Micromolar Zn2+ potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: Possible disturbance of intracellular Ca2+ and Zn2+ homeostasis

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn²⁺ at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn²⁺ permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn²⁺ at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn²⁺, we have cytometrically examined the effects of econazole, ZnCl₂, and their combination on rat thymocytes. ZnCl₂ at concentrations ranging from 1 μM to 30 μM significantly increased the lethality induced by 10 μM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 μM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl₂, suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl₂. Econazole at 0.3 μM or more in the presence of ZnCl₂ increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca²⁺. Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn²⁺, was also augmented by econazole at 0.1 μM or more in the presence of ZnCl₂. Results suggest that the combination of submicromolar econazole with micromolar ZnCl₂ may increase the intracellular concentration of Ca²⁺ and Zn²⁺, leading to disturbance of intracellular Ca²⁺ and Zn²⁺ homeostasis that triggers cytotoxic action.     

Effect of honokiol on erythrocytes

Honokiol ((3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol), a component of Magnolia officinalis, stimulates apoptosis and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and by breakdown of cell membrane phosphatidylserine asymmetry with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored, whether honokiol elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. As a result, a 48 h exposure to honokiol was followed by a slight but significant increase of [Ca²⁺]_i (15 μM), significant decrease of forward scatter (5 μM), significant increase of annexin-V-binding (5 μM) and significant increase of ceramide formation (15 μM). Honokiol further induced slight, but significant hemolysis. Honokiol (15 μM) induced annexin-V-binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. In conclusion, honokiol triggers suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of Ca²⁺ entry and ceramide formation.     

Effects of 17 β-estradiol on lipopolysacharride-induced intracellular adhesion molecule-1 mRNA expression and Ca2+ homeostasis alteration in human endothelial cells

Recent evidence showed that 17 β-estradiol (E₂) decreased cytokine-induced expression of cell adhesion molecules (CAM). Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been shown to be associated with CAM expression in endothelial cells. Here, the effects of E₂ (1 μM, 24 h) on the expression of intracellular adhesion molecule-1 (ICAM-1) and [Ca²⁺]_i were investigated in a lipopolysaccharide (LPS) (100 ng/mL, 18 h)-stimulated human endothelial cell line, EA.hy926, using real-time PCR and spectrofluorometry, respectively. PCR analysis revealed a significant increase in ICAM-1 expression in calcium ionophore A23187 (1 nM)- or LPS-stimulated cells. Pretreatment of cells with E₂ significantly inhibited LPS-induced ICAM-1 mRNA expression. [Ca²⁺]_i was monitored in Fura-2 AM-loaded cells in the presence and absence of extracellular Ca²⁺ with thapsigargin (TG, 1 μM), a sarco/endoplasmic reticulum ATPase inhibitor or ATP (100 μM). The extent of TG- or ATP-induced [Ca²⁺]_i increase was significantly higher in LPS-stimulated cells than in control cells. Pre-treatment of LPS-stimulated cells with E₂ limited the Ca²⁺ response to the same level as in control cells. Furthermore, ICI 182,780, an estrogen receptor antagonist, attenuated the inhibitory actions of E₂ on ICAM-1 mRNA expression and Ca²⁺ responses, suggesting that estrogen receptors mediate, at least in part, the effects of estrogen. These data suggest a potential underlying mechanism for the protective effect of E₂ against atherosclerosis.     

Emodin inhibits human sperm functions by reducing sperm [Ca2+]i and tyrosine phosphorylation

Emodin, a bioactive anthraquinone widely used in Chinese traditional medicine, disrupts mouse testicular gene expression in vivo. In this study, we investigated the toxicity of emodin to human sperm in vitro. Different doses of emodin (25, 50, 100, 200 and 400 μM) were applied to ejaculated human sperm. The results indicated that 100, 200 and 400 μM emodin significantly inhibited the total motility, progressive motility and linear velocity of human sperm. In addition, sperm's ability to penetrate viscous medium together with progesterone induced capacitation and acrosome reaction was also adversely affected by emodin. In contrast, emodin did not affect sperm viability. Furthermore, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tyrosine phosphorylation, which serve as key regulators of sperm function, were dose-dependently reduced by emodin (50–400 μM). These results suggest that emodin inhibits human sperm functions by reducing sperm [Ca²⁺]_i and suppressing tyrosine phosphorylation in vitro.     

Effect of the environmental pollutant bisphenol A dimethacylate (BAD) on Ca2+ movement and viability in OC2 human oral cancer cells

The environmental pollutant bisphenol A dimethacylate (BAD) has been used as a dental composite. The effect of BAD on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in OC2 human oral cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. BAD induced [Ca²⁺]_i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. BAD-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, and SK&F96365. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished BAD-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter BAD-induced [Ca²⁺]_i rise. At 10–30 μM, BAD inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. BAD (20–30 μM) also induced apoptosis. Collectively, in OC2 cells, BAD induced a [Ca²⁺]_i rise by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. BAD also caused apoptosis.     

Anti-allergic activity of emodin on IgE-mediated activation in RBL-2H3 cells

BackgroundEmodin (1,3,8-trihydroxy-6-methylanthraquinone) is a Chinese herbal anthraquinone derivative from the rhizome of rhubarb (Rheum palmatum L.) that exhibits numerous biological activities, such as antitumor, antibacterial, antiinflammatory, and immunosuppressive. In the present studies, the anti-allergic activities of emodin were investigated to elucidate the underlying active mechanisms.MethodsThe inhibitory effects of emodin on the IgE-mediated allergic response in rat basophilic leukemia (RBL-2H3) cells were evaluated by measuring the release of granules and cytokines. The Ca²⁺ mobilization in RBL-2H3 cells loaded with the Ca²⁺-reactive fluorescent probe Fluo-4 AM was also measured by laser scanning confocal microscope.ResultsEmodin inhibited the release of β-hexosaminidase (β-HEX; IC₅₀ = 5.5 μM) and tumor necrosis factor (TNF)-α (IC₅₀ = 11.5 μM) from RBL-2H3 cells induced by 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) and displayed stronger inhibition of β-HEX release than ketotifen fumarate salt (IC₅₀ = 63.8 μM). Emodin at a concentration of 12.5 μM also inhibited the DNP-BSA-induced influx of extracellular Ca²⁺ in RBL-2H3 cells.ConclusionsThese results suggested that emodin likely exhibits anti-allergic activities via increasing the stability of the cell membrane and inhibiting extracellular Ca²⁺ influx.     

Screening quality for Ca2 +-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin

IntroductionIonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca^2 + concentration ([Ca^2 +]_i). This difficulty prevents obtaining high-quality data regarding Ca^2 +-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca^2 +]_i in the IonWorks Quattro automated patch clamp system to evaluate BK channels.MethodsElectrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca^2 +]_i, ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca^2 + chelator, BAPTA-AM (20 μM).ResultsBK current activated by step pulses to + 100 mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11 nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1 Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (> 20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10 μM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30 μM.DiscussionThe main finding of this study is that the [Ca^2 +]_i variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca^2 +-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca^2 +-activated ion channels such as SK channels.     

Arsenite promotes apoptosis and dysfunction in microvascular endothelial cells via an alteration of intracellular calcium homeostasis

Vascular endothelium has been considered as a target for arsenic-induced cardiovascular toxicity. The present study demonstrated that arsenite caused slow and sustained elevation of intracellular free calcium levels ([Ca²⁺]_i) in HMEC-1, a human microvessel-derived endothelial cell line, in a concentration-dependent manner. Pretreatment with U-73122 (a specific PLC inhibitor) or 2-APB (a specific IP₃ receptor antagonist) attenuated this effect, suggesting that PLC/IP₃ signaling cascade is involved in arsenite-induced elevation of [Ca²⁺]_i. Cytotoxic concentrations of arsenite (5 and 10 μM) significantly enhanced endothelial nitric oxide synthase (eNOS) phosphorylation, nitric oxide (NO) production and apoptosis after 24-h exposure. Additionally, 2-APB attenuated eNOS phosphorylation and apoptosis induced by arsenite, indicating that Ca²⁺-mediated eNOS activation participates in arsenite-induced endothelial cell apoptosis. Moreover, we also found that non-apoptotic concentrations of arsenite (0.5 and 1 μM) dramatically mitigated thrombin-induced rapid transient rise of [Ca²⁺]_i, eNOS phosphorylation and NO production, suggesting functional disruption of endothelial by arsenite, and these effects occurred without an alteration of PLC-β₁ and thrombin receptor levels. Altogether, the results reveal that arsenite induces apoptotic cell death and endothelial dysfunction as indicated by the reduction of thrombin responses, particularly related to an alteration of intracellular Ca²⁺ homeostasis.     

Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation

Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10 μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48 h exposure to 10 μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca²⁺]_i level was peaked and persisted within 70 s in K562 cells whilst 50 s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia.     

Calcium signaling and β2-adrenergic receptors regulate 1-nitropyrene induced CXCL8 responses in BEAS-2B cells

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread environmental pollutants, generated from reactions between PAHs and nitrogen oxides during combustion processes. In the present study we have investigated the mechanisms of CXCL8 (IL-8) responses induced by 1-nitropyrene (1-NP) in human bronchial epithelial BEAS-2B cells, with focus on the possible importance of Ca²⁺-signaling and activation of β2-adrenergic receptors (β2AR). Ca²⁺-chelator treatment obliterated 1-NP-induced CXCL8 (IL-8) responses. 1-NP at 10 μM (but not 1 μM) induced a rapid and sustained increase in intracellular Ca²⁺-levels ([Ca²⁺]_i). The early but not the later, sustained phase of 1-NP-induced [Ca²⁺]_i was suppressed by beta-blocker treatment (carazolol). Moreover, inhibition of β2AR by blocking-antibody, beta-blocker treatment (ICI 118551) or siRNA transfection attenuated CXCL8 responses induced by 1-NP. The results confirm that PAHs may induce Ca²⁺-signaling also in BEAS-2B cells, at least partly through activation of β2AR, and suggest that both β2AR- and Ca²⁺-signaling may be involved in 1-NP-induced CXCL8 responses in bronchial epithelial cells.     

Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2 + activity in the vascular smooth muscle cell line A10

Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca^2 + indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca^2 + activity [Ca^2 +]_i in A10 cells. This effect was absent in a Ca^2 +-free medium, indicating that OlyA/PlyB-induced [Ca^2 +]_i increase was dependent on Ca^2 + influx into cells. The increase in [Ca^2 +]_i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La^3 +, ii) the inhibitor of the sodium–calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca^2 +-ATPase inhibitor thapsigargin reduced the [Ca^2 +]_i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K⁺ in the medium did not prevent OlyA/PlyB-induced [Ca^2 +]_i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca^2 +]_i is due to an influx of Ca^2 + through a variety of co-existing plasma membrane Ca^2 +-permeable channels, Ca^2 + entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca^2 + release, altogether leading to disturbed Ca^2 + homeostasis in A10 cells.     

A Ca2+ chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression

Hexavalent chromium could result in cell malfunctions. Intracellular Ca²⁺ ([Ca²⁺]_i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca²⁺]_i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca²⁺]_i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4 μM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4 μM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca^2± overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.     

Effects of phenyl saligenin phosphate on cell viability and transglutaminase activity in N2a neuroblastoma and HepG2 hepatoma cell lines

The main aim of this study was to determine whether sub-lethal concentrations of the organophosphate compound phenyl saligenin phosphate (PSP) could disrupt the activity of the Ca²⁺-activated enzyme tissue transglutaminase (TGase 2) from cultured cell lines of neuronal (N2a) and hepatic (HepG2) origin.The results indicated that PSP added directly to cytosol extracts from healthy cells was able to inhibit TGase 2 activity by 40–60% of control levels at sub-lethal concentrations (?0.1 μM) that were approximately 100-fold lower than their IC₅₀ values in cytotoxicity assays. Following 24 h exposure of N2a cells to 0.3 and 3 μM PSP in situ, a similar reduction in activity was observed in subsequent assays of TGase 2 activity. However, significantly increased activity was observed following in situ exposure of HepG2 cells to PSP (ca. 4-fold at 3 μM). Western blotting analysis indicated slightly reduced levels of TGase 2 in N2a cells compared to the control, whereas an increase was observed in the level of TGase 2 in HepG2 cells. We suggest that TGase 2 represents a potential target of organophosphate toxicity and that its response may vary in different cellular environments, possibly affected by its expression pattern.     

Effect of miltefosine on erythrocytes

BackgroundMiltefosine, an alkylphosphocholine drug with antiparasite, antibacterial, antifungal and antineoplastic potency, is the only oral drug that can be used to treat visceral and cutaneous leishmaniasis. The effect of miltefosine is at least partially due to triggering of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-level ([Ca²⁺]_i). The present study explored, whether miltefosine elicits eryptosis.MethodsCell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence.ResultsA 48 h exposure to miltefosine (?4.9 μM) was followed by significant decrease of forward scatter and significant increase of annexin-V-binding. The effect was paralleled by significant increase of [Ca²⁺]_i. The annexin-V-binding following miltefosine treatment was significantly blunted in the nominal absence of extracellular Ca²⁺.ConclusionMiltefosine stimulates eryptosis, an effect at least partially due to stimulation of Ca²⁺ entry.     

KMUP-1 inhibits pulmonary artery proliferation by targeting serotonin receptors/transporter and NO synthase,inactivating RhoA and suppressing AKT/ERK phosphorylation

KMUP-1 inhibits monocrotaline (MCT)-induced pulmonary artery (PA) proliferation by targeting serotonin (5-HT) receptors, inactivating RhoA and reducing phosphorylation of AKT/ERK. In MCT-treated rats, KMUP-1 f (5 mg/kg p.o.; 1 mg/kg i.p. × 21 days) decreased proliferation (PCNA-positive) cells and 5-HTT-expression in lung and 5-HT levels in plasma. In isolated PA, KMUP-1 and simvastatin (0.1–100 μM) inhibited 5-HT (10 μM)-induced PA constriction. l-NAME-pretreatment reduced KMUP-1-induced relaxation. In pulmonary arterial smooth muscle cells (PASMCs), KMUP-1 (1–100 μM) and simvastatin (10 μM) inhibited 5-HT-induced cell migration and proliferation and KMUP-1 (1–100 μM) inhibited 5-HT-induced Ca²⁺ influx. Similar to Y27632, KMUP-1 (1–100 μM) inhibited 5-HT-induced RhoA/ROCK expression, while KMUP-1, Y27632 and simvastatin at 10 μM inhibited 5-HT-induced 5-HTT expression and KMUP-1 inhibited 5-HT-induced phosphorylation of AKT and ERK1/2 in PASMCs. In human pulmonary arterial endothelial cell (HPAEC), KMUP-1 (1–100 μM) increased the expression of eNOS and 5-HT_2B and also at 10 μM augmented eNOS expression and production of nitric oxide (NO) in 5-HT-treated HPAEC. In radioligand binding, the IC₅₀/K_i values of KMUP-1 for 5-HT_2A, 5-HT_2B and 5-HT_2C receptors were 0.34/0.0971, 0.04/0.0254, and 0.408/0.214 μM respectively. In conclusion, KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT_2A, 5-HT_2B and 5-HT_2C receptors, increasing endothelial eNOS/5-HT_2B receptor expression and NO release and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation. KMUP-1 is suggested to be useful in the treatment of 5-HT-induced pulmonary artery proliferation.     

Effects of Zizyphus lotus L. (Desf.) polyphenols on Jurkat cell signaling and proliferation

We assessed the effects of Zizyphus lotus L. (Desf.) polyphenols (ZLP) on T-cell signaling and proliferation. Our results showed that ZLP exerted no effect on the increases in intracellular free calcium concentrations, [Ca^2 +]_i, in human Jurkat T-cells. However, ZLP modulated the thapsigargin-induced increases in [Ca^2 +]_i in these cells. ZLP treatment was found to decrease the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, ZLP induced a rapid (t_1/2 = 33 s) and dose-dependent decrease in intracellular pH (pH_i) in human Jurkat T-cells. Furthermore, ZLP significantly curtailed T-cell proliferation by diminishing their progression from S to G2/M phase of cell cycle, and the expression of interleukin-2 (IL-2) mRNA. Taken together, the results of the present study demonstrate that ZLP modulate cell signaling and exert immunosuppressive effects in human T-cells.     

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose

ObjectiveTo investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition.MethodsThe hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmol L⁻¹), the PPD low dose group (1 μmol L⁻¹, PPD-L), the PPD middle dose group (5 μmol L⁻¹, PPD −M) and the PPD high dose group (10 μmol L⁻¹, UCN-H). The GMC were incubated for 48 h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca²⁺]_і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method.ResultsThe viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P < 0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P < 0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca²⁺]_і transient was reduced (P < 0.05 or P < 0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups.ConclusionThe protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca²⁺]_і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.     

New insights into the mechanism of neurolathyrism: L-β-ODAP triggers [Ca2+]i accumulation and cell death in primary motor neurons through transient receptor potential channels and metabotropic glutamate receptors

Neurolathyrism is a motor neuron (MN) disease caused by β-N-oxalyl-L-α,β-diaminopropionic acid (L-β-ODAP), an AMPA receptor agonist. L-β-ODAP caused a prolonged rise of intracellular Ca²⁺ ([Ca²⁺]_i) in rat spinal cord MNs, and the [Ca²⁺]_i accumulation was inversely proportional to the MN’s life span. The [Ca²⁺]_i rise induced by L-β-ODAP or (S)-AMPA was antagonized completely by NBQX, an AMPA-receptor blocker. However, blocking the L-type Ca²⁺ channel with nifedipine significantly lowered [Ca²⁺]_i induced by (S)-AMPA, but not that by L-β-ODAP. Tetrodotoxin completely extinguished the [Ca²⁺]_i rise induced by (S)-AMPA or kainic acid, whereas that induced by L-β-ODAP was only attenuated by 65.6 ± 6% indicating the prominent involvement of voltage-independent Ca²⁺ entry. The tetrodotoxin-resistant [Ca²⁺]_i induced by L-β-ODAP was blocked by 2-APB, Gd³⁺, La³⁺, 1-(β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF-96365) and flufenamic acid, which all are blockers of the transient receptor potential (TRP) channels. Blockers of group I metabotropic glutamate receptors (mGluR I), 7-(hydroxyiminocyclopropan[b]chromen-1α-carboxylate ethyl ester (CPCCPEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) also lowered the [Ca²⁺]_i rise by L-β-ODAP. MN cell death induced by L-β-ODAP was prolonged significantly with SKF-96365 as well as NBQX. The results show the involvement of TRPs and mGluR I in L-β-ODAP-induced MN toxicity through prolonged [Ca²⁺]_i mobilization, a unique characteristic of this neurotoxin.     

5-HT-induced depression of the spinal monosynaptic reflex potential utilizes different types of 5-HT receptors depending on Mg2+ availability

Receptor subtypes involved in the 5-hydroxytryptamine (5-HT)-induced depression of synaptic transmission in neonatal rat spinal cords in vitro were evaluated in the absence or presence of Mg²⁺ in the medium. Stimulation of a dorsal root evoked monosynaptic reflex potential (MSP) and polysynaptic reflex potential (PSP) in the segmental ventral root in Mg²⁺-free medium where the voltage-dependent blockade of NMDAreceptors is absent. The 5-HT (0.3–50 μM) in the Mg²⁺-free medium depressed the MSPand PSP in a concentration-dependent manner. At 30 μM of 5-HT, the depression was 57% and 95% for MSP and PSP, respectively, and no further depression was seen at 50 μM. The 5-HT-induced depression of the reflexes in the Mg²⁺-free medium was blocked by ondansetron (5-HT₃ receptor antagonist), but not by spiperone (5-HT_2A/2C antagonist). In the Mg²⁺-free medium, phenylbiguanide (5-HT₃ agonist) also depressed the MSPand PSP in a concentration-dependent manner and was blocked by ondansetron. Addition of Mg²⁺ (1.3 mM) to the medium abolished the PSP and decreased the MSPby 30%. In the presence of Mg²⁺, 5-HT (1–50 μM) also depressed the MSP in a concentration-dependent manner. At 10 μM of 5-HT, there was approximately 20% depression and at 50 μM the depression was 100%. The 5-HT-induced depression of MSP in the Mg²⁺-containing medium was antagonized by spiperone (p < 0.05, two-way ANOVA), but not by ondansetron. The results indicate that the 5-HT-induced depression of MSPinvolves 5-HT₃ receptors in the Mg²⁺-free medium and 5-HT_2A/2C in the presence of Mg²⁺ when NMDA receptors are in the closed state.     

Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells

The aims of this study were to examine the effects of CdCl₂ on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl₂ was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl₂ for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl₂ in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl₂ caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd^2 + also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1–12 μM Cd^2 + is attainable in vivo, our findings are consistent with the possibility that Cd^2 + induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.