Antagonism of neurosteroid modulation of native gamma-aminobutyric acid receptors by (3alpha,5alpha)-17-phenylandrost-16-en-3-ol

Abstract

Endogenous pregnane neurosteroids are allosteric modulators at gamma-aminobutyric acid type-A (GABAA) receptors at nanomolar concentrations. There is direct evidence for multiple distinct neurosteroid binding sites on GABAA receptors, dependent upon subunit composition and stoichiometry. This view is supported by the biphasic kinetics of various neuroactive steroids, enantioselectivity of some neurosteroids, selective mutation studies of recombinantly expressed receptors and the selectivity of the neurosteroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA) on 5alpha-pregnane steroid effects on recombinant GABAA receptors expressed in Xenopus oocytes and native receptors in dissociated neurons. However, it is unclear whether this antagonist action is present in a mature mammalian system. The present study evaluated the antagonist activity of 17PA on neurosteroid agonists both in vivo and in vitro by examining the effects of 17PA on 5alpha-pregnane-induced sedation in rats, native mature GABAA receptor ion channels utilizing the chloride flux assay and further studies in recombinant alpha1beta2gamma2 receptors. The data show that 17PA preferentially inhibits 3alpha,5alpha-THP vs. alphaxalone in vivo, preferentially inhibits 3alpha,5alpha-THDOC vs. alphaxalone potentiation of GABA-mediated Cl- uptake in adult cerebral cortical synaptoneurosomes, but shows no specificity for 3alpha,5alpha-THDOC vs. alphaxalone in recombinant alpha1beta2gamma2 receptors. These data provide further evidence of the specificity of 17PA and the heterogeneity of neurosteroid recognition sites on GABAA receptors in the CNS.

Figure 1

Effects of 17PA on 3α,5α-THP-induced loss of righting reflex (LORR). a. I.c.v. administration of the neurosteroid antagonist 17PA attenuated the sedative/hypnotic action of the physiological neurosteroid, 3α,5α-THP. Pre-treatment with 17PA (10 nmoles) induced a significant rightward shift in the percentage of animals exhibiting loss of righting reflex to intravenous administration of 3α,5α-THP (0.625 – 5.0 mg/kg). b I.c.v. administration of 17PA significantly reduced 3α,5α-THP-induced loss of righting reflex duration as revealed by a one-way repeated measures ANOVA (F = 6.9, df =1,135, P < 0.05). A Tukey’s multiple comparison post-hoc analyses showed that 17PA pre-treatment significantly attenuated loss of righting reflex time induced by 3α,5α-THP at 2.0 and 2.5 mg/kg (P <0.05).

Figure 2

Effects of 17PA on alphaxalone-induced loss of righting reflex. a. I.c.v. administration of 17PA did not significantly alter sedative/hypnotic action of the synthetic neurosteroid, alphaxalone. Pre-treatment with 17PA (10 nmoles) did not produce a significant shift in the percentage of animals exhibiting loss of righting reflex to intravenous administration of alphaxalone (1.25 – 10.0 mg/kg). b. I.c.v. administration of 17PA does not significantly reduce alphaxalone-induced loss of righting reflex duration as revealed by a two-way repeated measures ANOVA (F = 0.58, df =1,104, P = 0.46).

Figure 3

Effect of 17PA on basal or muscimol-stimulated ³⁶Cl^- uptake. a. 17PA (0.1 μM – 100 μM) did not significantly alter basal ³⁶Cl^- uptake in synaptoneurosome preparations (One-way ANOVA, F = 0.2, df = 10, p = 0.89). b. 17PA did not significantly alter ³⁶Cl^- uptake stimulated by muscimol (1.5 μM) (F = 0.62, df = 11, p = 0.62) demonstrating no direct antagonist action at native GABA_A receptors.

Figure 4

The antagonist action of 17PA on 3α,5α-THP and 3α,5α-THDOC potentiation of muscimol-stimulated ³⁶Cl^- uptake. a. 17PA antagonism was assessed across increasing 3α,5α-THP concentrations in rat cerebral cortical synaptoneurosomes. 17PA (open circles) did not produce a significant rightward shift in the concentration-response curve compared to vehicle (closed circles). However, 17PA significantly reduced 3α,5α-THP E_max (P < 0.05), therefore attenuating maximal 3α,5α-THP potentiation of muscimol-stimulated ³⁶Cl^- uptake. b. 17PA antagonism assessed across increasing 3α,5α-THDOC concentrations in rat cortical synaptoneurosomes. 17PA (open circles) did not produce a significant rightward shift in the concentration-response curve for 3α,5α-THDOC compared to vehicle (closed circles) but significantly reduced the E_max of 3α,5α-THDOC potentiation of muscimol-stimulated ³⁶Cl^- uptake (P < 0.01), thus attenuating maximal 3α,5α-THDOC potentiation in the synaptoneurosome preparation.

Figure 5

Comparison of the antagonist action of 17PA on 5α-reduced steroid potentiation of muscimol-stimulated ³⁶Cl^- uptake in rat cortical synaptoneurosomes. a. 3α,5α-THP (3 μM) potentiation of muscimol-stimulated (1.5 μM) ³⁶Cl^- uptake relative to basal (P<0.0001, inset panel). Note that 17PA completely blocked 3α,5α-THP potentiation of muscimol-stimulated ³⁶Cl^- uptake (IC₅₀ = 30.17 μM). b. Synthetic neurosteroid alphaxalone (3 μM) potentiation of muscimol-stimulated (1.5 μM) ³⁶Cl^- uptake (P<0.001, inset panel). Note that 17PA partially attenuated alphaxalone potentiation of muscimol stimulation (IC₅₀ = 11.23 μM). c. 3α,5α-THDOC (1 μM) potentiation of muscimol-stimulated (1.5 μM) ³⁶Cl^- uptake (P<0.001, inset panel). 17PA partially attenuated 3α,5α-THDOC potentiation of muscimol-stimulated ³⁶Cl^- uptake (IC₅₀ = 6.08 μM). d. pregnenolone sulfate inhibition of 3α,5α-THDOC potentiation (IC₅₀ = 21.64 μM).

Figure 6

Responses of Xenopus oocytes expressing recombinant rat α1β2γ2 subunits to GABA co-applied with potentiating steroids and 17PA. a. Effect of 17PA on 3α,5α-THDOC potentiation. b. Effect of 17PA on alphaxalone potentiation. Horizontal bars indicate the duration of drug exposure. Oocytes were clamped at -70 mV and drug concentrations were GABA (2 μM), 3α,5α-THDOC (0.5 μM), alphaxalone (0.5 μM) and 17PA (10 μM). Responses in the two panels were obtained from two separate oocytes. c. Inhibition curve for 17PA against 3α,5α-THDOC in oocytes expressing recombinant receptors. 3α,5α-THDOC concentration was constant at 0.5 μM. Potentiation refers to the fractional increase over the baseline GABA response (1.0 denotes a doubling of the response). Error bars denote standard error of the mean. The solid line is a fit predicting an IC₅₀ of 25 μM. d. Similar inhibition curve for alphaxalone (0.5 μM) predicts an IC₅₀ of 36 μM. e. Effects of 17PA on alphaxalone concentration response parameters. The graphs are taken from fitting a concentration response function to 8 oocytes challenged with 0.3 – 10 μM alphaxalone in the absence and presence of 10 μM 17PA. The only significant change was a shift to the right in the alphaxalone EC₅₀, consistent with a competitive or pseudocompetitive mechanism of 17PA.