General anesthetic actions on GABAA receptors in vivo are reduced in phospholipase C-related catalytically inactive protein knockout mice.

PURPOSE: The aim of this study was to investigate the action of general anesthetics in phospholipase C-related catalytically inactive protein (PRIP)-knockout (KO) mice that alter GABAA receptor signaling. METHODS: PRIP regulates the intracellular trafficking of ß subunit-containing GABAA receptors in vitro. In this study, we examined the effects of intravenous anesthetics, propofol and etomidate that act via ß subunit-containing GABAA receptors, in wild-type and Prip-KO mice. Mice were intraperitoneally injected with a drug, and a loss of righting reflex (LORR) assay and an electroencephalogram analysis were performed. RESULTS: The cell surface expression of GABAA receptor ß3 subunit detected by immunoblotting was decreased in Prip-knockout brain compared with that in wild-type brain without changing the expression of other GABAA receptor subunits. Propofol-treated Prip-KO mice exhibited significantly shorter duration of LORR and had lower total anesthetic score than wild-type mice in the LORR assay. The average duration of sleep time in an electroencephalogram analysis was shorter in propofol-treated Prip-KO mice than in wild-type mice. The hypnotic action of etomidate was also reduced in Prip-KO mice. However, ketamine, an NMDA receptor antagonist, had similar effects in the two genotypes. CONCLUSION: PRIP regulates the cell surface expression of the GABAA receptor ß3 subunit and modulates general anesthetic action in vivo. Elucidation of the involved regulatory mechanisms of GABAA receptor-dependent signaling would inform the development of safer anesthetic therapies for clinical applications.

Antiallodynic action of 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), a betaine/GABA transporter inhibitor.

The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 µM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 µM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.

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.

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

BACKGROUND: An 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. FINDINGS: To 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 PRIPs 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. CONCLUSIONS: Suppressed 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.

Inhibitory action of antidepressants on mouse Betaine/GABA transporter (BGT1) heterologously expressed in cell cultures.

Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [(3)H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1.

[A case of metastatic breast cancer resistant to anastrozole treatment responding to high-dose toremifene].

The patient was a 56-year-old female. At the age of 35 years, she had under gone left mastectomy and axillary lymph node dissection for breast cancer. After surgery, hormonal therapy was continued for 3 years. Then, no treatment was performed. In this study, single therapy with an AI agent was started to treatbilateral supraclavicular fossa/mediastinal lymphnode metastases. After 6 months, a partial response(PR)was achieved. However, progression of the disease(PD)was noted after 1 year. Thereafter,the regimen was switched to single high-dose(120mg/day)TOR therapy. CT revealed the disappearance of the bilateral supraclavicular fossa lymphnodes and a marked reduction of the other lymphnodes. Currently, the patient is being treated, with an interval of 10 months from the start of TOR therapy.

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.

[Breast cancer during pregnancy--a case report].

Pregnancy-associated breast carcinoma is generally defined as cancer that occurs during pregnancy or within 1 year of delivery, although treatment options are the most complicated when the disease is diagnosed during pregnancy. We report the case of a 30-year-old woman who was diagnosed with breast cancer at her 9th week of pregnancy. The patient initially had mastectomy with axillary lymph node dissection. She began adjuvant therapy with 3 courses of epirubicin/cyclophosphamide at 19 weeks of gestation. After delivery of a healthy child, she received one course of epirubicin/cyclophosphamide and 4 courses of docetaxel. Although the data are limited, pregnant patients with cancer can be treated with systemic chemotherapy with minimal risks to the fetus during the second or third trimester. Management of breast cancer during pregnancy requires an interdisciplinary care team and careful consideration of the patient's stage of disease, the gestational age of the fetus, and the preferences of the patient and her family.

Novel hyperthermia for metastatic bone tumors with magnetic materials by generating an alternating electromagnetic field.

We have developed a novel hyperthermic treatment modality using magnetic materials for metastatic bone tumors. The purpose of this study is to show the results of novel hyperthermia for metastatic bone tumors. This novel hyperthermic treatment modality was used for 15 patients with 16 metastatic bone lesions. In seven lesions, after curettage of the metastatic lesion followed by reinforcement with a metal intramedullary nail or plate, calcium phosphate cement (CPC) containing powdery Fe3O4 was implanted into the cavity. In one lesion, prosthetic reconstruction was then performed after an intralesional tumor excision. For the remaining eight lesions, metal intramedullary nails were inserted into the affected bone. Hyperthermic therapy was started at 1 week postoperatively. To comparatively evaluate the radiographic results of patients who underwent hyperthermia (HT group), we also assessed eight patients who received a palliative operation without either radiotherapy or hyperthermia (Op group), and 22 patients who received operation in combination with postoperative radiotherapy (Op + RT group). In HT group, all patients had an acceptable limb function with pain relief without any complications. On radiographs, 87, 38, and 91% were, respectively, considered to demonstrate an effective treatment outcome in HT group, Op group, and Op + RT group. The patients in HT group showed a statistically better radiographic outcome than the patients in Op group (P = 0.0042). But when compared between HT group and Op + RT group, there were no significant difference (P = 0.412). This first series of clinical hyperthermia using magnetic materials achieved good local control of metastatic bone lesion.

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.

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.

Reduction of diuretics and analysis of water and muscle volumes to prevent falls and fall-related fractures in older adults.

AIM: In an attempt to decrease the incidence of falls and fall-related fractures at a special geriatric nursing home, we endeavored to reduce diuretic doses, and examined the relationship between the effectiveness of this approach with the body compositions and activities of daily living of the study cohort. METHODS: We enrolled 93 participants living in the community, 60 residents of an intermediate geriatric nursing home and 50 residents of the 100-bed Kandayama Yasuragien special geriatric nursing home. We recorded body composition using a multifrequency bioelectrical impedance analyzer. Daily loop diuretic and other diuretic regimens of those in the special geriatric nursing home were reduced or replaced with "NY-mode" diuretic therapy, namely, spironolactone 12.5 mg orally once on alternate days. RESULTS: The incidence of falls fell from 53 in 2011 to 29 in 2012, and there were no fall-related proximal femoral fractures for 3 years after the introduction of NY-mode diuretic therapy. We also found statistically significant differences in muscle and intracellular water volumes in our elderly participants: those with higher care requirements or lower levels of independence had lower muscle or water volumes. CONCLUSIONS: We found that reducing or replacing daily diuretics with NY-mode therapy appeared to reduce the incidence of falls and fall-related proximal femoral fracture, likely by preserving intracellular and extracellular body water volumes. Low-dose spironolactone (12.5 mg on alternate days) appears to be an effective means of treating elderly individuals with chronic heart failure or other edematous states, while preventing falls and fall-related fractures. Geriatr Gerontol Int 2017; 17: 262-269.

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.

[A case of recurrent breast cancer with life-threatening multiple lung metastasis markedly responding to CMF as third-line chemotherapy].

A 35-year-old woman with taxane and anthracycline-resistant recurrent breast cancer with multiple lung metastasis and carcinomatous lymphangitis was treated with CMF as third-line chemotherapy. A complete response was achieved,and the toxicities were tolerable. We thought that CMF could be a useful regimen as third-line chemotherapy for metastatic breast cancer.

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.