculline: suppression by NMDA antagonists and GABA mimetics.

Slices of rat cerebral cortex developed spontaneous paroxysmal discharges when superfused with Krebs medium containing zero Mg2+ or 50 microM bicuculline. In both situations, the N-methyl-D-aspartate (NMDA) antagonists APV, 100 microM, and ketamine, 100 microM substantially reduced the frequency of the paroxysmal events, the reduction being greater in zero Mg2+. gamma-Aminobutyric acid (GABA) 1 mM, the GABA-A agonist muscimol 2 microM and the GABA-B receptor agonist baclofen 10 microM, each reduced the frequency of events in zero Mg2+ while muscimol and GABA also reduced the amplitude of the events. GABA and baclofen were similarly effective against bicuculline-induced events but the muscimol concentration required was 5-10-fold higher. These results suggest that, under our vitro conditions, neocortical cells are normally restrained from paroxysmal discharges by Mg2+. Inhibition by GABA through GABA-A receptors and inhibition by GABA through GABA-B receptors, may also contribute to this restraint.

Effects of acute and chronic ethanol on the γ-aminobutyric acid system in rat brain

Abstract The effects of acute and chronic ethanol treatment have been investigated on the cerebral concentration of γ-aminobutyric acid (GABA). In addition, the activities of both l -glutamate 1-carboxylyase (GAD) and 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-T) have been measured and also the rate of 3 H-GABA disappearance following its intracisternal injection into rat brain. Acute ethanol administration caused no significant change in the GABA concentration. There was, however, an increase in GAD activity, but no change in GABA-T activity or the rate of disappearance of 3 H-GABA. Chronic ethanol treatment, on the other hand, caused a 48 per cent increase in GABA concentration. There was no simultaneous change in GAD activity but the GABA-T activity was significantly increased. Again there was no change in the rate of disappearance of 3 H-GABA. It is suggested that these effects may be due to an action of ethanol on the metabolic pool of GABA as distinct from the transmitter pool.

Modulation of the GABAA receptor complex by steroids in slices of rat cuneate nucleus.

1 Several derivatives of pregnane and androstane that have hypnotic properties have been investigated for their ability to potentiate responses to the GABAA receptor agonist muscimol and to reduce the effect of the non‐competitive GABAA antagonist picrotoxin. 2 Depolarizing responses to muscimol in slices of rat cuneate nucleus were potentiated most potently by 3α‐hydroxy, 5α‐pregnane‐11,20‐dione (alphaxalone), which gave half‐maximal potentiation at 0.15 μm. The 5β isomer of alphaxalone had little effect up to 3 μm but in analogues lacking an 11‐keto substituent (pregnanolones), both the 5α‐ and 5β‐isomers potentiated with potencies 20 and 10 times lower, respectively, than that of alphaxalone. The a configuration of the 3‐hydroxy was essential in alphaxalone, the 3β‐hydroxy isomer being inactive. However, there was little difference between the potencies of the 3α‐ and 3β‐hydroxy configurations in the pregnanolones, although the maximal effects of the 30‐hydroxy isomers were rather lower than those of the 3α‐hydroxy isomers. 3 Reductions in the effect of picrotoxin as an antagonist of muscimol were caused most potently by the 3α‐hydroxy pregnanolones, with a ten fold reduction in picrotoxin potency at 1 μm concentrations of these steroids. Alphaxalone and its 5β‐isomer were about half as potent. Androsterone was about 10 times less potent and the 3β‐hydroxy pregnanolones were ineffective. 4 This difference in the structure‐activity relationships for steroidal potentiation of muscimol and reduction in picrotoxin antagonism of muscimol is reminiscent of an analogous distinction found with the barbiturates.

Effects of acute and chronic pentobarbitone on the γ-aminobutyric acid system in rat brain

Abstract The effects of an acute anaesthetic dose and of chronic administration of pentobarbitone have been investigated on the GABA system in rat brain. This was done by estimating the cerebral γ-aminobutyric acid (GABA) concentration, the activities of both l -glutamate 1-carboxylyase (GAD) and 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-T), and also the rate of 3H-GABA disappearance following its intracisternal injection. Neither acute nor chronic barbiturate administration caused any marked changes in the GABA concentrations or the enzyme activities. The exponential disappearance curves observed after injection of a pulse label of 3H-GABA were resolved into “fast” and “slow” components. Acute pentobarbitone was found to significantly decrease the rate constant of the fast component, whilst chronic pentobarbitone significantly decreased the rate constant of the slow component. The possible morphological correlates of these components are discussed. It is concluded that pentobarbitone has distinct actions on the GABA system which are different from those of other central nervous system depressants.