AHR/TET2/NT5E axis downregulation is associated with the risk of systemic lupus erythematosus and its progression.

The prognosis of systemic lupus erythematosus (SLE) is unpredictable. This study aimed to examine the regulatory mechanism of the AHR/TET2/NT5E pathway during SLE progression. The AHR, TET2 and NT5E expression levels were examined in T regulatory cells (Tregs) of patients with SLE. The correlation of AHR, TET2 or NT5E expression levels with the immunosuppressive functions of Tregs was analysed. In patients with SLE, the number of CD4+ IL2RA- FOXP3+ T cell subset was positively correlated with the SLE disease activity index value and negatively correlated with the AHR and TET2 expression levels in CD4+ IL2RA+ FOXP3+ Tregs. Transcriptional profiles of 79 patients with SLE obtained from the Gene Expression Omnibus database (GSE61635 dataset) revealed a significant positive correlation between the mRNA expression levels of AHR and TET2. In silico analysis predicted that the TET2 promoter comprises an AHR-binding site. Kynurenine (KYN) promoted the binding of AHR to the TET2 promoter in Tregs of patients with SLE and Jurkat T cell lines. Furthermore, NT5E expression was significantly downregulated in Tregs of patients with SLE, which can be attributed to the dysregulation of NT5E promoter methylation status induced by downregulated TET2 activity. Furthermore, the Treg immunosuppressive activity, which is mediated through the TET2 and A2AR-adenosine pathways, in the KYN-treated group was approximately two-fold higher than that in the control group. The AHR/TET2/NT5E axis mediates the Treg immunosuppressive activity. These findings provide novel insights for the development of therapeutic approaches for SLE and related autoimmune diseases.

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol], a natural phenolic compound found in the olive, alters Ca2+ signaling and viability in human HepG2 hepatoma cells.

Hepatotoma is the leading type of primary liver cancer in adults and third cause of death in the world. Hydroxytyrosol is a natural phenol existing in olive (Olea europaea L.). Hydroxytyrosol is the chief ingredient of olive oil, which was early deemed to be the most robust antioxidant in olive oil. Hydroxytyrosol is known to inhibit various types of cancer by different methods. This study was aimed to delineate the action of hydroxytyrosol on viability and [Ca2+]i in HepG2 hepatoma cells. Fura-2 was used to detect [Ca2+]i, and WST-1 assays were applied to explore cell cytotoxicity. Hydroxytyrosol elicited [Ca2+]i raises. Eliminating external Ca2+ diminished the Ca2+ signal by 30%. Hydroxytyrosol-evoked Ca2+ influx was diminished by 20% by three inhibitors of store-operated Ca2+ channels and by a protein kinase C activator and an inhibitor. In the absence of Ca2+, thapsigargin eradicated hydroxytyrosol-provoked [Ca2+]i raises. Suppression of phospholipase C (PLC) with U73122, a PLC inhibitor, did not inhibit hydroxytyrosol-elicited [Ca2+]i raises. Hydroxytyrosol reduced cell viability. This cytotoxic action was not reversed by preincubation with BAPTA/AM, a cytosolic Ca2+ binder. In sum, in HepG2 hepatoma cells, hydroxytyrosol elicited [Ca2+]i raises by provoking PLC-unrelated discharge of Ca2+ from ER and Ca2+ influx through PKC-sensitive store-operated Ca2+ entry. In addition, hydroxytyrosol elicited Ca2+-dissociated cytotoxicity.

Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells.

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 Ca2+ levels ([Ca2+]i) and cytotoxicity in renal tubular cells is unknown. Our research explored if tectorigenin changed Ca2+ signal transduction and viability in Madin-Darby Canine Kidney (MDCK) renal tubular cells. [Ca2+]iin suspended cells were measured by applying the fluorescent Ca2+-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 [Ca2+]irises. Ca2+ removal reduced the signal by approximately 20%. Tectorigenin (50 µM) induced Mn2+ influx suggesting of Ca2+ entry. Tectorigenin-induced Ca2+ entry was inhibited by 10% by three inhibitors of store-operated Ca2+ channels, namely, nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited 83% of tectorigenin-evoked [Ca2+]irises. Conversely, treatment with tectorigenin abolished thapsigargin-evoked [Ca2+]irises. Inhibition of phospholipase C with U73122 inhibited 50% of tectorigenin-induced [Ca2+]irises. Tectorigenin at concentrations between 10 and 60 µM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ 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 [Ca2+]irises and induced cell death that was not associated with [Ca2+]irises. Therefore, tectorigenin may be a Ca2+-independent cytotoxic agent for kidney cells.

Effect of Protriptyline on [Ca²âº]i and Viability in MDCK Renal Tubular Cells.

Protriptyline has been used as an antidepressant. Clinically it has been prescribed in the auxiliary treatment of cancer patients. However, its effect on Ca²âº signaling and related physiology is unknown in renal cells. This study examined the effect of protriptyline on cytosolic free Ca²âº concentrations ([Ca²âº]i) and viability in Madin-Darby canine kidney (MDCK) tubular cells. Protriptyline induced [Ca²âº]i rises concentration-dependently. The response was reduced by 20% by removing extracellular Ca²âº. Protriptyline-induced Ca²âº entry was not altered by protein kinase C (PKC) activity but was inhibited by 20% by three modulators of store-operated Ca²âº channels: nifedipine, econazole and SKF96365. In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5- di-tert-butylhydroquinone (BHQ) or thapsigargin partially inhibited protriptyline-evoked [Ca²âº]i rises. Conversely, treatment with protriptyline inhibited partially BHQ or thapsigargin-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 did not change protriptyline-induced [Ca²âº]i rises. Protriptyline at 5-200 µM decreased cell viability, which was not reversed by pretreatment with the Ca²âº chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/ AM). Together, in MDCK cells, protriptyline induced [Ca²âº]i rises by evoking PLC-independent Ca²âº release from the endoplasmic reticulum and other unknown stores, and Ca²âº entry via PKCinsensitive store-operated Ca²âº entry. Protriptyline also caused Ca²âº-independent cell death.

Effect of NPC15199 on [Ca²âº]i and viability in SCM1 human gastric cancer cells.

NPC15199 is a synthesized compound that inhibits inflammation in some models. However, whether NPC15199 affects Ca²âº homeostasis in human gastric cancer is unclear. This study examined the effect of NPC15199 on cytosolic free Ca²âº concentrations ([Ca²âº]i) and viability in SCM1 human gastric cancer cells. The Ca²âº-sensitive fluorescent dye fura-2 was used to measure [Ca²âº]i. NPC15199 evoked [Ca²âº]i rises concentration-dependently. The response was reduced by removing extracellular Ca²âº. NPC15199-evoked Ca²âº entry was not inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365) and protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA), or PKC inhibitor (GF109203X). In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished NPC15199-evoked [Ca²âº]i rises. Conversely, treatment with NPC15199 also nearly abolished thapsigargin or BHQ-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 did not affect NPC15199-evoked [Ca²âº]i rises. NPC15199 at concentrations of 100-900 µM induced concentration-dependent, Ca²âº-independent decrease in viability. Together, in SCM1 cells, NPC15199 induced [Ca²âº]i rises that involved Ca²âº entry through PKC-insensitive non-store-operated Ca²âº channels and PLC-independent Ca²âº release from the endoplasmic reticulum. NPC15199 also induced Ca²âº-independent cell death.

Effect of thymol on Ca2+ homeostasis and viability in MG63 human osteosarcoma cells.

AIMS: The effect of the natural product thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was examined. METHODS: The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). RESULTS: Thymol at concentrations of 200-1,000 µmol/l induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Thymol-induced Ca(2+) entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. When extracellular Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited the thymol-induced [Ca(2+)](i) rise. Incubation with thymol also inhibited the thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished the thymol-induced [Ca(2+)](i) rise. At concentrations of 100-600 µmol/l, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM. Annexin V/propidium iodide staining data suggest that thymol (200 and 400 µmol/l) induced apoptosis in a concentration-dependent manner. Thymol (200 and 400 µmol/l) also increased levels of reactive oxygen species. CONCLUSIONS: In MG63 cells, thymol induced a [Ca(2+)](i) rise by inducing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis via mitochondrial pathways.

Effect of diindolylmethane on Ca(2+) movement and viability in HA59T human hepatoma cells.

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol in cruciferous vegetables, on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca(2+)](i) in HA59T cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 1-50 µM evoked a [Ca(2+)](i) rise in a concentration-dependent manner. The signal was reduced by removing Ca(2+). Diindolylmethane-induced Ca(2+) influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 10-75 µM, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25-50 µM) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca(2+)](i) rise by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.

Better short-term clinical response to etanercept in Chinese than Caucasian patients with active ankylosing spondylitis.

Tumor necrosis factor-alpha (TNF-α) inhibitors including etanercept have been demonstrated to be very effective in severe ankylosing spondylitis (AS) in Caucasian patients. However, clinical efficacy of etanercept to treat active AS in Chinese patients has not been reported. In this study, a prospective, open-label trial of etanercept (25 mg BIW), involving 46 AS patients from 16 medical centers of Taiwan, was conducted. Questionnaire was utilized to record demographic data and clinical parameters, including Bath AS Disease Activity Index (BASDAI), Bath AS Functional Index (BASFI), Bath AS Global Index (BASGI), Assessment in Ankylosing Spondylitis (ASAS) 20, 50, and 70, and others, before and at different time intervals after etanercept treatment. Laboratory tests including blood chemistry, hematology, urine analysis, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were done at baseline and at weeks 4, 8, and 12. In this 12-week study, etanercept demonstrated rapid and significant improvement in the ASAS20 response criteria (91.3%), at as early as 2 weeks of therapy (71.3%). Partial remission of AS was achieved in 49.3% of patients after 12 weeks of treatment. Disease activity (BASDAI) and function (BASFI) were also significantly improved after 12 weeks etanercept treatment (p < 0.0001 and p < 0.0001, respectively). In addition, significant increase of chest expansion (2.77 ± 1.69 cm versus 3.56 ± 1.82 cm, p = 0.0004) and lumbar flexion (2.11 ± 2.76 cm versus 2.58 ± 3.42 cm, p = 0.0075) and significant reduction of occiput-to-wall distance (6.59 ± 7.14 cm versus 5.32 ± 6.65 cm, p = 0.0006) were also demonstrated. Both ESR and CRP declined significantly after patients were treated with etanercept. There were no severe adverse effects during the treatment period. Etanercept is generally safe, well tolerated, and effective in Chinese patients with severe AS. Clinical efficacy, including partial remission and BASDAI, is even better in Chinese than in Caucasian patients. Further study is required to assess long-term efficacy and safety in Chinese patients with AS.

Systemic lupus erythematosus complicated with posterior reversible encephalopathy syndrome and intracranial vasculopathy.

Posterior reversible encephalopathy syndrome (PRES) is a neurotoxic condition characterized by reversible vasogenic edema on neuroimaging. It is associated with various neurological manifestations, including headaches, vomiting, seizures, visual loss, altered mental status and focal neurological deficits. PRES mainly occurs in the setting of eclampsia, hypertension, uremia, malignancy, transplantation, autoimmune diseases and/or use of immunosuppressive drugs. This syndrome has been described in patients with systemic lupus erythematosus (SLE). PRES is a potentially reversible clinical-radiological entity; however, it can be complicated with vasculopathy, infarction or hemorrhage. Vasculopathy has been demonstrated to be a common finding in patients with SLE. We report the case of a woman with lupus nephritis and PRES whose diffuse vasculopathy was present on initial neuroimaging. Subsequent brain computed tomography scan demonstrated interval development of intraparenchymal hemorrhage and subarachnoid hemorrhage. To our knowledge, this unique brain image pattern has not been reported in SLE patients.

Effects of Antrodia camphorata extracts on the viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation of OC2 human oral cancer cells.

The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ regulation of OC2 human oral cancer cells. AC at a concentration of 25 microM induced an increase in cell viability, but AC at concentrations > or = 50 microg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200 microg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25 microg/ml) did not alter basal [Ca2+]i, but decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinin-induced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargin-induced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca2+]i, induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

Hemolytic uremic syndrome and pericarditis as early manifestations of primary Sjögren's syndrome.

Hemolytic uremic syndrome (HUS) consists of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Autoimmune diseases have seldom been reported to be the etiology of HUS. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease affecting primarily the exocrine glands. Symptomatic pericarditis and pulmonary hemorrhage are rare manifestations in pSS patients. We describe the unusual case of a pSS patient with the initial presentation of HUS and pericarditis and a fatal progression of pulmonary hemorrhage. A renal biopsy established the diagnosis of HUS with histologically proven arterial thrombotic microangiopathy and glomerular and tubular ischemic necrosis. The relationships between HUS and pericarditis and pSS will be discussed in this report.

Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells.

The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent apoptotic pathway.

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells.

The effect of calmidazolium on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca2+]i and caused cell death in HA59T cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations > or =1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1.5 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Calmidazolium induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to L-type Ca2+ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), calmidazolium-induced [Ca2+]i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change calmidazolium-induced [Ca2+]i rises. At concentrations between 1 and 15 microM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via protein kinase C-regulated Ca2+ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.

Econazole-evoked [Ca2+]i rise and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC).

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca(2+) concentrations ([Ca(2+)](i)) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations > or = 1 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The econazole-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 20 microM econazole, [Ca(2+)](i) rises induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change econazole-induced [Ca(2+)](i) rises. At concentrations between 10 and 80 microM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 microM econazole was not reversed by prechelating cytosolic Ca(2+) with BAPTA. This shows that in SIRC cells econazole induces [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca(2+)](i) rise.

Hypereosinophilic syndrome with recurrent strokes: a case report.

PURPOSE: Hypereosinophilic syndrome is a rare disorder which can cause ischemic stroke. Although cardioembolism is acknowledged as the most common etiology for stroke, the underlying pathogenesis of hypereosinophilic syndrome could be heterogeneous. Herein we describe a patient with persistent hypereosnophillia with recurrent strokes focusing on the pathogenetic mechanism of stroke. CASE REPORT: A 43-year-old male patient with persistent primary eosinophilia presented with ischemic stroke which persisted for three weeks. Magnetic resonance imaging showed bilateral multiple cerebral infarctions over both anterior and posterior vascular territories. Segmental stenosis of the right posterior cerebral artery was revealed with magnetic resonance angiography and computed tomography angiography. Extensive laboratory workup ruled out other etiologies for the strokes except eosinophilia, which responded well to corticosteroid therapy. CONCLUSIONS: Cerebrovascular wall damage inflicted by eosinophilia may be the pathogenesis of the thromboembolic strokes in this case.

Diethylstilbestrol-induced estrogen receptor-dependent [Ca2+]i rises and apoptosis in Chinese hamster ovary (CHO) cells.

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations>or=1 proportional, variant increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. In Ca2+-free medium, after pretreatment with 50 proportional, variant DES, 1 proportional, variant thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca2+]i rises. At a concentration of 5 proportional, variant, DES increased cell viability. At concentrations of 100-200 microM, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 microM DES on viability was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 microM)-induced [Ca2+]i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 microM). ICI-182,780 did not affect 5 microM DES-induced increase in viability but partly reversed 100 microM DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca2+]i rises by stimulating estrogen receptors leading to Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca2+-dependent pathway.

Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells.

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.

Tamoxifen-induced [Ca2+]i rises and Ca2+-independent cell death in human oral cancer cells.

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) and cell viability in OC2 human oral cancer cells. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The tamoxifen-induced Ca(2+) influx was sensitive to blockade of L-type Ca(2+) channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 1 muM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), tamoxifen-induced [Ca(2+)](i) rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change tamoxifen-induced [Ca(2+)](i) rises. At concentrations between 10 and 50 microM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 microM tamoxifen was not reversed by prechelating cytosolic Ca(2+) with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca(2+)](i) rises, in a nongenomic manner, by causing Ca(2+) release from the endoplasmic reticulum, and Ca(2+) influx from L-type Ca(2+) channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca(2+)](i) rise.

Ketoconazole-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC).

The effect of ketoconazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca2+ levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 microM and above increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The ketoconazole-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 50 microM ketoconazole, thapsigargin-(1 microM)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change ketoconazole-induced [Ca2+]i rises. At concentrations between 5 and 100 microM, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 microM ketoconazole was not reversed by prechelating cytosolic Ca2+ with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca2+]i rise.

The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells.

The effect of the carcinogen safrole on intracellular Ca2+ movement in renal tubular cells has not been explored previously. The present study examined whether safrole could alter Ca2+ handling in Madin-Darby canine kidney (MDCK) cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 33 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 400 microM. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+, but was not affected by nifedipine, verapamil, or diltiazem. Addition of Ca2+ after safrole had depleted intracellular Ca(2+)-induced dramatic Ca2+ influx, suggesting that safrole caused store-operated Ca2+ entry. In Ca(2+)-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca 2+. Inhibition of phospholipase C with 2 microM U73122 did not affect safrole-induced Ca2+ release. Trypan blue exclusion assays revealed that incubation with 650 microM safrole for 30 min did not kill cells, but killed 70% of cells after incubation for 60 min. Collectively, the data suggest that in MDCK cells, safrole induced a [Ca2+] increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent fashion, and by inducing Ca2+ influx via store-operated Ca2+ entry. Furthermore, safrole can cause acute toxicity to MDCK cells.