The protective role of selenium against dental amalgam-induced intracellular oxidative toxicity through the TRPV1 channel in DBTRG glioblastoma cells.

OBJECTIVE: The exposure to mercury (Hg) from dental amalgams is a suspected causative factor in neurological diseases. This study investigated the toxic effects of two different amalgam compositions related to Hg and the protective effects of selenium against the toxic effects of Hg through the TRPV1 channel in the human DBTRG glioblastoma cell line. METHODOLOGY: Six groups of the cells were organized. Analyses of cell viability, apoptosis, caspase 3 and caspase 9 activities, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, and Western Blotting for protein expression levels were performed. RESULTS: Cell viability values were lower in amalgam with high copper (HCu) and low copper (LCu) groups independently of time but were increased by selenium and capsazepine (p<0.001 and p<0.05). Conversely, apoptosis rates, caspase 3 and caspase 9 expression, ROS formation, mitochondrial membrane depolarization, and protein expression levels were higher in the HCu and LCu groups but were decreased by selenium (p<0.001 and p<0.05). CONCLUSIONS: Selenium combined with an amalgam of either HCu or LCu decreases the toxic effects created by Hg in human DBTRG glioblastoma cells.

The protective role of selenium against dental amalgam-induced intracellular oxidative toxicity through the TRPV1 channel in DBTRG glioblastoma cells

Abstract Objective The exposure to mercury (Hg) from dental amalgams is a suspected causative factor in neurological diseases. This study investigated the toxic effects of two different amalgam compositions related to Hg and the protective effects of selenium against the toxic effects of Hg through the TRPV1 channel in the human DBTRG glioblastoma cell line. Methodology Six groups of the cells were organized. Analyses of cell viability, apoptosis, caspase 3 and caspase 9 activities, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, and Western Blotting for protein expression levels were performed. Results Cell viability values were lower in amalgam with high copper (HCu) and low copper (LCu) groups independently of time but were increased by selenium and capsazepine (p<0.001 and p<0.05). Conversely, apoptosis rates, caspase 3 and caspase 9 expression, ROS formation, mitochondrial membrane depolarization, and protein expression levels were higher in the HCu and LCu groups but were decreased by selenium (p<0.001 and p<0.05). Conclusions Selenium combined with an amalgam of either HCu or LCu decreases the toxic effects created by Hg in human DBTRG glioblastoma cells.

Resveratrol diminishes bisphenol A-induced oxidative stress through TRPM2 channel in the mouse kidney cortical collecting duct cells.

Bisphenol A (BisPH-A) is a latent danger that threatens our health, which we frequently exposure in our modern life (e.g. the widespread use of drinking water in plastic pet bottles). But the BisPH-A induced transient receptor potential melastatin 2 (TRPM2)-mediated oxidative stress and apoptosis in these cells has not been studied yet. Calcium (Ca2+) plays an important role in a versatile intracellular signal transduction that works over a wide range to regulate oxidative stress processes. TRPM2 is activated by oxidative stress and it has emerged as an important Ca2+ signaling mechanism in a variety of cells, contributing many cellular functions including cell death. Resveratrol (RESV), which belongs to the polyphenol group, acts as an antioxidant, eliminating cellular oxidative stress and increasing the body's resistance to diseases. The current study aimed to elucidate the effect of antioxidant resveratrol on TRPM2-mediated oxidative stress induced by BisPH-A exposure in the mouse kidney cortical collecting duct cells (mpkCCDcl4). The cells were divided into four groups as control, resveratrol (50 µM for 24 h), BisPH-A (100 µM for 24 h) and BisPH-A + RESV. Intracellular free Ca2+ concentrations and TRPM2 channel currents were high in BisPH-A treated cells, but decreased with resveratrol treatment. In addition, BisPH-A induced mitochondrial membrane depolarization, reactive oxygen species (ROS), caspase 3, caspase 9 and apoptosis values were decreased by the resveratrol treatment. In conclusion, resveratrol protected cells from BisPH-A induced oxidative damage. In this study, we showed that TRPM2 channel mediates this protective effect of resveratrol.

Albumin evokes Ca2+-induced cell oxidative stress and apoptosis through TRPM2 channel in renal collecting duct cells reduced by curcumin.

In proteinuric nephropathies of chronic kidney disease, the epithelial cells of the nephron including the collecting duct are exposed to high concentrations of luminal albumin. Albumin is taken up from collecting duct cells by endocytosis causing excessive reactive oxygen species (ROS) production and a proinflammatory response. Curcumin used in the traditional medicine possesses anti-inflammatory and antioxidant effects. ROS and ADP-ribose (ADPR) activate the cation channel TRPM2. We hypothesize, that albumin-induced cell stress and proinflammatory response are mediated by Ca2+ and can be reduced by curcumin. The cortical collecting duct (CCD) cells mpkCCDc14 exhibit spontaneous and inducible Ca2+ oscillations, which can be blocked by pre-treatment with curcumin. Curcumin accumulates in plasma membrane and intracellular vesicles, where it interferes with TRPM2 and decreases the influx of Ca2+. Albumin reduces cell viability and increases apoptosis, NF-κB activation, and mitochondrial membrane depolarization via Ca2+-dependent signaling, which results in increased ROS production. Albumin-induced cell stress is diminished by the inhibition of TRPM2 after administration of curcumin and ADPR (PARP1) inhibitors. Curcumin did not reduce the Ca2+ elevation induced by thapsigargin in Ca2+-free medium, but it reduced the function of store-operated Ca2+ channels and ATP-evoked Ca2+ response. In conclusion, albumin-induced oxidative stress is mediated by Ca2+-dependent signaling via TRPM2 and leads to cell damage and a proinflammatory response, strengthening the role of CCD cells in the progression of chronic kidney disease.

Inhibitions of anandamide transport and FAAH synthesis decrease apoptosis and oxidative stress through inhibition of TRPV1 channel in an in vitro seizure model.

The expression level of TRPV1 is high in hippocampus which is a main epileptic area in the brain. In addition to the actions of capsaicin (CAP) and reactive oxygen species (ROS), the TRPV1 channel is activated in neurons by endogenous cannabinoid, anandamide (AEA). In the current study, we investigated the role of inhibitors of TRPV1 (capsazepine, CPZ), AEA transport (AM404), and FAAH (URB597) on the modulation of Ca2+ entry, apoptosis, and oxidative stress in in vitro seizure-induced rat hippocampus and human glioblastoma (DBTRG) cell line. The seizure was induced in the hippocampal and DBTRG neurons using in vitro 4-aminopyridine (4-AP) to trigger a seizure-like activity model. CPZ and AM404 were fully effective in reversing 4-AP-induced intracellular free Ca2+ concentration of the hippocampus and TRPV1 current density of DBTRG. However, AEA and CAP did not activate TRPV1 in the URB597-treated neurons. Hence, we observed TRPV1 blocker effects of URB597 in the DBTRG neurons. In addition, the AM404 and CPZ treatments decreased intracellular ROS production, mitochondrial membrane depolarization, apoptosis, caspases 3 and 9 values in the hippocampus. In conclusion, the results indicate that inhibition of AEA transport, FAAH synthesis, and TRPV1 activity can result in remarkable neuroprotective effects in the epileptic neurons. Possible molecular pathways of involvement of capsazepine (CPZ) and AM4040 in anandamide and capsaicin (CAP)-induced apoptosis, oxidative stress, and Ca2+ accumulation through TRPV1 channel in the seizure-induced rat hippocampus and human glioblastoma neurons. The TRPV1 channel is activated by different stimuli including reactive oxygen species (ROS), anandamide (AEA), and CAP and it is blocked by capsazepine (CPZ). Cannabinoid receptor type 1 (CB1) is also activated by AEA. The AEA levels in cytosol are decreased by fatty acid amide hydrolase (FAAH) enzyme. Inhibition of FAAH through URB597 induces stimulation of CB1 receptor through accumulation AEA. URB597 acts antiepileptic effects through inhibition of TRPV1. Overloaded Ca2+ concentration of mitochondria can induce an apoptotic program by stimulating the release of apoptosis-promoting factors such as caspases 3 and caspase 9 by generating ROS due to respiratory chain damage. AM404 and CPZ reduce TRPV1 channel activation and Ca2+ entry in the in vitro 4-AP seizure model-induced hippocampal and glioblastoma neurons.

Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells.

Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50-500µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50-500µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells.

Involvement of TRPM2 and TRPV1 channels on hyperalgesia, apoptosis and oxidative stress in rat fibromyalgia model: Protective role of selenium.

Fibromyalgia (FM) results in pain characterized by low selenium (Se) levels, excessive Ca2+ influx, reactive oxygen species (ROS) production, and acidic pH. TRPM2 and TRPV1 are activated by ROS and acid; nevertheless, their roles have not been elucidated in FM. Therefore, we investigated the contribution of TRPM2 and TRPV1 to pain, oxidative stress, and apoptosis in a rat model of FM and the therapeutic potential of Se. Thirty-six rats were divided into four groups: control, Se, FM, and FM + Se. The Se treatment reduced the FM-induced increase in TRPM2 and TRPV1 currents, pain intensity, intracellular free Ca2+, ROS, and mitochondrial membrane depolarization in the sciatic (SciN) and dorsal root ganglion (DRGN) neurons. Furthermore, Se treatment attenuated the FM-induced decrease in cell viability in the DRGN and SciN, glutathione peroxidase, and reduced glutathione and α-tocopherol values in the DRGN, SciN, brain, muscle, and plasma; however, lipid peroxidation levels were decreased. Se also attenuated PARP1, caspase 3, and 9 expressions in the SciN, DRGN, and muscle. In conclusion, Se treatment decreased the FM-induced increase in hyperalgesia, ROS, apoptosis, and Ca2+ entry through TRPM2 and TRPV1 in the SciN and DRGN. Our findings may be relevant to the elucidation and treatment of FM.

Targeting breast cancer cells by MRS1477, a positive allosteric modulator of TRPV1 channels.

There is convincing epidemiological and experimental evidence that capsaicin, a potent natural transient receptor potential cation channel vanilloid member 1 (TRPV1) agonist, has anticancer activity. However, capsaicin cannot be given systemically in large doses, because of its induction of acute pain and neurological inflammation. MRS1477, a dihydropyridine derivative acts as a positive allosteric modulator of TRPV1, if added together with capsaicin, but is ineffective, if given alone. Addition of MRS1477 evoked Ca2+ signals in MCF7 breast cancer cells, but not in primary breast epithelial cells. This indicates that MCF7 cells not only express functional TRPV1 channels, but also produce endogenous TRPV1 agonists. We investigated the effects of MRS1477 and capsaicin on cell viability, caspase-3 and -9 activities and reactive oxygen species production in MCF7 cells. The fraction of apoptotic cells was increased after 3 days incubation with capsaicin (10 µM) paralleled by increased reactive oxygen species production and caspase activity. These effects were even more pronounced, when cells were incubated with MRS1477 (2 µM) either alone or together with CAPS (10 µM). Capsazepine, a TRPV1 blocker, inhibited both the effect of capsaicin and MRS1477. Whole-cell patch clamp recordings revealed that capsaicin-evoked TRPV1-mediated current density levels were increased after 3 days incubation with MRS1477 (2 µM). However, the tumor growth in MCF7 tumor-bearing immunodeficient mice was not significantly decreased after treatment with MRS1477 (10 mg/ kg body weight, i.p., injection twice a week). In conclusion, in view of a putative in vivo treatment with MRS1477 or similar compounds further optimization is required.

Selenium potentiates the anticancer effect of cisplatin against oxidative stress and calcium ion signaling-induced intracellular toxicity in MCF-7 breast cancer cells: involvement of the TRPV1 channel.

BACKGROUND: In breast cancers, calcium signaling is a main cause of proliferation and apoptosis of breast cancer cells. Although previous studies have implicated the transient receptor potential vanilloid 1 (TRPV1) cation channel, the synergistic inhibition effects of selenium (Se) and cisplatin in cancer and the suppression of ongoing apoptosis have not yet been investigated in MCF-7 breast cancer cells. This study investigates the anticancer properties of Se through TRPV1 channel activity in MCF-7 breast cancer cell line cultures when given alone or in combination with cisplatin. MATERIALS: The MCF-7 cells were divided into four groups: the control group, the Se-treated group (200 nM), the cisplatin-treated group (40 µM) and the Se + cisplatin-treated group. RESULTS: The intracellular free calcium ion concentration and current densities increased with TRPV1 channel activator capsaicin (0.01 mM), but they decreased with the TRPV1 blocker capsazepine (0.1 mM), Se, cisplatin, and Se + cisplatin incubations. However, mitochondrial membrane depolarization, apoptosis, and the caspase 3, and caspase 9 values increased in the Se-treated group and the cisplatin-treated group, although Western blot (procaspase 3 and 9) results and the cell viability levels decreased with the Se and Se + cisplatin treatments. Apoptosis and caspase-3 were further increased with the Se + cisplatin treatment. Intracellular reactive oxygen species production increased with the cisplatin treatment, but not with the Se treatment. CONCLUSION: This study's results report, for the first time, that at a cellular level, Se and cisplatin interact on the same intracellular toxic cascade, and the combination of these two drugs can result in a remarkable anticancer effect through modulation of the TRPV1.

Synergic Effects of Doxorubicin and Melatonin on Apoptosis and Mitochondrial Oxidative Stress in MCF-7 Breast Cancer Cells: Involvement of TRPV1 Channels.

Transient receptor transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+)-permeable channel gated by oxidative stress and capsaicin (CAP) and modulated by melatonin (MEL) and capsazepine (CPZ). A combination of doxorubicin (DOX) and MEL may offer a potential therapy for breast cancer by exerting antitumor and anti-apoptotic effects and modulating Ca(2+) influx and TRPV1 activity. We aimed to investigate the effects of MEL and DOX on the oxidative toxicity of MCF-7 human breast cancer cells, in addition to the activity of the TRPV1 channel and apoptosis. The MCF-7 cells were divided into the following six treatment groups: control, incubated with MEL (0.3 mM), incubated with 0.5 µM DOX, incubated with 1 µM DOX, incubated with MEL + 0.5 µM DOX, or incubated with MEL + 1 µM DOX. The intracellular free Ca(2+) concentration was higher in the DOX groups than in the control, and the concentration was decreased by MEL. The intracellular free Ca(2+) concentration was further increased by treatment with the TRPV1 channel activator CAP (0.01 mM), and it was decreased by the CPZ (0.1 mM). The intracellular production of reactive oxygen species, mitochondrial membrane depolarization, apoptosis level, procaspase 9 and PARP activities, and caspase 3 and caspase 9 activities were higher in the DOX and MEL groups than in the control. Apoptosis and the activity of caspase 9 were further increased in the DOX plus MEL groups. Taken together, the findings indicate that MEL supported the effects of DOX by activation of TRPV1 and apoptosis, as well as by inducing MCF-7 cell death. As the apoptosis and caspase activity of cancer cells increase because of their elevated metabolism, MEL may be useful in supporting their apoptotic capacity.

The efficiency of Poly(ADP-Ribose) Polymerase (PARP) cleavage on detection of apoptosis in an experimental model of testicular torsion.

The aim of this study was to evaluate the histopathological and apoptotic changes occurring in the rat ipsilateral and contralateral testes, after experimental spermatic cord torsion, and to explore and the role of poly(ADP-ribose) polymerase (PARP) cleavage in testicular torsion-detorsion injury. A total of 37 Wistar albino rats were subjected to 720° unilateral spermatic cord torsion for 1, 2 and 4 h, followed by 4-h reperfusion, or else to a sham operation (control group). Histology of the testicle was evaluated using haematoxylin-eosin (H&E) staining and Johnsen's scoring system. Germ cell apoptosis was evaluated via active caspase-3 immunostaining, and PARP expression levels were evaluated via Western blotting. The mean Johnsen's tubular biopsy scores (JTBS) of the ipsilateral testicles were lower for all torsion groups than for the controls (P < 0.05), but the JTBS of the contralateral testicles were only lower in the 4-h torsion group (P < 0.05). The mean apoptosis score (AS) of the ipsilateral and contralateral testicles was significantly higher in the torsion groups than in the sham group. AS increased correlatively with torsion time, in both testicles. The effect of testicular torsion on PARP cleavage was time dependent, with the highest effect observed after 4 h of testicular torsion (P < 0.05). Testicular torsion caused time-dependent histological changes, apoptosis and increases in PARP cleavage. Our results suggest that testicular torsion-detorsion injury caused cell damage and germ cell apoptosis that apparently involved cleavage of PARP. Increased PARP cleavage could, in turn, lead to enhanced apoptosis.

Investigation of the effects of distance from sources on apoptosis, oxidative stress and cytosolic calcium accumulation via TRPV1 channels induced by mobile phones and Wi-Fi in breast cancer cells.

TRPV1 is a Ca2+ permeable channel and gated by noxious heat, oxidative stress and capsaicin (CAP). Some reports have indicated that non-ionized electromagnetic radiation (EMR)-induces heat and oxidative stress effects. We aimed to investigate the effects of distance from sources on calcium signaling, cytosolic ROS production, cell viability, apoptosis, plus caspase-3 and -9 values induced by mobile phones and Wi-Fi in breast cancer cells MCF-7 human breast cancer cell lines were divided into A, B, C and D groups as control, 900, 1800 and 2450 MHz groups, respectively. Cells in Group A were used as control and were kept in cell culture conditions without EMR exposure. Groups B, C and D were exposed to the EMR frequencies at different distances (0 cm, 1 cm, 5 cm, 10 cm, 20 cm and 25 cm) for 1h before CAP stimulation. The cytosolic ROS production, Ca2+ concentrations, apoptosis, caspase-3 and caspase-9 values were higher in groups B, C and D than in A group at 0 cm, 1 cm and 5 cm distances although cell viability (MTT) values were increased by the distances. There was no statistically significant difference in the values between control, 20 and 25 cm. Wi-Fi and mobile phone EMR placed within 10 cm of the cells induced excessive oxidative responses and apoptosis via TRPV1-induced cytosolic Ca2+ accumulation in the cancer cells. Using cell phones and Wi-Fi sources which are farther away than 10 cm may provide useful protection against oxidative stress, apoptosis and overload of intracellular Ca2+. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Selenium reduces mobile phone (900 MHz)-induced oxidative stress, mitochondrial function, and apoptosis in breast cancer cells.

Exposure to mobile phone-induced electromagnetic radiation (EMR) may affect biological systems by increasing free oxygen radicals, apoptosis, and mitochondrial depolarization levels although selenium may modulate the values in cancer. The present study was designed to investigate the effects of 900 MHz radiation on the antioxidant redox system, apoptosis, and mitochondrial depolarization levels in MDA-MB-231 breast cancer cell line. Cultures of the cancer cells were divided into four main groups as controls, selenium, EMR, and EMR + selenium. In EMR groups, the cells were exposed to 900 MHz EMR for 1 h (SAR value of the EMR was 0.36 ± 0.02 W/kg). In selenium groups, the cells were also incubated with sodium selenite for 1 h before EMR exposure. Then, the following values were analyzed: (a) cell viability, (b) intracellular ROS production, (c) mitochondrial membrane depolarization, (d) cell apoptosis, and (e) caspase-3 and caspase-9 values. Selenium suppressed EMR-induced oxidative cell damage and cell viability (MTT) through a reduction of oxidative stress and restoring mitochondrial membrane potential. Additionally, selenium indicated anti-apoptotic effects, as demonstrated by plate reader analyses of apoptosis levels and caspase-3 and caspase-9 values. In conclusion, 900 MHz EMR appears to induce apoptosis effects through oxidative stress and mitochondrial depolarization although incubation of selenium seems to counteract the effects on apoptosis and oxidative stress.

2.45-Gz wireless devices induce oxidative stress and proliferation through cytosolic Ca²âº influx in human leukemia cancer cells.

PURPOSE: Electromagnetic radiation from wireless devices may affect biological systems by increasing free radicals. The present study was designed to determine the effects of 2.45 GHz radiation on the antioxidant redox system, calcium ion signaling, cell count and viability in human leukemia 60 cells. MATERIALS AND METHODS: Twelve cell cultures were equally divided into two main groups as controls (n = 6) and irradiated (n = 6) and then subdivided into four different subgroups depending on the duration of exposure, namely 1, 2, 12 and 24 hours. The samples were analyzed immediately after the experimental period. RESULTS: The extent of lipid peroxidation, cytosolic free Ca²âº and cell numbers were higher in 2.45 GHz groups than in the controls. The increase of cytosolic free Ca²âº concentrations was radiation time-dependent and was highest at 24-h exposure. The reduced glutathione, glutathione peroxidase, vitamin C and cell viability values did not show any changes in any of the experimental groups. 2-aminoethyl diphenylborinate inhibits Ca²âº ions influx by blockage of the transient receptor potential melastatin 2. CONCLUSIONS: 2.45 GHz electromagnetic radiation appears to induce proliferative effects through oxidative stress and Ca²âº influx although blocking of transient receptor potential melastatin 2 channels by 2-aminoethyl diphenylborinate seems to counteract the effects on Ca²âº ions influx.

Melatonin potentiates chemotherapy-induced cytotoxicity and apoptosis in rat pancreatic tumor cells.

Melatonin has antitumor activity via several mechanisms including its antiproliferative and proapoptotic effects in addition to its potent antioxidant action. Thus, melatonin has proven useful in the treatment of tumors in association with chemotherapeutic drugs. This study was performed to evaluate the effect of melatonin on the cytotoxicity and apoptosis induced by three different chemotherapeutic agents, namely 5-fluorouracil (5-FU), cisplatin, and doxorubicin in the rat pancreatic tumor cell line AR42J. We found that both melatonin and the three chemotherapeutic drugs induce a time-dependent decrease in AR42J cell viability, reaching the highest cytotoxic effect after 48 hr of incubation. Furthermore, melatonin significantly augmented the cytotoxicity of the chemotherapeutic agents. Consistently, cotreatment of AR42J cells with each of the chemotherapeutic agents in the presence of melatonin increased the population of apoptotic cells, elevated mitochondrial membrane depolarization, and augmented intracellular reactive oxygen species (ROS) production compared to treatment with each chemotherapeutic agent alone. These results provide evidence that in vitro melatonin enhances chemotherapy-induced cytotoxicity and apoptosis in rat pancreatic tumor AR42J cells and, therefore, melatonin may be potentially applied to pancreatic tumor treatment as a powerful synergistic agent in combination with chemotherapeutic drugs.

Colchicine modulates oxidative stress in serum and neutrophil of patients with Behçet disease through regulation of Ca²âº release and antioxidant system.

Behçet disease (BD) is a chronic, inflammatory, and multisystemic condition with an uncertain pathogenesis. One of the major immunologic findings in BD pathogenesis is increase in activity of neutrophil. An increase in the cytosolic free Ca²âº[Ca²âº](i) concentration that induces Ca²âº signaling is an important step that participates in the neutrophil activation and reactive oxygen species production that leads to tissue damage in body cells. We aimed to investigate the effects of colchicine on oxidative stress and Ca²âº release in serum and neutrophil of BD patients with active and inactive periods. Twelve Behçet patients (6 active and 6 inactive) and 6 control subject were included in the study. Disease activity was considered by clinical findings. Serum and neutrophil samples were obtained from the patients and control subjects. Neutrophils from patients with active BD were divided into three subgroups and were incubated with colchicine, verapamil + diltiazem, and colchicine + verapamil + diltiazem, respectively. Erythrocyte sedimentation rate, leucocytes counts, serum C-reactive protein, neutrophil, and serum lipid peroxidation and intracellular Ca²âº release levels were higher in active and inactive groups than in the control group, although their levels were lower in active group than in inactive group. However, neutrophil Ca²âº release levels were decreased in colchicine, verapamil + diltiazem, and colchicine + verapamil + diltiazem groups group compared to active group. Serum glutathione, vitamin A, vitamin E, and ß-carotene concentrations were lower in active and inactive groups than in the control group, although serum vitamin E and ß-carotene concentrations were higher in the inactive group than in the active group. Neutrophil and serum glutathione peroxidase activity within the three groups did not change. In conclusion, we observed the importance of Ca²âº influx into the neutrophils and oxidative stress in the pathogenesis and activation of the patients with BD. Colchicine induced protective effects on oxidative stress by modulating Ca²âº influx in BD patients.

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons.

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H(2)O(2) or BSO. TRPM2 channels current densities and cytosolic free Ca(2+) content of the neurons were higher in BSO and H(2)O(2) groups than in control. However, the current densities and cytosolic Ca(2+) release were also higher in the BSO + H(2)O(2) group than in the H(2)O(2) alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H(2)O(2) or rotenone. BSO and H(2)O(2)-induced Ca(2+) gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca(2+) influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.

Aminoethoxydiphenyl borate and flufenamic acid inhibit Ca2+ influx through TRPM2 channels in rat dorsal root ganglion neurons activated by ADP-ribose and rotenone.

Exposure to oxidative stress causes health problems, including sensory neuron neuropathy and pain. Rotenone is a toxin used to generate intracellular oxidative stress in neurons. However, the mechanism of toxicity in dorsal root ganglion (DRG) neurons has not been characterized. Melastatin-like transient receptor potential 2 (TRPM2) channel activation and inhibition in response to oxidative stress, ADP-ribose (ADPR), flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) in DRG neurons are also not clear. We tested the effects of FFA and 2-APB on ADPR and rotenone-induced TRPM2 cation channel activation in DRG neurons of rats. DRG neurons were freshly isolated from rats and studied with the conventional whole-cell patch-clamp technique. Rotenone, FFA and 2-APB were extracellularly added through the patch chamber, and ADPR was applied intracellularly through the patch pipette. TRPM2 cation currents were consistently induced by ADPR and rotenone. Current densities of the neurons were higher in the ADPR and rotenone groups than in control. The time courses (gating times) in the neurons were longer in the rotenone than in the ADPR group. ADPR and rotenone-induced TRPM2 currents were totally blocked by 2-APB and partially blocked by FFA. In conclusion, TRPM2 channels were constitutively activated by ADPR and rotenone, and 2-APB and FFA induced an inhibitory effect on TRPM2 cation channel currents in rat DRG neurons. Since oxidative stress is a common feature of neuropathic pain and diseases of sensory neurons, the present findings have broad application to the etiology of neuropathic pain and diseases of DRG neurons.