O

10" 10" 10'1° 10"9 10"8 10-7 10-6 105 101 Concentration [M]

Fig. 3. Concentration-effect curves of certain neuroactive steroids in freshly isolated adult rat hypothalamic ventromedial nulceus (VMN) neurons. Each data point represents the mean± S.E.M. of 5-6 neurons for each compound; see Fig. 2 for details. Tetrahydrodeoxycorticosterone and medroxyprogesterone acetate had an IC = 673 and 4 nM, respectively, and an nH = 0.7 and 0.5, respectively. Progesterone and pregnenolone sulfate had a maximal inhibition of 7 ± 2% and 6.9 ± 2%, respectively.

10" 10" 10'1° 10"9 10"8 10-7 10-6 105 101 Concentration [M]

Fig. 3. Concentration-effect curves of certain neuroactive steroids in freshly isolated adult rat hypothalamic ventromedial nulceus (VMN) neurons. Each data point represents the mean± S.E.M. of 5-6 neurons for each compound; see Fig. 2 for details. Tetrahydrodeoxycorticosterone and medroxyprogesterone acetate had an IC = 673 and 4 nM, respectively, and an nH = 0.7 and 0.5, respectively. Progesterone and pregnenolone sulfate had a maximal inhibition of 7 ± 2% and 6.9 ± 2%, respectively.

Subsequently, other neuroactive steroids were reported to also inhibit voltage-gated Ca2+ channels, such as the glucocorticosteroids cortisol and corticosterone in freshly isolated hippocampal CA1 neurons (12), megestrol acetate in VMN neurons (13), estradiol, estriol and 4-hydroxyestradiol in freshly isolated and cultured neostriatal neurons (14). The steroid anesthetic alfaxolone, which potently (30 nM) potentiates the GABA current (15), at higher concentrations inhibited a fraction of the total Ca2+ channel current in hippocampal CA1 neurons (Fig. 2). All of these neuroactive steroids had a rapid and direct action and appeared to exert their inhibitory effect via an extracellular membrane receptor (11-14).

However, PROG, which is also derived from PREG in the brain, had no effect on the Ca2+channel current in either guinea-pig hippocampal CA1 neurons (Figs. 1 and 2) (11) or rat VMN neurons (Fig. 3) (13). Other studies have shown that PROG enhances the GABAA-induced responses in Purkinjie and spinal chord neurons (16,17). Megestrol acetate, a synthetic orally active progesterone derivative, is utilized clinically for the treatment of metastatic breast cancer and endometrial cancer (18) and also to induce appetite stimulation and weight gain in cancer/AIDS patients with anorexia/cachexia (19). Although its mechanism of action is unknown, megestrol acetate was found to inhibit potently (IC50 = 2 nM and an nH = 0.5) a fraction (24 ± 3%) of the total VGCCs in freshly isolated adult rat VMN neurons with a maximal saturating concentration of 1 and 10 ^M (13). The megestrol acetate analogue, medroxyprogesterone acetate (MP) also inhibited the VGCCs in VMN neurons (Figs. 1C and 3). MP showed an identical potency of inhibition to megestrol acetate with a maximal inhibition of 23 ± 3% at 10 ^M of the total VGCCs with an IC50 = 4 nM and an nH = 0.5 (13). In contrast, medroxyprogesterone weakly inhibited the VGCCs with a 7% inhibition at 10 mM (13). Furthermore, even in the presence of 10 ^M PROG, megestrol acetate showed an identical inhibition of the VGCCs to megestrol acetate alone, suggesting a novel membrane receptor (13).

While inhibiting the Ca2+ channel current, some neuroactive steroids also modulated certain parameters of Ca2+ channel gating. One commonality observed with those neuroactive steroids was a voltage-dependence of Ca2+ channel current inhibition. PREGS, 3a,5a-THDOC, megestrol acetate, and cortisol all showed a voltage-dependent inhibition, specific for negative voltages—that is, greater inhibition at more negative test potentials—typically between -60 and 0 mV (11-13). Another common observation to these same neuroactive steroids was that although there was a lack of effect on the voltage-dependence of activation, there was a slowing of the rate of activation (11-13). The rate or time-course of Ca2+ channel activation was slowed by PREGS, 3a,5a-THDOC, megestrol acetate, and cortisol; however, deactivation was slowed by PREGS, 3a,5a-THDOC, and megestrol acetate, but not cortisol (11-13).

With regard to which Ca2+ channels these neuroactive steroids inhibited, there was indeed a reasonable lack of specificity. PREG, 3a,5a-THDOC and cortisol were nonselective in that they inhibited both the N- and L-type Ca2+ channels, although 3a,5a-THDOC appeared to be more selective in inhibiting the N-type Ca2+ channels (11,12). In contrast, in VMN neurons, megestrol acetate selectively inhibited a fraction of the resistant or R-type Ca2+ channel current (13). Calcium channel selectivity was also demonstrated with estradiol, which specifically inhibited the L-type Ca2+ channel in neostriatal neurons (14).

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