Propofol Causes Sustained Ca2+ Elevation in Endothelial Cells by Stimulating Ryanodine Receptor and Suppressing Plasmalemmal Ca2+ Pump.

ABSTRACT: Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We here investigated the effects of propofol on Ca2+ transport and pain mediator release in human umbilical vein endothelial cells (EA.hy926). Propofol mobilized Ca2+ from cyclopiazonic acid (CPA)-dischargeable pool but did not cause Ca2+ release from the lysosomal Ca2+ stores. Propofol-elicited Ca2+ release was suppressed by 100 µM ryanodine, suggesting the participation of ryanodine receptor channels. Propofol did not affect ATP-triggered Ca2+ release but abolished the Ca2+ influx triggered by ATP; in addition, propofol also suppressed store-operated Ca2+ entry elicited by CPA. Ca2+ clearance during CPA-induced Ca2+ discharge was unaffected by a low Na+ (50 mM) extracellular solution, but strongly suppressed by 5 mM La3+ (an inhibitor of plasmalemmal Ca2+ pump), suggesting Ca2+ extrusion was predominantly through the plasmalemmal Ca2+ pump. Propofol mimicked the effect of La3+ in suppressing Ca2+ clearance. Propofol also stimulated release of pain mediators, namely, reactive oxygen species and bradykinin. Our data suggest propofol elicited Ca2+ release and repressed Ca2+ clearance, causing a sustained cytosolic [Ca2+]i elevation. The latter may cause reactive oxygen species and bradykinin release, resulting in pain.

Lipotoxicity in human lung alveolar type 2 A549 cells: Mechanisms and protection by tannic acid.

Palmitic acid (PA) is a saturated free fatty acid which, when being excessive, accounts for lipotoxicity. Using human lung A549 cells as a model for lung alveolar type 2 epithelial cells, we found that challenge of A549 cells with PA resulted in apoptotic cell death, as reflected by positive annexin V and PI staining, and also appearance of cleaved caspase-3. PA treatment also caused depletion of intracellular Ca2+ store, endoplasmic reticulum (ER) stress, and oxidative stress. Tannic acid (TA), a polyphenol present in wines and many beverages, alleviated PA-induced ER stress, oxidative stress and apoptotic death. Thus, our results suggest PA lipotoxicity in lung alveolar type 2 epithelial cells could be protected by TA.

Characterization of Ca2+-Sensing Receptor-Mediated Ca2+ Influx in Microvascular bEND.3 Endothelial Cells.

Ca2+-sensing receptors (CaSR), activated by elevated concentrations of extracellular Ca2+, have been known to regulate functions of thyroid cells, neurons, and endothelial cells (EC). In this report, we studied CaSR-mediated Ca2+ influx in mouse cerebral microvascular EC (bEND.3 cells). Cytosolic free Ca2+ concentration and Mn2+ influx were measured by fura-2 microfluorometry. High (3 mM) Ca2+ (CaSR agonist), 3 mM spermine (CaSR agonist), and 10 µM cinacalcet (positive allosteric modulator of CaSR) all triggered Ca2+ influx; however, spermine, unlike high Ca2+ and cinacalcet, did not promote Mn2+ influx and its response was poorly sensitive to SKF 96365, a TRP channel blocker. Consistently, 2-aminoethoxydiphenyl borate and ruthenium red (two other general TRP channel blockers) suppressed Ca2+ influx triggered by cinacalcet and high Ca2+ but not by spermine. Ca2+ influx triggered by high Ca2+, spermine, and cinacalcet was similarly suppressed by A784168, a potent and selective TRPV1 antagonist. Our results suggest that CaSR activation triggered Ca2+ influx via TRPV1 channels; intriguingly, pharmacological, and permeability properties of such Ca2+ influx depended on the stimulating ligands.

Tannic acid, a vasodilator present in wines and beverages, stimulates Ca2+ influx via TRP channels in bEND.3 endothelial cells.

Tannic acid (TA) is a polyphenol compound present in wines and many beverages. Although previous works have shown that TA could cause vasodilation in an endothelial cell (EC)-dependent manner, there is hitherto no report showing whether TA could raise EC cytosolic Ca2+ concentration. In this work we examined the effects of TA on cytosolic Ca2+ of mouse brain bEND.3 EC. TA (1-30 µM) caused a slow elevation in cytosolic Ca2+ level in a concentration-dependent manner. At 30 µM, TA triggered Ca2+ influx without causing intracellular Ca2+ release. TA-triggered Ca2+ influx was suppressed by Ni2+ (a non-specific Ca2+ channel blocker), ruthenium red and SKF 96365 (non-specific TRP channel blockers), CBA (a selective TRPM4 inhibitor) and M 084 (a selective TRPC4/C5 blocker). However, TA-triggered Ca2+ influx pathway was not permeable to Mn2+. Our results suggest TA activated TRP channels, possibly TRPM4 and TRPC4/C5, to promote influx of Ca2+.

Gossypol stimulates opening of a Ca2+ - and Na+ -permeable but Ni2+ - and Co2+ -impermeable pore in bEND.3 endothelial cells.

Gossypol, a polyphenolic dialdehyde toxin isolated from cotton seed, has anti-cancer properties and has recently shown some success in the treatment of glioma. Its effects on brain neurons and blood vessels are poorly understood. In this work we examined the effects of gossypol on cytosolic Ca2+ concentration ([Ca2+ ]i ) of mouse brain bEND.3 endothelial cells. Cell viability tests revealed that after 3 hour and 18 hour exposures, 10 µmol/L gossypol caused 23% and 65% cell death, respectively; 3 µmol/L gossypol caused no and 21% cell death, respectively. [Ca2+ ]i was raised concentration-dependently by 1-10 µmol/L gossypol. We then explored the Ca2+ signalling triggered by 3 µmol/L gossypol, which inflicted minimal toxicity: the Ca2+ signal was composed largely of Ca2+ influx and to a small extent, intracellular Ca2+ release. Such Ca2+ influx was much larger than store-operated Ca2+ influx triggered by maximal Ca2+ pool depletion. The Ca2+ influx triggered by 3 and 10 µmol/L gossypol caused NO release and cell death, respectively. Gossypol also triggered influx of Mn2+ and Na+ , but not Ni2+ and Co2+ . Gossypol-triggered Ca2+ signal was inhibited only by 14% and 37% by 100 µmol/L La3+ and 10 µmol/L nimodipine, respectively; and not suppressed at all by 5 mmol/L Ni2+ . Gossypol-triggered Ca2+ signal was suppressed by 78% by 30 µmol/L ruthenium red, suggesting gossypol may act on TRPV channels. Our results suggest gossypol triggered opening of a non-selective cation pore, possibly a member of the TRPV family.

Voltage-gated K+ channels promote BT-474 breast cancer cell migration.

OBJECTIVE: A variety of ion channels have been implicated in breast cancer proliferation and metastasis. Voltage-gated K+ (Kv) channels not only cause repolarization in excitable cells, but are also involved in multiple cellular functions in non-excitable cells. In this study we investigated the role of Kv channels in migration of BT474 breast cancer cells. METHODS: Transwell technique was used to separate migratory cells from non-migratory ones and these two groups of cells were subject to electrophysiological examinations and microfluorimetric measurements for cytosolic Ca2+. Cell migration was examined in the absence or presence of Kv channel blockers. RESULTS: When compared with non-migratory cells, migratory cells had much higher Kv current densities, but rather unexpectedly, more depolarized membrane potential and reduced Ca2+ influx. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of Kv1.1, Kv1.3, Kv1.5, Kv2.1, Kv3.3, Kv3.4 and Kv4.3 channels. Cell migration was markedly inhibited by tetraethylammonium (TEA), a delayed rectifier Kv channel blocker, but not by 4-aminopyridine, an A-type Kv channel blocker. CONCLUSIONS: Taken together, our results show that increased Kv channel expression played a role in BT474 cell migration, and Kv channels could be considered as biomarkers or potential therapeutic targets for breast cancer metastasis. The mechanism(s) by which Kv channels enhanced migration appeared unrelated to membrane hyperpolarization and Ca2+ influx.

Perturbation of Akt Signaling, Mitochondrial Potential, and ADP/ATP Ratio in Acidosis-Challenged Rat Cortical Astrocytes.

Cells switch to anaerobic glycolysis when there is a lack of oxygen during brain ischemia. Extracellular pH thus drops and such acidosis causes neuronal cell death. The fate of astrocytes, mechanical, and functional partners of neurons, in acidosis is less studied. In this report, we investigated the signaling in acidosis-challenged rat cortical astrocytes and whether these signals were related to mitochondrial dysfunction and cell death. Exposure to acidic pH (6.8, 6.0) caused Ca2+ release and influx, p38 MAPK activation, and Akt inhibition. Mitochondrial membrane potential was hyperpolarized after astrocytes were exposed to acidic pH as soon as 1 h and lasted for 24 h. Such mitochondrial hyperpolarization was prevented by SC79 (an Akt activator) but not by SB203580 (a p38 inhibitor) nor by cytosolic Ca2+ chelation by BAPTA, suggesting that only the perturbation in Akt signaling was causally related to mitochondrial hyperpolarization. SC79, SB203580, and BAPTA did not prevent acidic pH-induced cell death. Acidic pH suppressed ROS production, thus ruling out the role of ROS in cytotoxicity. Interestingly, pH 6.8 caused an increase in ADP/ATP ratio and apoptosis; pH 6.0 caused a further increase in ADP/ATP ratio and necrosis. Therefore, astrocyte cell death in acidosis did not result from mitochondrial potential collapse; in case of acidosis at pH 6.0, necrosis might partly result from mitochondrial hyperpolarization and subsequent suppressed ATP production. J. Cell. Biochem. 118: 1108-1117, 2017. © 2016 Wiley Periodicals, Inc.

Propofol produces preventive analgesia via GluN2B-containing NMDA Receptor/ERK1/2 Signaling Pathway in a rat model of inflammatory pain.

Abstract: Propofol, an intravenous anesthetic, has been shown to offer superior analgesic effect clinically. Whether propofol has preventive analgesic property remains unexplored. The present study investigated the antinociceptive effect of propofol and underlying molecular and cellular mechanisms via pre-emptive administration in a formalin-induced inflammatory pain model in rats. Male adult Sprague­Dawley rats were randomly allocated into four groups: naïve (Group Naïve), formalin injection only (Group Formalin), and formalin injection at 30 min (Group P-30 min) or 2 h (Group P-2 h) after intravenous infusion of propofol (0.6 mg kg−1 min−1) for 1 h. Nociceptive responses and protein expression of phosphorylated- or pan-GluN2B, ERK1/2, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in the spinal dorsal horn were evaluated. Alteration of intracellular Ca2+ concentration induced by N-methyl-D-aspartate (NMDA) receptor agonists with or without pre-treatment of propofol was measured using fluorometry in SH-SY5Y cells while neuronal activation in the spinal dorsal horn by immunofluorescence. Pre-emptive propofol reduced pain with a delayed response to formalin and a reduction in hypersensitivity that lasted at least for 2 h. The formalin-induced activation of spinal GluN2B and ERK1/2 but not p38 or c-Jun N-terminal kinase was also diminished by propofol treatment. Preconditioning treatment with 3 µM and 10 µM of propofol inhibited Ca2+ influx mediated through NMDA receptors in SH-SY5Y cells. Propofol also reduced the neuronal expression of c-Fos and p-ERK induced by formalin. This study shows that pre-emptive administration of propofol produces preventive analgesic effects on inflammatory pain through regulating neuronal GluN2B-containing NMDA receptor and ERK1/2 pathway in the spinal dorsal horn.

Parthenolide-Induced Cytotoxicity in H9c2 Cardiomyoblasts Involves Oxidative Stress.

BACKGROUND: Cardiac cellular injury as a consequence of ischemia and reperfusion involves nuclear factor-κB (NF-κ B), amongst other factors, and NF-κ B inhibitors could substantially reduce myocardial infarct size. Parthenolide, a sesquiterpene lactone compound which could inhibit NF-κ B, has been shown to ameliorate myocardial reperfusion injury but may also produce toxic effects in cardiomyocytes at high concentrations. The aim of this study was to examine the cytotoxic effects of this drug on H9c2 cardiomyoblasts, which are precursor cells of cardiomyocytes. METHODS: Cell viability and apoptosis were examined by MTT and TUNEL assay, respectively, and protein expression was analyzed by western blot. Reactive oxygen species (ROS) production was measured using DCFH-DA as dye. Cytosolic Ca(2+) concentration and mitochondrial membrane potential were measured microfluorimetrically using, respectively, fura 2 and rhodamine 123 as dyes. RESULTS: Parthenolide caused apoptosis at 30 µ M, as judged by TUNEL assay and Bax and cytochrome c translocation. It also caused collapse of mitochondrial membrane potential and endoplasmic reticulum stress. Parthenolide triggered ROS formation, and vitamin C (antioxidant) partially alleviated parthenolide-induced cell death. CONCLUSIONS: The results suggested that parthenolide at high concentrations caused cytotoxicity in cardiomyoblasts in part by inducing oxidative stress, and demonstrated the imperative for cautious and appropriate use of this agent in cardioprotection. KEY WORDS: Cardiomyoblast; Endoplasmic reticulum stress; Oxidative stress; Parthenolide; Reperfusion injury.

Ca2+ store depletion and endoplasmic reticulum stress are involved in P2X7 receptor-mediated neurotoxicity in differentiated NG108-15 cells.

P2X7 receptor (P2X7R) activation by extracellular ATP triggers influx of Na(+) and Ca(2+), cytosolic Ca(2+) overload and consequently cytotoxicity. Whether disturbances in endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress are involved in P2X7R-mediated cell death is unknown. In this study, a P2X7R agonist (BzATP) was used to activate P2X7R in differentiated NG108-15 neuronal cells. In a concentration-dependent manner, application of BzATP (10-100 µM) immediately raised cytosolic Ca(2+) concentration ([Ca(2+)]i) and caused cell death after a 24-h incubation. P2X7R activation for 2 h did not cause cell death but resulted in a sustained reduction in ER Ca2+ pool size, as evidenced by a diminished cyclopiazonic acid-induced Ca(2+) discharge (fura 2 assay) and a lower fluorescent signal in cells loaded with Mag-fura 2 (ER-specific Ca(2+)-fluorescent dye). Furthermore, P2X7R activation (2 h) led to the appearance of markers of ER stress [phosphorylated α subunit of eukaryotic initiation factor 2 (p-eIF2α) and C/EBP homologous protein (CHOP)] and apoptosis (cleaved caspase 3). Xestospongin C (XeC), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor (IP3R), strongly inhibited BzATP-triggered [Ca(2+)]i elevation, suggesting that the latter involved Ca(2+) release via IP3R. XeC pretreatment not only attenuated the reduction in Ca(2+) pool size in BzATP-treated cells, but also rescued cell death and prevented BzATP-induced appearance of ER stress and apoptotic markers. These novel observations suggest that P2X7R activation caused not only Ca(2+) overload, but also Ca(2+) release via IP3R, sustained Ca(2+) store depletion, ER stress and eventually apoptotic cell death.

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer.

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/ß(Tyr270/216) phosphorylation and reduced GSK3α/ß(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/ß activation in mediating the MCPP-induced human colon cancer cell apoptosis.

Dependence of 6beta-acetoxy-7alpha-hydroxyroyleanone block of Kv1.2 channels on C-type inactivation.

Voltage-gated K(+) (Kv) channels exhibit slow or C-type inactivation during continuous depolarization. A selective pharmacological agent targeting C-type inactivation is hitherto lacking. Here, we report that 6beta-acetoxy-7alpha-hydroxyroyleanone (AHR), a diterpenoid compound isolated from Taiwania cryptomerioides, can selectively modify C-type inactivation of Kv1.2 channels. Extracellular, but not intracellular, AHR (50 muM) dramatically accelerated the slow decay of Kv currents and left-shifted the steady-state inactivation curve. AHR blocked Kv currents with an IC(50) of 17.7 muM. AHR did not affect the kinetics and voltage-dependence of Kv1.2 channel activation. Channel block by AHR was independent of intracellular K(+) concentration. In addition, effect of AHR was much attenuated in a Kv1.2 V370G mutant defective in C-type inactivation. Therefore, block of Kv1.2 channels by AHR did not appear to involve direct occlusion of the outer pore but depended on C-type inactivation. AHR could thus be a probe targeting Kv channel C-type inactivation gate.

Perturbation of Ca2+ stores and store-operated Ca2+ influx by lidocaine in neuronal N2A and NG108-15 cells.

In this report we examined the effects of lidocaine on Ca2+ homeostasis of neuronal cells using microfluorimetric measurement of cytosolic Ca2+ with fura 2 as probe. In mouse neuroblastoma N2A cells, 10 mM lidocaine caused Ca2+ release from the cyclopiazonic acid (CPA)-dischargeable pool and abolished ATP-triggered Ca2+ release. Lidocaine-triggered Ca2+ release was not affected by xestospongin C (XeC), an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor. N2A cells did not have functional ryanodine receptors (RYR) (absence of caffeine response) and we used differentiated NG108-15 cells (presence of caffeine response) for further experiments. Caffeine-triggered Ca2+ release was unaffected by a brief lidocaine exposure, but was eliminated after a prolonged treatment of lidocaine, suggesting lidocaine abolished caffeine action possibly not by interfering caffeine binding but via Ca2+ store depletion. Lidocaine-elicited Ca2+ release was unaffected by XeC or a high concentration of ryanodine, suggesting Ca2+ release was not via IP3R or RYR. Lidocaine did not affect nigericin-dischargeable lysosomal Ca2+ stores. Lastly, we observed that lidocaine suppressed CPA-induced store-operated Ca2+ influx in both N2A cells and differentiated NG108-15 cells. Our results suggest two novel actions of lidocaine in neuronal cells, namely, depletion of Ca2+ store (via an IP3R- and RYR-independent manner) and suppression of store-operated Ca2+ influx.

Bradykinin-induced cell migration and COX-2 production mediated by the bradykinin B1 receptor in glioma cells.

Bradykinin is produced and acts at the site of injury and inflammation. Recent reports have also shown that bradykinin selectively modulates blood-tumor barrier permeability. However, the molecular mechanisms and pathologic roles underlying bradykinin-induced glioma migration remain unclear. Glioma is the most common primary adult brain tumor, with a poor prognosis because of the ease with which tumor cells spread to other regions of the brain. In this study, we found that bradykinin increases the cell migration and expression of cyclo-oxygenase-2 (COX-2) in glioma cells. Bradykinin-mediated migration was attenuated by the selective COX-2 inhibitor NS-398. Moreover, increased motility of glioma cells and expression of COX-2 were mimicked by a bradykinin B1 receptor (B1R) agonist and markedly inhibited by a B1R antagonist. Bradykinin-mediated migration was attenuated by phosphoinositide 3-kinase (PI-3 kinase)/AKT inhibitors LY 294002 and wortmannin. Bradykinin stimulation also increased the phosphorylation of the p85 subunit of PI-3 kinase and serine 473 of AKT. Treatment of bradykinin with AP-1 inhibitors Tanshinone IIA and curcumin also reduced COX-2 expression and glioma cell migration. Moreover, treatment of bradykinin also induced phosphorylation of c-Jun in glioma cells. AP-1 promoter analysis in the luciferase reporter construct showed that bradykinin increased AP-1 transcription activity and was inhibited by LY 294002 and wortmannin. One mechanism underlying bradykinin-directed migration is transcriptional up-regulation of COX-2 and activation of the B1R receptor, PI-3 kinase, AKT, c-Jun, and AP-1 pathways.

Propofol depresses angiotensin II-induced cell proliferation in rat cardiac fibroblasts.

BACKGROUND: Propofol may have beneficial effects on the prevention of angiotensin II (Ang II)-induced cardiac fibroblast proliferation via its antioxidative properties. The authors hypothesized that propofol may alter Ang II-induced cell proliferation and aimed to identify the putative underlying signaling pathways in rat cardiac fibroblasts. METHODS: Cultured rat cardiac fibroblasts were pretreated with propofol then stimulated with Ang II; cell proliferation and endothelin-1 gene expression were examined. The effect of propofol on Ang II-induced nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity were also examined. The effect of propofol on nitric oxide production and protein kinase B and endothelial nitric oxide synthase phosphorylations were also tested to elucidate the intracellular mechanism of propofol in proliferation. RESULTS: Ang II (100 nm) increased cell proliferation and endothelin-1 expression, which were partially inhibited by propofol (10 or 30 microm). Propofol also inhibited Ang II-increased nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity. Propofol was also found to increase nitric oxide generation and protein kinase B and nitric oxide synthase phosphorylations. Nitric oxide synthase inhibitor (N-nitro-L-arginine methylester) and the short interfering RNA transfection for protein kinase B or endothelial nitric oxide synthase markedly attenuated the inhibitory effect of propofol on Ang II-induced cell proliferation. CONCLUSIONS: The authors' results suggest that propofol prevents cardiac fibroblast proliferation by interfering with the generation of reactive oxygen species and involves the activation of the protein kinase B-endothelial nitric oxide synthase-nitric oxide pathway.

Osthol is a use-dependent blocker of voltage-gated Na+ channels in mouse neuroblastoma N2A cells.

Osthol, a Chinese herbal compound, has been shown to possess vasorelaxant and neuroprotective properties. Not much is known about the effects of osthol on ionic channels, activities of which are implicated in vasorelaxation and neuroprotection. In this work we report that osthol could inhibit voltage-gated Na (+) currents with state-dependence in mouse neuroblastoma N2A cells (IC (50) = 12.3 microM and 31.5 microM at holding potentials of - 70 mV and - 100 mV, respectively). Current blockade was equally effective in both extracellular and intracellular application of osthol. Osthol (18 microM) did not significantly affect the kinetics and voltage-dependence of Na (+) channel activation, but left-shifted the steady-state inactivation curve (V (1/2) = - 60.5 mV and - 78.7 mV in the absence and presence of osthol, respectively). Osthol also mildly but significantly retarded channel recovery from inactivation (recovery time constant = 19.9 ms and 35.6 ms in the absence and presence of osthol, respectively). In addition, osthol blocked Na (+) currents in a frequency-dependent fashion: blockades of 17 %, 34 % and 49 % when currents were triggered at 0.33 Hz, 1 Hz and 3.33 Hz, respectively. Taken together, our results therefore suggest that osthol blocked voltage-gated Na (+) channels intracellularly with state- and frequency-dependence.

Polyamine stimulation perturbs intracellular Ca2+ homeostasis and decreases viability of breast cancer BT474 cells.

Intracellular polyamines such as spermine and spermidine are essential to cell growth in normal and especially in cancer cells. However, whether extracellular polyamines affect cancer cell survival is unknown. We therefore examined the actions of extracellular polyamines on breast cancer BT474 cells. Our data showed that spermine, spermidine, and putrescine decreased cell viability by apoptosis. These polyamines also elicited Ca2+ signals, but the latter were unlikely triggered via Ca2+-sensing receptor (CaSR) as BT474 cells have been demonstrated previously to lack CaSR expression. Spermine-elicited Ca2+ response composed of both Ca2+ release and Ca2+ influx. Spermine caused a complete discharge of the cyclopiazonic acid (CPA)-sensitive Ca2+ pool and, expectedly, endoplasmic reticulum (ER) stress. The Ca2+ influx pore opened by spermine was Mn2+-impermeable, distinct from the CPA-triggered store-operated Ca2+ channel, which was Mn2+-permeable. Spermine cytotoxic effects were not due to oxidative stress, as spermine did not trigger reactive oxygen species formation. Our results therefore suggest that spermine acted on a putative polyamine receptor in BT474 cells, causing cytotoxicity by Ca2+ overload, Ca2+ store depletion, and ER stress.

Quercetin depletes intracellular Ca2+ stores and blunts ATP-triggered Ca2+ signaling in bEnd.3 endothelial cells.

Quercetin is a flavonol polyphenol widely found in many vegetables, grains, and fruits. Quercetin has been shown to inhibit proliferation and invasion of various glioma cells and is regarded as a potential anticancer agent against glioma. However, whether and how this drug could affect brain blood vessels and endothelial cells (EC) are less understood. Further, there is hitherto no report on how quercetin affects brain EC Ca2+ homeostasis. In this report, we investigated the effects of quercetin on Ca2+ homeostasis in mouse brain bEnd.3 EC. We demonstrated that quercetin raised cytosolic Ca2+ level in a concentration-dependent manner. Quercetin-triggered Ca2+ signal composed of both internal Ca2+ release and extracellular Ca2+ influx. Quercetin caused Ca2+ release from the endoplasmic reticulum, and consistently, inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) by xestospongin C (XeC) suppressed quercetin-triggered Ca2+ release. Quercetin also caused Ca2+ release from lysosomes, an observation in concordance with the inhibition of quercetin-triggered Ca2+ release by trans-Ned-19, a blocker of two-pore channels. As quercetin depleted intracellular Ca2+ storage, it suppressed ATP-induced Ca2+ release and thereby blunted ATP-triggered Ca2+ signaling. In addition, quercetin co-treatment significantly suppressed ATP-stimulated nitric oxide release. Our work therefore showed, for the first time, quercetin perturbed intracellular Ca2+ stores and strongly suppressed ATP-triggered response in bEnd.3 cells.

Hypoxia-induced matrix metalloproteinase-13 expression in astrocytes enhances permeability of brain endothelial cells.

Matrix metalloproteinase-13 (MMP-13) is involved in the degradation of extracellular matrix in many kinds of tissues. Here we found that hypoxia increased MMP-13 protein and mRNA levels in primary rat astrocyte cultures. Hypoxia stimulation also increased the secretion of MMP-13 from astrocytes, as shown by zymographic analysis. In addition, exposure to hypoxia up-regulated the expression of c-Fos and c-Jun time-dependently. Hypoxia-induced MMP-13 overexpression was antagonized by transfection with antisense oligodeoxynucleotides (AS-ODN) of c-Fos or c-Jun. Furthermore, hypoxic-conditioned medium (Hx-CM) collected from astrocytes exposed to hypoxia increased paracellular permeability of adult rat brain endothelial cells (ARBECs). Administration of MMP-13 neutralizing antibody antagonized Hx-CM-induced paracellular permeability of ARBECs. Furthermore, pre-transfection of astrocytes with AS-ODN of c-Fos, c-Jun or MMP-13-shRNA significantly decreased hyperpermeability of ARBECs induced by Hx-CM. The arrangement of tight junction protein (TJP) zonular occludens-1 (ZO-1) of ARBECs disorganized in response to Hx-CM. Administration of Hx-CM to ARBECs also resulted in the production of proteolytic fragments of ZO-1, which was antagonized by transfection of MMP-13-shRNA in primary astrocytes. Administration of MMP-13 recombinant protein to ARBECs led to the disorganization and fragmentation of ZO-1 protein and also increased paracellular permeability. These results suggest that hypoxia-induced MMP-13 expression in astrocytes is regulated by c-Fos and c-Jun. MMP-13 is an important factor leading to the disorganization of ZO-1 and hyperpermeablility of blood-brain barrier in response to hypoxia.

Effects of propofol on cyclic strain-induced endothelin-1 expression in human umbilical vein endothelial cells.

BACKGROUND: Propofol is one of the most popular intravenous induction agents of general anesthesia. Experimental results revealed that propofol exerted hypotensive and antioxidative effects. However, the intracellular mechanism of propofol remains to be delineated. The aims of this study were to test the hypothesis that propofol may alter strain-induced endothelin-1 (ET-1) secretion and nitric oxide production, and to identify the putative underlying signaling pathways in human umbilical vein endothelial cells. METHODS: Cultured human umbilical vein endothelial cells were exposed to cyclic strain in the presence of propofol, and ET-1 expression was examined by Northern blotting and enzyme-linked immunosorbent assay kit. Activation of extracellular signal-regulated protein kinase, endothelial nitric oxide synthase, and protein kinase B were assessed by Western blot analysis. RESULTS: The authors show that propofol inhibits strain-induced ET-1 expression, strain-increased reactive oxygen species formation, and extracellular signal-regulated protein kinase phosphorylation. On the contrary, nitric oxide production, endothelial nitric oxide synthase activity, and protein kinase B phosphorylation were enhanced by propofol treatment. Furthermore, in the presence of PTIO, a nitric oxide scavenger, and KT5823, a specific inhibitor of cyclic guanosine monophosphate-dependent protein kinase, the inhibitory effect of propofol on strain-induced extracellular signal-regulated protein kinase phosphorylation and ET-1 release was reversed. CONCLUSIONS: The authors demonstrate for the first time that propofol inhibits strain-induced ET-1 secretion and enhances strain-increased nitric oxide production in human umbilical vein endothelial cells. Thus, this study delivers important new insight into the molecular pathways that may contribute to the proposed hypotensive effects of propofol in the cardiovascular system.