Dopamine Release from Rat Striatum Via Σ Receptors

Home , BD1063, Sigma receptor

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, dehydroepiandrosterone 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, dehydroepiandrosterone; 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-[(dimethylamino)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-benzofuran-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. pressed as radioactivity released above baseline during the collection interval as a fraction of the total radioactivity in the tissue at the beginning of the collection interval (fractional release, percentage) or Materials and Methods as a percentage of the radioactivity released by the control stimulus (percentage of control-stimulated release). Data are presented as a Drugs and Reagents. The following chemicals were kindly pro- percentage of control-stimulated release for facilitation of compari- vided by or obtained from the following sources: domperidone and son across experiments. Under the experimental conditions used, the nomifensine (Sigma/RBI, Natick, MA); L-ascorbic acid, pregnenolone, released radioactivity has been shown to be primarily dopamine testosterone, progesterone, dihydoisoandosterone, cholesterol, and (Werling et al., 1988). All statistical analyses were performed by mifepristone (Sigma-Aldrich, St. Louis, MO); [3H]dopamine (Amer- one-way factorial analysis of variance with post hoc Dunnett’s. Sta- sham Biosciences Inc., Piscataway, NJ); LY379196 (gift of Dr. Robin tistical significance is considered at p Ͻ 0.05. Bowman, Eli Lilly & Co., Indianapolis, IN); trilostane (gift of Dr. J. Puddefoote; Queen Mary and Westfield College, London, UK); DuP734 (DuPont Merck Pharmaceutical Co., Wilmington, DE); and Results Lu28-179 (H. Lundbeck, Copenhagen, Denmark). 3 To examine the possibility that steroids could act similarly Measurement of Stimulated [ H]Dopamine Release from ␴ Striatal Slices. All experiments were carried out in accordance with to identified agonists or antagonists at receptors, we tested the guidelines and the approval of the George Washington Univer- several steroids for their ability to regulate NMDA-stimu- 3 sity Institutional Animal Use and Care Committee. Male Sprague- lated [ H]dopamine release via ␴ receptors from rat striatal Dawley rats (Hilltop Lab Animals, Scottsdale, PA), weighing 200 to tissue. We first tested single concentrations of progesterone, 225 g, were sacrificed by decapitation, and brains removed to ice. pregnenolone, DHEA, and testosterone to determine whether Striata were dissected, chopped in two planes at right angles into they had any effect on NMDA-stimulated [3H]dopamine re- 250 ϫ 250 ␮m strips with a Sorvall T-2 tissue sectioner, and sus- ␮ ␴ lease. As seen in Fig. 1, neither DHEA at 10 M(Ki at pended in modified Krebs-HEPES buffer (MKB; 127 mM NaCl, 5 mM receptors ϭ 3.7 ␮M, undifferentiated for subtype; Klein and KCl, 1.3 mM NaH PO , 2.5 mM CaCl , 15 mM HEPES, 10 mM 2 4 2 Musacchio, 1994) nor testosterone at 3 or 10 ␮M(K ϭ 1 ␮M, glucose, pH adjusted to 7.4 with NaOH) by titration through a plastic i undifferentiated for subtype; Su et al., 1988) showed any pipette. Buffers were oxygenated throughout the experiments and brain slices were kept at a constant temperature of 37°C. After three significant difference from control-stimulated release. ␮ washes in MKB, tissue was resuspended in 20 ml of MKB and In preliminary experiments, progesterone at 3 M and incubated for 30 min with 0.1 mM ascorbic acid and 15 nM [3H]do- pregnenolone at 1 ␮M showed an inhibition of NMDA-stim- 3 pamine. Tissue was then washed twice with 20 ml of MKB and once ulated [ H]dopamine release. We then constructed concen- in 20 ml of MKB containing 10 ␮M nomifensine and 1 ␮M domperi- tration-response curves for both these steroids. As seen in 936 Nuwayhid and Werling

Fig. 1. Lack of effect of 10 ␮M DHEA, 3 or 10 ␮M testosterone on NMDA-stimulated [3H]dopamine release. Release of [3H]dopamine from slices of rat striatal tissue was stimulated by 25 ␮M NMDA or 25 ␮M NMDA in the presence of DHEA or testosterone as indicated. Statistical Downloaded from analysis by analysis of variance, performed on untransformed data (ratios of S2/S1) indicated no significant difference in 25 ␮M NMDA alone compared with 25 ␮M NMDA in the presence of DHEA or testosterone. Data are expressed as percentage of control NMDA-stimulated release. n ϭ 3 independent experiments in which each treatment was tested in triplicate. Note break in y-axis. jpet.aspetjournals.org Fig. 2A, progesterone inhibited NMDA-stimulated [3H]dopamine release in a concentration-dependent matter. Proges- ␴ terone has a Ki value of 270 nM at receptors, undifferentiated for subtype (Su et al., 1988). The IC50 for inhibition of release was similar to this value, lying between 100 and 300 nM. Progesterone significantly inhibited release at 300 nM and 1 ␮M. At 1 ␮M, the highest concentration tested, pro- Fig. 2. Concentration-response curves for progesterone (A) and preg- at ASPET Journals on March 13, 2015 gesterone inhibited release approximately 30%, similar to nenolone (B) on NMDA-stimulated [3H]dopamine release, and reversal by the maximum inhibition produced by (ϩ)-pentazocine that ␴ receptor antagonists. Release of [3H]dopamine from slices of rat striatal was attributable to ␴ receptors (Gonzalez-Alvear and Wer- tissue was stimulated by 25 ␮M NMDA alone or 25 ␮M NMDA in the presence of progesterone or pregnenolone as indicated, with or without ling, 1994). To confirm whether the inhibition by progester- the ␴ antagonist DuP734 (100 nM) or the ␴ antagonist Lu28-179 (1 nM). ␴ ␴ 1 2 ,ء .one was mediated through receptors, we tested the 1 Data are expressed as percentage of control NMDA-stimulated release ␴ significantly different from NMDA alone at p Ͻ 0.05. #, significantly receptor-selective antagonist DuP734 (100 nM) and the 2 receptor-selective antagonist Lu28-179 (1 nM) against 1 ␮M different from NMDA in the presence of progesterone or pregnenolone as indicated, p Ͻ 0.05. n Ն 3 independent experiments in which each progesterone (Fig. 2A). Both DuP734 and Lu2-1798 com- treatment was tested in triplicate. Note breaks in y-axes. pletely reversed the inhibition of release by progesterone to values slightly above control-stimulated release. The eleva- testosterone (3 ␮M) had any effect on the inhibition of [3H]do- tion above control is not significant for Lu28-179, and al- pamine release seen by either progesterone (Fig. 3A) or preg- though the value for DuP734 achieves statistical significance nenolone (Fig. 3B). as differing for NMDA alone, is associated with a high error To determine whether PKC␤ is involved in the inhibition of value. NMDA-stimulated [3H]dopamine release, we constructed Next, we constructed a concentration-response curve for dose-response curves for the PKC␤-selective inhibitor

pregnenolone. As seen in Fig. 2B, the IC50 value for inhibi- LY379196 against both progesterone and pregnenolone. We tion of release was between 300 nM and 1 ␮M. Pregnenolone have previously shown that LY379196 blocked (ϩ)-pentazo- at 3 ␮M inhibited release approximately 25%. As with pro- cine-mediated inhibition of NMDA-stimulated [3H]dopamine gesterone, we also tested pregnenolone (3 ␮M) with both the release in a concentration-dependent manner (Nuwayhid ␴ ␴ ␤ ␤ 1 antagonist DuP734 (100 nM) and the 2 antagonist Lu28- and Werling, 2003). LY379196 has Ki values at I and II 179 (1 nM). Both DuP734 and Lu28-179 completely reversed isozymes of 18 and 16 nM, and all other isozymes, including the inhibition by pregnenolone (Fig. 2B) to levels not differ- PKC␣, PKC␦, PKC␥, PKC⑀, and PKC␩ Ͼ300 nM (Louis Vig- ent from control-stimulated release. nati, personal communication). As seen in Fig. 4A, LY379196 Because DHEA and testosterone have affinity for ␴ recep- completely reversed the inhibition of release by 1 ␮M proges- tors, but did not show any inhibition of NMDA-stimulated terone at both 30 and 100 nM. At 30 nM, LY379196 com- [3H]dopamine release, we tested whether they might behave pletely reversed the inhibition of release by 3 ␮M preg- as ␴ antagonists in our assay. We tested DHEA and testos- nenolone (Fig. 4B). terone in combination with both progesterone and preg- Progesterone and pregnenolone are synthesized from cho- nenolone to examine whether they reversed the inhibition of lesterol, as demonstrated in neural cells in culture (Guarneri NMDA-stimulated [3H]dopamine release seen by both pro- et al., 2000). We therefore tested cholesterol to determine gesterone and pregnenolone. Neither DHEA (10 ␮M) nor whether it, as the parent compound, displayed any effects on Steroids Modulate Dopamine Release via ␴ Receptors 937 Downloaded from jpet.aspetjournals.org at ASPET Journals on March 13, 2015

Fig. 3. Lack of effect of testosterone or DHEA on the inhibition of Fig. 4. Reversal of steroid-mediated inhibition of NMDA-stimulated NMDA-stimulated [3H]dopamine release by 1 ␮M progesterone (A) and 3 [3H]dopamine release by the PKC␤-selective inhibitor LY379196. Release ␮M pregnenolone (B). Release of [3H]dopamine from slices of rat striatal of [3H]dopamine from rat striatal slices was stimulated by 25 ␮M NMDA tissue was stimulated by 25 ␮M NMDA alone or 25 ␮M NMDA in the alone or 25 ␮M NMDA in the presence of 1 ␮M progesterone (A) or 3 ␮M presence of agonist steroid, as indicated, with or without potential an- pregnenolone (B), as indicated, with or without the indicated concentra- tagonist steroid testosterone (3 ␮M) or DHEA (10 ␮M). Data are ex- tion of LY379196. Data are expressed as percentage of control NMDA- significantly different from NMDA alone at p Ͻ ,ء .signifi- stimulated release ,ء .pressed as percentage of control NMDA-stimulated release cantly different from NMDA alone at p Ͻ 0.05. Neither the inclusion of 0.05, n ϭ 3 independent experiments. #, significantly different from 1 ␮M testosterone nor DHEA significantly changed the inhibition produced by progesterone or 3 ␮M pregnenolone without PKC inhibitor at p Ͻ 0.05, progesterone or pregnenolone alone. n ϭ 3 independent experiments in n ϭ 3 independent experiments in which each treatment was tested in which each treatment was tested in triplicate. Note breaks in y-axes. triplicate. Note breaks in y-axes.

3 NMDA-stimulated [ H]dopamine release. Cholesterol (25 and not progesterone receptors by testing 1 ␮M progesterone ␮ 3 M) did not affect NMDA-stimulated release of [ H]dopam- in the presence of 10 ␮M mifepristone, a progesterone recep- ine (Fig. 5). tor antagonist. As seen in Fig. 7, there was no difference in Last, to verify the effects seen by pregnenolone on NMDA- ␮ 3 the inhibition seen by 1 M progesterone alone compared stimulated [ H]dopamine release were attributed to preg- with 1 ␮M progesterone in the presence of 10 ␮M mifepris- nenolone and not dependent upon its conversion to proges- tone. terone or other metabolite, we tested pregnenolone in the presence of 20 ␮M trilostane. Trilostane is an inhibitor of 3␤-hydroxysteriod dehydrogenase, an enzyme that converts ϭ Discussion pregnenolone to progesterone (Ki 50 nM; Takahashi et al., 1990). As seen in Fig. 6, inhibition of [3H]dopamine release The identity of the endogenous ligand for ␴ receptors has by 3 ␮M pregnenolone in the presence of trilostane was not been equivocal. Su et al. (1988) found that progesterone, deoxy- 3 ␴ significantly different compared with the inhibition of cortisone, and testosterone competed for [ H]SKF10,047 at 1 [3H]dopamine release by 3 ␮M pregnenolone alone. We also sites in guinea pig brain tissue and proposed steroids as the verified that progesterone was acting through ␴ receptors endogenous ligands. Several studies have now demonstrated 938 Nuwayhid and Werling

Fig. 5. Lack of effect of cholesterol on NMDA-stimulated [3H]dopamine release. Release of [3H]dopamine from slices of rat striatal tissue was Fig. 7. Lack of effect of mifepristone on the inhibition of NMDA-stimu- Downloaded from stimulated by 25 ␮M NMDA alone or 25 ␮M NMDA in the presence 25 lated [3H]dopamine release by 1 ␮M progesterone. Release of [3H]dopam- ␮M cholesterol, as indicated. There was no significant difference in re- ine from slices of rat striatal tissue was stimulated by 25 ␮M NMDA lease of [3H]dopamine stimulated by 25 ␮M NMDA alone compared with alone or 25 ␮M NMDA in the presence 1 ␮M pregnenolone, as indicated, 25 ␮M NMDA in the presence of 25 ␮M cholesterol. Data are expressed as with or without mifepristone. Data are expressed as percentage of control significantly different from NMDA alone at ,ء .percentage of control NMDA-stimulated release. n ϭ 3 independent ex- NMDA-stimulated release periments in which each treatment was tested in triplicate. Note break in p Ͻ 0.05, n ϭ 3 independent experiments in which each treatment was y-axis. tested in triplicate. Note break in y-axis. jpet.aspetjournals.org

ited NMDA-stimulated [3H]dopamine release. Both inhibited release in a concentration-dependent manner, with a maximum of about 25 to 30%, similar to the inhibition seen by (ϩ)-pentazocine in studies by Gonzalez-Alvear and Werling

(1994, 1995). The IC50 value of progesterone (300 nM) for inhibition of NMDA-stimulated [3H]dopamine release was at ASPET Journals on March 13, 2015 similar to its Ki value of 270 nM in competing for binding to ␴ receptors (Su et al., 1988). Pregnenolone showed an IC50 ␮ ␴ value in the range of 300 nM to 1 M. A Ki value at receptors in brain tissue has not been reported, but we found ␴ that pregnenolone competed for 1 binding with a Ki value of 980 Ϯ 340 nM in SH-SY5Y cells (Werling, 2002). Su et al. ␮ (1988) reported a Ki value of 3.2 M for pregnenolone sulfate ␴ binding to 1 receptors. These findings support that pregnenolone acts via ␴ receptors to inhibit NMDA-stimulated [3H]dopamine release. Fig. 6. Lack of significant effect of trilostane on pregnenolone-mediated The hypothesis that progesterone and pregnenolone act as inhibition of NMDA-stimulated [3H]dopamine. Release of [3H]dopamine ␴ agonists in our assay was confirmed by the action of ␴ ␮ from slices of rat striatal tissue was stimulated by 25 M NMDA alone or antagonists. The inhibition of NMDA-stimulated [3H]dopam- 25 ␮M NMDA in the presence 3 ␮M pregnenolone, as indicated, with or without 25 ␮M trilostane. Data are expressed as percentage of control ine release by 1 ␮M progesterone and 3 ␮M pregnenolone ␴ ϭ ء NMDA-stimulated release. , significantly different from NMDA alone at was fully reversed by the 1 antagonist DuP734 (Ki 10 nM) p Ͻ 0.05, n ϭ 3 independent experiments in which each treatment was ␴ (Culp et al., 1992) at 100 nM and the 2 antagonist Lu28-179 tested in triplicate. Note break in y-axis. ϭ (Ki 0.12 nM) (Moltzen et al., 1995) at 1 nM. The reversal seen by 100 nM DuP734 was somewhat above control, al- that steroids are synthesized in the brain (Hu et al., 1987; though associated with a relatively high error determination. Jung-Testas et al., 1989; Guarneri et al., 2000). It is possible that there is antagonism of tone exerted by Steroids exhibit genomic and nongenomic effects (Ruppert endogenous steroids. However, the antagonists had no effect and Holsboer, 1999). In the current study, we examined on basal release, which argues against this possibility. The presumably nongenomic effects of steroids on NMDA-stimu- action seen by both DuP734 and Lu28-179 suggest that the lated [3H]dopamine release in rat striatal tissue via ␴ recep- inhibition of [3H]dopamine release is mediated through both ␴ ␴ tors. The effects on regulation of dopamine release occur 1 and 2 receptor subtypes. This is a contrast to our findings within a relatively short time frame because the length of (Gonzalez-Alvear and Werling, 1994, 1995) in which compo- exposure of tissue to steroid is 12 min, not likely sufficient to nents of (ϩ)-pentazocine-mediated inhibition were clearly ␴ ␴ produce changes in protein expression. If steroids are endog- attributable to either 1 or 2 receptors based on reversal by enous ligands for ␴ receptors they should behave similarly to subtype-selective antagonists. However, in our assays exam- ϩ ␴ prototypic ligands, such as ( )-pentazocine. ining the effects of 2 agonists on amphetamine-stimulated We found that both progesterone and pregnenolone inhib- [3H]dopamine release from rat striatal slices, we found sim- Steroids Modulate Dopamine Release via ␴ Receptors 939 ␴ ␴ ilar antagonism of effect by 1 and 2 antagonists (Liu et al., late dopamine release from striatum are located on dopami- ␴ 2001) to that seen for the current steroid experiments. The 1 nergic nerve terminals, but those regulating norepinephrine ␴ ␴ ϭ and 2 receptors are relatively small ( 1 28 kDa, Hanner et release are not. Therefore, the strength of the stimulus and ␴ ϭ al., 1996; 2 22 kDa, Hellewell and Bowen, 1990). Perhaps the circuitry involved in hippocampus is likely to be much upon activation of ␴ receptor subtypes by progesterone and more complex. Monnet et al. (1995) used a concentration of pregnenolone the receptors interact with each other, which in NMDA 4 times that used by us in the current experiment and turn inhibits [3H]dopamine release. In this situation, com- in our previous experiments on norepinephrine. The higher ␴ ␴ peting off the steroid agonist with either 1 or 2 antagonists NMDA concentration could activate multiple other neurons would prevent regulation of dopamine release. Reports in upon which sigma receptors are located. Monnet et al. (1995) which ␴ receptor protein was purified and detected via pho- identified that the effects observed for some ␴ agonists were toaffinity labeling (Schuster et al., 1995) or with antibody mediated by different populations of ␴ receptor subtypes selective for ␴ receptor (Hanner et al., 1996), show a band depending upon the agonist used. It seems that our responses ␴ ␴ with a molecular weight of approximately 60 kDa, which are due to activation of only 1 and 2 subtypes only as ␴ ␴ could be a dimer of 1 and/or 2 receptors. Neuroactive ste- identified by selective antagonists. roids are known to modulate the actions of GABAA and Other groups have also reported effects of steroids that NMDA receptors (Ruppert and Holsboer, 1999). Although it were mediated by ␴ receptors. PREG S increase spontaneous

cannot be absolutely excluded that the effects of the steroids glutamate release via activation of a presynaptic Gi/o-coupled ␴ observed in the current study involve actions via one of these receptor (Meyer et al., 2002). Because steroid sulfotrans- Downloaded from receptors, the regulation of dopamine release by the steroids ferases and sulfatases are present in the central nervous is presumably via actions at the dopaminergic nerve termi- system (Rajkowski et al., 1997), both sulfated and nonsul- nal, because ␴ receptors regulating dopamine release have fated forms could be present in the brain and modulate been localized to that location (Gonzalez-Alvear and Werling, neurotransmitter release. However, the permeability of ste- ␴ 1995). Regardless of whether GABAA or NMDA receptors roids is reduced by the addition of sulfate; if receptors are contribute in some way to the overall response, the effects of intracellular, as suggested by McCann and Su (1994) and jpet.aspetjournals.org progesterone and pregnenolone are both completely reversed Vilner and Bowen (2000), the actions of sulfated steroids in the current study by ␴ receptor antagonists. would be limited. Unsulfated steroids would have to enter The concentrations of DHEA and testosterone tested were the cell and be subsequently sulfated to become active. It is

chosen based on their reported Ki values from binding stud- also possible that sulfated steroids could be concentrated and ies. Although both competed for ␴ receptor binding (Su et al., stored in vesicles (Gibbs and Farb, 2000). However, the weak 1988; Klein and Musacchio, 1994), our data do not indicate activities of sulfatases and sulfotransferases make the con- that the ␴ receptor subtype(s) involved in the regulation of version of steroids in vivo doubtful (Baulieu and Robel, 1996). at ASPET Journals on March 13, 2015 dopamine release are sensitive to these steroids. Neither Studies have now implicated the PKC signaling system in showed an inhibition of stimulated dopamine release, and ␴ receptor mediated processes. GF109203x, a PKC inhibitor, ␴ ␴ when tested for potential antagonist activity, neither re- abolished 2 receptor-mediated regulation of dopamine versed the inhibition of [3H]dopamine release by progester- transporter activity (Derbez et al., 2002). Morin-Surin et al. one or pregnenolone. DHEA and testosterone may bind to ␴ (1999) showed a parallel translocation of ␴ receptors and receptors but not have either agonist or antagonist properties PKC␤I and PKC␤II from the cytosol to the plasma membrane in our system. It is possible that higher or lower concentra- upon ␴ agonist application. They also showed a time-depen- tions could have produced effects, although this would not be dent desensitization in the ability of (ϩ)-pentazocine to re- predicted based on reported affinities at ␴ receptors. Further duce the firing rate in rat hypoglossal neurons. This desen- confirming that ␴ agonist properties are conferred only upon sitization may have been attributed to the desensitization of specific steroids, and not the heterocyclic steroidal structure PKC itself. In our studies, LY379196, a PKC␤-selective in- in general, was the finding that cholesterol, the parent com- hibitor, blocked the inhibition of [3H]dopamine release pro- pound from which steroids are derived, had no effect on duced by (ϩ)-pentazocine (Nuwayhid and Werling, 2003). NMDA-stimulated [3H]dopamine release. U73122, a PLC inhibitor, also blocked (ϩ)-pentazocine-medi- We confirmed that the effects displayed by pregnenolone ated regulation. This suggests that in order for (ϩ)-pentazo- were due to pregnenolone itself and not its metabolite pro- cine to exert its inhibition of dopamine release the PLC/PKC gesterone, using trilostane, an inhibitor of 3␤-hydroxysteroid system must be intact. PKC activation has also been linked dehydrogenase enzyme that converts pregnenolone into pro- to ␴ inhibition of NMDA-stimulated Ca2ϩ changes in cere- gesterone. Therefore, both progesterone and pregnenolone bellar granular cells (Snell et al., 1994). seem to act as ␴ receptors agonists in the regulation of In the current study, we determined that the inhibition of dopamine release. NMDA-stimulated [3H]dopamine release by steroids is de- Monnet et al. (1995) showed that DHEA S potentiated pendent upon PKC activation. Steroids are known to interact [3H]norepinephrine release from rat hippocampal slices and with the PKC pathway. Progesterone can rapidly stimulate this response was reversed by both ␴ antagonists haloperidol phosphatidylinositol bisphosphate hydrolysis, leading to the and BD1063. In a later article (Monnet et al., 1996), they also formation of diacylglycerol and inositol triphosphate, pre- showed regulation of norepinephrine release by other ste- sumably due to the action of Ca2ϩ-dependent PKC (Thomas roids, but with a different pharmacology than ours. Our data and Meizel, 1989). Estrogen increased inositol triphosphate show regulation of NMDA-stimulated [3H]dopamine release concentrations and stimulated PKC␣ levels in membrane by steroids is inhibitory. This could be due to the difference in fraction of HEPG2 cells through a nonclassical steroid mech- the neurotransmitter studied or other experimental vari- anism (Marino et al., 1998). Last, in studies by Condliffe et ables. We have previously shown that ␴ receptors that regu- al. (2001), 17-␤-estradiol regulated ClϪ secretion in rat co- 940 Nuwayhid and Werling

lonic epithelium, and regulation is blocked by the chelation of oligodendrocyte mitochondria convert cholesterol to pregnenolone. Proc Natl Acad 2ϩ Sci USA 84:8215–8219. Ca with BAPTA or the PKC inhibitor chelerythrine. In our Izenwasser S, Thompson-Montgomery DT, Deben SE, Chowdhury IN, and Werling study, steroids exhibited the same effects of (ϩ)-pentazocine LL (1998) Modulation of amphetamine-stimulated (transporter-mediated) dopa- ␤ mine release by sigma2 receptor agonists and antagonists in vitro. Eur J Pharma- in the presence of LY379196, an inhibitor of PKC isozymes. col 346:189–196. This reinforces the possibility that steroids have nongenomic Jung-Testas Z, Hu ZY, Baulieu EE, and Robel P (1989) Neurosteroids: biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells. Endocri- actions and can signal through PLC/PKC via their actions at nology 125:2083–2091. ␴ receptors. Klein M and Musacchio JM (1994) Effects of cytochrome P-450 ligands on the binding 3 In conclusion, both progesterone and pregnenolone have of [ H]dextromethorphan and sigma ligands on to guinea pig brain, in Sigma Receptors (Itzhak Y ed) pp 243–262, Academic Press, San Diego. been identified as ␴ receptor agonists in the modulation of Liu X, Nuwayhid S, Christie M, Kassiou M, and Werling LL (2001) Trishmocubanes: NMDA-stimulated [3H]dopamine release. We found them to novel ␴ ligands modulate amphetamine-stimulated [3H]dopamine release. Eur J Pharmacol 422:39–45. behave similarly to prototypic ligands, such as (ϩ)-pentazo- Marino PJ, Pallottini V, and Trentalance A (1998) Estrogens cause rapid activation cine. In addition, we have shown that the inhibition of of IP3-PKC signal transduction in HEPG2 cells. Biochem Biophys Res Commun 3 245:254–258. NMDA-stimulated [ H]dopamine release mediated by pro- Martin WR, Eades CG, Thompson JA, Huppler RE, and Gilbert PE (1976) The effects gesterone and pregnenolone involves a PKC signaling system of morphine- and nalorphine-type drugs in the non-dependent and morphine- ␤ dependent chronic spinal dog. J Pharmacol Exp Ther 197:517–532. most likely to involve the PKC isozyme. Our findings in this Maurice T, Phan VL, Urani A, and Guillemain (2001) Differential involvement of the study further support steroids as candidates for endogenous sigma-1 receptor in the anti-amnesic effect of neuroactive steroids as demon- ␴ strated using an antisense oligonucleotide strategy in the mouse. Br J Pharmacol ligands at receptors. 134:1731–1741. Maurice T, Phan VL, Urani A, Kamei H, Noda Y, and Nabeshima T (1999) Neuro- Downloaded from active steroids as endogenous effectors for the sigma1 (␴1) receptor: pharmacolog- Acknowledgments ical evidence and therapeutic opportunities. Jpn J Pharmacol 81:125–155. McCann DM and Su TP (1994) Sigma1 and sigma2 sites in rat brain: comparison of We thank Dr. John Puddefoot (Queen Mary and Westfield College) regional, ontogenetic and subcellular patterns. Synapse 17:182–189. for the gift of trilostane, Dr. Rob Zaczek (DuPont Merck) for the gift Meyer DA, Carta M, Partridge D, Covey DF, and Valenzuela CF (2002) Neuros- of DuP734, and Dr. Connie Sanchez (H. Lundbeck) for the gift of teroids enhance spontaneous glutamate release in hippocampal neurons: possible ␴ role of metabotropic 1 like receptors. J Biol Chem 277:28725–28732. Lu28-179. We also thank Dr. Nancy Pilotte (National Institute on Moltzen EK, Perregard TA, and Meier E (1995) ␴ ligands with subnanomolar affinity Drug Abuse) for helpful suggestions regarding experimental design and preference for the ␴ binding site. 2. Spiro-joined benzofuran, isobenzofuran

2 jpet.aspetjournals.org using steroids. and benzopyran piperidines. J Med Chem 38:2009–2017. Monnet FP, Mahe V, Robel P, and Baulieu EE (1995) Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by NMDA in the rat References hippocampus. Proc Natl Acad Sci USA 92:3774–3778. Bastianetto S, Monnet F, Junien J-L, and Quirion R (1999) Steroidal modulation of Morin-Surin MP, Collin T, Denavit-Saubie M, Baulieu EE, and Monnet FP (1999) sigma receptor function, in Contemporary Endocrinology: Neurosteroids: A New Intracellular sigma1 receptor modulates phospholipase C and protein kinase C Regulatory Function on the Nervous System (Baulieu EE, Robel P, and Schuma- activities in the brainstem. Proc Natl Acad Sci USA 96:8196–8199. cher M eds) pp 191–205, Humana Press, Totowa, NJ. Nuwayhid S and Werling LL (2003) Sigma1 receptor agonist-mediated regulation of 3 Baulieu EE and Robel P (1996) Dehydroepiandrosterone and dehydroepiandros- NMDA-stimulated [ H]dopamine release in dependent upon protein kinase C. terone sulfate as neuroactive steroids. J Endocrinol 150 (Suppl):S221–S239. J Pharmacol Exp Ther 304:364–369. Bergeron R, de Montigny C, and Debonnel G (1996) Potentiation of neuronal NMDA Rajkowski KM, Robel P, and Baulieu EE (1997) Hydroxysteroid sulfotransferase at ASPET Journals on March 13, 2015 response induced by dehydroepiandrosterone and its suppression by progesterone: activity in the rat brain and liver as a function of age and sex. Steroids 62:427–436. effects mediated via sigma receptors. J Neurosci 16:1193–1202. Ruppert R and Holsboer F (1999) Neuroactive steroids: mechanisms of action and Condliffe SB, Doolan CM, and Harvey BJ (2001) 17-␤-Estradiol acutely regulates ClϪ neuropsychopharmacological perspectives. Trends Neurosci 22:410–416. secretion in rat distal colonic epithelium. J Physiol (Lond) 530:47–54. Schuster DI, Arnold FJ, and Murphy RB (1995) Purification, pharmacological char- Culp SG, Rominger D, Tam SW, and De Souza EB (1992) [3H]DuP 734 [1-(cyclopro- acterization and photoaffinity labeling of sigma receptors from rat and bovine pylmethyl)-4-(2Ј-(4Љ-fluorophenyl)-2Ј-oxoethyl)-piperidine HBr]: a receptor binding brain. Brain Res 670:14–28. profile of a high-affinity novel sigma receptor ligand in guinea pig brain. J Phar- Snell LD, Iorio KR, Tbakoff B, and Hoffman PL (1994) Protein kinase C activation macol Exp Ther 263:1175–1187. attenuates NMDA-induced increases in intracellular calcium in cerebellar granule cells. J Neurochem 62:1783–1789. Derbez AE, Moody RM, and Werling LL (2002) Sigma2 receptor regulation of dopamine transporter activity via protein kinase C. J Pharmacol Exp Ther 301:306– Su TP, London ED, and Jaffe JH (1988) Steroid binding at sigma receptors suggest 314. a link between endocrine, nervous and immune systems. Science (Wash DC) Falkenstein E, Tillmann H-C, Christ M, Feuring M, and Wehling M (2000) Multiple 240:219–221. actions of steroid hormones—a focus on rapid, nongenomic effects. Pharm Rev Swartz S, Pohl P, and Zhou GZ (1989) Steroid binding at ␴-“opioid” receptors. Science 52:513–555. (Wash DC) 246:1635–1637. Gibbs T and Farb D (2000) Dueling engimas: neurosteroids and sigma receptors in Takahashi M, Luu-The V, and Labrie F (1990) Inhibitory effect of synthetic proges- the limelight. Sci STKE Nov 28 2000(60):PE1. tins, 4-MA and cyanoketone on human placental 3␤-hydroxysteroid dehydroge- Gonzalez-Alvear GM and Werling LL (1994) Regulation of [3H]dopamine release nase/5–4-ene isomerase activity. J Steroid Biochem Mol Biol 37:231–236. from rat striatal slices by sigma receptor ligands. J Pharmacol Exp Ther 271:212– Thomas P and Meizel S (1989) Phosphatidylinositol 4,5 biphosphate hydrolysis in 219. human sperm stimulated with follicular fluid or progesterone is dependent on upon Ca2ϩ influx. J Biochem 264:539–546. Gonzalez-Alvear GM and Werling LL (1995) Sigma1 receptors in rat striatum regulate NMDA stimulated [3H]dopamine release via a presynaptic mechanism. Eur Vilner BJ and Bowen WD (2000) Modulation of cellular calcium by sigma-2 recep- J Pharmacol 294:713–719. tors: release from intracellular stores in human SK-N-SH neuroblastoma cells. Guarneri P, Cascio C, Piccoli T, Piccoli F, and Guarneri R (2000) Human neuroblas- J Pharmacol Exp Ther 292:900–911. toma SH-SY5Y cell line: neurosteroid-producing cell line relying on cytoskeletal Werling LL (2002) Sigma receptor-active steroids and neurotransmitter release. Int organization. J Neurosci Res 60:656–665. J Psychopharmacol 5:S28. Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J, Kemper E, and Werling LL, Frattali A, Portoghese PS, Takemori AE, and Cox BM (1988) Kappa Glossman H (1996) Purification, molecular cloning and expression of the mamma- receptor regulation of dopamine release from striatum and cortex of rat and guinea pig. J Pharmacol Exp Ther 246:282–286. lian sigma1 binding site. Proc Natl Acad Sci USA 93:8072–8077. Hellewell SB and Bowen WD (1990) A sigma-like binding site in rat pheochromocy- toma (PC12): decreased affinity for (ϩ)benzomorphans and lower molecular weight Address correspondence to: Dr. Linda L. Werling, Department of Pharma- suggest a different ␴ receptor from that of guinea pig brain. Brain Res 527:244– cology, The George Washington University, 2300 Eye St., NW, Washington, 253. DC 20037. E-mail: [email protected] Hu ZY, Bourreau E, Jung-Testas I, Robel P, and Baulieu EE (1987) Neurosteroids: