0022-3565/03/3063-934–940$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 52324/1083036 JPET 306:934–940, 2003 Printed in U.S.A. Steroids Modulate N-Methyl-D-aspartate-Stimulated [3H]Dopamine Release from Rat Striatum via ␴ Receptors SAMER J. NUWAYHID and LINDA L. WERLING Department of Pharmacology, George Washington University Medical Center, Washington, DC Received March 31, 2003; accepted May 13, 2003 ABSTRACT Steroids have been proposed as endogenous ligands at ␴ indol-3-yl]-1-butyl]spiro[iso-benzofuran-1(3H), 4Јpiperidine] Downloaded from receptors. In the current study, we examined the ability of (Lu28-179). Lastly, to determine whether a protein kinase C (PKC) steroids to regulate N-methyl-D-aspartate (NMDA)-stimulated signaling system might be involved in the inhibition of NMDA- [3H]dopamine release from slices of rat striatal tissue. We found stimulated [3H]dopamine release, we tested the PKC␤-selective that both progesterone and pregnenolone inhibit [3H]dopamine inhibitor 5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4– release in a concentration-dependent manner similarly to pro- 1][1,8]diacyclohexadecine-18,20(19H)-dione,8-[(dimethylamin- totypical agonists, such as (ϩ)-pentazocine. The inhibition seen o)methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate (9Cl) jpet.aspetjournals.org by both progesterone and pregnenolone exhibits IC50 values (LY379196) against both progesterone and pregnenolone. We consistent with reported Ki values for these steroids obtained in found that LY379196 at 30 nM reversed the inhibition of release by ␴ binding studies, and was fully reversed by both the 1 antagonist both progesterone and pregnenolone. These findings support ste- 1-(cyclopropylmethyl)-4–2Ј-4Љflurophenyl)-2Јoxoethyl)piperidine roids as candidates for endogenous ligands at ␴ receptors. ␴ Ј HBr (DuP734) and the 2 antagonist 1 -[4-[1-(4-fluorophenyl)-1-H- at ASPET Journals on March 13, 2015 Since their proposal in 1976 by Martin et al. (1976), ␴ vived when studies showed that steroids mediated many receptors have been characterized pharmacologically in bio- effects through a nongenomic mechanism (Falkenstein et al., assays and radioligand binding studies. ␴ receptors bind a 2000) and that steroids were synthesized in the brain (Hu et wide array of drugs from various classes, including benzo- al., 1987; Jung-Testas et al., 1989; Guarneri et al., 2000), morphans, guanidines, morphinans, antipsychotics, and co- along with pharmacological studies on regulation of trans- caine. However, none of these drugs is endogenous to the mitter release (Monnet et al., 1995) and responses in hip- brain or in cells in culture. To establish a relevance of ␴ pocampal neurons (Bergeron et al., 1996) to steroids. The receptors to physiological function, it is important to identify steroids the Su group found that competed best for an endogenous ligand. 3 ␴ [ H]SKF10,047 at 1 sites were progesterone, deoxycorti- Steroids were originally proposed as endogenous ligands at sone, and testosterone. More recently, McCann and Su (1994) ␴ receptors by Su et al. (1988). Swartz et al. (1989) ques- ␴ examined steroid competition at 1 sites and found both tioned this proposal because they assumed steroids could not ␴ ␴ progesterone and testosterone had an affinity for 1 and 2 be produced in the brain, and the concentration of steroids subtypes. ␴ crossing the blood-brain barrier would not suffice to occupy Monnet et al. (1995) described the modulation of NMDA- receptors. Interest in steroid-␴ receptor interactions was re- stimulated [3H]norepinephrine release by progesterone, de- hydroepiandrosterone sulfate (DHEA S), and pregnenolone This work was supported by a grant from National Institute on Drug Abuse (DA06667) and a Faculty Enhancement Research Award from George Wash- sulfate (PREG S) in rat hippocampal slices. The effects were ington University Medical Center (to L.L.W.). blocked by ␴ receptor antagonists and progesterone acted as Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. an antagonist to DHEA S and PREG S. Most recently, Meyer DOI: 10.1124/jpet.103.052324. et al. (2002) showed that PREG S enhances glutamate re- ABBREVIATIONS: NMDA, N-methyl-D-aspartate; DHEA S, dehydroepiandrosterone sulfate; PREG S, pregnenolone sulfate; DHEA, dehydroepi- androsterone; PKC, protein kinase C; PLC, phospholipase C; MKB, modified Krebs-HEPES buffer; S1, first stimulus; ISI, inter stimulus interval; S2, second stimulus; LY379196, 5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4-1][1,8]diacyclohexadecine-18,20(19H)-dione,8-[(dimeth- ylamino)methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate (9Cl); BD1063, 1[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine; DuP734, 1-(cyclopropylmethyl)-4–2Ј-4Љflurophenyl)-2Јoxoethyl)piperidine HBr; Lu28-179, 1Ј-[4-[1-(4-fluorophenyl)-1-H-indol-3-yl]-1-butyl]spiro[iso-benzo- furan-1(3H),4Јpiperidine]; GF109203x, 3[1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-1-H-pyrprole-2,5-dione; BAPTA, 1,2-bis(2-aminophe- noxy)ethane-N,N,NЈ,NЈ-tetraacetic acid; SKF10,047, n-allylnormetazocine; BD737, 1S,2R-(Ϫ)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)-cyclyohexylamine; U73122, 1(6-((17-b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione. 934 Steroids Modulate Dopamine Release via ␴ Receptors 935 ␴ lease from hippocampal slices via a 1-like receptor. In stud- done. These drugs were included in all subsequent steps to prevent ies reviewed by Bastianetto et al. (1999) and Maurice et al. reuptake of and feedback inhibition by the released [3H]dopamine. (1999), PREG S, DHEA S, and allotetrahydroxycorticoste- Tissue was suspended a final time in 7.5 ml of MKB, containing 10 rone show antistress, anxiolytic, and antiamnesiac activity ␮M nomifensine and 1 ␮M domperidone, and distributed in 275-␮l that is blocked by ␴ antagonists, as well as antisense oligo- aliquots between glass fiber discs into chambers of a superfusion apparatus (Brandel, Inc., Gaithersburg, MD). MKB was superfused nucleotides to ␴ receptors (Maurice et al., 2001). Taking all 1 over tissue at a rate of 0.6 ml/min. A low stable baseline release of these studies in consideration, steroids at this time are likely approximately 1.3%/ 2 min collection interval was established over a ␴ candidates for endogenous receptor ligands. 30-min period. Tissue was then stimulated by a 2-min exposure to 25 Gonzalez-Alvear and Werling (1994) first demonstrated ␮M NMDA (S1). The mean fractional release (percentage) produced 3 regulation of NMDA-stimulated [ H]dopamine release from in the S1 stimulus interval was 11.9 Ϯ 1.2%. Inflow was then re- rat striatum by ␴ receptor ligands, including (ϩ)-pentazo- turned to nonstimulating buffer during a 10-min interstimulus in- cine, (ϩ)-SKF10,047, and BD737. The inhibition produced by terval (ISI). If a steroid, ␴ antagonist, cholesterol, mifepristone, or ␴ trilostane was being tested, it was included at this time. The inclu- low concentrations of these ligands was reversed by the 1 antagonist DuP734 (Gonzalez-Alvear and Werling, 1994, sion of ␴ antagonist drug in the buffer did not significantly affect Ϯ 1995). A second phase of inhibition produced by higher con- basal release (per 2-min collection interval: no antagonist 1.3 0.33, ϩ n ϭ 3; 100 nM DuP734, 1.2 Ϯ 0.24, n ϭ 3; 1 nM Lu28-179 1.4 Ϯ 0.14, centrations of ( )-pentazocine was reversed by nonsubtype- ϭ ␴ n 3). Neither did inclusion of steroid affect basal release signifi- selective receptor antagonists, indicating the participation cantly (progesterone, 1.3 Ϯ 0.24, n ϭ 4; pregnenolone, 1.6 Ϯ 0.09, n ϭ ␴ of 2 receptors. Studies by Izenwasser et al. (1998) revealed 3). Tissue was then exposed to a second stimulus (S2) identical to the Downloaded from 3 that amphetamine-stimulated [ H]dopamine release can be first except in the presence of a steroid, trilostane, mifepristone, or ␴ modulated by 2 receptor agonists and antagonists in vitro. cholesterol, as appropriate. In the experiments testing the PKC In our current study, we examined the ability of steroids to inhibitor LY379196, the drug was present throughout S1, ISI, and regulate NMDA-stimulated [3H]dopamine release from rat S2. Inflow was once again returned to nonstimulating buffer before striatal slices via ␴ receptors. If steroids are indeed the extraction of the remaining radioactivity in the tissue by a 45-min endogenous ligand for ␴ receptors, they should affect neuro- exposure to 0.2 N HCl at a reduced flow rate. Superfusates were jpet.aspetjournals.org transmitter release and signaling similarly to prototypical ␴ collected at 2-min intervals in scintillation vials with the glass fiber ␴ filter discs and tissue collected into the final vials. Released radio- ligands. We have previously demonstrated that 1 agonist- 3 activity was determined by liquid scintillation spectroscopy. mediated inhibition of NMDA-stimulated [ H]dopamine re- All data were statistically analyzed as ratios (S2/S1) before con- lease is mediated by a PKC signaling system, likely involving version to percentage of control values for presentation. The ratio of the ␤ isoform. ␴ receptor regulation of release is abolished by S2/S1 in the absence of any test drug was 0.54 Ϯ 0.07 (n ϭ 10). An pretreatment with phorbol 12-myristate 13-acetate, as well enhancement by test drug would result in a higher ratio and an by treatment with a PKC␤ or a PLC inhibitor (Nuwayhid and inhibition in a lower ratio. In this way, differences in response at ASPET Journals on March 13, 2015 Werling, 2003). Therefore, we tested whether the same PKC between tissue samples are taken into account and therefore, do not pathway is involved in regulation of stimulated dopamine affect the comparison of treatments. In the results, data are ex- release by steroids via ␴ receptors by using a PKC␤ inhibitor.