Introduction

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Sigma receptors are unique drug binding sites with pharmacological profiles and potential functions that are unrelated to other known receptors (for reviews, see Walker et al., 1990; Itzhak, 1994). Two pharmacologically defined subclasses of sigma-receptor are now widely accepted, termed sigma-1 and sigma-2 (Hellewell and Bowen, 1990; Quirion et al., 1992). Although both subtypes bind haloperidol and 1,3-di-o-tolylguanidine (DTG) with high affinity, sigma-1 receptors bind (+)-benzomorphans and (+)-morphinans with high affinity, whereas sigma-2 receptors exhibit low to negligible affinity for these compounds (Hellewell and Bowen, 1990; Quirion et al., 1992).

Sigma ligands have been found to produce a wide array of effects, implicating sigma-receptors in various biochemical, physiological, and behavioral processes. Notable among these are several modulatory actions demonstrated in vitro, including the modulation of basal neurotransmitter synthesis or release, modulation of N-methyl-D-aspartate (NMDA)-glutamate receptor-stimulated neuronal activity, modulation of NMDA-glutamate receptor-stimulated dopamine and norepinephrine release, modulation of muscarinic receptor-stimulated phosphoinositide turnover, and modulation of potassium channel conductances (Walker et al., 1990; cf. Itzhak, 1994). Some behavioral effects of sigma-receptors include the regulation of movement and posture and involvement in learning and memory processes (Walker et al., 1990; cf. Itzhak, 1994). Sequence similarity and pharmacological overlap of the cloned sigma-1 receptor with the fungal sterol isomerase enzyme have suggested a role in sterol biosynthesis (Hanner et al., 1996). However, sigma-1 receptor protein lacks any enzymatic activity, and a clear role in sterol metabolism has yet to be demonstrated.

Little is known of the signal transduction mechanisms occurring at the cellular level that might underlie functional effects of sigma receptors. Ca²⁺ serves as an important intracellular signal for cellular processes such as growth and differentiation, regulation of gene expression, and cellular stimulus-secretion coupling (Clapham, 1995; Simpson et al., 1995; Berridge et al., 1998). Ca²⁺ is also known to be toxic to cells and is involved in the triggering of events leading to excitotoxic cell death in neurons, as well as apoptosis in various cell types (McConkey and Orrenius, 1996; Berridge et al., 1998). Maintenance of cellular Ca²⁺ homeostasis involves a coordinated control of several processes that change the intracellular Ca²⁺ concentration in the cytosol ([Ca²⁺]_i). [Ca²⁺]_i can be altered by entry of Ca²⁺ from outside of the cell through 1) voltage-dependent channels, 2) voltage-independent channels or pores, and 3) ligand-gated Ca²⁺ channels. [Ca²⁺]_i can also be altered by changing the activity of the energy-dependent mechanisms that pump Ca²⁺ out of the cell (plasma membrane Ca²⁺-ATPases) or those that concentrate Ca²⁺ from the cytoplasm into the endoplasmic reticular, sarcoplasmic reticular, and mitochondrial intracellular storage pools. Ca²⁺ can be released from intracellular storage pools by certain signals such as electrical stimulation in the case of the sarcoplasmic reticulum and by activation of inositol-1,4,5-trisphosphate (IP₃) receptors in the case of endoplasmic reticulum. The latter is the result of activation of G protein-coupled hormone or neurotransmitter receptors that are linked to activation of phospholipase-C and production of inositol phosphates and diacylglycerol. Mitochondria and other intracellular organelles can also be involved in Ca²⁺ signaling by playing a role in cytoplasmic Ca²⁺ buffering, thus helping to maintain Ca²⁺ homeostasis (Pozzan et al., 1994; Clapham, 1995; Simpson et al., 1995).

We have previously shown that sigma receptors mediate morphological changes in neuronal and non-neuronal cells and ultimately produce cell death on continued exposure in culture (Bowen and Vilner, 1994; Vilner et al., 1995a). In an attempt to determine the mechanisms that might be involved in the cytotoxic effects of sigma ligands and potential signaling pathways used by sigma-receptors in normal cell functions, we investigated whether sigma ligands can modulate the levels of intracellular Ca²⁺. Previous experiments revealed that sigma ligands from various structural classes produce a dual modulation of [Ca²⁺]_i: release of Ca²⁺ from intracellular stores and blockade of depolarization-dependent influx of Ca²⁺ (Vilner and Bowen, 1995). Here, we further characterize the sigma ligand-induced intracellular release of Ca²⁺ in human SK-N-SH neuroblastoma cells with respect to specificity, time course, Ca²⁺ source, and pharmacological profile and show that it is mediated via sigma-2 receptors. Portions of this work have been published previously in abstract form (Vilner and Bowen, 1995; Bowen et al., 1996).

	Materials and Methods

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Cell Culture. SK-N-SH line cell (human neuroblastoma, HTB-11; American Type Culture Collection, Manassas, VA) were grown in 75-cm² culture flasks (Costar, Cambridge, MA) in Dulbecco's modified Eagle's medium (DMEM), enriched with 10% FBS in a humidified atmosphere of 5% CO₂/95% air at 37°C. Once cells formed a confluent monolayer, they were washed in Dulbecco's phosphate-buffered saline [DPBS; 136.9 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl₂, 8.10 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 0.904 mM CaCl₂, 5.55 mM D-(+)-glucose, 0.33 mM sodium pyruvate, 0.05 g/l streptomycin sulfate, pH 7.2] and dispersed with cell dissociation solution (2 ml/flask for 10 min at 37°C). Cells were then harvested by centrifuging and resuspending in DMEM/10% FBS at a density of 50,000 to 100,000 cells/ml.

[Ca²⁺]_i Measurement in Single Cells. For [Ca²⁺]_i measurement, cover glasses containing cells were washed twice in DPBS and then incubated in DPBS containing 2.0 to 3.3 µM Indo-1 AM and 0.066% Pluronic F-127 (Molecular Probes, Inc., Eugene, OR). After incubation for 60 to 75 min at room temperature in darkness, cultures were washed twice in DPBS and allowed to stand at room temperature in the dark for 30 min until use in the experiments. Cells could be maintained this way for up to 5 h before use, with no differences in basal Ca²⁺ levels or results.

Sigma Receptor Binding. Sigma receptor binding was carried out in membranes from SK-N-SH neuroblastoma cells. For binding assays, cells were plated at a density of 2.5 × 10⁶ cells/75-cm² plastic culture flasks and cultured under the conditions described above. The medium was changed once a week, and the cells were allowed to grow to near confluence. Cells were harvested and membranes were prepared as described previously (Vilner et al., 1995b), with the following modifications. Cells were washed in situ with DPBS before detachment and then detached by scraping in DPBS. The final membrane pellet was resuspended to a protein concentration of 10 to 15 mg/ml in 10 mM Tris · HCl, pH 7.4.

Chemicals. (+)-[³H]Pentazocine (51.7 Ci/mmol) was synthesized as described previously (Bowen et al., 1993a). [³H]DTG (35.2 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA).

	Results

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Acute Effect of Sigma Ligands on Intracellular Ca²⁺ Levels

Effect of Selective Sigma Ligands. Human SK-N-SH neuroblastoma cells, which have been shown to express both sigma-1 and sigma-2 receptors (Vilner et al., 1995b), were used to examine the direct effect of sigma ligands on intracellular Ca²⁺. Figure 1 shows time versus [Ca²⁺]_i traces for the effect of the selective sigma agonist BD737 (Bowen et al., 1992) and the benzylidene-5-phenylmorphan CB-64D (Bowen et al., 1995a) at 100 µM. Both BD737 and CB-64D produced a transient rise in [Ca²⁺]_i that began in seconds after the addition of drug to cells. The peak level of [Ca²⁺]_i was usually reached in 1.5 to 3 min and returned to a baseline level that was slightly higher than the original within 4 to 5 min after reaching the peak. Red HAL, a sigma-active metabolite of haloperidol, produced the same effect (plot not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Acute effect of sigma ligands on [Ca2+]i in human SK-N-SH neuroblastoma cells. Sigma ligands (100 µM) were added to cells in normal DPBS at the indicated time point, and the effect on [Ca2+]i was monitored until a return to stable baseline. Ratios of 410:485 nm were converted to concentration (nM) using the FASTINCA program as described in Materials and Methods. Representative time-versus-[Ca2+]i traces for BD737 (A) and CB-64D (B) are shown. The sigma ligands produced a transient rise in [Ca2+]i that began within seconds after drug addition and returned to baseline within 7 to 10 min.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Dose-response for ability of sigma ligands to increase [Ca2+]i. Various concentrations (3, 10, 30, and 100 µM) of BD737, Red HAL, and CB-64D were added to cells in normal DPBS, and time-versus-[Ca2+]i traces were obtained as shown in Fig. 1. The percent increase in [Ca2+]i produced by the ligand was calculated for each individual trace by determining the peak (maximum) Ca2+ concentration relative to the starting baseline level. One cell per dish was chosen for study, and each cell was used only once. The initial (basal) [Ca2+]i was 80 to 120 nM. Cells with higher levels of basal [Ca2+]i were not used in this study. Values are the averages of no fewer than three cells ±S.E.

Sigma-1 and Sigma-2 Receptors of SK-N-SH Neuroblastoma Cells and Pharmacological Profile of Effect on [Ca²⁺]_i. As previously reported (Vilner et al., 1995b), the sigma-1 receptor probe (+)-[³H]pentazocine labeled a single population of sites with a K_d value of 28.0 nM and a B_max value of 975 fmol/mg protein in membranes from SK-N-SH neuroblastoma cells. The sigma-2 receptor probe [³H]DTG (in the presence of dextrallorphan to mask sigma-1 sites) labeled a single population of sites with a K_d value of 32.4 nM and a B_max value of 944 fmol/mg protein.

View this table: [in this window] [in a new window]   TABLE 1 Sigma Receptor Binding in SK-N-SH Neuroblastoma Cells and Pharmacological Profile of Effect of Sigma Ligands on [Ca2+]i Sigma receptor binding assays were carried out under the conditions described in Materials and Methods. Twelve concentrations of unlabeled test ligand ranging from 0.05 to 10,000 nM or 0.5 to 100,000 nM were incubated with SK-N-SH neuroblastoma cell membranes and 10 nM [3H](+)-pentazocine (sigma-1 receptors) or 10 nM [3H]DTG in the presence of 1 µM dextrallorphan (sigma-2 receptors). IC50 values were determined using the iterative curve-fitting program GraphPAD Prism (San Diego, CA). IC50 values were then converted to apparent Ki values using the Cheng-Prusoff equation and radioligand Kd values determined previously (Vilner et al., 1995b). Ki values are the averages of two to three experiments ±S.E.M. Each experiment was carried out in duplicate. Values for some compounds have been published previously (Vilner et al., 1995b). The various compounds were examined for their ability to affect [Ca2+]i at a concentration of 100 µM. Experiments were carried out and data analyzed as described in the legend to Fig. 2. All data are the averages from three cells ±S.E.M. The following ligands for other receptors, which lack affinity for sigma-1 or sigma-2 receptors produced little or no change (<10%) in [Ca2+]i (n = 2-3 cells): morphine (30 and 100 µM), DAMGO (1 µM), ()-naltrexone (100 µM), atropine (100 µM), ()-sulpiride (30 and 100 µM), MK-801 (30 and 100 µM), and mianserin (10 µM).

Specificity of Effect on [Ca²⁺]_i for Sigma Receptors. The specificity of the effect for sigma-2 receptors was further investigated. BD1006 is an ethylene diamine that lacks an aromatic ring moiety. As shown in Table 1, this compound is devoid of sigma receptor affinity and thus is a control for possible nonspecific effects of diamines. CB-53D is a close analog of the 5-phenylmorphans CB-64D and CB-64L but lacks the benzylidene moiety that imparts sigma binding affinity (Bertha et al., 1995). Noribogaine is the O-desmethyl derivative of ibogaine and has markedly reduced sigma receptor affinity relative to ibogaine (Bowen et al., 1995b). All three of these compounds produced <10% increase in [Ca²⁺]_i at a concentration of 100 µM (Table 1). Thus, structurally related compounds that lack significant sigma receptor affinity failed to produce robust effects. This supports the notion that the activity of the arylethylenediamines BD737, LR172, BD1008, and JL-II-147; of the benzylidine phenylmorphans CB-64D and CB-64L; and of the iboga alkaloid ibogaine is related to the sigma-2 receptor binding affinity rather than to a nonspecific effect.

Blockade of Ligand-Induced Rise in [Ca²⁺]_i by Sigma Receptor Antagonists BD1047 and BD1063. The arylethylene diamines BD1047 and BD1063 have been demonstrated as highly selective sigma receptor antagonists (Matsumoto et al., 1995). BD1047 binds to sigma-1 and sigma-2 receptors of human SK-N-SH neuroblastoma cells with a K_i value of 36.5 ± 2.2 and 587 ± 29 nM, respectively. BD1063 binds to SK-N-SH sigma-1 and sigma-2 receptors with a K_i value of 8.80 ± 0.1 and 332 ± 40 nM, respectively. The effects of BD1047 and BD1063 on [Ca²⁺]_i were investigated.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of the sigma antagonists BD1047 and BD1063 on ability of CB-64D to increase [Ca2+]i. BD1047 (30 µM) or BD1063 (30 µM) was added to SK-N-SH neuroblastoma cells simultaneously with 10, 30, or 100 µM CB-64D. Effect on [Ca2+]i was then monitored for 10 min, and percent increase in [Ca2+]i was calculated as described in the legend to Fig. 2. Values are the averages of no fewer than three cells ±S.E. These experiments were carried out in DPBS at pH 8.2. Under these conditions, the rise in [Ca2+]i produced by 30 µM BD1047 and BD1063 alone was 31.4 ± 5.06 and 25 ± 2.18%, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of sigma antagonists on action of CB-64D and BD737 after preincubation of cells with antagonists. Cells were treated with 30 µM BD1047 for 10 min before the addition of 100 µM CB-64D (top) or with 30 µM BD1063 for 10 min before the addition of 100 µM BD737 (bottom) in DPBS at pH 8.2. Effect on [Ca2+]i was then monitored for 10 min after the addition of the agonist. Antagonists remained present during incubation with agonists. Values were compared with cells treated with either antagonist alone (30 µM) or agonist alone (100 µM), with [Ca2+]i monitored for 10 min after the addition in each case. Percent increase in [Ca2+]i was calculated as described in the legend to Fig. 2. Values are the averages from two to five cells ±S.E.

Origin of Transient Increase in [Ca²⁺]_i Induced by Sigma Ligands. To determine whether extracellular Ca²⁺ contributed to the transient rise in [Ca²⁺]_i, the effect of removing extracellular Ca²⁺ was investigated as shown in Table 2. Depolarization of SK-N-SH cells with 55 mM KCl in normal DPBS (which contains ~1 mM CaCl₂) produced a robust increase (565%) in [Ca²⁺]_i due to a large influx of Ca²⁺ through voltage-gated Ca²⁺ channels. Placement of cells in Ca²⁺-free DPBS completely eliminated this influx, confirming the absence of extracellular Ca²⁺ under these conditions. However, removal of extracellular Ca²⁺ had little or no effect on the ability of 100 µM BD737, Red HAL, or CB-64D to increase [Ca²⁺]_i. This shows that sigma ligands produce a rise in [Ca²⁺]_i by release from intracellular stores.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Time-versus-[Ca2+]i traces showing effect of thapsigargin pretreatment on ability of sigma ligands to transiently increase [Ca2+]i. Thapsigargin (150 nM) was added to cells in either normal DPBS or Ca2+-free DPBS at the indicated time point, and the effect on [Ca2+]i was monitored as described in Materials and Methods and the legend to Fig. 1. Representative time-versus-[Ca2+]i traces are shown. Thapsigargin produced a transient rise in [Ca2+]i that began within seconds after the addition and returned to baseline within 10 to 13 min, indicative of depletion of Ca2+ from the endoplasmic reticulum and subsequent removal from cell. At the peak, the thapsigargin-induced rise in [Ca2+]i was 155 ± 9% (n = 58 cells) relative to the initial basal level. After the return to baseline, 100 µM BD737 (A) or CB-64D (B) was then added as indicated by the arrow, and [Ca2+]i was continually monitored. The effect of thapsigargin on BD737 was carried out in normal DPBS, whereas the effect on CB-64D was carried out in Ca2+-free DPBS. No effect on [Ca2+]i was seen after the addition of sigma ligand to thapsigargin-treated cells.

Effect of Prolonged Exposure to Sigma Ligands on [Ca²⁺]_i

Effect of Selective Sigma Ligands and Pharmacological Profile. The transient rise in [Ca²⁺]_i produced by sigma ligands as characterized above occurred within seconds of the addition of the compound, and in most cells, it had returned to near baseline within ~10 min. Observation of the cells by phase contrast microscopy at the time of maximum rise in [Ca²⁺]_i or shortly after the return to near baseline showed no sign of morphological changes. Because longer-term exposure of cells to sigma ligands produces changes in morphology (Bowen and Vilner, 1994; Vilner et al., 1995a), we investigated the effect of prolonged exposure on [Ca²⁺]_i.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of prolonged exposure of SK-N-SH neuroblastoma cells to sigma ligand. The sigma ligand was added to SK-N-SH neuroblastoma cells at the point indicated, and the [Ca2+]i was monitored continuously for 5 min or until Ca2+ levels returned to near original baseline. The shutter was then closed. After ~15 min had elapsed since drug addition, the [Ca2+]i was determined every 10 min for up to 70 min by taking readings over a few seconds. This diminished bleaching of Indo-1 dye due to prolonged exposure to light. Cell movement and/or premature detachment often hindered observation of cells for prolonged periods. This was particularly problematic in Ca2+-free DPBS, where cells are attached less strongly than in normal DPBS. This was remedied by growing the cells on coverslips in collagen gel instead of on dry collagen as described in Materials and Methods. Alternatively, cells grown on dry collagen could be stabilized by placement under a small piece of glass wool after selection in phase contrast mode. These procedures did not affect the normal response of the cells. Experiments with 100 µM CB-64D were carried out in normal DPBS (n = 5 cells) and in Ca2+-free DPBS (n = 6 cells) with similar results. Shown is a representative time-versus-[Ca2+]i plot for CB-64D (100 µM), carried out in Ca2+-free DPBS. The horizontal bars represent points at which readings were taken, and the [Ca2+]i (nM) is displayed beside each bar. Similar results were also obtained with 100 µM BD737 and 100 µM Red HAL (normal DPBS, n = 3 cells each). However, neither 100 µM (+)-pentazocine nor 100 µM DTG (normal DPBS, n = 3 cells each) had any effect on [Ca2+]i for up to 60 min.

Origin of Sustained Increase in [Ca²⁺]_i Induced by Sigma Ligands. Figure 7 shows a direct comparison of the effect of extracellular Ca²⁺ removal and thapsigargin pretreatment relative to the normal condition on both the transient and sustained rise in [Ca²⁺]_i produced by CB-64D. The sustained rise, like the transient rise, was observed in the absence of extracellular Ca²⁺. This shows that the source of the sustained rise in [Ca²⁺]_i is also intracellular. However, unlike the transient rise, the sustained rise in [Ca²⁺]_i was not affected by thapsigargin pretreatment, indicating that the intracellular source is not the endoplasmic reticulum. In fact, thapsigargin pretreatment had no effect on the sigma ligand-induced sustained rise in [Ca²⁺]_i even when extracellular Ca²⁺ was excluded. Taken together, these results suggest that in addition to a rapid and transient release of Ca²⁺ from the endoplasmic reticulum, prolonged exposure of cells to sigma-2 receptor ligands results in a latent and sustained release of Ca²⁺ from other intracellular stores that are thapsigargin-insensitive. This latent release of Ca²⁺ may be associated with alterations in cellular morphology.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effect of thapsigargin pretreatment and removal of extracellular Ca2+ on ability of sigma ligands to induce a latent rise in [Ca2+]i. Experiments were carried out essentially as described in the legend of Fig. 6. For the data shown, cells in normal DPBS were treated with vehicle for 10 min () or with 150 nM thapsigargin for 10 min () before the addition of 100 µM CB-64D at the time point indicated by the arrow. The effect of removal of extracellular Ca2+ (without thapsigargin pretreatment) is included for comparison (), with CB-64D added at the point shown by the arrow. Values of the individual points are the averages of three experiments ±S.E. After producing an initial, transient rise in [Ca2+]i itself, thapsigargin alone (150 nM) produced no further effect on [Ca2+]i, with Ca2+ levels remaining at or near the normal basal level for up to 70 min in the absence of a sigma ligand (n = 9 cells in normal DPBS; data not shown). Thapsigargin pretreatment () abolished the rapid transient Ca2+ signal produced by CB-64D but had no effect on the latent sustained rise in [Ca2+]i. Except for a more rapid return to baseline for the transient rise, removal of extracellular Ca2+ () had no effect on the magnitude of either the transient rise or the latent, sustained rise. It should be noted that similar results were obtained in thapsigargin-pretreated cells regardless of whether extracellular Ca2+ was present when cells were exposed to CB-64D: thapsigargin experiments with 100 µM CB-64D were carried out in normal DPBS (n = 9 cells) or in Ca2+-free DPBS (n = 6 cells) with similar results. Furthermore, thapsigargin pretreatment gave similar results with 100 µM BD737 and 100 µM Red HAL (n = 3 cells each in normal DPBS; n = 3 cells each in Ca2+-free DPBS).

	Discussion

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Here, we further characterized sigma-1 and sigma-2 receptors in human SK-N-SH neuroblastoma cells and demonstrated the presence of sigma-2 sites with properties somewhat distinct from those of rodent tissues (Table 1). It is not clear at the present whether this represents sigma-2 receptor heterogeneity or simple species differences. However, recent studies on the modulation of NMDA receptor function by sigma ligands in rat brain has provided functional evidence indicating possible sigma-2 receptor heterogeneity (Couture and Debonnel, 1998).

Sigma receptor ligands from various structural classes that include arylethylene diamine, butyrophenone-related, 5-phenylmorphan, and indole alkaloid produced a dose-dependent, transient rise in [Ca²⁺]_i in SK-N-SH neuroblastoma cells. Despite a requirement for micromolar concentrations, the effect was highly specific for ligands with sigma-2 binding affinity. Importantly, the action of sigma ligands was blocked by two sigma antagonists, BD1047 and BD1063 (Matsumoto et al., 1995). Thus, the sigma ligand-induced rise in [Ca²⁺]_i was specifically mediated by sigma-2 receptors, and active ligands are acting as sigma-2 receptor agonists. The only anomaly was the weak activity of DTG, a known sigma agonist with high sigma-2 affinity (Hellewell and Bowen, 1990; Matsumoto et al., 1995).

The removal of extracellular Ca²⁺ had no effect on the percent increase in [Ca²⁺]_i produced by sigma ligands, whereas pretreatment of the cells with thapsigargin completely eliminated this effect (Tables 2 and 3). The results showed that thapsigargin, carbachol, and sigma ligands all release Ca²⁺ from a common intracellular storage pool: the endoplasmic reticulum (Table 3 and Fig. 5). A comparison of the effect of CB-64D in the presence and absence of extracellular Ca²⁺ (Fig. 7) reveals that although the peak level of [Ca²⁺]_i reached is the same under both conditions, the return to near baseline occurs more quickly in the Ca²⁺-free medium. Thus, the broader peak of [Ca²⁺]_i that occurs in the presence of extracellular Ca²⁺ is actually composed of two components: a component derived solely from the endoplasmic reticulum and a component derived extracellularly. This indicates that the activation of sigma-2 receptors may result in capacitative Ca²⁺ entry, where the release of Ca²⁺ from the endoplasmic reticulum subsequently triggers the opening of Ca²⁺ release-activated channels in the plasma membrane to allow the entry of extracellular Ca²⁺ to replenish the intracellular store (Putney, 1990). The possible involvement of Ca²⁺ release-activated channels in the actions of sigma-2 receptors in these cells deserves further investigation.

Sigma receptor ligands also induced a sustained rise in [Ca²⁺]_i (Figs. 6 and 7). This was apparent only after longer-term exposure of cells to sigma ligands and may account for the incomplete return to baseline observed with the transient phase (Fig. 1). The latent, sustained rise in [Ca²⁺]_i persisted in the absence of extracellular Ca²⁺ and in cells pretreated with thapsigargin. Mitochondria are the most likely source for this Ca²⁺. However, release could also occur from thapsigargin-insensitive Ca²⁺ storage pools known to be present in other organelles such as Golgi apparatus, secretory vesicles, or nucleus (Pozzan et al., 1994; Clapham, 1995; Simpson et al., 1995). The pharmacological profile of the sustained rise in [Ca²⁺]_i was identical to that of the transient Ca²⁺ rise. Furthermore, as with the transient rise in [Ca²⁺]_i, DTG was anomalously inactive. This indicates that these two effects on [Ca²⁺]_i are closely related processes. The exact source and mechanism of release require further investigation.

The mechanism by which sigma-2 receptor ligands cause intracellular release of Ca²⁺ from the endoplasmic reticulum in SK-N-SH neuroblastoma cells is not known. It was recently shown that nanomolar concentrations of sigma ligands cause increases in IP₃ formation and modulation of Ca²⁺ transients in adult rat cardiac myocytes (Novakova et al., 1998). However, these effects in cardiac myocytes appear to involve sigma-1 receptors, because (+)-pentazocine produces potent effects (Ela et al., 1994). Furthermore, unlike the case with cardiac cells, sigma ligand-induced stimulation of inositol phosphate production could not be detected in brain synaptoneurosomes or in neuronal cell lines (Walker et al., 1990; Bowen et al., 1992, 1993b; Cutts et al., 1993). In fact, sigma ligands [e.g., (+)-pentazocine, DTG, haloperidol, Red HAL, BD737, BD1008] blocked the ability of muscarinic agonists to stimulate phosphoinositide turnover in these systems, including SK-N-SH neuroblastoma cells. It is therefore unlikely that sigma ligands would gate Ca²⁺ via IP₃ production in neuronal cells. However, it is possible that production of low levels of IP₃ went undetected in previous studies, and the effect of selective sigma-2 receptor agonists on phosphoinositide turnover in SK-N-SH neuroblastoma cells will be reinvestigated.

Sigma-2 receptors could potentially influence Ca²⁺ release from the endoplasmic reticulum by influencing the activity of either the IP₃-gated Ca²⁺ channel (IP₃ receptor) or ryanodine-gated Ca²⁺ channel (ryanodine receptors). Interestingly, sigma-1 receptors were recently shown to potentiate the IP₃-induced Ca²⁺ release resulting from the stimulation of NG-108 cells with bradykinin, apparently by enhancing the action of IP₃ at IP₃ receptors (Su et al., 1998). Because the activity of IP₃ and ryanodine receptors is susceptible to modulation by effectors such as protein kinases and other endogenous molecules, it is at least conceivable that sigma-2 receptor activation could result in activation of these Ca²⁺ channels by a downstream signaling mechanism in the absence of IP₃ production (IP₃ receptor) or membrane depolarization (ryanodine receptor).

Because sigma-2 ligands and thapsigargin produced similar Ca²⁺ release profiles (compare Figs. 1 and 5), another possibility is that sigma compounds inhibit the SERCA pump. Thapsigargin exhibited no affinity (K_i > 10 µM) for either sigma-1 sites or sigma-2 sites (B.J.V. and W.D.B., unpublished observation), suggesting that currently defined sigma binding sites are not synonymous with a thapsigargin-sensitive Ca²⁺-ATPase. Furthermore, the effect of sigma-2 receptor ligands was blocked by sigma receptor antagonists, whereas sigma receptor antagonists had no effect on the ability of thapsigargin to release Ca²⁺. Thus, if SERCA pump inhibition is involved, it is unlikely that sigma-2 ligands and thapsigargin would act via the same mechanism. The data support a receptor-mediated mechanism for the sigma ligands. It is possible, however, that activation of sigma-2 receptors might result in negative modulation of Ca²⁺-ATPase activity by a downstream signaling mechanism.

Sigma receptors have been localized in high density to subcellular fractions of brain and liver tissue, particularly endoplasmic reticular fractions (McCann and Su, 1990; Basile et al., 1992) and mitochondrial fractions (Itzhak et al., 1991), and in lower density in plasma membrane fractions (McCann and Su, 1990). Furthermore, the cloned sigma-1 receptor has been found to contain an endoplasmic reticulum localization motif at the amino terminus (Hanner et al., 1996). The localization of sigma receptors in subcellular fractions known to store Ca²⁺ could suggest an intracellular site of action for sigma-2 ligands to gate Ca²⁺.

An intracellular site of action for sigma-2 ligands could explain some apparent inconsistencies in the data. 1) Although the sigma-2 binding affinities for the active ligands are in the nanomolar range, micromolar concentrations are required for effects on [Ca²⁺]_i. If the site of action is intracellular, the ligands may have difficulty crossing the cell membrane, thus requiring a high extracellular concentration to achieve a nominally effective intracellular concentration. 2) Ligands with similar sigma-2 binding affinity do not always have similar potencies. This may be due to differences in the ability to penetrate the cell membrane. 3) DTG, despite high sigma-2 affinity, is practically inactive. DTG might be expected to show little or no activity because, being a guanidine, it is highly charged at physiological pH and might have great difficulty entering cells. Several recent observations support an intracellular site of action for sigma-2 ligands (Bowen et al., 1999). The potency of sigma-2 ligands at increasing [Ca²⁺]_i and inducing cytotoxicity was directly related to the hydrophobicity (LgP value) and was increased when the extracellular pH was raised from 7.2 to 8.2. This suggests that deprotonation and the resultant increased lipid solubility enhance the activity of the compounds. Also, the relatively hydrophilic sigma antagonists BD1047 and BD1063 were effective blockers of ligand-stimulated rises in [Ca²⁺]_i at pH 8.2, as shown in Figs. 3 and 4, but were largely inactive at pH 7.2. This is consistent with deprotonation leading to increased intracellular access of the antagonists to the site of agonist action. The greater blockade with antagonist pretreatment (Fig. 4) likely reflects time needed for antagonist to adequately diffuse into the cell.

A rise in [Ca²⁺]_i is known to play a central role in cellular toxicity (Berridge et al., 1998). This results from activation of several Ca²⁺-dependent processes, including Ca²⁺-dependent proteases and nitric oxide production. Ca²⁺ has also been implicated in triggering apoptosis via activation of Ca²⁺-dependent endonucleases and other enzymes involved in cell destruction (McConkey and Orrenius, 1996). We have shown that certain compounds that exhibit sigma affinity produce profound morphological changes and cell death in human SK-N-SH neuroblastoma cells, rat C6 glioma cells, and several other neuronal and non-neuronal cell lines that contain sigma-1 and sigma-2 receptors (Vilner et al., 1995a). Similar effects occur in rat cerebellar granule cells and primary cultures of other areas of rat nervous system (Bowen and Vilner, 1994). We have now demonstrated that the prolonged activation of sigma-2 receptors results in apoptotic death in these cells (Vilner and Bowen, 1997; Vilner et al., 1998). Furthermore, it has been shown that Red HAL induces apoptosis and a prolonged rise in [Ca²⁺]_i in MCF-7 adenocarcinoma and WIDr colon carcinoma cells (Brent et al., 1996). Therefore, it is feasible that the observed sigma-2 receptor-mediated rises in [Ca²⁺]_i might be linked in some way to sigma-2 receptor-mediated morphological changes and induction of apoptosis in various kinds of cells. It is also feasible that the transient, sigma ligand-induced rise in [Ca²⁺]_i might subserve a normal signaling role in the varied modulatory actions observed with sigma ligands, such as neurotransmitter synthesis and release and electrophysiological activity.