Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells by Ifenprodil: The Role of Sigma-1 and IP3 Receptors

In addition to both the α1 adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor antagonists, ifenprodil binds to the sigma receptor subtypes 1 and 2. In this study, we examined the effects of ifenprodil on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Ifenprodil significantly potentiated NGF-induced neurite outgrowth, in a concentration-dependent manner. In contrast, the α1 adrenergic receptor antagonist, prazosin and the NMDA receptor NR2B antagonist, Ro 25-6981 did not alter NGF-induced neurite outgrowth. Potentiation of NGF-induced neurite outgrowth mediated by ifenprodil was significantly antagonized by co-administration of the selective sigma-1 receptor antagonist, NE-100, but not the sigma-2 receptor antagonist, SM-21. Similarly, ifenprodil enhanced NGF-induced neurite outgrowth was again significantly reduced by the inositol 1,4,5-triphosphate (IP3) receptor antagonists, xestospongin C and 2-aminoethoxydiphenyl borate (2-APB) treatment. Furthermore, BAPTA-AM, a chelator of intracellular Ca2+, blocked the effects of ifenprodil on NGF-induced neurite outgrowth, indicating the role of intracellular Ca2+ in the neurite outgrowth. These findings suggest that activation at sigma-1 receptors and subsequent interaction with IP3 receptors may mediate the pharmacological effects of ifenprodil on neurite outgrowth.

PC12 cells, a cell line derived from a pheochromocytoma of rat adrenal medulla, have been widely used as a model system for nerve growth factor (NGF)-induced neuronal differentiation. It has been reported that sigma-1 receptor agonists, such as (+)pentazocine, imipramine, fluvoxamine, donepezil, and dehydroepiandrosterone-sulfate (DHEA-S), potentiate NGF-induced neurite outgrowth in PC12 cells, and that the selective sigma-1 receptor antagonist NE-100 significantly attenuated the efficacy of these drugs, suggesting a role for sigma-1 receptors in neuronal plasticity [16][17][18]. However, the precise cellular mechanisms of the sigma-2 receptor are unclear, as this receptor has not yet been cloned.
Ifenprodil (CerocralH) has been used as a cerebral vasodilator in a limited number of countries, including Japan and France. It acts as a prototypical antagonist of the N-methyl-D-aspartate (NMDA) receptor, NR2B subunit [19,20]. As well as binding to the a1 adrenergic receptor and NMDA receptor antagonists, ifenprodil also binds to sigma-1 and sigma-2 receptors [21][22][23]. Despite the current knowledge, there are no reports describing the effects of ifenprodil on neuronal plasticity. In addition, it remains unclear whether ifenprodil acts as an agonist or antagonist on the sigma receptor subtypes.
In this study, we examined whether ifenprodil could potentiate NGF-induced neurite outgrowth in PC12 cells. We also examined the roles of sigma receptor subtypes 1 and 2, the a1 adrenergic receptor, and the NR2B subtype of the NMDA receptor in ifenprodil mediated, NGF-induced neurite outgrowth. Moreover, we looked at the role of IP 3 receptors on NGF-induced neurite outgrowth by ifenprodil, since sigma-1 receptor associated neuronal plasticity utilizes IP 3 receptors [1,2,17,18].

Effects of ifenprodil on NGF-induced neurite outgrowth
Ifenprodil (0.1, 1.0, or 10 mM) increased the number of cells with neurite outgrowth in NGF (2.5 ng/ml) treated PC12 cells, in a concentration-dependent manner ( Figure 1). MAP-2 and GAP-43 immunocytochemistry have been used as a useful indicator of neuronal differentiation in PC12 cells [17,18,24]. MAP-2 and GAP-43 immunocytochemistry revealed that the addition of ifenprodil (10 mM) increased the number of cells with neurite outgrowth in PC12 cells compared with controls ( Figure 2).
To assess the role of the a1 adrenergic receptor and the NMDA receptor, NR2B subunit, we examined the effects of the selective a1 adrenergic receptor antagonist prazosin and the selective NR2B antagonist Ro 25-6981 [27] on the potentiation of NGFinduced neurite outgrowth by ifenprodil (10 mM). Neither the a1 adrenergic receptor antagonist, prazosin (10 mM) nor the NR2B antagonist Ro 25-6981 (10 mM) altered the number of cells with NGF induced neurite outgrowth (Figure 3), suggesting that these receptors do not play a role in the mechanisms of ifenprodil potentiation of neurite outgrowth.

Role of IP 3 receptor and intracellular Ca 2+ in the mechanisms of potentiation of NGF-induced neurite outgrowth by ifenprodil
Next, we examined the effects of IP 3 receptor antagonists, xestospongin C (a selective, reversible membrane-permeable inhibitor of IP 3 receptors) [28] and 2-APB (a membranepermeable IP 3 receptor antagonist) [29,30] on ifenprodil potentiation of neurite outgrowth. ANOVA analysis revealed significant differences among the four groups (F (3,20) = 44.02, p,0.001) ( Figure 4A). Co-administration of xestospongin C (1.0 mM) significantly reduced neurite outgrowth by ifenprodil (10 mM) ( Figure 4A). ANOVA analysis revealed that the data among the  To assess the role of intracellular Ca 2+ in the cells, we examined the effects of the BAPTA-AM, a chelator of intracellular Ca 2+ [31,32], on the potentiation of NGF-induced neurite outgrowth by ifenprodil (10 mM). ANOVA analysis revealed significant differences among the four groups (F (3,20) = 56.06, p,0.001) ( Figure 5). Administration of BAPTA-AM (5.0 mM) significantly reduced neurite outgrowth by ifenprodil (10 mM) ( Figure 5). In addition, BAPTA-AM (5.0 mM) alone significantly blocked NGFinduced neurite outgrowth. These findings suggest that the intracellular Ca 2+ plays an important role in the mechanism of NGF-induced neurite outgrowth.

Discussion
In this study, we found that ifenprodil potentiated NGF-induced neurite outgrowth in PC12 cells, and that the effects of ifenprodil   were blocked by treatment with the selective sigma-1 receptor antagonist, NE-100 [25], but not the sigma-2 receptor antagonist, SM-21 [26]. Furthermore, the effects of ifenprodil were also blocked by treatment with two IP 3 receptor antagonists. This is a first paper demonstrating a role for sigma-1 and IP 3 receptors in ifenprodil mediated potentiation of NGF-induced neurite outgrowth. Recently, we reported that sigma-1 receptor agonists such as SA4503, fluvoxamine, DHEA-S and donepezil, could potentiate NGF-induced neurite outgrowth in PC12 cells, and that this potentiation could be antagonized by co-administration of NE-100 or xestospongin C [17,18]. Taken together, it is likely that both sigma-1 and IP 3 receptors are involved in the potentiation of NGF-induced neurite outgrowth by ifenprodil. In contrast, the sigma-2 receptor antagonist SM-21, failed to alter this enhanced outgrowth, suggesting a lack of involvement in this process.
It is likely that sigma-1 receptors interact with IP 3 receptors on the ER, as well as regulate intracellular Ca 2+ release. Recently, Hayashi and Su [1] identified sigma-1 receptors as novel, ligandoperated chaperones that specifically target the mitochondrionassociated ER membrane. When the sigma-1 receptor forms a complex with the ER chaperone, immunoglobulin heavy chain binding protein, BiP, activity is minimized. In contrast, when dissociated from BiP, the sigma-1 receptor exerts maximum chaperone activity. In addition, several synthetic agonists of the sigma-1 receptor promote its dissociation from BiP, thus stimulating the chaperone activity of this receptor [1][2][3]. In this study, we found that the cell-permeable Ca 2+ chelator BAPTA-AM blocked the effects of ifenprodil on NGF-induced neurite outgrowth. In addition, BAPTA-AM alone blocked NGF-induced neurite outgrowth in PC12 cells, consistent with previous reports [31,32]. These findings suggest that the intracellular Ca 2+ plays an important role in the neurite outgrowth mediated by NGF. Therefore, it is likely that the therapeutic activity of sigma-1 receptor agonists, such as ifenprodil, could be mediated through modulation of intracellular Ca 2+ signaling.
Recently, we reported that ifenprodil was effective in treating emotional incontinence in patients with vascular dementia (Kishimoto et al., submitted), and flashbacks in female posttraumatic stress disorder (PTSD) patients, with a history of childhood sexual abuse [33]. To our knowledge, this represents the first demonstration of a beneficial effect for ifenprodil in these groups of patients. However, the precise mechanisms underlying the therapeutic effects of ifenprodil are currently unclear. Given the role of sigma-1 receptors in modulating neurite outgrowth, it is likely that these receptors may at least play a partial a role in the beneficial effects of ifenprodil seen in these patients, although further detailed studies are needed. Accumulating evidence suggests a role for glutamatergic neurotransmission via the NMDA receptors, in the pathophysiology of PTSD [34,35]. With its high affinity for both the NMDA and sigma-1 receptors, it is likely that ifenprodil acts on these receptors to alleviate emotional incontinence as well as flashbacks in these patients ( [33], Kishimoto et al., submitted). Nonetheless, further large scale clinical studies will be needed to further support this initial finding.
In conclusion, this study suggests that ifenprodil can potentiate NGF-induced neurite outgrowth in PC12 cells, and that both the sigma-1 receptor and IP 3 receptors play a role in the mechanisms of this potentiation. Therefore, it is likely that stimulation at sigma-1 receptors may be involved in the pharmacological action of ifenprodil in humans.
Four days after incubation with NGF (2.5 ng/ml) and test drugs, morphometric analysis was performed on digitized images of live cells, taken under phase contrast illumination, using an inverted microscope linked to a camera. Images of three fields per well were taken, with an average of 100 cells per field. The number of differentiated cells was determined by visually examining the field, and counting cells with at least one neurite equivalent to the length of a cell body diameter. This number was expressed as a percentage of the total number of cells in the field. Counting was performed in a blind manner as described previously [17,18,36,37].

Statistical analysis
Data are expressed as the mean 6 standard error of the mean (S.E.M.). Statistical analysis was performed by one-way analysis of variance (ANOVA) and post hoc Bonferroni/Dunn testing. Values of p less than 0.05 were considered statistically significant.

Author Contributions
Conceived and designed the experiments: KH. Performed the experiments: TI. Analyzed the data: TI KH. Contributed reagents/materials/analysis tools: TI KH. Wrote the paper: TI KH.