[steroid M

Fig. 1. The water soluble steroid Org 20599 potently enhances GABA-evoked currents mediated by human GABA receptors with the subunit composition a p y expressed in Xenopus oocytes. Left, Each pair oftraces illustrates the augmentation of inward current responses evoked by the bath application of GABA (at a concentration that gives a response 10% of the maximum (EC10) by 1 3a-hydroxy-5a-pregnan-20-one (3a,5a-THPROG), 10 ^M Org 20599, and 10 alphaxalone. The increases in current amplitude illustrated are examples of those evoked by maximally effective concentrations of the steroids. The periods of drug application are denoted by the horizontal lines above each trace. The recordings for each steroid were made from different oocytes voltage-clamped at a holding potent of -60 mV. Calibration bars: vertical 200 nA; Horizontal 1 min. Right: Graphical depiction of the concentration-dependent potentiation of GABA-evoked currents by 3a,5a-THPROG: (•); Org 20599, (■); and alphaxalone, (▼). Currents are expressed as a percentage of the maximal GABA current. Each point represents the mean data obtained from four oocytes. Error bars indicate the S.E.M.

C20 Substitution

The presence of a carbonyl group at C20 of the acetyl side chain was initially deemed essential to the activity of pregnane steroids at the GABAa receptor (26). However, subsequent studies have revealed 20-keto reduced analogues of 3a,5a-THPROG and 3a,5 P-THPROG (i.e., pregnanediols) to modulate GABAA receptor activity in a manner consistent with partial agonism (see Fig. 1). The potency and efficacy of such pregnanediols are dependent on structural determinants that include cis or trans fusion of the A and B rings and the orientation (a or P) or the 20-hydroxyl moiety (41,60).

C21 Substitution

The presence of a hydroxyl group at C21 (as the in naturally occurring 3 a,5 a-THDOC) or its esterification to the acetate or mesylate produces only modest reductions in activity (53). Similarly, from studies conducted with a series of water-soluble morpholinyl steroids (see section "Water Soluble Steroids"), it appears that the steroid binding site of the

Table 2 The Selectivity of Action of the Anesthetic Steroids Alphaxalone and Minaxolone




2.2 ± 0.3 ^M

0.5 ± 0.1 ^M

(78 ± 3%)

(93 ± 5%)

60 ^M

11 ± 1 ^M

No effect

(89 ± 4%)

60 \iM

100 ^M

No effect

No effect

30 ^M

30 ^M

No effect

No effect

5 ± 1 ^Mb

19 ± 3 ^M

13 ± 2 ^Mb

11 ± 1 ^M

~50 ^Mb

(alP2Y2L: EC50) Glycine

(Rat spinal cord/a^: EC50) AMPA/kainate

(Rat cerebellum: IC) NMDA

(Rat cerebellum: IC50) Nicotinic

(Rat a4p2: ICJ


All experiments were performed on oocytes voltage-clamped at a holding potential of -60 mV. The sources of receptor were: GABAa, human a^^L' glycine, human ^ rat p for alphaxalone, and rat spinal cord mRNA for minaxolone; kainate and NMDA, rat cerebellar mRNA; neuronal nicotinic, rat a,B and chick a„; 5-HT, human 5-HT

All experiments upon GABAa and glycine receptors utilized the EC10 concentration of the natural agonist. For the other receptors, the appropriate agonist EC50 was used. For GABAa and glycine receptors, the steroid EC50 and the maximum potentiation produced in parenthesis are given. For kainate, NMDA, nicotinic, and 5-HT A receptors, the IC

values are given where appropriate.

Denotes betaxalone to be equieffective with alphaxalone. Where quantified, all data are the mean ± S.E.M. of observations made from 3-5 oocytes.

GABAA receptor can accept a range of functional groups, including hydroxyl, chloride, acetate, thioacetate, thiocyanate, and azide moieties (61).

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