Previous investigations have established that aromatase acti
Previous investigations have established that aromatase activity is regulated via two different pathways in a tissue-specific manner. The first is a slower transcription regulatory pathway that involves an alteration in gene transcription and represents what is classically thought of as the way by which sex steroids affect nociception in mammals (Balthazart et al., 2011). The second represents a nongenomic pathway that causes rapid changes in estrogen bioavailability and allows estrogens to control nociception based at least in part on aromatase phosphorylation/dephosphorylation (Balthazart et al., 2006). Thus, this latter pathway allows for rapid changes in enzymatic activity and is associated with quick changes in the local production of estrogen, which can then induce various biological modifications in adjacent cells (Woolley, 2007, Wu et al., 2012). Because the first phase of formalin-induced nociceptive responses typically occurs within the first minute after injection and the second phase within 20 min after injection it is reasonable to assume that formalin injection into the hindpaw mediates aromatase activation through a rapid nongenomic pathway. It has been reported that phosphorylated 2665 aromatase shows a rapid decrease in enzymatic activity (Balthazart et al., 2001). In particular, the serine-118 and serine-497 residues of aromatase have been identified as key phosphorylation sites that regulate both aromatase activity and protein stability (Evrard, 2006, Miller et al., 2008). Similar to these previous reports, our immunoprecipitation and Western blot analyses indicate that formalin-induced noxious stimuli are associated with significant decreases in phospho-serine levels of aromatase compared to aromatase phospho-serine levels in sham animals. On the other hand, the expression levels of spinal aromatase were not changed by formalin injection. Collectively, these results suggest that formalin injection produces peripheral noxious afferent inputs to the spinal cord that trigger decreases in phospho-serine levels of aromatase, resulting in the rapid activation of aromatase. Since the changes of phosphorylation status in aromatase has been shown to be controlled by an intracellular Ca2+-dependent pathway (Balthazart et al., 2003), this raises critical questions concerning the specific mechanisms that are responsible for the increase in the intracellular Ca2+ concentration that induces the dephosphorylation of aromatase in formalin mice. Several studies have demonstrated that intraplantar injection of formalin produces the acute nociception through ATP release from astrocytes, following activation of P2X receptors (Tsuda et al., 1999). Because NMDA-stimulated ATP release facilitates the influx of Ca2+ through the NMDA receptor (Werry et al., 2006), formalin-induced astrocytic ATP release can be one possible mechanism that underlies the increase in astrocyte intracellular Ca2+ concentration. Moreover, activated sigma-1 receptors are suggested as another possible mechanism which potentiates glutamate-induced intracellular Ca2+ influx through NMDA receptors (Monnet et al., 2003). In the present study, we have focused on spinal sigma-1 receptors as possible contributors to the dephosphorylation of aromatase via an intracellular Ca2+ mechanism. Sigma-1 receptors have been demonstrated to serve as an important trigger protein which contributes to nociceptive processes by inducing Ca2+-dependent second messenger cascades in astrocytes (Moon et al., 2014, Choi et al., 2016). In particular, activated sigma-1 receptors mediate the dephosphorylation of nNOS associated with an increase in nitric oxide production, resulting in nitric oxide-induced mechanical and thermal hypersensitivity (Roh et al., 2011). These studies imply that sigma-1 receptors facilitate the activity of Ca2+-dependent phosphatases which modulate the function of other proteins or enzymes through a dephosphorylation processes. Consistent with this previous work, the present findings demonstrated that antagonism of sigma-1 receptors blocked the formalin-induced decrease in aromatase-specific phospho-serine levels when compared to those of vehicle-treated formalin mice, suggesting that sigma-1 receptors play a critical modulatory role in the rapid dephosphorylation of spinal aromatase associated with formalin injection. Moreover, our behavioral and immunohistochemical data demonstrate that i.t. co-administration of a sub-effective dose of BD1047 together with a sub-effective dose of LET significantly inhibits both formalin-induced nociception and formalin-induced spinal Fos expression. These synergistic effects of the two drugs support our hypothesis that sigma-1 receptor-mediated activation of aromatase plays an important role in the development of acute inflammatory nociception. On the other hand, several studies have reported that sigma-1 receptor is located on neurons, but not on astrocytes, and further suggesting the neuroprotective effects of sigma-1 receptor in neuronal degenerative diseases, like amyotrophic lateral sclerosis (ALS) (Luty et al., 2010, Peviani et al., 2014). Although the discrepancy for cellular distribution of sigma-1 receptor is probably attributable to the difference in antibodies used for immunohistochemistry, the further investigations should be examined to clarify the cellular location of sigma-1 receptor and their role in pathological conditions.