Tomoya Kitayama

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide (ORG25543) and (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine) (ALX1393), or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor α3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain

Injury to peripheral or spinal nerves following either trauma or disease has several consequences including the development of neuropathic pain. This syndrome is often refractory against conventional analgesics; and thus, novel medicaments are desired for its treatment. Recent studies have emphasized that dysfunction of inhibitory neuronal regulation of pain signal transduction may be relevant to the development of neuropathic pain. Glycinergic neurons are localized in specific brain regions and the spinal cord, where they play an important role in the prevention of pathological pain symptoms. Thus, an enhancement of glycinergic control in the spinal cord is a promising strategy for pain relief from neuropathic pain. Glycine transporter (GlyT) 1 and GlyT2, which are located in glial cells and neurons, respectively play important roles by clearing synaptically released glycine or supplying glycine to glycinergic neurons to regulate glycinergic neurotransmission. Thus, an inhibition of GlyTs could be used to modify pain signal transmission in the spinal cord. Recently developed specific inhibitors of GlyTs have made this possibility a reality. Both GlyT1 and GlyT2 inhibitors produced potential anti-nociceptive effect in various neuropathic pain models, chronic and acute inflammatory models in animals. Their anti-allodynia effects are mediated by the inhibition of GlyTs following activation of spinal glycine receptor alpha3. These results established GlyTs as target molecules for medicaments for neuropathic pain. Moreover, the phase-dependent anti-allodynia effects of GlyT inhibitors have provided important information on effective therapeutic strategies and also understanding the underlying molecular mechanisms of the development of neuropathic pain.

P2X7 receptor stimulation in primary cultures of rat spinal microglia induces downregulation of the activity for glutamate transport

It has been shown that spinal microglia expressing certain types of glutamate transporters function in the modulation of neuropathogenesis. In this study, the effect of ATP, potentially able to mediate the communication between neurons and glial cells in the spinal cord on the transport of glutamate in cultured spinal microglia, was investigated. Both GLAST and GLT‐1 were detected in the cells. Preincubation with ATP or 2′‐3′‐O‐(4‐benzoyl‐benzoyl) ATP (BzATP), a selective agonist for the P2X7 receptor, significantly blocked the uptake of glutamate. The effect of BzATP was reversed by pretreatment with brilliant blue G or oxidized ATP, each a selective antagonist for P2X7. The inhibitory effect of P2X7 receptor activation also occurred in the absence of extracellular Na+ or Ca2+, suggesting that the receptor regulates glutamate transport by a metabotropic pathway. Furthermore, pretreatment with inhibitors of mitogen‐activated protein kinase kinase, or antioxidants, significantly reversed the inhibitory effect of BzATP on the uptake of glutamate. Incubation with BzATP led to a marked decrease in the Vmax, but not the Km, of glutamate transport. However, treatment with BzATP did not induce the trafficking of glutamate transporters. These results suggest that the activation of P2X7 receptors in spinal microglia is important in the regulation of glutamate transport via activation of the extracellular signal‐regulated kinase cascade and production of oxidants.

Glycinergic mediation of tactile allodynia induced by platelet-activating factor (PAF) through glutamate-NO-cyclic GMP signalling in spinal cord in mice.

&NA; Our previous study showed that intrathecal (i.t.) injection of platelet‐activating factor (PAF) induced tactile allodynia, suggesting that spinal PAF is a mediator of neuropathic pain. The present study further examined the spinal molecules participating in PAF‐induced tactile allodynia in mice. I.t. injection of l‐arginine, NO donor (5‐amino‐3‐morpholinyl‐1,2,3‐oxadiazolium (SIN‐1) or 3,3‐bis(aminoethyl)‐1‐hydroxy‐2‐oxo‐1‐triazene (NOC‐18)) or cGMP analog (8‐(4‐chlorophenylthio)‐guanosine 3′,5′‐cyclic monophosphate; pCPT‐cGMP) induced tactile allodynia. PAF‐ and glutamate‐ but not SIN‐1‐ or pCPT‐cGMP‐induced tactile allodynia was blocked by an NO synthase inhibitor. NO scavengers and guanylate cyclase inhibitors protected mice against the induction of allodynia by PAF, glutamate and SIN‐1, but not by pCPT‐cGMP. cGMP‐dependent protein kinase (PKG) inhibitors blocked the allodynia induced by PAF, glutamate, SIN‐1 and pCPT‐cGMP. To identify signalling molecules through which PKG induces allodynia, glycine receptor α3 (GlyR α3) was knocked down by spinal transfection of siRNA for GlyR α3. A significant reduction of GlyR α3 expression in the spinal superficial layers of mice treated with GlyR α3 siRNA was confirmed by immunohistochemical and Western blotting analyses. Functional targeting of GlyR α3 was suggested by the loss of PGE2‐induced thermal hyperalgesia and the enhancement of allodynia induced by bicuculline, a GABAA receptor antagonist in mice after GlyR α3 siRNA treatment. pCPT‐cGMP, PAF, glutamate and SIN‐1 all failed to induce allodynia after the knockdown of GlyR α3. These results suggest that the glutamate–NO–cGMP–PKG pathway in the spinal cord may be involved in the mechanism of PAF‐induced tactile allodynia, and GlyR α3 could be a target molecule through which PKG induces allodynia.

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Cα in C6 glioma cells

Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCalpha, PKCdelta, and PKCvarepsilon of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca(2+). Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCdelta and PKCvarepsilon, did not affect the transport of glycine. Silencing of PKCdelta expression by using RNA interference or pretreatment with the inhibitor peptide for PKCvarepsilon had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCalpha to be a crucial factor in the regulation of glycine transport in C6 cells.

Pain-releasing action of Platelet-activating factor (PAF) antagonists in neuropathic pain animal models and the mechanisms of action

Platelet‐activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF‐R) antagonists have varied anti‐allodynia effects by route of administration and nerve injury models in rats.

Down-regulation of norepinephrine transporter function induced by chronic administration of desipramine linking to the alteration of sensitivity of local anesthetics-induced convulsions and the counteraction by co-administration with local anesthetics.

Alterations of norepinephrine transporter (NET) function by chronic inhibition of NET in relation to sensitization to seizures induce by cocaine and local anesthetics were studied in mice. Daily administration of desipramine, an inhibitor of the NET, for 5 days decreased [(3)H]norepinephrine uptake in the P2 fractions of hippocampus but not cortex, striatum or amygdalae. Co-administration of lidocaine, bupivacaine or tricaine with desipramine reversed this effect. Daily treatment of cocaine increased [(3)H]norepinephrine uptake into the hippocampus. Daily administration of desipramine increased the incidence of appearance of lidocaine-induced convulsions and decreased that of cocaine-induced convulsions. Co-administration of lidocaine with desipramine reversed the changes of convulsive activity of lidocaine and cocaine induced by repeated administration of desipramine. These results suggest that down-regulation of hippocampal NET induced by chronic administration of desipramine may be relevant to desipramine-induced sensitization of lidocaine convulsions. Inhibition of Na(+) channels by local anesthetics may regulate desipramine-induced down-regulation of NET function. Repeated administration of cocaine induces up-regulation of hippocampal NET function. Desipramine-induced sensitization of lidocaine seizures may have a mechanism distinct from kindling resulting from repeated administration of cocaine.

Phospholipase C-related but catalytically inactive protein modulates pain behavior in a neuropathic pain model in mice

BackgroundAn inositol 1,4,5-trisphosphate binding protein, comprising 2 isoforms termed PRIP-1 and PRIP-2, was identified as a novel modulator for GABAA receptor trafficking. It has been reported that naive PRIP-1 knockout mice have hyperalgesic responses.FindingsTo determine the involvement of PRIP in pain sensation, a hind paw withdrawal test was performed before and after partial sciatic nerve ligation (PSNL) in PRIP-1 and PRIP-2 double knockout (DKO) mice. We found that naive DKO mice exhibited normal pain sensitivity. However, DKO mice that underwent PSNL surgery showed increased ipsilateral paw withdrawal threshold. To further investigate the inverse phenotype in PRIP-1 KO and DKO mice, we produced mice with specific siRNA-mediated knockdown of PRIP s in the spinal cord. Consistent with the phenotypes of KO mice, PRIP-1 knockdown mice showed allodynia, while PRIP double knockdown (DKD) mice with PSNL showed decreased pain-related behavior. This indicates that reduced expression of both PRIPs in the spinal cord induces resistance towards a painful sensation. GABAA receptor subunit expression pattern was similar between PRIP-1 KO and DKO spinal cord, while expression of K+-Cl--cotransporter-2 (KCC2), which controls the balance of neuronal excitation and inhibition, was significantly upregulated in DKO mice. Furthermore, in the DKD PSNL model, an inhibitor-induced KCC2 inhibition exhibited an altered phenotype from painless to painful sensations.ConclusionsSuppressed expression of PRIPs induces an elevated expression of KCC2 in the spinal cord, resulting in inhibition of nociception and amelioration of neuropathic pain in DKO mice.

Down-regulation of norepinephrine transporter expression on membrane surface induced by chronic administration of desipramine and the antagonism by co-administration of local anesthetics in mice

We have previously shown that chronic administration of the antidepressant desipramine, a norepinephrine transporter (NET) inhibitor to mice markedly enhanced convulsions induced by local anesthetics and that behavioral sensitization may be relevant to decreased [(3)H]norepinephrine uptake by the isolated hippocampus. The co-administration of local anesthetics with desipramine reversed the behavioral sensitization and down-regulation of NET function induced by desipramine. The present study aimed to elucidate whether chronic treatment with desipramine regulates the expression of NET protein examined in membrane fractions in various brain regions and whether co-administration of local anesthetics affects the desipramine-induced alteration of NET expression. Desipramine with or without local anesthetics was injected intraperitoneally once a day for 5 days. The animals were decapitated 48 h after the last administration of drugs and the whole cell fraction, membrane fraction and cell-surface protein fraction were prepared. [(3)H]nisoxetine binding was significantly reduced in the P2 fraction of the hippocampus by chronic administration of desipramine, and the reduction was overcome by co-administration of lidocaine with desipramine. Immunoreactive NET was detected by SDS-PAGE and immunoblotting in the murine hippocampus. NET protein expression in the whole cell fraction and membrane fraction was not affected by treatment with any drugs. However, administration of desipramine significantly reduced the amount of immunoreactive NET in the cell-surface protein fraction. This reduction was blocked by simultaneous injection of lidocaine, bupivacaine or tricaine. These results indicate that the NET down-regulation indicated by the reduction of [(3)H]nisoxetine binding was induced by administration of desipramine via decrease of NET localization on the cell surface. The antagonistic actions of local anesthetics against NET down-regulation by desipramine were related to alterations of the cell-surface localization of NET.

Phospholipase C-related catalytically inactive protein (PRIP) controls KIF5B-mediated insulin secretion

ABSTRACT We previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.