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Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein-protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.

Objective Volatile anesthetics are widely used for general anesthesia during surgical operations. Voltage-gated Na+ channels expressed in central neurons are major targets for volatile anesthetics; but it is unclear whether these drugs modulate native tetrodotoxin-resistant (TTX-R) Na+ channels, which are involved in the development and maintenance of inflammatory pain. Methods In this study, we examined the effects of sevoflurane on TTX-R Na+ currents (INa) in acutely isolated rat dorsal root ganglion neurons, using a whole-cell patch-clamp technique. Results Sevoflurane slightly potentiated the peak amplitude of transient TTX-R INa but more potently inhibited slow voltage-ramp-induced persistent INa in a concentration-dependent manner. Sevoflurane (0.86 ± 0.02 mM) (1) slightly shifted the steady-state fast inactivation relationship to hyperpolarizing ranges without affecting the voltage-activation relationship, (2) reduced the extent of use-dependent inhibition of Na+ channels, (3) accelerated the onset of inactivation and (4) delayed the recovery from inactivation of TTX-R Na+ channels. Thus, sevoflurane has diverse effects on TTX-R Na+ channels expressed in nociceptive neurons. Conclusions The present results suggest that the inhibition of persistent INa and the modulation of the voltage dependence and inactivation might be, at least in part, responsible for the analgesic effects elicited by sevoflurane.

Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.

Neural stem/progenitor cells (NSPCs) are self-renewing, multipotent cells located in the embryonic and adult central nervous system (CNS). Extensive preclinical and clinical studies have shed light on the potential of stem cell replacement therapy for various neurodegenerative diseases. The key prerequisite for the success of these clinical applications is the procurement of a sufficient number of high-quality NSPCs. In this study, we explored the biological activity of Quadrella incana leaf in NSPC homeostasis. We showed that the leaf extract of Quadrella incana upregulated NSPC marker and proliferative potential. On the other hand, Quadrella incana leaf suppressed spontaneous unintended NSPC differentiation. Mechanistically, Quadrella incana leaf contributed to the maintenance of NSPCs by upregulating glycolytic flux and redox potential.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC

Glioblastoma is an actively growing and aggressive brain tumor with a high propensity of recurrence. Although the surgical removal of tumor mass is the primary therapeutic option against glioblastoma, supportive pharmacotherapy is highly essential due to incredibly infiltrative characteristic of glioblastoma. Temozolomide, an FDA-approved alkylating agent, has been used as a first-line standard pharmacological approach, but several evident limitations were repeatedly reported. Despite additional therapeutic options suggested, there are no medications that successfully prevent a recurrence of glioblastoma and increase the five-year survival rate. In this study, we tested the possibility that finasteride has the potential to be developed as an anti-glioblastoma drug. Finasteride, an FDA-approved medication for the treatment of benign prostate hyperplasia and androgenic alopecia, is already known to pass through the blood–brain barrier and possess antiproliferative activity of prostate epithelial cells. We showed that finasteride inhibited the maintenance of glioma stem-like cells and repressed the proliferation of glioblastoma. Mechanistically, finasteride lowered intracellular ROS level by upregulating antioxidant genes, which contributed to inefficient β-catenin accumulation. Downregulated β-catenin resulted in the reduction in stemness and cell growth in glioblastoma.

Sevoflurane, which is preferentially used as a day-case anesthetic based on its low blood solubility, acts on the central nervous system and exerts analgesic effects. However, it still remains unknown whether sevoflurane affects the excitability of nociceptive sensory neurons. Therefore, we conducted this study to examine the effects of sevoflurane on the excitability of small-sized dorsal root ganglion (DRG) neurons of rats using the whole-cell patch-clamp technique. In a voltage-clamp condition, sevoflurane elicited the membrane current in a concentration-dependent manner, in which the reversal potential was similar to the equilibrium potential of K

RalBP1 associated EPS domain containing 1 (REPS1) is conserved from Drosophila to humans and implicated in the endocytic system. However, an exact role of REPS1 remains largely unknown. Here, we demonstrated that mitogen activated protein kinase kinase (MEK)-p90 ribosomal S6 Kinase (RSK) signaling pathway directly phosphorylated REPS1 at Ser709 upon stimulation by epidermal growth factor (EGF) and amino acid. While REPS2 is known to be involved in the endocytosis of EGF receptor (EGFR), REPS1 knockout (KO) cells did not show any defect in the endocytosis of EGFR. However, in the REPS1 KO cells and the KO cells reconstituted with a non-phosphorylatable REPS1 (REPS1 S709A), the recycling of transferrin receptor (TfR) was attenuated compared to the cells reconstituted with wild type REPS1. Collectively, we suggested that the phosphorylation of REPS1 at S709 by RSK may have a role of the trafficking of TfR. [BMB Reports 2021; 54(5): 272-277].

Background Glioblastoma (GBM) is the most aggressive tumor residing within the central nervous system, with extremely poor prognosis. Although the cytotoxic effects of ginsenoside F2 (GF2) on GBM were previously suggested, the precise anti-GBM mechanism of GF2 remains unclear. The aim of this study was to explore the anti-cancer molecular mechanism of GF2 toward human GBM. Methods GF2-driven cellular toxicity was confirmed in two different GBM cells, U373 and Hs683. To test mitochondrial impairment driven by GF2, we examined the mitochondrial membrane potential, OCR, and ATP production. An intracellular redox imbalance was identified by measuring the relative ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), glutaredoxin (GLRX) mRNA expression, intracellular NAD+ level, and AMPK phosphorylation status. Results GF2 increased the percentage of cleaved caspase 3-positive cells and γH2AX signal intensities, confirming that GF2 shows the cytotoxicity against GBM. GO enrichment analysis suggested that the mitochondrial function could be negatively influenced by GF2. GF2 reduced the mitochondrial membrane potential, basal mitochondrial respiratory rate, and ATP production capacity. Our results showed that GF2 downregulated the relative GSH/GSSG, intracellular NAD+ level, and GLRX expression, suggesting that GF2 may alter the intracellular redox balance that led to mitochondrial impairment. Conclusion GF2 reduces mitochondrial membrane potential, inhibits cellular oxygen consumption, activates AMPK signaling, and induces cell death. Our study examined the potential vulnerability of mitochondrial activity in GBM, and this may hold therapeutic promise.

Epilepsy is a common chronic neurological disease characterized by recurrent epileptic seizures. A seizure is an uncontrolled electrical activity in the brain that can cause different levels of behavior, emotion, and consciousness. One-third of patients fail to receive sufficient seizure control, even though more than fifty FDA-approved anti-seizure drugs (ASDs) are available. In this study, we attempted small molecule screening to identify potential therapeutic agents for the treatment of seizures using seizure-induced animal models. Through behavioral phenotype-based screening, 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one (GM-90432) was identified as a prototype. GM-90432 treatment effectively decreased seizure-like behaviors in zebrafish and mice with chemically induced seizures. These results were consistent with decreased neuronal activity through immunohistochemistry for pERK in zebrafish larvae. Additionally, electroencephalogram (EEG) analysis revealed that GM-90432 decreases seizure-specific EEG events in adult zebrafish. Moreover, we revealed the preferential binding of GM-90432 to voltage-gated Na+ channels using a whole-cell patch clamp technique. Through pharmacokinetic analysis, GM-90432 effectively penetrated the blood-brain barrier and was distributed into the brain. Taken together, we suggest that GM-90432 has the potential to be developed into a new ASD candidate.

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized β-transducin repeat-containing protein 1 (βTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-X(L) or BCL2L1). This binding disrupted an interaction between Bcl-X(L) and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-X(L). Interestingly, the structure of the Bcl-X(L)–p73-TAD16 peptide complex revealed a novel mechanism of Bcl-X(L) recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-X(L), comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-X(L) recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.

Migraine is characterized by recurrent and disabling headaches; therefore, several drugs have been widely prescribed to prevent acute migraine attacks. Amitriptyline, a tricyclic antidepressant, is among the most commonly administered. It is poorly known, however, whether amitriptyline modulates the excitability of dural afferent neurons that transmit pain signals from the dura mater. In this study, the effects of amitriptyline on tetrodotoxin-resistant (TTX-R) Na+ channels were examined in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from medium-sized DiI-positive neurons using a whole-cell patch clamp technique. Amitriptyline (3 μM) slightly reduced the peak component of transient INa and induced a marked decrease in the steady-state component of transient TTX-R INa, as well as in the slow ramp-induced TTX-R INa. Our findings suggest that amitriptyline specifically inhibits persistent Na+ currents mediated by TTX-R Na+ channels. While amitriptyline had minor effects on voltage-activation/inactivation, it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Amitriptyline also affected the inactivation kinetics of TTX-R Na+ channels by significantly accelerating the inactivation of TTX-R Na+ channels and slowing the subsequent recovery. Amitriptyline decreased the number of action potentials by increasing the threshold for their generation. In conclusion, the amitriptyline-mediated diverse modulation of TTX-R Na+ channels would be, at least in part, responsible for its prophylactic efficacy for migraine attacks.

Introduction To investigate the correlation between serum anti-ABO immunoglobulin G (IgG) and IgG subclasses, anti-ABO IgG subclasses were measured by flow cytometry (FCM) in ABO-incompatible (ABOi) kidney transplant recipients. We also evaluated baseline anti-ABO C1q antibody. Method Baseline anti-ABO IgG titers were measured by both FCM and column agglutination technique methods in 18 ABOi kidney transplant recipients. The mean florescence intensity (MFI) ratios of baseline anti-ABO IgG subclasses and anti-ABO C1q antibody were obtained by FCM and followed-up after rituximab treatment, each plasmapheresis (PP) session, and kidney transplantation. Correlation between the values of IgG subclass and total IgG titer was analyzed. Results The baseline MFI ratios of total IgG, IgG1, IgG2, IgG3, and IgG4 were 202.46, 62.41, 30.01, 1.04, and 1.13, respectively. The MFI ratios of IgG1, IgG2, and total IgG measured at baseline and pre-PP were positively correlated with the baseline ABO titer was measured using the column agglutination technique. The numbers of PP sessions to reach the target titer were correlated with the baseline IgG and IgG1 levels. IgG1 and IgG2 as well as total IgG were removed effectively after serial PP. Anti-ABO C1q antibody was neither detected nor correlated with total IgG and any IgG subclasses. Conclusions Our findings suggest that IgG1 and IgG2 are the dominant IgG subclass in ABOi kidney transplant recipients. Baseline levels of IgG1 and IgG2 were correlated with baseline total IgG titer. However, anti-ABO C1q antibody was not detected in the present study.

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. For example, miRNAs repress gene expression by recruiting the miRNA-induced silencing complex (miRISC), a ribonucleoprotein complex that contains miRNA-engaged Argonaute (Ago) and the scaffold protein GW182. Recently, ubiquitin–protein ligase E3 component N-recognin 5 (UBR5) has been identified as a component of miRISC. UBR5 directly interacts with GW182 proteins and participates in miRNA silencing by recruiting downstream effectors, such as the translation regulator DEAD-box helicase 6 (DDX6) and transducer of ERBB2,1/2,2 (Tob1/2), to the Ago–GW182 complex. However, the regulation of miRISC-associated UBR5 remains largely elusive. In the present study, we showed that UBR5 down-regulates the levels of TNF receptor-associated factor 3 (TRAF3), a key component of Toll-like receptor signaling, via the miRNA pathway. We further demonstrated that p90 ribosomal S6 kinase (p90RSK) is an upstream regulator of UBR5. p90RSK phosphorylates UBR5 at Thr637, Ser1227, and Ser2483, and this phosphorylation is required for the translational repression of TRAF3 mRNA. Phosphorylated UBR5 co-localized with GW182 and Ago2 in cytoplasmic speckles, which implies that miRISC is affected by phospho-UBR5. Collectively, these results indicated that the p90RSK–UBR5 pathway stimulates miRNA-mediated translational repression of TRAF3. Our work has added another layer to the regulation of miRISC.

Sevoflurane, which is preferentially used as a day-case anesthetic based on its low blood solubility, acts on the central nervous system and exerts analgesic effects. However, it still remains unknown whether sevoflurane affects the excitability of nociceptive sensory neurons. Therefore, we conducted this study to examine the effects of sevoflurane on the excitability of small-sized dorsal root ganglion (DRG) neurons of rats using the whole-cell patch-clamp technique. In a voltage-clamp condition, sevoflurane elicited the membrane current in a concentration-dependent manner, in which the reversal potential was similar to the equilibrium potential of K+. In a current-clamp condition, sevoflurane directly depolarized the membrane potentials in a concentration-dependent manner. Moreover, at a clinically relevant concentration, sevoflurane decreased the threshold for action potential generation. These findings suggest that sevoflurane acts on the leak K+ channels to increase the excitability of DRG neurons. Sevoflurane increased the half-width of single action potentials, which resulted from the inhibition of voltage-gated K+ currents, including the fast inactivating A-type and non-inactivating delayed rectifier K+ currents. Our study indicates that sevoflurane could exhibit pronociceptive effects on nociceptive sensory neurons by inhibiting K+ conductances.

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. Proton sensing by ASICs has been known to mediate pain, mechanosensation, taste transduction, learning and memory, and fear. In this study, we investigated the differential subcellular localization of ASIC2a and ASIC3 in heterologous expression systems. While ASIC2a targeted the cell surface itself, ASIC3 was mostly accumulated in the ER with partial expression in the plasma membrane. However, when ASIC3 was co-expressed with ASIC2a, its surface expression was markedly increased. By using bimolecular fluorescence complementation (BiFC) assay, we confirmed the heteromeric association between ASIC2a and ASIC3 subunits. In addition, we observed that the ASIC2a-dependent surface trafficking of ASIC3 remarkably enhanced the sustained component of the currents. Our study demonstrates that ASIC2a can increase the membrane conductance sensitivity to protons by facilitating the surface expression of ASIC3 through herteromeric assembly. [BMB Reports 2016; 49(10): 542-547].

New, more effective pharmacologic treatments for epilepsy are needed, as a substantial portion of patients (>30%) are refractory to currently available anti-epileptic drugs. Cenobamate (YKP3089) is an investigational anti-epileptic drug in clinical development. Two completed adequate and well-controlled studies demonstrated a significant reduction in focal seizures with cenobamate in patients with epilepsy. In this study, we characterized the effects of cenobamate on voltage-gated Na+ channels in acutely isolated rat hippocampal CA3 neurons using a whole-cell patch-clamp technique. While cenobamate had little effect on the peak component of transient Na+ current (INaT) induced by brief depolarizing step pulses, it potently inhibited the non-inactivating persistent component of INa (INaP). In addition, cenobamate potently inhibited the current by slow voltage-ramp stimuli. Cenobamate significantly shifted the steady-state fast inactivation relationship toward a hyperpolarizing range, indicating that cenobamate binds to voltage-gated Na+ channels at the inactivated state with a higher affinity. Cenobamate also accelerated the development of inactivation and retarded recovery from inactivation of voltage-gated Na+ channels. In current clamp experiments, cenobamate hyperpolarized membrane potentials in a concentration-dependent manner, and these effects were mediated by inhibiting the INaP. Cenobamate also increased the threshold for generation of action potentials, and decreased the number of action potentials elicited by depolarizing current injection. Given that the INaP plays a pivotal role in the repetitive and/or burst generation of action potentials, the cenobamate-mediated preferential blockade of INaP might contribute to anti-epileptic activity.

Acid-sensing ion channels (ASICs) are widely expressed in both the peripheral and central nervous system, and contribute to the modulation of central nociceptive transmission under both physiological and pathophysiological conditions. In this study, we characterized the proton-induced membrane currents in acutely isolated rat substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis using the whole cell patch-clamp technique. Exposure to acidic conditions (pH

Purine nucleotides, such as ATP and ADP, activate ionotropic P2X and metabotropic P2Y receptors to regulate neurotransmitter release in the peripheral as well as central nervous system. Here we report another type of ATP-induced presynaptic modulation of glutamate release in rat medullary dorsal horn neurons. Glutamatergic excitatory postsynaptic currents (EPSCs) induced by electrical stimulation of trigeminal tract were recorded from horizontal brain stem slices using a whole-cell patch clamp technique. ATP decreased the amplitude of glutamatergic EPSCs in a reversible and concentration dependent manner and increased the paired-pulse ratio. In addition, ATP reduced the frequency of miniature EPSCs without affecting the current amplitude, suggesting that ATP acts presynaptically to reduce the probability of glutamate release. The ATP-induced decrease in glutamatergic EPSCs was not affected by P2X and P2Y receptor antagonists, but was completely blocked by DPCPX, a selective adenosine A1 receptor antagonist. The ATP-induced decrease in glutamatergic EPSCs was also inhibited by an inhibitor of tissue nonspecific alkaline phosphatase but not by inhibitors of other enzymes such as ecto-nucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidases. The results suggest that exogenously applied purine nucleotides are rapidly converted to adenosine by specific enzymes, and subsequently act on presynaptic A1 receptors to inhibit glutamate release from primary afferent terminals. This type of modulation mediated by purine nucleotides may play an important role in regulating nociceptive transmission from orofacial tissues.

Background The aim of this study was to compare anti-ABO antibody levels as measured by means of flow cytometry (FCM) with the levels measured with the use of the column agglutination test (CAT), and to evaluate the clinical outcome as it relates to the baseline mean fluorescence intensity (MFI) ratio obtained by FCM. Methods We reviewed 21 cases of ABO-incompatible kidney transplantation (ABO-i KT). In these patients, baseline IgG titers were measured with the use of both FCM and CAT methods. We investigated the correlation between levels measured by FCM and those by CAT with the use of correlation coefficients. Patients were classified into a high MFI ratio group (≥200; n = 7) or low MFI ratio group ( Results The MFI ratio for the FCM-based method was highly correlated with the titer measured by CAT (r = 0.890; P = .01). The relationship between MFI ratio and CAT titer can be expressed as follows: log (MFI ratio) = 0.879 × log (CAT titer) + 0.298. The number of pre-transplantation rounds of plasmapheresis significantly increased as the baseline MFI ratio increased. The allograft function was immediately recovered and stable. A single case of acute cellular rejection was observed in the low MFI ratio group. Conclusions Anti-ABO antibody levels measured by means of the FCM-based method were highly correlated with the levels measured with the use of CAT in cases of ABO-i KT. The decreased level of anti-ABO antibody measured by means of FCM after plasmapheresis suggests its potential as an effective and objective method for assessment of anti-ABO antibody levels.

Background The objective of this study was to investigate the clinical impact of BK virus surveillance on graft injury in kidney transplantation. Methods BK viremia in kidney transplant recipients was evaluated by use of plasma quantitative polymerase chain reaction. The prevalence of BK viremia and BK virus–associated nephropathy (BKVAN) and the clinical impact of BK viremia on graft outcomes were assessed. Results This study took place between January 2008 and June 2013. A total of 213 kidney transplant recipients were included. The prevalence of BK viremia and high BK viremia (≥1 × 10 4 copies/mL) was 66.7% (142/213) and 17.4% (37/213), respectively. A diagnosis of BKVAN was confirmed by means of allograft biopsy in 9 patients (4.2%). The estimated glomerular filtration rate after transplantation was similar in both the low BK viremia ( 4 copies/mL) and non–BK viremia groups but was significantly lower in the high BK viremia group after 18 months. In receiver operating characteristic curve analysis, the area under the curve value of plasma polymerase chain reaction was 0.980. We found that a viral load >92,850 copies/mL was able to predict BKVAN with 89% sensitivity and 94.6% specificity. The risk factors for viral loads ≥1 × 10 4 copies/mL were cytomegalovirus infection, steroid pulse therapy, and acute rejection. Conclusions High BK viremia was associated with poor graft function after kidney transplantation. The serial monitoring of BK viremia in kidney transplant recipients was helpful in predicting BKVAN and might prevent further progression.

The roles of persistent Na(+) currents (INaP) in intrinsic membrane properties were examined in rat substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. In a voltage-clamp mode, riluzole inhibited the slow voltage ramp-induced INaP but had little effect on the peak amplitude of transient Na(+) currents in SG neurons. In a current-clamp mode, most SG neurons exhibited spontaneous action potentials and tonic firing pattern. Riluzole reduced both spontaneous and elicited action potentials in a concentration-dependent manner. The present results suggest that the riluzole-sensitive INaP plays an important role in the excitability of SG neurons and are thus, likely to contribute to the modulation of nociceptive transmission from the orofacial tissues.

Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.

Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during apoptosis. [BMB Reports 2013; 46(12): 588-593]

Screening for latent tuberculosis infection (LTBI) before kidney transplantation (KT) is an indispensable process, purposes of this study were to compare the QuantiFERON-TB Gold In-Tube test (QFT-GIT) with the tuberculin skin test (TST) for screening of LTBI in kidney transplant recipients (KTRs).We compared prospectively the results of QFT-GIT with TST in 97 KTRs screened for LTBI between July 2008 and July 2012. Isoniazid (INH) prophylaxis was applied to KTRs with a positive TST or positive QFT-GIT or clinical risk factors for LTBI. Post-transplant tuberculosis (TB) was diagnosed by clinical evidence.The mean patients follow-up was 24.6 ± 14.4 months. Positive results on QFT-GIT and TST was obtained among 19 (20.4%) and 12 (12.9%) subjects, respectively, an overall agreement of 79.3% (κ = 0.27, 95% confidence interval [CI] -0.03-0.50; P .014). The incidence of TB was 0.52 per 100 person-years (95% CI 0.02-3.68). None of the patients in the INH prophylaxis group developed TB, whereas 1 in the no prophylaxis group developed disease at 14 months after KT. Sensitivity of the 2 tests could not be compared because patients who showed positive results on QFT-GIT or TST did not develop TB. The difference of specificity between QFT-GIT (79.3%) and TST (86.9%) was not significant (P = .l67). Abnormal chest radiographs (odds ratio [OR] 27.94, 95% CI 1.22-636.61, P = .037) and positive TST (OR 7.65, 95% CI 1.75-33.30, P = .007) showed significant associations with positive QFT-GIT results. Only positive QFT-GIT (OR 6.03, 95% CI 1.51-24.01, P = .011) showed an association with positive TST results.QFT-GIT and TST for diagnosis of LTBI in KTRs showed reasonable concordance but no superiority of either test.

Cell bodies of trigeminal mesencephalic nucleus (Vmes) neurons are located within the central nervous system, and therefore, peripheral as well as central acidosis can modulate the excitability of Vmes neurons. Here, we report the effect of acidic pH on voltage-gated Na channels in acutely isolated rat Vmes neurons using a conventional whole-cell patch clamp technique. Acidic pH (pH 6.0) slightly but significantly shifted both the activation and steady-state fast inactivation relationships toward depolarized potentials. However, acidic pH (pH 6.0) had a minor effect on the inactivation kinetics of voltage-gated Na channels. Less sensitivity of voltage-gated Na channels to acidic pH may allow Vmes neurons to transduce the precise proprioceptive information even under acidic pH conditions.

BACKGROUND AND PURPOSE Although 5-HT1B receptors are expressed in trigeminal sensory neurons, it is still not known whether these receptors can modulate nociceptive transmission from primary afferents onto medullary dorsal horn neurons. EXPERIMENTAL APPROACH Primary afferent-evoked EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices using a conventional whole-cell patch clamp technique under a voltage-clamp condition. KEY RESULTS CP93129, a selective 5-HT1B receptor agonist, reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, CP93129 reduced the frequency of spontaneous miniature EPSCs without affecting the current amplitude. The CP93129-induced inhibition of EPSCs was significantly occluded by GR55562, a 5-HT1B/1D receptor antagonist, but not LY310762, a 5-HT1D receptor antagonist. Sumatriptan, an anti-migraine drug, also decreased EPSC amplitude, and this effect was partially blocked by either GR55562 or LY310762. On the other hand, primary afferent-evoked EPSCs were mediated by the Ca2+ influx passing through both presynaptic N-type and P/Q-type Ca2+ channels. The CP93129-induced inhibition of EPSCs was significantly occluded by ω-conotoxin GVIA, an N-type Ca2+ channel blocker. CONCLUSIONS AND IMPLICATIONS The present results suggest that the activation of presynaptic 5-HT1B receptors reduces glutamate release from primary afferent terminals onto medullary dorsal horn neurons, and that 5-HT1B receptors could be, at the very least, a potential target for the treatment of pain from orofacial tissues. LINKED ARTICLE This article is commented on by Connor, pp. 353–355 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01963.x

The effect of icilin, a potent agonist of transient receptor potential ankyrin 1 (TRPA1) and TRPM8, on glycinergic transmission was examined in mechanically isolated rat medullary dorsal horn neurons by use of the conventional whole-cell patch-clamp technique. Icilin increased the frequency of glycinergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in a dose-dependent manner. Either allyl isothiocyanate(AITC) or cinnamaldehyde, other TRPA1 agonists, also increased mIPSC frequency, but the extent of facilitation induced by AITC or cinnamaldehyde was less than that induced by icilin. However, menthol, a TRPM8 agonist, had no facilitatory effect on glycinergic mIPSCs. The icilin-induced increase in mIPSC frequency was significantly inhibited by either HC030031, a selective TRPA1 antagonist, or ruthenium red, a non-selective transient receptor potential channel blocker. Icilin failed to increase glycinergic mIPSC frequency in the absence of extracellular Ca(2+), suggesting that the icilin-induced increase in mIPSC frequency is mediated by the Ca(2+) influx from the extracellular space. In contrast, icilin still increased mIPSC frequency either in the Na(+) -free external solution or in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are permeable to Ca(2+), to enhance glycinergic transmission onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the peripheral tissues.

Amitriptyline, a representative tricyclic antidepressant, has been widely used for the treatment of neuropathic pain, such as post-herpetic and trigeminal neuralgia. In the present study, we investigated the effect of amitriptyline on glycinergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in acutely isolated medullary dorsal horn neurons by use of a conventional whole-cell patch-clamp technique. Amitriptyline (30 μM) significantly increased mIPSC frequency without affecting the current amplitude, suggesting that amitriptyline acts presynaptically to increase the probability of glycine release. Amitriptyline also directly inhibited the glycine receptor-mediated Cl(-) currents induced by lower concentrations of glycine. The amitriptyline-induced increase in mIPSC frequency was not affected either in the Na(+)-free external solutions or in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker, indicating that amitriptyline is unlikely to elicit a presynaptic depolarization. In addition, amitriptyline still increased mIPSC frequency even in the absence of extracellular Ca(2+). In contrast, the depletion of intracellular Ca(2+) stores with thapsigargin significantly reduced the extent of amitriptyline-induced increase in mIPSC frequency. These data suggest that amitriptyline increases spontaneous glycine release onto acutely isolated medullary dorsal neurons by increasing the intraterminal Ca(2+) concentration, which might be mediated by the Ca(2+) release from the Ca(2+) stores rather than the Ca(2+) influx from the extracellular space. The amitriptyline-induced modulation of glycinergic transmission could have a broad impact on the excitability of medullary dorsal neurons, and this mechanism would contribute, at least in part, to the anti-allodynic action of amitriptyline.

Background Tacrolimus is a substrate of cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp), encoded by the CYP3A and ATP-binding cassette subfamily B member 1 (ABCB1) genes, respectively. This study was aimed to investigate the impact of CYP3A and ABCB1 polymorphisms on the tacrolimus pharmacokinetics and clinical outcomes in Korean renal transplant recipients. Methods We analyzed data from a cohort of 70 renal transplant recipients receiving tacrolimus. CYP3A4*4, CYP3A4*5, CYP3A4*18, CYP3A5*3, ABCB1 C1236>T, ABCB1 G2677>T/A, and ABCB1 C3435>T polymorphisms were genotyped and correlated to dose-adjusted tacrolimus trough concentration at months 1, 3, 6, and 12 after transplantation. Results Patients with the CYP3A5*3 alleles showed higher dose-adjusted tacrolimus concentrations for 12 months and higher trough levels until 6 months after transplantation. ABCB1 polymorphisms and haplotypes were not associated with tacrolimus concentrations. In a multivariate analysis, the presence of ≥1 CYP3A5*3 allele was a significant independent variable affecting dose-adjusted tacrolimus concentrations. Glomerular filtration rate, acute rejection, opportunistic infection, and graft survival were not affected by CYP3A5 polymorphisms. Calcineurin inhibitor toxicity, which showed higher tendency in patients with CYP3A5*1 alleles, might be associated with higher tacrolimus dose per kilogram. Conclusions The CYP3A5 genotype is a major factor in determining the dose requirement of tacrolimus, and genotyping may be of value in individualization of immunosuppressive therapy of renal transplant patients.

BACKGROUND AND PURPOSE Although 3α-hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone (AlloP) and tetrahydrodeoxycorticosterone, are endogenous positive modulators of postsynaptic GABAA receptors, the functional roles of endogenous neurosteroids in synaptic transmission are still largely unknown. EXPERIMENTAL APPROACH In this study, the effect of AlloP on spontaneous glutamate release was examined in mechanically isolated dentate gyrus hilar neurons by use of the conventional whole-cell patch-clamp technique. KEY RESULTS AlloP increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in a dose-dependent manner. The AlloP-induced increase in sEPSC frequency was completely blocked by a non-competitive GABAA receptor blocker, tetrodotoxin or Cd2+, suggesting that AlloP acts on presynaptic GABAA receptors to depolarize presynaptic nerve terminals to increase the probability of spontaneous glutamate release. On the other hand, γ-cyclodextrin (γ-CD) significantly decreased the basal frequency of sEPSCs. However, γ-CD failed to decrease the basal frequency of sEPSCs in the presence of a non-competitive GABAA receptor antagonist or tetrodotoxin. In addition, γ-CD failed to decrease the basal frequency of sEPSCs after blocking the synthesis of endogenous 5α-reduced pregnane steroids. Furthermore, γ-CD decreased the extent of muscimol-induced increase in sEPSC frequency, suggesting that endogenous neurosteroids can directly activate and/or potentiate presynaptic GABAA receptors to affect spontaneous glutamate release onto hilar neurons. CONCLUSIONS AND IMPLICATIONS The modulation of presynaptic GABAA receptors by endogenous neurosteroids might affect the excitability of the dentate gyrus-hilus-CA3 network, and thus contribute, at least in part, to some pathological conditions, such as catamenial epilepsy and premenstrual dysphoric disorder.

We have investigated the adenosine-mediated presynaptic inhibition of primary afferent-evoked glutamate release in rat substantia gelatinosa neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. Adenosine reversibly and concentration dependently decreased the amplitude of glutamatergic excitatory postsynaptic currents and increased the paired-pulse ratio, indicating that adenosine acts presynaptically to reduce glutamate release from primary afferents. The adenosine-induced inhibition of excitatory postsynaptic currents was occluded by a selective A₁ receptor antagonist, DPCPX, and was mimicked by a selective A₁ receptor agonist CPA. The results suggest that presynaptic A₁ receptors decrease action potential-dependent glutamate release from trigeminal primary afferents onto medullary dorsal horn neurons, and thus adenosine A₁ receptors could be a potential target for the treatment of pain of orofacial tissues.

We have recently reported that tyramine acts on putative presynaptic trace amine receptors to inhibit glycinergic transmission in substantia gelatinosa (SG) neurons of the rat trigeminal subnucleus caudalis. However, it is still unknown how tyramine elicits presynaptic inhibition of glycine release. In the present study, therefore, we investigated cellular mechanisms underlying the tyramine-induced inhibition of glycinergic transmission in SG neurons using a conventional whole-cell patch clamp technique. Tyramine (100 μM) reversibly and repetitively decreased the amplitude of action potential-dependent glycinergic inhibitory postsynaptic currents (IPSCs), and increased the paired-pulse ratio. Pharmacological data suggest that the tyramine-induced decrease in glycinergic IPSCs was not mediated by the modulation of adenylyl cyclase, protein kinase A and C, or G-protein coupled inwardly rectifying K + channels. On the other hand, glycinergic IPSCs were mainly mediated by the Ca 2+ influx passing through presynaptic N-type and P/Q-type Ca 2+ channels. The tyramine-induced decrease in glycinergic IPSCs was completely blocked by ω-conotoxin GVIA, an N-type Ca 2+ channel blocker, but not ω-agatoxin IVA, a P/Q-type Ca 2+ channel blocker. The results suggest that tyramine acts presynaptically to decrease action potential-dependent glycine release onto SG neurons via the selective inhibition of presynaptic N-type Ca 2+ channels. This tyramine-induced inhibition of glycinergic transmission in SG neurons might affect the process of orofacial nociceptive signals.

Dopamine (DA)-induced pre-synaptic inhibition of glycinergic transmission was studied from substantia gelatinosa (SG) neurons of the trigeminal nucleus pars caudalis using a conventional whole-cell patch clamp technique. The action potential-dependent glycinergic inhibitory post-synaptic currents (IPSCs) were recorded from SG neurons in the presence of 3 mM kynurenic acid and 10 μM 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid HBr (SR95531). In these conditions, bath applied DA (100 μM) reduced the amplitude of glycinergic IPSCs and increased the paired-pulse ratio, suggesting that DA acts pre-synaptically to reduce the probability of glycine release. However, the inhibitory action of DA on glycinergic IPSCs was not blocked by SCH23390 (10 μM) and spiperone (1 μM), selective D(1) - and D(2) -like receptor antagonists, respectively. In addition, either SKF38393 (100 μM), a selective D(1) -like receptor agonist, or quinpirole (100 μM), a selective D(2) -like receptor agonist, had no pre-synaptic effect on glycinergic IPSCs. The results suggest that both D(1) - and D(2) -like receptors are not involved in the DA-induced decrease in glycinergic IPSCs. On the other hand, tyramine (100 μM), one of representative trace amines, reduced the amplitude of glycinergic IPSCs and increased the paired-pulse ratio, suggesting that tyramine acts pre-synaptically to reduce the probability of glycine release. Considering that DA can activate trace amine (TA) receptors and that TA receptors are exclusively expressed on the trigeminal nucleus pars caudalis, DA might act on putative pre-synaptic TA receptors, rather than classical DA receptors, to inhibit glycinergic transmission onto SG neurons of the trigeminal nucleus pars caudalis.

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na+ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na+ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na+ currents, it strongly inhibited steady-state currents of transient as well as persistent Na+ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na+ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na+ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na+ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.

cAMP is known to regulate neurotransmitter release via protein kinase A (PKA)-dependent and/or PKA-independent signal transduction pathways at a variety of central synapses. Here we report the cAMP-mediated long-lasting enhancement of glycinergic transmission in developing rat spinal substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, elicited a long-lasting increase in the amplitude of nerve-evoked glycinergic inhibitory postsynaptic currents (IPSCs), accompanied by a long-lasting decrease in the paired-pulse ratio in immature substantia gelatinosa neurons, and this forskolin-induced increase in glycinergic IPSCs decreased with postnatal development. Forskolin also decreased the failure rate of glycinergic IPSCs evoked by minimal stimulation, and increased the frequency of glycinergic miniature IPSCs. All of these data suggest that forskolin induces the long-lasting enhancement of glycinergic transmission by increasing in the presynaptic release probability. This pre-synaptic action of forskolin was mediated by hyperpolarization and cyclic nucleotide-activated cation channels and an increase in intraterminal Ca(2+) concentration but independent of PKA. The present results suggest that cAMP-dependent signal transduction pathways represent a dynamic mechanism by which glycinergic IPSCs could potentially be modulated during postnatal development.

Although it has been well established that GABA(A) receptors are molecular targets of a variety of allosteric modulators, such as benzodiazepines, the pharmacological properties of presynaptic GABA(A) receptors are poorly understood. In this study, the effects of diazepam and Zn(2+) on presynaptic GABA(A) receptors have been investigated by measuring the GABA(A) receptor-mediated facilitation of spontaneous glutamate release in mechanically dissociated rat CA3 pyramidal neurons. Diazepam significantly enhanced the muscimol-induced facilitation (particularly at submicromolar concentrations) of spontaneous glutamate release and shifted the concentration-response relationship for muscimol toward the left, whereas Zn(2+) (OR= 100 muM) had little effect on the muscimol-induced facilitation of spontaneous glutamate release. In contrast, Zn(2+) significantly suppressed the muscimol-induced currents mediated by GABA(A) receptors expressed on dentate gyrus granule cells, which are parent neurons of mossy fibers, whereas the effect of diazepam on GABA(A) receptors expressed on dentate gyrus granule cells was lesser than that on presynaptic GABA(A) receptors. The results suggest that the pharmacological properties of GABA(A) receptors differ considerably between presynaptic (axon terminals) and somatic regions in the same granule cell and that presynaptic GABA(A) receptors should be considered as one of the important pharmacological targets of many drugs affecting GABA(A) receptors.

Purpose Transforming growth factor-β1 (TGF-β1) has been associated with the promotion of renal allograft interstitial fibrosis and thereby chronic allograft nephropathy (CAN). The literature on TGF-β1 polymorphisms and their importance in graft survival and CAN is not conclusive. Methods TGF-β1 gene polymorphisms (C-509T and T869C) were examined in a group of 207 Korean renal transplant recipients using real-time polymerase chain reaction assays. The CAN group ( n = 18) was defined by a typical biopsy confirming CAN or chronic calcineurin inhibitor nephrotoxicity. The rest of the patients were classified into the No CAN group ( n = 189). Results No significant differences were observed in the genotype distributions of both C-509T and T869 polymorphisms between the two groups. Allele frequencies and age-, sex-, HLA mismatch–adjusted odds ratio of each genotype as assessed by logistic regression analysis were also not significantly different between the two groups. Linkage disequilibrium coefficients between polymorphisms indicated that investigated polymorphisms of TGF-β1 (D′ = 0.98) were in tight linkage. However, there were no significant differences in the frequencies of the reconstructed haplotypes between the two groups. Kaplan-Meier method and log-rank tests did not indicate any statistically significant effects of TGF-β1 gene polymorphisms on graft survival. Conclusion TGF-β1 gene polymorphisms (C-509T, T869C) are not significantly associated with an increased risk of development of CAN and graft survival in Korean renal transplant recipients.

Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippocampus, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch clamp techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl − currents in a concentration-dependent manner. The glycine-induced current ( I Gly ) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01–1 µM), ICS-205,930 potentiated I Gly , but at higher concentrations (> 10 µM) it inhibited I Gly . These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing α2 subunits. Furthermore, at lower concentrations (1–30 µM), Zn 2+ potentiated I Gly , but at higher concentrations (> 100 µM) it inhibited I Gly . Considering that Zn 2+ is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn 2+ modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.

Although anticonvulsant drugs that block voltage-dependent Na+ channels have been widely used for neuropathic pain, including peripheral nerve injury-induced pain, much less is known about the actions of these drugs on immature trigeminal ganglion (TG) neurons. Here we report the effects of carbamazepine (CBZ) and amitriptyline (ATL) on tetrodotoxin-resistant (TTX-R) Na' channels expressed on immature rat TG neurons. TTX-R Na+ currents (I(Na)) were recorded in the presence of 300 nM TTX by use of a conventional whole-cell patch clamp method. Both CBZ and ATL inhibited TTX-R I(Na) in a concentration-dependent manner, but ATL was more potent. While CBZ and ATL did not affect the overall voltage-activation relationship of TTX-R Na+ channels, both drugs shifted the voltage-activation relationship to the left, indicating that they inhibited TTX-R Na+ channels more efficiently at depolarized membrane potentials. ATL showed a profound use-dependent blockade of TTX-R I(Na), but CBZ had little effect. The present results suggest that both CBZ and ATL, common drugs used for treating neuropathic pain, efficiently inhibit TTX-R Na+ channels expressed on immature TG neurons, and that these drugs might be useful for the treatment of trigeminal nerve injury-induced neuropathic pain, as well as the inhibition of ongoing central sensitization, even during immature periods.