Differential Blockade of g-Aminobutyric Acid Type A Receptors by the Neuroactive Steroid Dehydroepiandrosterone Sulfate in Posterior and Intermediate Pituitary

Dehydroepiandrosterone sulfate (DHEAS) is a neuroactive steroid with antagonist action at g-aminobutyric acid type A (GABAA) receptors. Patch-clamp techniques were used to investigate DHEAS actions at GABAA receptors of the rat pituitary gland at two distinct loci: posterior pituitary nerve terminals and intermediate pituitary endocrine cells. The GABA responses in these two regions were quite different, with posterior pituitary responses having smaller amplitudes and desensitizing more rapidly and more completely. DHEAS blockade of GABAA receptors in the two regions also was different. In posterior pituitary, a site with an apparent dissociation constant of 15 mM accounted for most of the blockade, but a small fraction of blockade may be related to a site with a dissociation constant in the nanomolar range. In the intermediate lobe, DHEAS sensitivities in the nanomolar and micromolar ranges were clearly evident, in proportions that varied widely from cell to cell. Regardless of whether the GABA response of a cell was highly sensitive or weakly sensitive to DHEAS, GABA alone evoked currents that were indistinguishable in terms of amplitude, desensitization kinetics, and GABA sensitivity. Thus, the structural elements responsible for DHEAS blockade have a highly selective impact on receptor function. GABAA receptors with nanomolar sensitivity to DHEAS have not been described previously. This suggests that DHEAS may have an important role in the modulation of neuropeptide secretion, and the diverse properties of GABAA receptors in the rat pituitary provide mechanisms for selective regulation of the different peptidergic systems of this gland. In endocrine cells of the pituitary intermediate lobe (IL), g-aminobutyric acid type A (GABAA) receptor-specific agonists modulate the release of a-melanocyte-stimulating hormone (Tomiko et al., 1983; Taraskevich and Douglas, 1985). In the nerve terminals of the posterior pituitary (PP), GABAA receptor activation alters the release of oxytocin and vasopressin (Dyball and Shaw, 1978; Fjalland et al., 1987; Saridaki et al., 1989). In both IL (Demeneix et al., 1986; Taleb et al., 1987; Schneggenburger and Konnerth, 1992) and PP (Zhang and Jackson, 1993), the receptors have many of the properties of classic neuronal GABAA receptors. The channels are selectively permeable to Cl, muscimol acts as an agonist, and the responses are blocked by bicuculline and picrotoxin. IL GABAA receptors possess a pure type 2 benzodiazepine-binding site, and ribonuclease protection assays have shown that the IL contains mRNA encoding for the a2, a3, b1, b3, g2s, and g1 GABAA receptor subunits (Berman et al., 1994). Sensitivity to benzodiazepines and insensitivity to zinc indicate that PP GABAA receptors contain g subunits (Zhang and Jackson, 1994b). These different molecular and pharmacological properties determine how GABA and GABAA receptor-specific drugs regulate different peptide systems in the pituitary gland. Neuroactive steroids modulate the responsiveness of GABAA receptors in many preparations, and this represents an important nongenomic action of steroids (Harrison and Simmonds, 1984; Paul and Purdy, 1992; Gee et al., 1995). Allopregnanolone (5a-pregnan-3a-hydroxy-20-one) and alphaxolone have been shown to act as positive GABAA receptor modulators in the PP (Zhang and Jackson, 1994a). In the IL, allopregnanolone also enhances GABAA receptor-mediated responses, and the neuroactive steroid pregnenolone sulfate (5-pregnen-3a-ol-20-one sulfate) antagonizes them (Poisbeau et al., 1997). These findings raise the possibility that neuroactive steroid modulation of neuropeptide release plays a role in some of the endocrinological transitions mediated by these peptidergic systems. Furthermore, neuroactive steroid potencies vary between brain regions and species (Gee et al., 1995; Nguyen et al., 1995). This adds another dimension to This work was supported by National Institutes of Health Grant NS30016 and by the Royal Danish School of Pharmacy. ABBREVIATIONS: DHEAS, dehydroepiandrosterone sulfate; GABA, g-aminobutyric acid; IL, intermediate lobe; PP, posterior pituitary; aCSF, artificial cerebrospinal fluid. 0026-895X/99/030489-08$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:489–496 (1999). 489 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from neuroactive steroid signaling by allowing for differential control over peptidergic versus synaptic processes and, possibly, differential control between various peptidergic systems. Dehydroepiandrosterone sulfate (DHEAS) is a neuroactive steroid that has been the subject of wide-ranging discussions regarding potential roles in cognitive function, aging, stress, and development (Baulieu and Robel, 1996; Bastianetto and Quirion, 1997). In contrast to many neuroactive steroids that enhance the responses of GABAA receptors, DHEAS blocks GABAA receptors in a number of preparations (Majewska et al., 1990; Spivak, 1994; Souza and Ticku, 1997). In the present study, we investigated the actions of DHEAS at GABAA receptors in slices prepared from the rat pituitary gland (Jackson et al., 1991; Schneggenburger and Konnerth, 1992), focusing on both the peptidergic nerve terminals of the PP and the endocrine cells of the IL. DHEAS inhibits GABA responses in both PP and IL but with a concentration dependence indicative of multiple binding sites. Variations in the DHEAS sensitivity of endocrine cells within the IL suggest the presence of multiple molecular forms of GABAA receptors. This would allow DHEAS to influence neuropeptide release over a broad range of concentrations and possibly modulate different peptide systems selectively. Materials and Methods Pituitary Slices. Slices of rat pituitary gland were prepared as described previously (Jackson et al., 1991). Male Sprague-Dawley rats 4 to 6 weeks old were sacrificed by decapitation after CO2induced narcosis. The pituitary gland was quickly removed and placed into ice-cold artificial cerebrospinal fluid (aCSF) consisting of 125 mM NaCl, 4 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 10 mM glucose, pH 7.3, bubbled with a mixture of 95% O2/5% CO2 (carbogen). The pituitary gland was glued to a cutting block, with the posterior lobe facing upward, and immersed in chilled aCSF. Slices 70 to 80 mm thick were then cut with a Vibratome. Slices were either kept in carbogen-bubbled aCSF or transferred to a recording chamber. Recordings were made with continuous perfusion of carbogen-bubbled aCSF, while being viewed with a DIC microscope at 6003. Nerve terminals in the PP ranging in size from 5 to 15 mm in diameter were selected for patch-clamp recording. IL endocrine cells at the perimeter of the slice were readily identified by appearance and location (Schneggenburger and Kon-