Unexpectedly under pathological pain conditions

Unexpectedly, under pathological pain conditions, inhibition of spinal glutamate transporter activity can produce antinociceptive effects. For example, intrathecal injection of the transportable inhibitor trans-pyrrolidine-2,4-dicarboxylic ML133 HCl (t-PDC) or antisense oligonucleotides reduced nociceptive behavior in the rat formalin test (Niederberger et al., 2003). A non-transportable blocker, DL-threo-β-benzyloxyaspartate (TBOA), resulted in significant reductions in complete Freund's adjuvant (CFA)- and formalin-induced inflammatory pain (Yaster et al., 2011). However, current knowledge about this paradoxical action of glutamate transporter inhibitors is limited. Mechanisms regarding the activation of inhibitory presynaptic group III metabotropic glutamate receptors (Yaster et al., 2011) and the modulation of glutamate release from presynaptic terminals (Yang et al., 2015) have been reported. However, other potential mechanisms, including postsynaptic desensitization of dorsal horn glutamate receptors, depletion of the glutamate-glutamine cycle in spinal glial cells, and inhibition of glutamate release by blocking inverse operation of the transporter, have not been investigated. Above all, as suggested by Tao et al., (Tao et al., 2005) determining the extracellular levels of glutamate in the spinal cord after glutamate transporter inhibition during pathological pain status, and thus clarifying the possible role of reversed transporter, may be an essential step in investigating the mechanism(s) of this phenomenon. In a previous report that studied glutamate release through reverse transport induced by ATP depletion, a transportable inhibitor enhanced, while a non-transportable inhibitor blocked, uptake reversal of glutamate (Anderson et al., 2001). Therefore the glutamate transporter in a reversed mode may be differentially affected by transportable and non-transportable inhibitors (Anderson et al., 2001, Roettger and Lipton, 1996).
Materials and methods
Results
Discussion In this study, we investigated the effects and mechanism(s) of glutamate transporter inhibitors using the rat formalin test. Intrathecally injected glutamate transporter inhibitors exhibited paradoxical antinociception, consistent with previous reports (Niederberger et al., 2003, Yaster et al., 2011). The microdialysis study was key in determining the mechanism of the paradoxical analgesia produced by glutamate transporter inhibitor in the pathological pain state. First, a non-transportable blocker, TBOA, increased glutamate levels in the normal state, as expected, but suppressed glutamate release in the formalin model, suggesting that the transporter might have worked in a reversed mode in the latter state. Second, a transportable inhibitor, tPDC, increased glutamate levels in control rats and unexpectedly further enhanced the formalin-induced increase in extracellular glutamate. However, blocking transporter function by preloading tPDC (Longuemare and Swanson, 1995) abolished the formalin-induced increase in extracellular glutamate, establishing that the increase occurs predominantly through glutamate transporters. Taken together, we suggest that glutamate transporters in the formalin-induced pain state are working inversely and can be blocked from releasing glutamate by TBOA and preloading with tPDC. To our knowledge, this is the first reported study that measured in vivo changes in extracellular glutamate levels to determine the role of inversely operating transporters in the paradoxical analgesia produced by glutamate transporter inhibitors in an inflammatory pain model. The transportable inhibitor, tPDC, and non-transportable blocker, TBOA, might have differentially affected the formalin-induced pain model in terms of glutamate levels. The glutamate transport mechanism by its transporter is thought to involve two half-cycles of conformational changes (Qu and Kanner, 2008). After binding of glutamate from the extracellular medium, the outward-facing glutamate-loaded conformation is formed. When the external gate is closed and the internal gate opened, the binding site is exposed to the cytoplasm, and the substrates dissociate into the cytoplasm. This process is coupled to the co-transport of three sodium ions and one proton and the counter-transport of one potassium ion (Levy et al., 1998). The transportable inhibitors act as potent competitive inhibitors (a substrate) of uptake and can themselves be transported into the cytoplasm (Anderson et al., 2001). Thus, they should block glutamate transport originating from the same side, but not the opposite side, of the membrane that they are located (Roettger and Lipton, 1996). If the transporters work inversely, tPDC delivered after formalin injection would be located on the side opposite to intracellular glutamate and would act as an additional substrate extracellularly to allow more rapid movement of the natural substrate glutamate from the intracellular side, thus further increasing the release of glutamate. This process has been termed heteroexchange (Griffiths et al., 1994). When administered 30min prior to formalin injection, the tPDC would have been preloaded intracellularly (Anderson et al., 2001) and might have inhibited the release of intracellular glutamate from the same side. However, when the non-transportable inhibitors bind from the extracellular medium, the transporters become locked in an outward-facing conformation, because transport is impossible (Qu and Kanner, 2008). Thus, intrathecally administered TBOA might have resulted in closure of the transporter to block the movement of glutamate from both sides. Blocking the transporter by TBOA under physiological conditions might have inhibited glutamate uptake from the extracellular space, while under the inverse condition, the release of intracellular glutamate might have been suppressed.