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.     

Zn2+, derived from cell preparation,partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore,in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca²⁺-dependent cell death by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn²⁺ and Fe²⁺ rather than Ca²⁺, trace metal cations in cell suspension may affect Ca²⁺-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1 mM EDTA while it was augmented by 50 μM EDTA and 10 μM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca²⁺]_i, was significantly reduced by 1 mM EDTA while it was not the case for 50 μM EDTA and 10 μM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn²⁺]_i, increased by A23187 was respectively reduced by 50 μM EDTA and 10 μM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1 mM EDTA is due to the chelation of extracellular Ca²⁺ and Zn²⁺ while the augmentation by 50 μM ETDA or 10 μM TPEN is due to the chelation of extracellular Zn²⁺. The Tyrode’s solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn²⁺, derived from cell preparation, partly attenuates the Ca²⁺-dependent cell death induced by A23187.     

A-1048400 is a novel, orally active, state-dependent neuronal calcium channel blocker that produces dose-dependent antinociception without altering hemodynamic function in rats

Blockade of voltage-gated Ca²⁺ channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca²⁺ channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC₅₀ = 0.8 μM and 1.4 μM, respectively) and T-type (IC₅₀ = 4.6 μM and 1.2 μM, respectively) Ca²⁺ channels in FLIPR based Ca²⁺ flux assays. A-686085 also potently blocked L-type Ca²⁺ channels (EC₅₀ = 0.6 μM), however, A-1048400 was much less active in blocking this channel (EC₅₀ = 28 μM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC₅₀ = 300–365 ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca²⁺ currents in rat dorsal root ganglion neurons (IC₅₀ = 3.0 μM and 1.6 μM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca²⁺ channels coupled with pharmacological selectivity vs. L-type Ca²⁺ channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function.     

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.     

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.     

Interaction of mephedrone with dopamine and serotonin targets in rats

IntroductionWe described a first approach to the pharmacological targets of mephedrone (4-methyl-methcathinone) in rats to establish the basis of the mechanism of action of this drug of abuse.Experimental proceduresWe performed in vitro experiments in isolated synaptosomes or tissue membrane preparations from rat cortex or striatum, studying the effect of mephedrone on monoamine uptake and the displacement of several specific radioligands by this drug.ResultsIn isolated synaptosomes from rat cortex or striatum, mephedrone inhibited the uptake of serotonin (5-HT) with an IC₅₀ value lower than that of dopamine (DA) uptake (IC₅₀ = 0.31 ± 0.08 and 0.97 ± 0.05 μM, respectively). Moreover, mephedrone displaced competitively both [³H]paroxetine and [³H]WIN35428 binding in a concentration-dependent manner (Ki values of 17.55 ± 0.78 μM and 1.53 ± 0.47 μM, respectively), indicating a greater affinity for DA than for 5-HT membrane transporters. The affinity profile of mephedrone for the 5-HT₂ and D₂ receptors was assessed by studying [³H]ketanserin and [³H] raclopride binding in rat membranes. Mephedrone showed a greater affinity for the 5-HT₂ than for the D₂ receptors.DiscussionThese results provide evidence that mephedrone, interacting with 5-HT and DA transporters and receptors must display a similar pattern of other psychoactive drugs such as amphetamine-like compounds.     

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.     

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.     

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC₅₀: 6.48 ± 2.74 μM) > celecoxib (IC₅₀: 14.92 ± 6.40 μM) > valdecoxib (IC₅₀: 161.4 ± 28.6 μM) > rofecoxib (IC₅₀: 238.4 ± 79.2 μM) > etoricoxib (IC₅₀: 405.1 ± 116.3 μM). Mechanism based inhibition of ATP synthesis (K_inact 0.078 min^− 1 and K_I 21.46 μM and K_inact/K_I ratio 0.0036 min^− 1 μM^− 1) was shown by lumiracoxib and data suggest that the opening of the MPT pore may not be the mechanism of toxicity. A positive correlation (with r² = 0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs and these metabolites did not alter the inhibition profile of ATP synthesis of the parent compound. The results suggest that coxibs themselves could be involved in the hepatotoxic action through inhibition of ATP synthesis.     

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.     

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.     

Characterization and structure-activity relationship of natural flavonoids as hERG K+ channel modulators

ObjectivesFlavonoids are present in varying concentrations in plant foods and have been reported to have numerous pharmacological activities, such as anti-cancer, antioxidant, anti-inflammatory, hepatoprotective, and vasodilator effects. We found that quercetin, fisetin, and some related flavonoid derivatives could inhibit human ether-à-go-go-related gene (hERG) K⁺ channels.Key findingsIn this study, we tested the effects of a series of flavonoids on the hERG K⁺ channel expressed in HEK293 cells. For the first time, we demonstrate that quercetin and fisetin (Fise) are potent hERG current blockers. The 50% inhibiting concentration (IC₅₀) and maximum efficacy (E_max) of quercetin were 11.8 ± 0.9 μM and 82 ± 2%, while those of fisetin were 38.4 ± 6 μM and 100 ± 6%, respectively. Luteolin (Lute) was a less potent inhibitor of hERG current (48 ± 1% at 100 μM). Galangin, kaempferol, and isorhamnetin (100 μM) showed weaker activity on the hERG currents.ConclusionThese results suggest that quercetin, fisetin, and luteolin are potent hERG K⁺ channel inhibitors and reveal the structure-activity relationship of natural flavonoids.     

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.     

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.     

Characterization of estrogenic and androgenic activity of phthalates by the XenoScreen YES/YAS in vitro assay

The presented study investigates and compares the estrogenic and androgenic activities of commonly used diesters of phthalic acid (phthalates) using the XenoScreen YES/YAS assay. Phthalates are commonly used plasticizers in polymers dedicated for i.e. food and drug containers. Since phthalates are not chemically bonded to the polymer, they can leach or migrate from the polymer. Therefore, phthalates are identified as contaminants in a variety of consumer products. Investigation of estrogenic and androgenic activities of phthalates (DEP, DBP, BBP, DEHP and DINP) showed no significant effect of tested substances either on hERα or hAR receptors. Phthalates exhibited strong anti-estrogenic (IC₅₀ for BBP = 8.66 μM, IC₅₀ for DEHP = 3.61 μM and IC₅₀ for DINP = 0.065 μM) and anti-androgenic (IC₅₀ for BBP = 5.30 μM, IC₅₀ for DEHP = 2.87 μM and IC₅₀ for DINP = 0.068 μM) activities.     

Zinc oxide nanoparticle disruption of store-operated calcium entry in a muscarinic receptor signaling pathway

The influences of ZnO nanoparticles on cellular responses to activation of muscarinic receptors were studied in Chinese hamster ovary cells expressing the human M3 muscarinic acetylcholine receptor. ZnO particles (20 nm) induced cytotoxicity in a time and concentration-dependent manner: following a 24 h exposure, toxicity was minimal at concentrations below 20 μg/ml but virtually complete at concentrations above 28 μg/ml. ZnO particles did not affect antagonist binding to M3 receptors or allosteric ligand effects, but increased agonist binding affinity while eliminating guanine nucleotide sensitivity. At a noncytotoxic concentration (10 μg/ml), ZnO increased resting [Ca²⁺]_i from 40 to 130 nM without compromising calcium homeostatic mechanisms. ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store-operated calcium entry (capacitive calcium entry). The latter effect was seen as (1) a decrease in the plateau phase of the response and (2) a decrease in Ca²⁺ entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50’s ≈ 2 μg/ml). Thus, ZnO nanoparticles interfere with two specific aspects of the M3 signaling pathway, agonist binding and store-operated calcium entry.     

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.     

Caffeine inhibits InsP3 responses and capacitative calcium entry in canine pulmonary arterial smooth muscle cells

Caffeine is a well described and characterized ryanodine receptor (RyR) activator. Previous evidence from independent research studies also indicate caffeine inhibits InsP₃ receptor functionality, which is important to activation of capacitative Ca²⁺ entry (CCE) in some cell types. In addition, RyR activation elicits excitatory-coupled Ca²⁺ entry (ECCE) in skeletal muscle myotubes. Recent studies by our group show that canine pulmonary arterial smooth muscle cells (PASMCs) have functional InsP₃ receptors as well as RyRs, and that CCE is dependent on InsP₃ receptor activity. The potential for caffeine to activate ECCE as well as inhibit InsP₃ receptor function and CCE was examined using fura-2 fluorescent imaging in canine PASMCs. The data show caffeine causes transient as well as sustained cytosolic Ca²⁺ increases, though this is not due to CCE or ECCE activity as evidenced by a lack of an increase in Mn²⁺ quench of fura-2. The experiments also show caffeine reversibly inhibits 5-HT elicited—InsP₃ mediated Ca²⁺ responses with an IC₅₀ of 6.87 × 10^? 4 M and 10 mM caffeine fully inhibits CCE. These studies provide the first evidence that caffeine is an inhibitor of InsP₃ generated Ca²⁺ signals and CCE in PASMCs.     

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.     

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.