Sigma receptors are putative targets for neuroprotection following ischemia; however, little is known on their mechanism of action. One of the key components in the demise of neurons following ischemic injury is the disruption of intracellular calcium homeostasis. Fluorometric calcium imaging was used to examine the effects of sigma receptor activation on changes in intracellular calcium concentrations ([Ca2+]i) evoked by in vitro ischemia in cultured cortical neurons from embryonic rats. The sigma receptor agonist, 1,3-di-o-tolyl-guanidine (DTG), was shown to depress [Ca2+]i elevations observed in response to ischemia induced by sodium azide and glucose deprivation. Two sigma receptor antagonists, metaphit [1-(1-(3-isothiocyanatophenyl)-cyclohexyl)-piperidine] and BD-1047 (N-[2–3,4-dichlorophenyl)-ethyl]-N-methyl-2-(dimethylamino)ethylamine), were shown to blunt the ability of DTG to inhibit ischemia-evoked increases in [Ca2+]i, revealing that the effects are mediated by activation of sigma receptors and not via the actions of DTG on nonspecific targets such as N-methyl-d-aspartate receptors. DTG inhibition of ischemia-induced increases in [Ca2+]i was mimicked by the σ-1 receptor-selective agonists, carbetapentane, (+)-pentazocine and PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], but not by the sigma-2-selective agonist, ibogaine, showing that activation of sigma-1 receptors is responsible for the effects. In contrast, DTG, carbetapentane, and ibogaine blocked spontaneous, synchronous calcium transients observed in our preparation at concentrations consistent with sigma receptor-mediated effects, indicating that both sigma-1 and sigma-2 receptors regulate events that affect [Ca2+]i in cortical neurons. Our studies show that activation of sigma receptors can ameliorate [Ca2+]i dysregulation associated with ischemia in cortical neurons and, thus, identify one of the mechanisms by which these receptors may exert their neuroprotective properties.
Sigma receptors are widely distributed in the mammalian brain, and these receptors recognize a diverse array of centrally acting substances including opiates, antipsychotics, antidepressants, phencyclidine (PCP)-related compounds, and neurosteroids (Walker et al., 1990; Bowen, 2000). Thus far, two sigma receptor subtypes have been identified on the basis of their pharmacological profile, with the sigma-1 receptor showing high affinity for the positive isomer of bezomorphas such as (+)-pentazocine and (+)-SKF-10,047, and the sigma-2 receptor having high affinity for ibogaine (Vilner and Bowen, 2000), but only the sigma-1 receptor has been cloned (Hanner et al., 1996). Sigma receptors have been implicated in numerous physiological and pathophysiological processes such as learning and memory (Senda et al., 1996), movement disorders (Matsumoto et al., 1990), and drug addiction (McCracken et al., 1999). These receptors are emerging as therapeutic targets for various diseases such neuropsychiatric disorders and cancer (Casellas et al., 2004; Hayashi and Su, 2004). Moreover, the observation that several sigma receptor ligands are neuroprotective in both in vivo and in vitro models of ischemia has generated interest in targeting these receptors to enhance neuronal survival following stroke (Lockhart et al., 1995; Takahashi et al., 1996). Recently, our laboratory has shown that activation of sigma receptors is neuroprotective at delayed time points in a rat model of ischemic stroke (Ajmo et al., 2006).
Dysregulation of intracellular calcium homeostasis greatly contributes to the demise of neurons following an ischemic insult in the central nervous system (Mattson, 2000). Elevation of intracellular calcium disrupts plasma membrane function via activation of calcium-sensitive ion channels (Murai et al., 1997) and triggers biochemical cascades that ultimately promote processes such as proteolysis, lipolysis, and the production of reactive oxygen species (Mattson, 2000). It has been suggested that the neuroprotective properties of sigma ligands depend in part on their ability to depress elevations in intracellular calcium associated with glutamate receptor-mediated excitotoxicity (Klette et al., 1995, 1997). However, the membrane dysfunction produced by ischemia can stimulate multiple plasma membrane calcium fluxes, including some that are independent of glutamate receptor activation (Tanaka et al., 1997). Sigma receptors have been shown to block both voltage-gated calcium channels and ionotropic glutamate receptors (Zhang and Cuevas, 2002; Monnet et al., 2003). Both of these ion channel types are believed to be involved in the dysregulation of intracellular calcium homeostasis accompanying ischemia, and selective inhibitors of these channels have been shown to provide some degree of neuroprotection (Schurr, 2004). Thus, one of the mechanisms by which sigma receptors may prevent these increases in calcium is via the inhibition of multiple plasma membrane calcium channels. However, the role of sigma receptors in the modulation of ischemia-induced elevations in intracellular calcium has not been unequivocally established because studies on the effects of sigma receptors on calcium homeostasis during neuronal injury have examined intracellular calcium changes in response to direct glutamate application rather than in vitro ischemia models. Given that nonglutamate-induced calcium fluxes are also a factor following ischemia, it is important to examine sigma receptor modulation of intracellular calcium using an ischemia model. Determining the role of sigma receptors in preserving calcium homeostasis is the first step toward establishing these receptors as a target for neuroprotection.
The studies of sigma receptor modulation of glutamate-evoked changes in intracellular calcium have also resulted in considerable controversy in the literature. There are conflicting reports as to whether sigma receptor ligands exert their effects via actions on sigma receptors (Hayashi et al., 1995; Monnet et al., 2003) or nonspecific interaction with other targets, in particular, NMDA receptors (Kume et al., 2002). To some extent, analysis and interpretation of the results has been confounded by limitations in the pharmacological approaches used. For example, sigma receptors and NMDA receptors both bind PCP and related compounds (e.g., MK-801) with high affinity (Sircar et al., 1987); thus, such drugs cannot be used to discriminate between direct and indirect effects. In addition, previous studies have not effectively used specific sigma receptor antagonists to confirm results.
Experiments were undertaken to determine whether activation of sigma receptors in cultured cortical neurons modulates elevations in intracellular calcium observed in response to in vitro ischemia. Activation of sigma receptors with specific sigma agonists was shown to depress the peak amplitude of ischemia-induced calcium transients, and this effect blocked sigma receptor-specific antagonists. Moreover, sigma receptor subtype-selective agonists showed that sigma-1 receptors are responsible for the observed depression of calcium elevations evoked by ischemia, whereas both sigma receptor subtypes regulate spontaneous calcium transients observed in cultured cortical neurons.
Materials and Methods
Preparation. The effects of sigma receptors on ischemia-induced changes in intracellular calcium concentrations were studied in cultured cortical neurons from embryonic (E18) rats. All procedures were done in accordance with the regulations of the University of South Florida Institutional Animal Care and Use Committee. Dams were euthanatized by decapitation, uterus removed, and embryos dissected out and placed in isotonic buffer containing 137 mM NaCl, 5 mM KCl, 0.2 mM NaH2PO4, 0.2 mM KH2PO4, 5.5 mM glucose, and 6 mM sucrose, pH 7.4 with NaOH. Cortexes were excised and minced, and tissue was digested in isotonic buffer containing 0.25% trypsin/EDTA for 10 min at 37°C and added to 3× volume of high-glucose culture media (Dulbecco's modified Eagle's medium; Invitrogen, Inc., Carlsbad, CA), 10% (v/v) fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were counted on a hemocytometer, plated (0.5 × 106 cells) on 18-mm coverslips coated with poly-l-lysine, and incubated at 37°C under a 95% air, 5% CO2 atmosphere. After 24 h, the media were replaced with Neurobasal (Invitrogen) medium supplemented with B27 (Invitrogen) and 0.5 mM l-glutamine to limit astrocyte proliferation in the cultures. Cells were used after 10 to 17 days in culture, unless otherwise indicated.
Microfluorometric Measurements. Intracellular free calcium was measured using the calcium-sensitive dye, fura-2, as described previously (DeHaven and Cuevas, 2004). Cells plated on coverslips were incubated for 1 h at room temperature in physiological saline solution (PSS) consisting of 140 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 7.7 mM glucose, and 10 mM HEPES, pH to 7.2 with NaOH, which also contained 1 μM concentration of the membrane-permeable ester form of fura-2, fura-2 AM, acetoxymethyl ester (AM), and 0.1% dimethyl sulfoxide. The coverslips were then washed in PSS (fura-2-AM free) before the experiments being carried out. All solutions were applied via a rapid application system identical to that described previously (Cuevas and Berg, 1998).
A DG-4 high-speed wavelength switcher (Sutter Instruments Co., Novato, CA) was used to apply alternating excitation light, and fluorescent emission was captured using a Sensicam digital CCD camera (Cooke Corporation, Auburn Hills, MI) and recorded with Slidebook 3.0 software (Intelligent Imaging Innovations, Denver, CO). Changes in [Ca2+]i were calculated using the Slidebook 3 software (Intelligent Imaging Innovations) from the intensity of the emitted fluorescence following excitation with 340 and 380 nm of light, respectively, using the Grynkiewicz equation: where R represents the fluorescence intensity ratio (F340/F380) as determined during experiments, Q is the ratio of Fmin to Fmax at 380 nm, and Kd is the Ca2+ dissociation constant for fura-2 (225 μM). The system was calibrated using a Fura-2 Calcium Imaging Calibration Kit (Molecular Probes, Eugene, OR), and values for Fmin/Fmax, Rmin, and Rmax were determined to be 23.04, 0.2, and 8.4, respectively.
In Vitro Ischemia. In vitro ischemia was achieved using the sodium azide/glucose deprivation model. This model for ischemic neuronal injury has been used effectively in numerous studies to mimic in vivo stroke in an in vitro environment and has been shown to elicit electrophysiological and neurochemical changes that are qualitatively identical to the oxygen/glucose deprivation model of ischemia (Murai et al., 1997; Finley et al., 2004). The major advantage of the sodium azide/glucose deprivation model over the oxygen/glucose deprivation is that it elicits neurochemical responses that are significantly more rapid and robust (Finley et al., 2004), thus facilitating the recording of changes in [Ca2+]i.
Data Analysis. Analyses of these data were conducted using the SigmaPlot 2000 program (SPSS Science, Chicago, IL). Data points represent means ± S.E.M. Statistical difference was determined using paired Student's t test for within-group experiments and unpaired Student's t test for between-group experiments. For multiple group comparison, an analysis of variance was used followed by post hoc analysis with a Dunn's test. Differences were considered significant if p < 0.05.
Solutions and Reagents. The control bath solution for all experiments was PSS containing 140 mM NaCl, 1.2 mM MgCl2, 3 mM KCl, 2.5 mM CaCl2, 7.7 mM glucose, and 10 mM HEPES, pH to 7.2 with NaOH. For experiments with no extracellular calcium, CaCl2 was excluded from the PSS. All drugs were applied in this solution unless otherwise noted. In vitro ischemia was induced by addition of the cytochrome oxidase inhibitor, NaN3 (4 mM), and removal of glucose from the PSS. For experiments in which multiple ischemic episodes were induced in a single cell, the order of drug application was alternatively reversed to compensate for any effects due to rundown, desensitization, or ischemic preconditioning. For experiments with metaphit, cells were preincubated in 50 μM metaphit during the last 15 min of fura-2 loading and immediately before experiments being conducted. The metaphit was washed off for 5 min in the bath using PSS.
All chemicals used in this investigation were of analytic grade. The following drugs were used: ruthenium red, tetrodotoxin (TTX), DTG, ibogaine, and metaphit (Sigma-Aldrich, St. Louis, MO); d-AP5, BD-1047, carbetapentane, and PRE-084 (Tocris Bioscience, Ellisville, MO); ryanodine and thapsigargin (Alomone Labs, Jerusalem, Israel); and fura-2-AM (Molecular Probes).
Experiments were conducted to characterize the changes in intracellular calcium evoked by the sodium-azide/glucose deprivation model of in vitro ischemia. Figure 1A shows representative traces of change in intracellular Ca2+ as a function of time evoked by rapid induction of chemical ischemia in two cortical neurons that had remained in culture for 3 or 14 days, respectively. After 3 days in culture, chemical ischemia elicited small, slow rising elevations in [Ca2+]i in the neurons, whereas after 14 days in culture, ischemia evoked rapid increases in [Ca2+]i. The increases in [Ca2+]i observed at later time points (7–21 days in culture) in response to ischemia were transient, and [Ca2+]i returned to control levels in >80% of the cells tested following washout with control PSS (n > 1000). A plot of mean peak change in [Ca2+]i shows that the response to chemical ischemia increased significantly from 3 to 14 days in culture, and the elevations in [Ca2+]i diminished when the neurons were in culture for over 14 days (Fig. 1B).
Previous studies have shown that chemical ischemia promotes the release of excitatory neurotransmitters that may elicit these elevations in [Ca2+]i (Djali and Dawson, 2001). Therefore, experiments were conducted to determine whether inhibition of voltage-activated Na+ channels and, consequently, neurotransmission, with TTX (200 nM) abolished the elevations in [Ca2+]i induced by chemical ischemia. Figure 1C shows representative [Ca2+]i traces of responses evoked by ischemia in the absence and presence of TTX. Inhibition of synaptic transmission with TTX depressed ischemia-evoked increases in [Ca2+]i relative to control. A plot of maximal increase in [Ca2+]i in the absence and presence of TTX is shown in Fig. 1D. The ischemia-evoked increase in [Ca2+]i was decreased in a significant manner by 76 ± 4% in the presence of TTX.
Further experiments were conducted to resolve the source of calcium mediating the elevations in [Ca2+]i observed in response to chemical ischemia. To determine whether extracellular calcium contributed to the increase in [Ca2+]i, ischemic conditions were induced in the absence and presence of extracellular calcium. Under both conditions, elevations in [Ca2+]i were noted (Fig. 2A), but in the absence of extracellular calcium, the peak increases in [Ca2+]i were significantly less than those observed in control experiments (Fig. 2B). Thus, a component of the ischemia-induced increase in [Ca2+]i depends on the presence of extracellular calcium. To gain further insight into the ion channel type mediating the influx of calcium following ischemia, we used the NMDA receptor-selective antagonist, d-AP5. d-AP5 (100 μM) had no effect on basal [Ca2+]i, but depressed peak [Ca2+]i following ischemia (Fig. 2C). However, the decrease in ischemia-induced increase in [Ca2+]i observed in the presence of d-AP5 was less than that produced by the nonselective calcium channel blocker La3+ (10 μM; Fig. 2D). Our data show that NMDA receptors are responsible for ∼35% of the calcium influx evoked in this ischemia model. Due to the fact that La3+ blocks ∼90% of the elevation in [Ca2+]i and that the concentration of La3+ used here (10 μM) blocks voltage-gated calcium channels, NMDA receptors, and kainate receptors, but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (Huettner et al., 1998), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors are likely to be minor contributors to the elevations in [Ca2+]i evoked by this chemical ischemia model.
Given that elimination of extracellular calcium did not abolish the increase in [Ca2+]i, we investigated the role of calcium release from intracellular stores in the response to ischemia. Ryanodine (10 μM) was used to selectively inhibit release from caffeine/ryanodine-sensitive calcium stores, whereas the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (10 μM), was used to deplete both ryanodine- and IP3-sensitive stores. Ruthenium red was used to block any calcium efflux from the mitochondria via reversal of the calcium uniporter. Ischemia increases in [Ca2+]i were observed in control, ryanodine, thapsigargin, and ruthenium red experiments (Fig. 2E). However, preincubation in thapsigargin decreased the peak elevation in [Ca2+]i, whereas ryanodine did not significantly alter the effects of ischemia on [Ca2+]i (Fig. 2F). Ruthenium red also failed to block ischemia-induced increases in [Ca2+]i and was associated with an increase in [Ca2+]i (Fig. 2F). This increase is probably due to ruthenium red preventing calcium uptake (buffering) by the mitochondria, which is known to occur in neurons in response to cytosolic elevations in calcium (Wang and Thayer, 2002). Taken together, these data suggest that elevations of [Ca2+]i observed in response to chemical ischemia are in part due to calcium release from IP3-sensitive stores but do not appear to involve liberation of calcium from ryanodine-sensitive stores or the mitochondria.
Experiments were carried out to determine whether stimulation of sigma receptors modulates the changes in [Ca2+]i evoked by in vitro ischemia. Figure 3A shows representative traces of [Ca2+]i recorded from a single neuron in response to ischemia in the absence (control) and presence of 50 μM DTG. The elevation in [Ca2+]i evoked by ischemia was abolished when the sigma receptor ligand was coapplied. A bar graph of ischemia-induced mean peak increase in [Ca2+]i observed in 13 neurons in the absence and presence of 50 μM DTG is shown in Fig. 3B and demonstrates that DTG decreases the rise in [Ca2+]i by ∼70%. This effect of DTG was statistically significant and was reversible upon washout of the sigma agonist (data not shown). Similar results were obtained for DTG inhibition of net change in [Ca2+]i (area under the curve) and [Ca2+]i amplitude at the end of the ischemic episode (data not shown).
The magnitude of the DTG-mediated block of elevations in [Ca2+]i suggests that sigma receptors are modulating calcium elevations evoked by the various components that contribute to the ischemia-induced dysregulation of [Ca2+]i. To test this possibility, DTG (100 μM) was applied under various conditions targeting specific components of the increase in [Ca2+]i. DTG significantly decrease ischemia-evoked elevations in [Ca2+]i when intracellular calcium stores were depleted by thapsigargin preincubation (10 μM, 1 h, 23°C) or when the mitochondrial calcium uniporter was blocked with ruthenium red (20 μM) (Fig. 3C). Likewise, DTG blocked calcium elevations evoked by ischemia following NMDA receptor inhibition with d-AP5 (100 μM) or when calcium influx was blocked by removal of extracellular calcium or application of the pan-specific calcium channel blocker, La3+ (10 μM) (Fig. 3C). However, DTG failed to block the residual calcium elevation evoked by ischemia in the presence of TTX. Thus, sigma receptor activation blocks calcium elevations associated with both calcium entry through the plasma membrane and calcium release from intracellular stores. However, the effects of sigma receptor activation appear to be limited to elevations in [Ca2+]i associated with synaptic transmission in this model of ischemia.
To confirm that the effects of DTG on [Ca2+]i are mediated via the activation of sigma receptors, the sigma receptor antagonist, metaphit, was used in a series of experiments. Cells were exposed to ischemia in the absence and presence of 50 μM DTG, with or without preincubation in metaphit (50 μM). Although DTG depressed the ischemia-induced elevation in [Ca2+]i in control cells, the responses observed in cells pretreated with metaphit were comparable in the absence and presence of DTG (Fig. 4A). Moreover, both responses (±DTG) observed in metaphit-pretreated cells were larger than the control response (no metaphit pretreatment). In similar experiments, cells not exposed to metaphit responded to DTG with a decrease in ischemia-induced elevations in [Ca2+]i from a control value of 291 ± 47 to 47 ± 9 nM in the presence of the sigma agonist (Fig. 4B). Cells preincubated in metaphit displayed a more robust increase in [Ca2+]i during ischemia (495 ± 68 nM) relative to control cells (Fig. 4B). Furthermore, neurons pretreated with metaphit continued to exhibit pronounced elevations in [Ca2+]i in the presence of DTG (237 ± 39 nM). These increases in [Ca2+]i were significantly greater (p < 0.01) than those observed in control neurons exposed to DTG and were comparable with those seen in control neurons not exposed to DTG. To confirm that the differences observed in responses to DTG and DTG following metaphit preincubation were not the result of metaphit augmentation of the [Ca2+]i responses, the responses were normalized to the mean of their respective controls. Figure 4C shows a bar graph of the relative change in [Ca2+]i observed in the presence of DTG in control neurons (DTG) and neurons preincubated in metaphit (MET + DTG). Although DTG decreased the elevation in [Ca2+]i evoked by ischemia in control cells by 83 ± 3%, the sigma receptor agonist only reduced the response by 52 ± 8% in cells preincubated in the irreversible sigma receptor antagonist.
A second sigma receptor-selective antagonist, BD-1047 (Matsumoto et al., 1995), was used to further support that DTG was acting via the stimulation of sigma receptors. Figure 5A shows intracellular calcium traces obtained from three neurons in response to chemical ischemia in the absence (control) and presence of 10 μM DTG (DTG) or 10 μM DTG following a 5-min preincubation in 10 μM BD-1047 (DTG + BD-1047). Although DTG reduced the ischemia-elicited elevations in [Ca2+]i, application of BD-1047 diminished the effectiveness of DTG. In similar experiments, the effects of DTG on ischemia-evoked calcium transients were blocked by 1 and 10 μM BD-1047 in a concentration-dependent and statistically significant manner (Fig. 5B). These two concentrations of BD-1047 reduced the effects of DTG by 15 and 55%, respectively.
DTG and metaphit are pan-selective sigma ligands, acting on both sigma-1 and sigma-2 receptors, and the concentrations of BD-1047 used here cannot definitively discriminate between the receptor subtypes. Therefore, experiments were conducted using sigma receptor subtype-selective agonists to determine the specific sigma receptor subtype(s) responsible for the depression of ischemia-induced increases in [Ca2+]i. Figure 6A shows representative traces of [Ca2+]i recorded from three neurons in the absence (control) and presence of the sigma-1-selective agonist, carbetapentane, at the indicated concentrations. Carbetapentane reduced the effect of ischemia on [Ca2+]i in a concentration-dependent manner, and this effect of carbetapentane was reversible upon washout of drug (data not shown). Figure 6B shows a plot of the relative ischemia-induced increases in [Ca2+]i as a function of carbetapentane concentration. A fit of the data using a Langmuir-Hill equation indicated that the sigma-1-selective ligand inhibits the effects of ischemia on [Ca2+]i with a half-maximal concentration of 13.3 μM and with a Hill coefficient of 0.8.
Additional experiments were conducted to determine whether sigma-2 receptors contribute to the DTG-mediated inhibition of ischemia-induced increases in [Ca2+]i. For these experiments, the sigma-2 receptor-selective agonist, ibogaine, was used. Figure 7A shows representative traces of [Ca2+]i recorded in response to ischemia in the absence and presence of 100 μM ibogaine. Unlike carbetapentane, ibogaine failed to inhibit the ischemia-induced elevations in [Ca2+]i. In similar experiments, ibogaine at a concentration range of 1 to 100 μM, which has been shown to block sigma-2-mediated events (Zhang and Cuevas, 2002), failed to inhibit the effects of ischemia on [Ca2+]i (Fig. 7B). This observation suggests that the sigma-1 receptor is primarily responsible for the depression of ischemia-induced increase in [Ca2+]i mediated by DTG.
To confirm that sigma-1 activation attenuates ischemia-induced increase in [Ca2+]i mediated by DTG and carbetapentane, neurons were treated with the sigma-1-selective agonists (+)-pentazocine and PRE-084. Figure 8A shows representative traces of [Ca2+]i recorded during ischemia from three neurons in the absence (control) and presence of (+)-pentazocine at the indicated concentrations. Peak elevations in [Ca2+]i were significantly depressed by (+)-pentazocine in a concentration-dependent manner. Application of 10 μM (+)-pentazocine decreased the ischemia-induced elevations in [Ca2+]i by 37 ± 5%, whereas 100 μM(+)-pentazocine depressed the change in [Ca2+]i by 49 ± 4% (Fig. 8B). Responses to ischemia were also blocked by application of PRE-084 (Fig. 8C). Both 10 and 100 μM PRE-084 decreased elevations in [Ca2+]i in a statistically significant manner. These decreases were 26 ± 4 and 58 ± 1%, respectively (Fig. 8D).
Spontaneous elevations in [Ca2+]i were frequently observed in our experiments (see Fig. 8C, control trace). These elevations in [Ca2+]i nearly always occurred in multiple neurons in the same visual field in a synchronized manner (data not shown). Although our data demonstrate that activation of sigma receptors decreased the ischemia-induced elevations of [Ca2+]i, further experiments were conducted to determine whether spontaneous increases in [Ca2+]i were also modulated by sigma receptors. Figure 9Ai shows traces of spontaneous activity recorded from a single cortical neuron in the absence (control) and presence of 100 μM DTG and following washout of drug (Wash). DTG was found to reversibly block spontaneous increases in [Ca2+]i in a statistically significant manner (Fig. 9B). To identify the subtype of sigma receptor involved in the modulation of spontaneous calcium transients, the sigma-2-selective agonist, ibogaine, was used. Traces of spontaneous activity recorded from a single cortical neuron in the absence (control, Wash) and presence of 50 μM ibogaine (IBO) are shown in Fig. 9Aii. In identical experiments, bath application of the sigma-2 agonist significantly decreased the number of spontaneous calcium events (Fig. 9C). Activation of sigma-1 receptors with carbetapentane (100 μM) also affected spontaneous activity (Fig. 9Aiii), resulting in a significant decrease in the number spontaneous of Ca2+ transients observed in the cells (Fig. 9D).
The major finding reported here is that activation of sigma-1 receptors depresses ischemia-induced dysregulation of intracellular calcium in cultured cortical neurons by affecting multiple pathways that contributing to the rise in intracellular calcium concentrations. This effect of sigma receptors is primarily due to the modulation of intracellular calcium increases resulting from the stimulation of synaptic transmission elicited by ischemia. Pharmacological studies using sigma-selective agonists and antagonists clearly show that the effects of the sigma ligands are mediated by actions on sigma receptors and are not the result of modulation of NMDA receptors by the drugs. Our data also indicate that tonic activation of sigma receptors or activation of sigma receptors upon induction of ischemia via an endogenous mechanism diminishes ischemia-induced elevations of intracellular calcium. Finally, our findings also demonstrate that although sigma-2 receptors do not appreciably influence ischemia-induced changes in [Ca2+]i, these receptors can decrease spontaneous calcium transients observed in cultured cortical neurons.
Previous studies have shown that the sigma ligands (+)SKF10047 (10 μM) and haloperidol (10 μM), but not carbetapentane (100 μM) or DTG (100 μM), inhibit calcium elevations evoked by glutamate application (Klette et al., 1995; Kume et al., 2002). Our studies show that DTG effectively blocks ischemia-induced elevations in [Ca2+]i at concentrations that have little or no effect on the rise in [Ca2+]i elicited by direct glutamate application (Klette et al., 1995). In contrast, similar concentrations of DTG (65 μM) have been shown to stimulate sigma receptor modulation of electrical activity in frog pituitary melanotrope cells (Soriani et al., 1998). Low concentrations of carbetapentane (10 μM) were shown to inhibit ∼50% of the peak ischemia-induced increases in [Ca2+]i, whereas 10-fold higher concentrations of this sigma receptor agonist failed to block glutamate-induced increases in [Ca2+]i (Kume et al., 2002). Moreover, DTG was shown to block a major component of the ischemia-induced increase in [Ca2+]i that is insensitive to the NMDA receptor antagonist, d-AP5. DTG even blocked a component of the ischemia-induced calcium elevation that was insensitive to La3+ (10 μM), which blocks voltage-gated calcium channels, NMDA receptors, kainate receptors, and other Ca2+ channel types at this concentration (Huettner et al., 1998). Taken together, these data indicate that the effects of sigma ligands on ischemia-induced increases in [Ca2+]i cannot be exclusively explained by the actions of these drugs on metabotropic and ionotropic glutamate receptors alone and are the result of their action on sigma receptors and, consequently, on effector targets of sigma receptors.
The role of sigma receptors in the depression of ischemia-elicited increases in [Ca2+]i is further supported by experiments using the sigma receptor-selective antagonists metaphit and BD-1047. Our laboratory has previously shown that metaphit is an irreversible inhibitor of sigma receptors and that preincubation of neurons in metaphit inhibits sigma-1 and sigma-2 receptor block of K+ and Ca2+ channels, respectively (Zhang and Cuevas, 2002, 2005). It is important to note that although metaphit, a PCP analog, can attenuate phencyclidine-induced antagonism of NMDA responses, it does not have any direct effects on NMDA-mediated responses, even at concentrations significantly greater than those used here (Wang and Lee, 1991). BD-1047 was also shown to block DTG-mediated inhibition of ischemia-related dysregulation of [Ca2+]i at concentrations selective for sigma receptors (Matsumoto et al., 1995). BD-1047 has also been used previously to show that sigma receptors mediate DTG-evoked hypothermia and the effects of cocaine on conditioned place preference (Rawls et al., 2002; Romieu et al., 2004).
The observation that the sigma-1-selective agonists (+)-pentazocine, PRE-084, and carbetapentane, but not the sigma-2-selective agonist, ibogaine, mimicked the effects of DTG on ischemia-induced elevations in [Ca2+]i indicates that sigma-1 receptors are responsible for the observed effects. Studies have shown that the affinity of sigma-1 receptors for carbetapentane is >30-fold greater than that of sigma-2 receptors, whereas the affinity of sigma-2 receptors for ibogaine is >40-fold greater than that of sigma-1 receptors (Vilner and Bowen, 2000). The calculated IC50 for carbetapentane inhibition of ischemia-evoked increases in [Ca2+]i (13 μM) is similar to values reported for carbetapentane inhibition of epileptiform activity via sigma receptors in rat hippocampal slices (38 μM) (Thurgur and Church, 1998). Furthermore, the affinity of sigma-1 receptors for (+)-pentazocine is ∼2000-fold greater than for ibogaine, whereas the affinity of sigma-2 receptors for ibogaine is ∼6-fold higher than for (+)-pentazocine (Vilner and Bowen, 2000). Here, we show that 10 μM (+)-pentazocine blocked ∼40% of ischemia-evoked increases in [Ca2+]i, which is consistent with the IC50 for (+)-pentazocine inhibition of delayed outwardly rectifying K+ channels (37 μM) and voltage-gated K+ channels (42 μM) via sigma-1 receptors in frog pituitary melanotrophs and rat intracardiac neurons, respectively (Soriani et al., 1998; Zhang and Cuevas, 2005). In contrast, in rat intracardiac neurons, sigma-2 receptors inhibit voltage-gated Ca2+, and the IC50 reported for ibogaine is 31 μM (Zhang and Cuevas, 2002). Even at 3-fold higher concentrations, ibogaine failed to affect the ischemia-induced elevations in [Ca2+]i. Thus, the attenuation of elevations in [Ca2+]i is primarily the result of sigma ligands acting on sigma-1 receptors in cortical neurons.
The mechanism by which sigma-1 receptors modulate ischemia-induced elevations in [Ca2+]i remains to be fully elucidated. However, several factors are probably involved due to the complex nature of the responses observed in our ischemia model. The dependence of the peak amplitude of ischemia-induced elevations in [Ca2+]i on the number of days the neurons are in culture coincides with the development of synapses in this preparation, suggesting that the phenomenon involves synaptic transmission. Also consistent with this hypothesis is the fact that either application of TTX or La3+ or removal of extracellular calcium significantly depressed [Ca2+]i responses. One possibility is that sigma-1 receptor activation is primarily exerting its effects by decreasing glutamate release evoked by ischemia. This would explain the observation that DTG fails to inhibit the small elevations in [Ca2+]i produced by ischemia when synaptic transmission is blocked with TTX. Studies have shown that DTG can decrease glutamate release evoked by oxygen and glucose deprivation from hippocampal brain slices (Lobner and Lipton, 1990). Alternatively, sigma-1 receptors may be modulating postsynaptic receptors and inhibiting neurotransmission. It has been suggested that sigma receptors may block calcium responses evoked by direct activation of both ionotropic and metabotropic glutamate receptors (Klette et al., 1997). The fact that sigma-1 receptor activation can eliminate the elevations in [Ca2+]i suggests that they are blocking calcium entry through the plasma membrane and calcium release from intracellular stores, consistent with inhibition of both glutamate receptor subtypes.
An interesting observation reported here is that inhibition of sigma receptors resulted in elevations in basal [Ca2+]i and potentiated the increases in [Ca2+]i evoked by ischemia. Thus, sigma receptors appear to be involved in calcium homeostasis in cortical neurons under control conditions. Previous studies have shown that sigma receptors can modulate various plasma membrane calcium channels, including voltage-gated Ca2+ channels and NMDA receptors (Hayashi et al., 1995; Zhang and Cuevas, 2002), and can regulate phosphatidylinositide turnover (Hayashi et al., 2000). These findings have led to the theory that one of the critical cellular functions of sigma receptors is the regulation of intracellular calcium levels (Hayashi et al., 2000). The observations reported here lend further support to this theory. Moreover, it appears that changes in intracellular calcium are modulated by both sigma-1 and sigma-2 receptors. Although sigma-1 receptors affect the ischemia-induced changes in [Ca2+]i, both sigma receptor subtypes can depress spontaneous calcium transients observed in cultured cortical neurons. Low concentrations of DTG and ibogaine depressed the genesis of these calcium transients, consistent with a sigma-2-mediated effect. However, the fact that carbetapentane also inhibited these spontaneous Ca2+ transients suggests that sigma-1 receptors may also regulate this phenomenon. It remains to be determined whether sigma-1 and sigma-2 receptors affect this spontaneous activity via actions on identical targets (e.g., ion channel, calcium store, etc.). These spontaneous increases in [Ca2+]i have a frequency that is similar to bursts of spontaneous action potentials observed in our preparation (data not shown), but the exact source and triggering mechanism for these calcium transients remains to be determined. Previous studies have reported these synchronous calcium transients, and they appear to be correlated with bursts of electrical activity, axon outgrowth, and synaptic development (Robinson et al., 1993; Tang et al., 2003).
In conclusion, our studies show that sigma-1 receptors mediate the depression of ischemia-induced elevations in [Ca2+]i. Findings reported here clearly establish that sigma ligands can affect cellular function during ischemia and the concomitant excitotoxicity by acting on sigma receptors, rather than through nonspecific effects on other molecular targets. Given that intracellular calcium dysregulation greatly contributes to the demise of cortical neurons following ischemic injury, sigma receptor-mediated neuroprotection, such as that reported by our laboratory (Ajmo et al., 2006), is probably due in part to the preservation of intracellular calcium homeostasis in these cells. Thus, sigma receptors are a viable target for neuroprotection following ischemia and possibly other neurodegenerative diseases involving excitotoxicity.
We thank Nivia Cuevas for comments on a draft of the manuscript.
This work was supported by American Heart Association Florida/Puerto Rico Affiliate Award Grant-in-aid (to J.C.).
A preliminary report of some of these results has been presented in abstract form (Zhang H, Guerrero W, DeMesquita D, Pennypacker K, and Cuevas J (2004) Sigma-1 receptor activation attenuates elevations in intracellular calcium evoked by chemical ischemia. Soc Neurosci Abstr 1019.1. Society for Neuroscience, Washington, DC.)
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PCP, phencyclidine; (+)-SKF-10,047, [2S-(2α,6α,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride; NMDA, N-methyl-d-aspartate; MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate; PSS, physiological saline solution; AM, acetoxymethyl ester; TTX, tetrodotoxin; DTG, 1,3-di-o-tolyl-guanidine; d-AP5, d-2-amino-5-phosphonopentanoic acid; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; BD-1047, N-[2–3,4-dichlorophenyl)-ethyl]-N-methyl-2-(dimethylamino)ethylamine; PRE-084, 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride; (+)-PTZ, (+)-pentazocine; IP3, inositol 1,4,5-trisphosphate.
↵1 These authors contributed equally to this work.
- Received May 9, 2006.
- Accepted September 12, 2006.
- The American Society for Pharmacology and Experimental Therapeutics