Relief of cancer pain by glycine transporter inhibitors.

BACKGROUND: Recent studies have revealed the antinociceptive effects of glycine transporter (GlyT) inhibitors in neuropathic pain models such as sciatic nerve-injured and diabetic animals. Bone cancer can cause the most severe pain according to complex mechanisms in which a neuropathic element is included. Bone cancer modifies the analgesic action of opioids and limits their effectiveness, and thus novel medicament for bone cancer pain is desired. METHODS: For the femur bone cancer model, NCTC 2472 tumor cells were injected into the medullary cavity of the distal femur of C3H/HeN mice. Effects of GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitors, ORG 25935, and knockdown of the expression of spinal GlyTs protein by GlyTs siRNA on pain-like behaviors, such as allodynia, withdrawal threshold, guarding behavior, and limb-use abnormality, were examined in the femur bone cancer model mice. Effects of morphine in combination with GlyT inhibitor were examined. RESULTS: GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitor ORG 25935 by IV or oral administration or knockdown of the expression of spinal GlyTs protein improved pain-like behaviors at 11 days after tumor transplantation. The pain-relief activity was potent and long lasting. Morphine at a dose with no analgesic activity combined with ORG 25543 further promoted the ORG 25543-induced pain-relief activity. Injection of ORG 25543 on the second day after tumor implantation caused 3 phases of pain responses; pain-like behaviors were initially accelerated (at 2-4 days) and subsequently almost disappeared (5-7 days) and then reappeared. Intrathecal injection of strychnine 1 day after injection of ORG 25543 transiently antagonized the pain-relief activity of ORG 25543. In control mice, strychnine improved pain-like behaviors 4 days after tumor implantation and aggravated the behaviors between 4 and 5 days. The evidence suggests that the different mechanisms are phase-dependently involved. CONCLUSIONS: GlyT inhibitors with or without morphine may be a new strategy for the treatment of bone cancer pain and lead to further investigations of the mechanisms underlying the development of bone cancer pain.

Noradrenaline induces clock gene Per1 mRNA expression in C6 glioma cells through beta(2)-adrenergic receptor coupled with protein kinase A - cAMP response element binding protein (PKA-CREB) and Src-tyrosine kinase - glycogen synthase kinase-3beta (Src-GSK-3beta).

Astrocytes in the hypothalamic suprachiasmatic nucleus, site of the master circadian pacemaker, play an essential role in the regulation of systemic circadian rhythms. To evaluate involvement of noradrenergic systems in regulation of circadian variation of clock-genes in astrocytes, we investigated effects of noradrenaline (NA) on expression of several clock genes in C6 glioma cells by using real-time PCR analysis. Treatment with NA (10 microM) induced transient expression of Per1 mRNA, but not of Per2, Bmal1, Clock, Cry1, or Cry2 mRNA, through activation of beta(2) adrenoceptors. Action of NA was partially blocked by H-89 [protein kinase A (PKA) inhibitor] or KG-501 [inhibitor of cAMP response element binding protein (CREB)]. We found that pretreatment with genistein or PP2 (general or Src tyrosine kinase inhibitors, respectively) or LiCl [inhibitor of glycogen synthase kinase-3beta (GSK-3beta)] significantly inhibited NA-induced Per1 mRNA expression. In addition, treatment with H-89 and either genistein or LiCl completely blocked NA stimulatory effects. NA markedly induced tyrosine phosphorylation of Src and GSK-3beta via activation of beta(2) adrenoceptors. Phosphorylation of GSK-3beta by NA was completely eliminated by genistein or PP2. These results primarily suggest that two distinct NA-mediating pathways, PKA-CREB and Src-GSK-3beta, play crucial roles in regulation of Per1 expression in astroglial cells.

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 alpha3. 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.

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Calpha 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.

C-terminal region regulates the functional expression of human noradrenaline transporter splice variants.

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

Riluzole, a glutamate release inhibitor, induces loss of righting reflex, antinociception, and immobility in response to noxious stimulation in mice.

BACKGROUND: The general anesthetic state comprises behavioral and perceptual components, including amnesia, unconsciousness, analgesia, and immobility. In vitro, glutamatergic excitatory neurons are important targets for anesthetic action at the cellular and microcircuits levels. Riluzole (2-amino-6-[trifluoromethoxy]benzothiazole) is a neuroprotective drug that inhibits glutamate release from nerve terminals in the central nervous system. Here, we examined in vivo the ability of riluzole to produce components of the general anesthetic state through a selective blockade of glutamatergic neurotransmission. METHODS: Riluzole was administered intraperitoneally in adult male ddY mice. To assess the general anesthetic components, three end-points were used: 1) loss of righting reflex (LORR; as a measure of unconsciousness), 2) loss of movement in response to noxious stimulation (as a measure of immobility), and 3) loss of nociceptive response (as a measure of analgesia). RESULTS: The intraperitoneal administration of riluzole induced LORR in a dose-dependent fashion with a 50% effective dose value of 27.4 (23.3-32.2; 95% confidence limits) mg/kg. The behavioral and microdialysis studies revealed that time-course changes in impairment and LORR induced by riluzole corresponded with decreased glutamate levels in the mouse brain. This suggests that riluzole-induced LORR (unconsciousness) could result, at least in part, from its ability to decrease brain glutamate concentrations. Riluzole dose-dependently produced not only LORR, but also loss of movement in response to painful stimulation (immobility), and loss of nociceptive response (analgesia) with 50% effective dose values of 43.0 (37.1-49.9), and 10.0 (7.4-13.5) mg/kg, respectively. These three dose-response curves were parallel, suggesting that the behavioral effects of riluzole may be mediated through a common site of action. CONCLUSIONS: These findings suggest that riluzole-induced LORR, immobility, and antinociception appear to be associated with its ability to inhibit glutamatergic neurotransmission in the central nervous system.

Increased gamma-aminobutyric acid levels in mouse brain induce loss of righting reflex, but not immobility, in response to noxious stimulation.

BACKGROUND: The general anesthetic state comprises behavioral and perceptual components, including amnesia, unconsciousness, and immobility. gamma-Aminobutyric acidergic (GABAergic) inhibitory neurotransmission is an important target for anesthetic action at the in vitro cellular level. In vivo, however, the functional relevance of enhancing GABAergic neurotransmission in mediating essential components of the general anesthetic state is unknown. Gabaculine is a GABA-transaminase inhibitor that inhibits degradation of released GABA, and consequently increases endogenous GABA in the central nervous system. Here, we examined, behaviorally, the ability of increased GABA levels to produce components of the general anesthetic state. METHODS: All drugs were administered systemically in adult male ddY mice. To assess the general anesthetic components, two end-points were used. One was loss of righting reflex (LORR; as a measure of unconsciousness); the other was loss of movement in response to tail-clamp stimulation (as a measure of immobility). RESULTS: Gabaculine induced LORR in a dose-dependent fashion with a 50% effective dose of 100 (75-134; 95% confidence limits) mg/kg. The behavioral and microdialysis studies revealed that the endogenous GABA-induced LORR occurred in a brain concentration-dependent manner. However, even larger doses of gabaculine (285-400 mg/kg) produced no loss of tail-clamp response. In contrast, all the tested volatile anesthetics concentration-dependently abolished both righting and tail-clamp response, supporting the evidence that volatile anesthetics act on a variety of molecular targets. CONCLUSIONS: These findings indicate that LORR is associated with enhanced GABAergic neurotransmission, but that immobility in response to noxious stimulation is not, suggesting that LORR and immobility are mediated through different neuronal pathways and/or regions in the central nervous system.

Assessing an eating disorder induced by 6-OHDA and the possibility of nerve regeneration therapy by transplantation of neural progenitor cells in rats.

Parkinson's disease is a neurodegenerative disorder of the substantia nigra accompanied by the depletion of dopamine levels. Symptoms of Parkinson's disease involve motor disorders, including dysphagia and aspiration. In this study, rats were injected with 6-hydroxydopamine (6-OHDA) in order to assess the eating disorder and evaluate the effect of transplantation of neural progenitor cells (NPCs). The administration of 6-OHDA resulted in an extension of feeding time and a marked increase in the amount of feed powder on the cage floor after feeding at 2 and 4 weeks after 6-OHDA. These rats had NPCs obtained from the brains of newborn rats transplanted into their striata 2 weeks after 6-OHDA injection. The treatment shortened the feeding time and decreased the amount of feed powder on the cage floor after feeding. The 6-OHDA injection decreased the number of tyrosine hydroxylase-positive cells in the striatum and substantia nigra, and NeuN in the solitary tract. A greater number of tyrosine hydroxylase-positive cells in the substantia nigra and NeuN-positive cells in the solitary tract were detected in the animals transplanted with NPCs than the 6-OHDA injected control. The NPCs labeled with 5-bromo-2'-deoxyuridine were detected in the striatum, but not in the substantia nigra and solitary tract. These results may suggest that the eating disorder induced by 6-OHDA may be related to neural damage to the substantia nigra and/or solitary tract. Transplantation of NPCs may cure 6-OHDA-induced eating disorders accompanied by the protection of neurons from the toxin.

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.

Analgesic action of nicotine on tibial nerve transection (TNT)-induced mechanical allodynia through enhancement of the glycinergic inhibitory system in spinal cord.

The activation of cholinergic pathways by nicotine elicits various physiological and pharmacological effects in mammals. For example, the stimulation of nicotinic acetylcholine receptors (nAChRs) leads to an antinociceptive effect. However, it remains to be elucidated which subtypes of nAChR are involved in the antinociceptive effect of nicotine on nerve injury-induced allodynia and the underlying cascades of the nAChR-mediated antiallodynic effect. In this study, we attempted to characterize the actions of nicotine at the spinal level against mechanical allodynia in an animal model of neuropathic pain, tibial nerve transection (TNT) in rats. It was found that the intrathecal injection of nicotine, RJR-2403, a selective alpha4beta2 nAChR agonist, and choline, a selective alpha7 nAChR agonist, produced an antinociceptive effect on the TNT-induced allodynia. The actions of nicotine were almost completely suppressed by pretreatment with mecamylamine, a non-selective nicotinic antagonist, or dihydro-beta-erythroidine, a selective alpha4beta2 nAChR antagonist, and partially reversed by pretreatment with methyllycaconitine, a selective alpha7 nAChR antagonist. Furthermore, pretreatment with strychnine, a glycine receptor antagonist, blocked the antinociception induced by nicotine, RJR-2403, and choline. On the other hand, the GABAA antagonist bicuculline did not reverse the antiallodynic effect of nicotine. Together, these results indicate that the alpha4beta2 and alpha7 nAChR system, by enhancing the activities of glycinergic neurons at the spinal level, exerts a suppressive effect on the nociceptive transduction in neuropathic pain.

Down-regulation of zinc transporter-1 in astrocytes induces neuropathic pain via the brain-derived neurotrophic factor - K+-Cl- co-transporter-2 signaling pathway in the mouse spinal cord.

We previously demonstrated, using a DNA microarray analysis, the down-regulated expression of the slc30a1 gene (zinc transporter 1, ZnT1) in a neuropathic pain model induced by partial sciatic nerve ligation (PSNL). Zinc is an essential trace mineral that plays important roles in physiological functions, and ZnT1 modulates intracellular zinc levels. In the present study, we examined the effects of the down-regulation of the ZnT1 gene in the spinal cord on tactile allodynia. The knockdown (KD) of ZnT1 by the intrathecal administration of siRNA against ZnT1 to mice induced allodynia, a characteristic syndrome of neuropathic pain, which persisted for at least one month. ZnT1 KD increased intracellular zinc concentrations in primary astrocyte cultures, and this was followed by enhanced PKCα membrane translocation and NFκB nuclear translocation as well as increases in the levels of IL-6 and BDNF expressed and the phosphorylation of CREB in vitro. Neuropathic pain induced by ZnT1 KD was inhibited by an IL-6, BDNF, and TrkB siRNA injection. The down-regulated expression of KCC2 in spinal cord was induced by ZnT1 KD and prevented by an intrathecal injection of IL-6, BDNF, and TrkB siRNA. These results indicate that PSNL via the down-regulated expression of ZnT1 increases intracellular zinc concentrations, enhances PKCα membrane translocation and NFκB nuclear translocation, up-regulates the expression of IL-6, increases the phosphorylation of CREB, and promotes the BDNF cascade reaction in astrocytes, thereby down-regulating the expression of KCC2 and inducing neuropathic pain in vivo. This mechanism is considered to be responsible for the activation of TrkB in neurons through the release of BDNF from astrocytes. The results of the present study also indicate that zinc signaling in astrocytes occurs upstream of the BDNF-TrkB-KCC2 cascade reaction.

Interleukin-1 inhibits voltage-dependent P/Q-type Ca2+ channel associated with the inhibition of the rise of intracellular free Ca2+ concentration and catecholamine release in adrenal chromaffin cells.

Effects of interleukin (IL) on intracellular free Ca2+ concentration ([Ca2+]i) rise and catecholamine (CA) release were examined in isolated, cultured bovine adrenal chromaffin cells. IL-1alpha and IL-1beta inhibited the rise of [Ca2+]i and CA release induced by acetylcholine (ACh) and excess KCl both in normal and in Ca2+-sucrose medium. Pretreatment by IL-1 receptor antagonist (IL-1RA) blocked the inhibitory actions of IL-1alpha. IL-1alpha reduced CA release induced by veratridine in normal medium but not in the presence of diltiazem. Analysis using specific blockers for voltage-operated Ca2+ channels (VOCC) revealed that IL-1alpha and IL-1beta specifically inhibited the P/Q-type Ca2+ channel to reduce [Ca2+]i rise induced by excess KCl. IL-1 did not affect [Ca2+]i rise induced either by bradykinin or caffeine in Ca2+-deprived medium or via activation of store-operated Ca2+ channel (SOC). The inhibitory effects of IL-1alpha were blocked by pretreatments with herbimycin A, U0126 and PD 98054, but not with SB202190, SP 600125 or pertussis toxin (PTX). These results demonstrated that IL-1 inhibits stimulation-evoked [Ca2+]i rise and CA release in chromaffin cells by blocking voltage-operated P/O-type Ca2+ channels. The inhibitory action of IL-1 may be mediated through the tyrosine kinase and MEK/ERK pathways.

Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT).

Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.

Inhibition of serotonin transporters by cocaine and meprylcaine through 5-TH2C receptor stimulation facilitates their seizure activities.

The present study examined whether the inhibition of serotonin transporters (SERT) contributes to cocaine- and other local anesthetics-induced convulsions, and which subtypes of 5-HT receptor are involved in the convulsions. For this purpose, cocaine, meprylcaine and lidocaine, all of which have different effects on SERT, were used as convulsants and the effects of serotonin reuptake inhibitors (SSRIs), specific agonists and antagonists for 5-HT receptor subtypes were evaluated in mice. Administration of SSRI, zimelidine, citalopram and fluoxetine, 5-HT(2A,2C) receptor agonist, R(-)-DOI and the 5-HT2C receptor agonists, mCPP, and MK212 resulted in a marked increase in incidence of convulsions and a reduction in the threshold of lidocaine-induced convulsions, while the 5-HT2B receptor agonist, BW723C86, had little influence. On the other hand, SSRI did not affect the measured parameters in meprylcaine- and cocaine-induced convulsions. R(-)-DOI, mCPP, and MK212 reduced the threshold of meprylcaine or cocaine with less extent than the reduction of lidocaine threshold. Incidence of cocaine- and meprylcaine-induced convulsions was significantly reduced by 5-HT(2A,2B,2C) antagonist, LY-53857, and 5-HT2C antagonist, RS 102221. The threshold of cocaine and meprylcaine was significantly increased by both antagonists. 5-HT2A antagonists MDL 11,939 and ketanserin, and 5-HT2B antagonist SB 204741 except at high doses had little effect on cocaine- and meprylcaine-induced convulsions. None of these antagonists altered the parameters of lidocaine-induced convulsions. Pretreatment with fluoxetine but not citalopram increased the plasma concentration of lidocaine. These results suggest that the increase of serotonergic neuronal activity through 5-HT2C receptor stimulation was responsible for increased activity of local anesthetics-induced convulsions and support the involvement of this mechanism in cocaine- and meprylcaine- but not in lidocaine-induced convulsions through their direct inhibitory action on central SERT.

Development of tactile allodynia and thermal hyperalgesia by intrathecally administered platelet-activating factor in mice.

Platelet-activating factor (PAF) is a potent inflammatory lipid mediator in peripheral tissues. However, its role in mediation of nociception in central nervous system is unknown. In the present study, whether PAF plays some role in pain transduction in the spinal cord was studied in mice. Intrathecal injection of PAF induced tactile pain, tactile allodynia at as low as 10 fg to 1 pg with a peak response at 100 fg, while lyso-PAF was without effect in the range of doses. Tactile allodynia induced by PAF was blocked by a PAF receptor antagonists, TCV-309, WEB 2086 and BN 50739. The expression of PAF receptor mRNA by RT-PCR was observed in DRG and spinal cord in mice. ATP P2X receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5-triphosphate, NMDA receptor antagonist, MK 801 and nitric oxide synthetase inhibitor, 7-nitroindazole blocked the PAF-induced tactile allodynia. PAF-induced tactile allodynia and thermal hyperalgesia disappeared in neonatally capsaicin-treated adult mice, while tactile allodynia but not thermal hyperalgesia induced by intrathecally injected alpha,beta-methylene ATP, a P2X receptor agonist, was capsaicin-insensitive. The present study demonstrated that PAF is a potent inducer of tactile allodynia and thermal hyperalgesia at the level of the spinal cord. PAF-evoked tactile allodynia is suggested to be mediated by ATP and the following NMDA and NO cascade through capsaicin-sensitive fiber, different from exogenously injected alpha,beta-methylene ATP which is insensitive to capsaicin treatment.

Identification and functional characterization of the novel isoforms of bovine norepinephrine transporter produced by alternative splicing.

Analyzing variation of bovine norepinephrine transporter (NET) at the 3'-region by RT-PCR in the adrenal glands and the brain revealed four isoforms of NET produced by alternative splicing of four cassettes (C0, C1, C2 and C3) encoded by exons 12-15, designated bNET1a (C0-C1-C2, formerly designated bNET1), bNET1b (C0-C2), bNET2a (C0-C1-C3) and bNET2b (C0-C3, formerly designated bNET2), respectively. Expression of these isoforms in COS-7 cells revealed that the isoforms that contain the C1 cassette encoded by exon 13 (bNET1a and bNET2a) showed a significant increase in [(3)H]norepinephrine uptake and [(3)H]nisoxetine binding, whereas the isoforms which lack the C1 cassette (bNET1b and bNET2b) failed to display those activities despite the selection of either exon 14 or exon 15. These results suggest that the region encoded by exon 13 is indispensable for NET functional expression.

Chronic inhibition of the norepinephrine transporter in the brain participates in seizure sensitization to cocaine and local anesthetics.

The involvement of chronic inhibition of monoamine transporters (MAT) in the brain with respect to sensitization to cocaine- and local anesthetic-induced seizures was studied in mice. Repeated administration of subconvulsive doses of meprylcaine as well as cocaine, both of which inhibit MAT, but not lidocaine, which does not inhibit MAT, increased seizure activity and produced sensitization to other local anesthetics. The effects of five daily treatments of monoamine transporter inhibitors on lidocaine-induced convulsions were examined 2 or 3 days after the last dose of the inhibitors. Daily treatments of GBR 12935, a specific inhibitor of dopamine uptake, significantly increased the incidence and the intensity of lidocaine-induced convulsions at 20 mg/kg and decreased the threshold of the convulsions. Daily treatments of desipramine and maprotiline, selective norepinephrine uptake inhibitors, markedly increased the incidence and intensity of lidocaine-induced convulsions, and decreased the threshold in a dose-dependent manner at between 5 and 20 mg/kg. Daily treatments of citalopram, a selective serotonin uptake inhibitor, at 10 and 20 mg/kg, produced no significant increase in the incidence or intensity of lidocaine-induced convulsions, but decreased the threshold of the convulsions. These results suggest that the chronic intermittent inhibition of monoamine uptake increases susceptibility to cocaine- and local anesthetic-induced seizures, and the norepinephrine transporter is an integral component of this sensitization.

Norepinephrine transporter splice variants and their interaction with substrates and blockers.

Norepinephrine transporter (NET), a member of the Na+/Cl--dependent neurotransmitter transporter family, displays species-specific isoforms produced by alternative RNA splicing. This occurs at 3'-flanking coding and noncoding regions, resulting in different carboxy-terminals. When these NET splice variants were expressed in cultured cell lines, the characteristics of substrate transport and the sensitivity to uptake inhibitors differed between isoforms. The different functional expression suggests the physiological importance of the action and interaction of NET splice variants in synaptic transmission.

Effects of GABAergic agents on anesthesia induced by halothane, isoflurane, and thiamylal in mice.

The effects of gamma-aminobutyric acid (GABA) receptor modulators and GABA uptake inhibitors on volatile and intravenous anesthetic-induced anesthesia were examined in male ICR mice, as assessed by the loss of righting reflex (LORR). The GABA uptake inhibitors, NO-711 and SKF89976A, which are permeable to the blood-brain barrier (BBB), but not nipecotic acid or guvacine, which poorly permeate BBB, shortened the onset of LORR but did not affect the duration of LORR induced by 1.5% halothane and 2% isoflurane. NO-711 and SKF89976A shortened the onset of and prolonged the duration of LORR induced by thiamylal (45 mg/kg i.p.). The GABA mimetics, muscimol and diazepam, shortened the onset of and prolonged the duration of LORR induced by halothane, isoflurane, and thiamylal. On the other hand, picrotoxin, a GABAA receptor antagonist, prolonged the onset of LORR induced by all anesthetics tested. Another GABAA receptor antagonist, bicuculline, prolonged the onset of LORR induced by halothane, but not by isoflurane or thiamylal. Both antagonists failed to affect the duration of LORR induced by halothane, isoflurane, or thiamylal. Baclofen, a GABAB receptor agonist, enhanced both volatile anesthetics- and thiamylal-induced anesthesia. These results suggest that anesthesia induced by volatile and intravenous anesthetics might be correlated with the modification of the pre- and/or postsynaptic GABAergic activities.

Regulation of dopamine and MPP+ transport by catecholamine transporters.

Following exocytotic release of the biogenic amine neurotransmitters, norepinephrine and dopamine, are removed from the synaptic cleft by the respective transporter, norepinephrine transporter (NET) and dopamine transporter (DAT) located on the plasma membrane. The catecholamine transporters are the molecular targets for psychoactive drugs as well as drugs of abuse such as cocaine and amphetamine and the Parkinsonism-inducing neurotoxin, MPP+. Nicotine regulates the transport of catecholamines and MPP+ and may exert self-medicating effects for depression, schizophrenia and attention deficit hyperactivity disorder, and neuroprotective effects against MPP+ through the regulation of the transporters. The availability of cDNAs of these transporters has permitted detailed pharmacological studies in heterologous expression systems for determining the mechanisms of action of nicotine on transporters. Moreover, functional analysis of the effect of single amino acid substitution suggests that specific residues in DAT molecules may play a significant role in interaction with MPP+ and cocaine, and thus would promise a development of novel drugs with diverse chemical structures.