Ca Signaling via s1-Receptors: Novel Regulatory Mechanism Affecting Intracellular Ca Concentration

The s1-receptor is a one-transmembrane endoplasmic reticulum protein that binds neurosteroids and dextrorotatory benzomorphans. The roles of s1-receptors in regulating intracellular Ca 21 in NG108 cells were examined in this study. s1-Ligands pregnenolone sulfate, (1)-pentazocine, and 2-(4-morpholino)ethyl-1phenylcyclohexane-1-carboxylate hydrochloride modulate Ca signaling in NG108 cells via two modes of action. First, nanomolar concentrations of the ligands, without effect by themselves, potentiated the bradykinin-induced increase of the cytosolic free Ca concentration in a bell-shaped manner. This effect of s1ligands was unaffected by depletion of Ca from perfusion buffer and was blocked by a 21-mer antisense oligodeoxynucleotide against the cloned s1-receptors. Second, after the cells were depleted of the endoplasmic reticulum Ca stores, the depolarization (75 mM KCl)-induced increase in cytosolic free Ca was potentiated by 2-(4-morpholino)ethyl-1-phenylcyclohexane-1carboxylate hydrochloride, whereas it was inhibited by pregnenolone sulfate and (1)-pentazocine. These effects, albeit opposite in direction, were blocked by both the 21-mer antisense oligodeoxynucleotide and pertussis toxin. Western blotting indicates that s1-receptors are increased on the plasma membrane and the nuclear membrane in the presence of s1-ligand. These results suggest that Ca signaling via s1-receptors may represent a novel mechanism that affects intracellular Ca concentrations. s1-Receptors are a subtype of “s-receptors” known to bind diverse classes of pharmacological agents with high affinity. These agents include neurosteroids, antipsychotics, and dextrorotatory benzomorphans (Snyder and Largent, 1989; Su, 1991). s-Receptors are not the “s/opioid” receptors originally proposed by Martin et al. (1976) to mediate psychotomimetic effects of benzomorphans. s1-Receptors are currently thought to be involved, among other functions, in learning and memory as well as in the analgesic processes of animals (Chien and Pasternak, 1994; Maurice et al., 1994, 1998; Bouchard et al., 1997). The s1-receptor has been cloned (Hanner et al., 1996; Prasad et al., 1998). The deduced amino acid sequence does not resemble that of any mammalian protein. The sequence of s1-receptors contains an endoplasmic reticulum (ER) retention signal close to the N terminus, a binding domain for steroid, and one putative transmembrane region. Although the cloned s1-receptor has a 30% identity and a 60% homology to a yeast C7-C8 sterol isomerase, s1-receptors apparently possess no similar enzymatic activity. Furthermore, the mammalian equivalent of the C7-C8 sterol isomerase has been cloned and the deduced amino acid sequence is different from that of both the yeast C7-C8 sterol isomerase and the s1-receptor (Silve et al., 1996). Thus, although the structure of s1-receptors is known, the biochemical basis subserving the action of s1-receptors remains elusive. Several lines of evidences have suggested that s-receptors may be related to the regulation of intracellular Ca. For example, exposure of cardiac myocytes to s-ligands was found to affect contractility, Ca influx, and beating rate (Ela et al., 1994) and to increase intracellular level of inositol 1,4,5-trisphosphate (IP3) in cultured myocytes (Novakova et al., 1998). s-Ligands affect intrasynaptosomal free Ca levels in rat forebrain synaptosomes and protein phosphorylation (Brent et al., 1997). Certain s-ligands also have been Received for publication October 6, 1999. 1 This study was supported by the Intramural Research Program of the National Institute on Drug Abuse/National Institutes of Health. The partial supports of the Division of Basic Research (Basic Neurobiology and Biological Systems Research Branch), National Institute on Drug Abuse/National Institutes of Health, and the Pharmacopsychiatry Research Foundation of Japan are appreciated. ABBREVIATIONS: ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; NMDA, N-methyl-D-aspartate; NE-100, N,N-dipropyl-2-[4methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride; AS ODN, antisense oligodeoxynucleotide; PS, pregnenolone sulfate; (1)PTZ, (1)-pentazocine; PRE-084; 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate hydrochloride; PT, pertussis toxin; BDK, bradykinin; TG, thapsigargin; HBSS, Hanks’ balanced salt solution; MS ODN, mismatched oligodeoxynucleotide; TBST, Tris-buffered saline/Tween 20; Prog, progesterone; GABA, g-aminobutyric acid; T 1 B 1 C, TG 1 BDK 1 caffeine; VDCC, voltage-dependent Ca channel. 0022-3565/00/2933-0788$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 293:788–798, 2000 /2173/823032 788 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from shown to affect N-methyl-D-aspartate (NMDA)-induced Ca signaling in rat primary neurons (Hayashi et al., 1995; Klette et al., 1997). However, none of these studies has definitively demonstrated a clear-cut agonist-antagonist relationship in which pharmacologically relevant concentrations of ligands were used and the antagonists by themselves produced no effect. Furthermore, none of the above-mentioned studies has definitively demonstrated which subtype of s-receptors (i.e., s1 or s2; Quirion et al., 1992; Hellewell et al., 1994) mediated these effects. With the availability of a selective s1-receptor antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) (Okuyama and Nakazato, 1996) and the successful demonstration of the use of an antisense oligodeoxynucleotide (AS ODN) directed against the cloned s1-receptors in a behavioral test (King et al., 1997), we decided to systematically investigate the potential involvement of s1-receptors in Ca 21 signaling with laser scanning confocal microscopic examinations in a neuroblastoma 3 glioma cell line (NG108) with two specific purposes. First, we wanted to establish a clear-cut agonist-antagonist relationship within the context of Ca signaling for s-receptors in a simple biological system; Second, we wanted to establish that the effects, if any, are mediated via s1-receptors. Because Ca signaling is regulated mainly by intracellular organelles such as the ER and by Ca influxes at the plasma membrane and because s-receptors have been shown to exist on both the ER and the plasma membrane (McCann and Su, 1990), this study examined the potential involvement of s1-receptors in Ca 21 signaling in both regards: via an intracellular site of action and via the plasma membrane. Three compounds, pregnenolone sulfate (PS), (1)-pentazocine [(1)-PTZ], and 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate hydrochloride (PRE-084), each representing a s1-ligand of different chemical class, were examined. Experimental Procedures Materials. PS, pertussis toxin (PT), MK-801 (dizocilpine), (1)bicuculline, and bradykinin (BDK) were purchased from Research Biochemicals International (Natick, MA). Thapsigargin (TG), Tween 20, phenylmethylsulfonyl fluoride, and aprotinin were from Calbiochem (San Diego, CA). (1)-PTZ was obtained from Research Triangle Institute (Research Triangle Park, NC). [H]IP3 and the IP3 assay kit were purchased from Amersham (Arlington Heights, IL). All other chemicals were from Sigma Chemical Co. (St. Louis, MO) Confocal Fluorescence Microscopic Examination. NG108 cells were cultured as described before (Hescheler et al., 1987) and maintained on petriPERM with a four-well FlexiPERM (Heraerus Instr., Am Kalkberg, Germany) attached to it. The cultured cells on petriPERM were washed with Hanks’ balanced salt solution (HBSS) (137 mM NaCl, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.4 mM KCl, 0.4 mM KH2PO4, 0.3 mM Na2HPO4, 3.0 mM NaHCO3, 1.5 mM CaCl2, 5.6 mM glucose, and 20 mM HEPES, pH 7.4) and loaded with 5 mM fluo-3/AM (Molecular Probes, Eugene, OR) in HBSS for 25 min at room temperature. Then uniform fluorescence intensities were observed in each cell. Permeabilization of cells with 10 mM digitonin in a Ca-free medium completely eliminated the fluorescence signals inside the cells, indicating that the observed fluorescence intensities represent free Ca in the cytosol (Golovina and Blaustein, 1997). After loading, cells were placed on the microscope stage and perfused continuously (2 ml/min) with HBSS at 37°C by using two peristaltic pumps (input and output) to maintain the test well volume at ;200 ml. One end of the solution input line was attached to a plastic pipette tip that was placed 1.5 mm above the cells. The other end of the input line was placed into beakers containing HBSS or HBSS with drugs at test concentrations. In the BDK experiments, HBSS containing s-ligands was perfused into the well for at least 10 min. Afterward, a solution containing both the s-ligands and 1 mM BDK was perfused into the well for additional 2 min. In the KCl experiments, the cells were first perfused with the HBSS containing a test drug (such as s-ligands) or a combination of several different drugs for 10 min. The same solution with an addition of 75 mM KCl was then applied to the cells for a duration of 30 s before the solution was replaced with the original solution containing only the test ligand(s). In this perfusion system, maximal response to KCl was reached within 3 to 5 s. Dye-loaded cells were perfused with 37°C HBSS and the cytosolic free Ca concentration ([Ca]cyt) was monitored with a laser scanning confocal system (Zeiss, Oberkochen, Germany) attached to a Zeiss Axiovert 135 inverted microscope. Laser scanning microscopic images were collected every 2 s with a Zeiss 40X achroplan water-immersion objective (0.75 numerical aperture), an argon laser (488 nm) excitation, and a 520-nm-long pass barrier filter. The confocal optical section was ;1.5 mm in thickness and the optical section of cells was adjusted to show both the cytosol and nucleus on the same section. Images (512 3 512 pixels) were collected digitally with the Zeiss Image Series software. Regions of interest were selected from the areas between the plasma membrane and nucleus. The nucleus was excluded from the Ca measurement because the intranucleosomal Ca dynamics might be different from that in the cytosol. [Ca]cyt was calibrated in each cell according to the equation [Ca] 5 Kd[F(t) 2 Fmin]/[Fmax 2 F(t)], as described by Kao et al. (1989), in which F(t) is the measured fluorescence intensity and Kd is the Ca-fluo-3 dissociation constant (390 nM). The Fmax equals (FMn 2 Fbkg)/0.2 2 Fbkg and the Fmin equals (Fmax 2 Fbkg)/40 2 Fbkg. FMn is the average of fluorescence intensities for 30 s with ionomycin (10 mM) and MnCl2 (2 mM), and Fbkg is the average of fluorescence intensities for 30 s with ionomycin (10 mM) and MnCl2 (2 mM) after lysis of cells with 40 mM digitonin. In this report, each experimental determination used a four-well FlexiPERM plate. An average of only three to nine cells per culture well, but never more than nine cells per well, was examined in each determination, which always included a control well. In each determination, a similar treatment condition was never repeated in other wells except occasionally the controls were repeated. Statistical significance was analyzed by comparing values from all cells receiving the same treatment condition in separate determinations. In this report, the number of determinations in each experimental group is indicated in parentheses after the number of cells examined. AS ODN Transfection. Genes of the s1-receptor were highly conserved from mouse to humans (Hanner et al., 1996; Prasad et al., 1998). Therefore, a 21-mer AS ODN, which was published based on the mouse sequence for s1-receptor (King et al., 1997), was synthesized with the Cyclone Plus DNA synthesizer (Milli Gen/biosearch, Bedford, MA), purified through an NAP-10 column (Pharmacia Biotech, Uppsala, Sweden), and used in this study. The sequences of the AS ODN and the mismatched oligodeoxynucleotide (MS ODN) control with three pairs of nucleotide substitutions were, respectively (King et al., 1997): 59-GAGTGCCCAGCCACAACCAGG-39 and 59GAGGTCCCGACCACACACAGG-39. When NG108 cells reached a 40 to 50% confluency, adherent cells were washed with culture medium containing reduced fetal calf serum (5%) without penicillin and streptomycin. For transfection, cells were treated with 200 ml of the same medium containing Lipofectin reagent (Life Technologies, Gaithersburg MD) and the AS ODN or MS ODN at 3.75 mg/ml. Cells were incubated at 37°C under 10% CO2 for 6 h and then 0.8 ml of Dulbecco’s modified Eagle’s medium with 10% fetal calf serum (without antibiotics) was added to each culture well. Cells were used for experiments 48 to 72 h after the transfection. The culture medium for the transfection experiment contains 0.1 mM hypoxanthine, 400 nM aminopterin, and 16 mM thymidine. 2000 Ca Signaling via s1-Receptors 789 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from Determination of IP3 Concentration. For the determination of contents of IP3, NG108 cells were washed with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.4) and resuspended in HBSS containing 10 mM LiCl at a density of 2 3 10 cells/ml. Cells (6 3 10) in 300-ml aliquots were preincubated for 10 min at 37°C and then stimulated with different concentrations of BDK, in the presence and absence of 100 nM (1)-PTZ, for 15 s. The reaction was terminated by adding 0.2 volumes of ice-cold 20% perchloric acid. The resultant samples were adjusted to pH 7.5 with KOH-HEPES buffer. Neutralized samples were centrifuged to remove KClO4, and the supernatant was used for the IP3 assay in a radioligand method with [H]IP3 as previously described in Katayama et al. (1994). Immunodetection of s1-Receptors by Western Blotting. A polyclonal antibody was raised in rabbit against a 20-amino acid peptide corresponding to the fragment 143 to 162 (synthesized by Neosystem, Strasbourg, France) of rat s1-receptor. The peptide was conjugated to BSA with glutaraldehyde (1 mg/peptide/5 mg of BSA) and the complex was dialyzed against saline. Three rabbits were subsequently subjected to a standard boost of the complex peptide with Freund’s adjuvant. For each rabbit, serum was collected before immunization and every 6 weeks after the third injection. The different sera obtained were screened by immunostaining with forebrain sections obtained from adult rats. The antiserum giving the most intense immunostaining was selected and purified against the antigenic peptide with the HiTrap N-hydroxysuccininide-activated affinity column (Pharamcia Biotech). The immunostaining was reduced by the antigen peptide. NG108 cells were washed and incubated with HBSS for 30 min. Cells were treated with or without s1-ligands. The membrane fractions from NG108 cells and rat hippocampus to be used for Western blotting were prepared as described previously and P1, P2, and P3 fractions were obtained accordingly (Furuichi et al., 1989; Miyawaki et al., 1991). Thirty micrograms of protein was dissolved in SDS gel sample buffer and separated by 12% SDS-polyacrylamide gel electrophoresis. For Western blotting, proteins were transferred to a polyvinylidene difluoride membrane (BioRad, Richmond, CA). The nonspecific sites were blocked with 5% (w/v) nonfat dry milk (Bio-Rad) in Tris-buffered saline/Tween 20 (TBST; 10 mM Tris-HCl, pH 8.0; 150 mM NaCl, and 0.05% Tween 20) at 4°C overnight. For immunodetection of s1-receptor, antibody was diluted 2000-fold in TBST containing 2% milk and incubated with membrane at 30°C for 2 h. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000 dilution; 1 h at room temperature; Amersham) in TBST with 5% milk. The peroxidase activity was revealed by using an enhanced chemiluminescence detection kit (Amersham). Statistical Analyses. ANOVA was first used to check the statistical tendency of experimental data. Differences between groups of data were then examined post hoc by Fisher’s protected least-significant difference test or Student’s t test. All data presented represent mean 6 S.E. The significance level was set at P , .05.