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Puerarin is a natural compound and has been used as herb medication in a number of countries, especially in Asia. The effect of puerarin on Ca 2+ signaling is unknown in renal cells. This study examined whether puerarin affected Ca 2+ physiology in MDCK renal tubular cells. Cytosolic free Ca 2+ levels ([Ca 2+ ] i ) were measured using the fluorescent dye fura-2. Cell viability was examined by using WST-1 assay. Puerarin induced [Ca 2+ ] i rises and the response was reduced by removing extracellular Ca 2+ . Puerarin-induced Ca 2+ entry was not altered by protein kinase C (PKC) activity, but was inhibited by nifedipine. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin partly inhibited puerarin-evoked [Ca 2+ ] i rises. Inhibition of phospholipase C (PLC) with U73122 did not change puerarin-induced [Ca 2+ ] i rises. Puerarin at 25–50 μM caused cytotoxicity, which was not reversed by pretreatment with the Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in MDCK cells, puerarin induced [Ca 2+ ] i rises by evoking PLC-independent Ca 2+ release from the endoplasmic reticulum and other unknown stores, and Ca 2+ entry via nifedipine-sensitive, PKC-insensitive Ca 2+ entry pathways. Puerarin also caused Ca 2+ -independent cell death. [ABSTRACT FROM AUTHOR]

Abstract: 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 10–30μM, BAD inhibited cell viability, which was not reversed by chelating cytosolic Ca2+. BAD (20–30μM) 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. [Copyright &y& Elsevier]

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca 2+ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca 2+ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca 2+ concentration ([Ca 2+ ] i ) was measured by the Ca 2+ -sensitive fluorescent dye fura-2. Theophylline (25-125 μM) induced [Ca 2+ ] i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca 2+ with BAPTA/AM. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin abolished theophylline-induced [Ca 2+ ] i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca 2+ ] i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca 2+ ] i rises. In Ca 2+ -containing medium, modulators of store-operated Ca 2+ channels inhibited 30% of the [Ca 2+ ] i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca 2+ influx was confirmed by Mn 2+ -induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca 2+ -associated cytotoxicity and induced Ca 2+ entry through PLC-independent Ca 2+ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca 2+ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Thioridazine, belonging to first-generation antipsychotic drugs, is a prescription used to treat schizophrenia. However, the effect of thioridazine on intracellular Ca 2+ concentration ([Ca 2+ ] i ) and viability in human liver cancer cells is unclear. This study examined whether thioridazine altered Ca 2+ signaling and viability in HepG2 human hepatocellular carcinoma cells. Ca 2+ concentrations in suspended cells were measured using the fluorescent Ca 2+ -sensitive dye fura-2. Cell viability was examined by WST-1 assay. Thioridazine at concentrations of 25-100 μM induced [Ca 2+ ] i rises. Ca 2+ removal reduced the signal by 20%. Thioridazine (100 μM) induced Mn 2+ influx suggesting of Ca 2+ entry. Thioridazine-induced Ca 2+ entry was inhibited by 20% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate) and inhibitor (GF109203X) and by three inhibitors of store-operated Ca 2+ channels: nifedipine, econazole, and SKF96365. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin (TG) abolished thioridazine-evoked [Ca 2+ ] i rises. On the other hand, thioridazine preincubation completely inhibited the [Ca 2+ ] i rises induced by TG. Furthermore, U73122 totally suppressed the [Ca 2+ ] i rises induced by thioridazine via inhibition of phospholipase C (PLC). Regarding cytotoxicity, at 30-80 μM, thioridazine reduced cell viability in a concentration-dependent fashion. This cytotoxicity was not prevented by preincubation with 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (a Ca 2+ chelator). To conclude, thioridazine caused concentration-dependent [Ca 2+ ] i rises in HepG2 human hepatoma cells by inducing Ca 2+ release from the endoplasmic reticulum via PLC-associated pathways and Ca 2+ influx from extracellular medium through PKC-sensitive store-operated Ca 2+ entry. In addition, thioridazine induced cytotoxicity in a Ca 2+ -independent manner., Competing Interests: None

Tectorigenin, a traditional Chinese medicine, is isolated from the flower of plants such as Pueraria thomsonii Benth. It is an O-methylated isoflavone, a type of flavonoid. Previous studies have shown that tectorigenin evoked various physiological responses in different models, but the effect of tectorigenin on cytosolic-free Ca 2+ levels ([Ca 2+ ] i ) and cytotoxicity in renal tubular cells is unknown. Our research explored if tectorigenin changed Ca 2+ signal transduction and viability in Madin-Darby Canine Kidney (MDCK) renal tubular cells. [Ca 2+ ] i in suspended cells were measured by applying the fluorescent Ca 2+ -sensitive probe fura-2. Viability was explored by using water-soluble tetrazolium-1 as a fluorescent dye. Tectorigenin at concentrations of 5-50 μM induced [Ca 2+ ] i rises. Ca 2+ removal reduced the signal by approximately 20%. Tectorigenin (50 μM) induced Mn 2+ influx suggesting of Ca 2+ entry. Tectorigenin-induced Ca 2+ entry was inhibited by 10% by three inhibitors of store-operated Ca 2+ channels, namely, nifedipine, econazole, and SKF96365. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin inhibited 83% of tectorigenin-evoked [Ca 2+ ] i rises. Conversely, treatment with tectorigenin abolished thapsigargin-evoked [Ca 2+ ] i rises. Inhibition of phospholipase C with U73122 inhibited 50% of tectorigenin-induced [Ca 2+ ] i rises. Tectorigenin at concentrations between 10 and 60 μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca 2+ with 1,2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl did not reverse tectorigenin's cytotoxicity. Our data suggest that, in MDCK cells, tectorigenin evoked [Ca 2+ ] i rises and induced cell death that was not associated with [Ca 2+ ] i rises. Therefore, tectorigenin may be a Ca 2+ -independent cytotoxic agent for kidney cells., Competing Interests: None

Chlorzoxazone is a skeletal muscle relaxant. However, the effect of chlorzoxazone on intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in oral cancer cells is unclear. This study examined whether chlorzoxazone altered Ca 2+ signaling and cell viability in OC2 human oral cancer cells. [Ca 2+ ] i in suspended cells was measured using the fluorescent Ca 2+ -sensitive dye fura-2. Cell viability was examined by water-soluble tetrazolium-1 assay. Chlorzoxazone (250-1000 μM) induced [Ca 2+ ] i rises in a concentration-dependent manner. Ca 2+ removal reduced the signal by approximately 50%. Mn 2+ has been shown to enter cells through similar mechanisms as Ca 2+ but quenches fura-2 fluorescence at all excitation wavelengths. Chlorzoxazone (1000 μM) induced Mn 2+ influx, suggesting that Ca 2+ entry occurred. Chlorzoxazone-induced Ca 2+ entry was inhibited by 20% by inhibitors of store-operated Ca 2+ channels and protein kinase C (PKC) modulators. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin (TG) inhibited chlorzoxazone-evoked [Ca 2+ ] i rises by 88%. Conversely, treatment with chlorzoxazone-suppressed TG-evoked [Ca 2+ ] i rises 75%. Chlorzoxazone induced [Ca 2+ ] i rises by exclusively releasing Ca 2+ from the endoplasmic reticulum. Inhibition of phospholipase C (PLC) with U73122 did not alter chlorzoxazone-induced [Ca 2+ ] i rises. PLC activity was not involved in chlorzoxazone-evoked [Ca 2+ ] i rises. Chlorzoxazone at 200-700 μM decreased cell viability, which was not reversed by pretreatment with Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl. In sum, in OC2 cells, chlorzoxazone induced [Ca 2+ ] i rises by evoking PLC-independent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via PKC-sensitive store-operated Ca 2+ entry. Chlorzoxazone also caused Ca 2+ -independent cell death. Since [Ca 2+ ] i rises play a triggering or modulatory role in numerous cellular phenomena, the effect of chlorzoxazone on [Ca 2+ ] i and cell viability should be taken into account in other in vitro studies., Competing Interests: None

This study explored the effect of deltamethrin, a pesticide, on free Ca(2+) concentration [Ca(2+)]i, viability, and apoptosis in Madin-Darby canine kidney (MDCK) canine renal tubular cells. Deltamethrin at concentrations between 10μM and 40μM evoked [Ca(2+)]i rises in a concentration-dependent manner. The Ca(2+) entry was inhibited by nifedipine, econazole, phorbol 12-myristate 13-acetate, and SKF96365. Treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) in a Ca(2+)-free medium abolished deltamethrin-induced [Ca(2+)]i rise. Treatment with deltamethrin also abolished BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) activity with U73122 abolished deltamethrin-evoked [Ca(2+)]i rise. Deltamethrin killed cells at 30-60μM in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Annexin V/propidium iodide staining data suggest that 30-50μM deltamethrin induced apoptosis. Together, in MDCK renal tubular cells, deltamethrin induced [Ca(2+)]i rises that involved Ca(2+) entry through protein kinase C-mediated store-operated Ca(2+) channels, and PLC-dependent Ca(2+) release from the endoplasmic reticulum. Deltamethrin also induced Ca(2+)-independent cell death that might involve apoptosis., (Copyright © 2014. Published by Elsevier Taiwan.)