I-1 I I ll)ll|-1 I I lllll|-1 I I 11 Hl|-1 I I I 11111-1 I I lllll|-1 I I IMU|

109 10-x 10'7 10-6 10"5 10-" 103 Concentration [M]

Fig. 2. Concentration-effect curves of certain neuroactive steroid-induced inhibition of the peak whole-cell Ca2+ channel current. Ca2+ channel currents were evoked in hippocampal CA1 neurons as described in Fig. 1. Each data point represents the mean ± S.E.M. 4-5 neurons for each compound; percent inhibition was calculated as previously described (11). Concentration-effect data were fitted with a nonlinear least-squares program according to the logistical equation B = 100/1 + (IC50/[DRUG]) , where [DRUG] is the drug concentration, IC50 is the concentration resulting in 50% inhibition, and nH is an empirical parameter that describes the steepness of the curve and has the same meaning as the Hill coefficient. The IC50 were 2.7, 10, and 3 for dihydroepiandosterone sulfate (DHEAS), pregnanolone, and alfaxolone, respectively; nH = 0.6, 0.95 and 1.5 for DHEAS, pregnanolone, and alfaxalone.

I-1 I I ll)ll|-1 I I lllll|-1 I I 11 Hl|-1 I I I 11111-1 I I lllll|-1 I I IMU|

109 10-x 10'7 10-6 10"5 10-" 103 Concentration [M]

Fig. 2. Concentration-effect curves of certain neuroactive steroid-induced inhibition of the peak whole-cell Ca2+ channel current. Ca2+ channel currents were evoked in hippocampal CA1 neurons as described in Fig. 1. Each data point represents the mean ± S.E.M. 4-5 neurons for each compound; percent inhibition was calculated as previously described (11). Concentration-effect data were fitted with a nonlinear least-squares program according to the logistical equation B = 100/1 + (IC50/[DRUG]) , where [DRUG] is the drug concentration, IC50 is the concentration resulting in 50% inhibition, and nH is an empirical parameter that describes the steepness of the curve and has the same meaning as the Hill coefficient. The IC50 were 2.7, 10, and 3 for dihydroepiandosterone sulfate (DHEAS), pregnanolone, and alfaxolone, respectively; nH = 0.6, 0.95 and 1.5 for DHEAS, pregnanolone, and alfaxalone.

nM, DHEAS essentially inhibited 80% of the inward current at 100 ^M (Fig. 2), which was irreversible; higher concentrations of DHEAS resulted in loss of recording, hence cell death (not shown).

The classical neurosteroids, pregnenolone (PREG) and its sulfate derivative, pregnenolone sulfate (PREGS), which are synthesized and found in intact brain and glial cultures (9,10), appear to have a multitude of actions on various membrane channels (6), including the inhibition of voltage-gated Ca2+ channels (11). Both PREG and PREGS inhibited a fraction of the total inward Ca2+ current with a maximal inhibition of 64 ± 3% and 57 ± 4%, respectively, at 100 ^M and IC50 values of 11 and 130 nM, respectively (11). Interestingly, in rat hypothalamic ventromedial nucleus (VMN) neurons, PREGS essentially had no inhibitory effect with a maximal inhibition of 6.5 ± 3% and 7 ± 2% at 1 and 10 ^M, respectively (Fig. 3). In contrast to PREGS, 3a,5a-THDOC inhibited the Ca2+ current with an IC50 = 673 nM and an nH = 0.4 (n = 6) (Fig. 3). The potency of inhibition in rat (VMN) was less than that in guinea-pig CA1 neurons.

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