Afatinib triggers a Ni2+ -resistant Ca2+ influx pathway in A549 non-small cell lung cancer cells.

Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1-30 µM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca2+ influx without causing Ca2+ release, and the Ca2+ influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca2+ response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn2+ influx. Ca2+ influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni2+ (a non-specific Ca2+ channel blocker). Afatinib caused an increase in mitochondrial Ca2+ level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24-48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca2+ condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca2+ influx pathway, mitochondrial Ca2+ overload, and potential collapse in part contribute to afatinib-induced cell death.

Suppression of Ca2+ oscillations by SERCA inhibition in human alveolar type 2 A549 cells: rescue by ochratoxin A but not CDN1163.

AIMS: Lung type 2 alveolar cells, by secreting surfactant to lower surface tension, contribute to enhance lung compliance. Stretching, as a result of lung expansion, triggers type 1 alveolar cell to release ATP, which in turn stimulates Ca2+-dependent surfactant secretion by neighboring type 2 cells. In this report, we studied ATP-triggered Ca2+ signaling in human alveolar type 2 A549 cells. MAIN METHODS: Ca2+ signaling was examined using microfluorimetric measurement with fura-2 as fluorescent dye. KEY FINDINGS: Ca2+ oscillations triggered by ATP relied on inositol 1,4,5-trisphosphate-induced Ca2+ release and store-operated Ca2+ entry. Pathological conditions such as influenza virus infection and diabetes reportedly inhibit sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that a very mild inhibition of SERCA by cyclopiazonic acid (CPA) sufficed to decrease Ca2+ oscillation frequency and the percentage of cells exhibiting Ca2+ oscillations. Ochratoxin A (OTA), an activator of SERCA, could prevent the suppressive effects by CPA. Inhibition of SERCA by hydrogen peroxide also suppressed Ca2+ oscillations. Interestingly, hydrogen peroxide-induced inhibition was prevented by OTA but aggravated by CDN1163, an allosteric activator of SERCA. CDN1163 also had an untoward effect of releasing intracellular Ca2+. SIGNIFICANCE: Different modes of activation of SERCA may determine the outcome of rescue of Ca2+ oscillations in case of SERCA inhibition in alveolar type 2 cells.

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.

A basal level of γ-linolenic acid depletes Ca2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells.

Gamma-linolenic acid (GLA), a natural fatty acid obtained from oils of various vegetables and seeds, has been demonstrated as an anticancer agent. In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. GLA at 30 µM, a concentration reportedly within the range of circulating concentrations in clinical studies, caused apoptotic cell death. GLA caused an elevation in mitochondrial Ca2+ level and a decrease in mitochondrial membrane potential. GLA treatment depleted cyclopiazonic acid (CPA)-sensitive Ca2+ store and triggered substantial Ca2+ influx. Intracellular Ca2+ release triggered by GLA was suppressed by 3 µM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 µM ryanodine (ryanodine receptor-channel blocker), suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel. Increased expressions of p-eIF2α and CHOP were observed in GLA-treated cells, suggesting GLA-treated cells had increased expressions of p-eIF2α and CHOP, which suggest endoplasmic reticulum (ER) stress. In addition, GLA elicited increased production of reactive oxygen species. Taken together, our results suggest a basal level of GLA induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel.

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.

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+.

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.

ARC 118925XX stimulates cation influx in bEND.3 endothelial cells.

In a previous publication when we studied the purinergic receptor with which ATP interacted in mouse brain bEND.3 endothelial cells, we observed addition of 3 µm ARC 118925XX (ARC; selective P2Y2 antagonist) strongly suppressed ATP-triggered Ca2+ release, suggesting the response was mediated via P2Y2 receptors. We here report ARC unexpectedly promoted substantial Ca2+ influx even when ATP-triggered Ca2+ release was largely inhibited. Since this large Ca2+ influx may have important pharmacological significance, we proceeded to investigate its mechanism. ARC did not trigger intracellular Ca2+ release thus suggesting Ca2+ influx triggered by ARC was not store-operated. ARC-triggered Ca2+ influx could be blocked by 1 mm Ni2+ , a general Ca2+ channel blocker, but not by SK&F 96365, a nonselective TRP channel blocker. Unexpectedly, ARC promoted influx of Na+ and La3+ , but not Mn2+ . This is a surprising finding, since Mn2+ is conventionally used as a Ca2+ surrogate ion (as it permeates Ca2+ channel), and La3+ is classically used as a potent Ca2+ channel antagonist. Electrophysiological examination showed ARC did not stimulate any cation currents. Therefore, ARC opened, rather than a cation channel pore, an unidentified Ca2+ influx pathway which was Na+ - and La3+ -permeable but Mn2+ -impermeable.

Antagonism of Ca2+-sensing receptors by NPS 2143 is transiently masked by p38 activation in mouse brain bEND.3 endothelial cells.

Ca2+-sensing receptors (CaSR) are G protein-coupled receptors which are activated by a rise in extracellular Ca2+. CaSR activation has been known to inhibit parathyroid hormone release and stimulate calcitonin release from parathyroid glands and thyroid parafollicular C cells, respectively. The roles of CaSR in other cell types including endothelial cells (EC) are much less understood. In this work, we demonstrated protein and functional expression of CaSR in mouse cerebral EC (bEND.3). Unexpectedly, CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by edelfosine or U73122 but strongly suppressed by SK&F 96365, ruthenium red, and 2-aminoethoxydiphenyl borate (2-APB), suggesting involvement of TRPV and TRPC channels but not Gq-phospholipase C. Acute application of NPS2143, a negative allosteric modulator of CaSR, suppressed CaSR response. However, a 40-min NPS2143 pre-treatment surprisingly enhanced CaSR response. After 4-24 h of application, this enhancement faded away and suppression of CaSR response was observed again. Similar results were obtained when La3+ and Sr2+ were used as CaSR agonists. The transient NPS 2143 enhancement effect was abolished by SB203580, a p38 inhibitor. Consistently, NPS 2143 triggered a transient p38 activation. Taken together, results suggest that in bEND.3 cells, NPS 2143 caused acute suppression of CaSR response, but then elicited a transient enhancement of CaSR response in a p38-dependent manner. NPS 2143 effects on CaSR in bEND.3 cells therefore depended on drug exposure time. These findings warrant cautious use of this agent as a CaSR modulator and potential cardiovascular drug.

Enhancing tetrandrine cytotoxicity in human lung carcinoma A549 cells by suppressing mitochondrial ATP production.

ATP depletion induced by inhibiting glycolysis or mitochondrial ATP production has been demonstrated to cause cancer cell death. Whether ATP depletion can enhance the efficacy and potency of anti-cancer effects of herbal compounds is so far unknown. We examined the enhancing effect of ATP depletion on anti-cancer actions of tetrandrine (TET) in human lung carcinoma A549 cells. A 24-h incubation of A549 cells with tetrandrine caused a concentration-dependent cytotoxic effect (LC50 = 66.1 µM). Co-incubation with 20 mM 2-deoxyglucose (2-DG, glycolysis inhibitor) caused only a very slight enhancement of tetrandrine cytotoxicity. By contrast, inhibiting mitochondrial ATP production with oligomycin (10 µM, ATP synthase inhibitor) and FCCP (30 µM, uncoupling agent) (thus, oligo-FCCP) on its own caused only slight cell cytotoxicity but strongly potentiated tetrandrine cytotoxicity (tetrandrine LC50 = 15.6 µM). The stronger enhancing effect of oligo-FCCP than 2-DG on TET toxicity did not result from more severe overall ATP depletion, since both treatments caused a similar ATP level suppression. Neither oligo-FCCP nor 2-DG synergized with tetrandrine in decreasing mitochondrial membrane potential. TET on its own triggered reactive oxygen species (ROS) production, and oligo-FCCP, but not 2-DG, potentiated TET in causing ROS production. Taken together, our results suggest that inhibiting ATP production from mitochondria, but not from glycolysis, appears to be a very effective means in augmenting TET-triggered ROS production and hence toxicity in A549 cells.

Valproic acid inhibits ATP-triggered Ca2+ release via a p38-dependent mechanism in bEND.3 endothelial cells.

Valproic acid (VA) is currently used to treat epilepsy and bipolar disorder. It has also been demonstrated to promote neuroprotection and neurogenesis. Although beneficial actions of VA on brain blood vessels have also been demonstrated, the effects of VA on brain endothelial cell (EC) Ca2+ signaling are hitherto unreported. In this report, we examined the effects of VA on agonist-triggered Ca2+ signaling in mouse cortical bEND.3 EC. While VA (100 µm) did not cause an acute inhibition of ATP-triggered Ca2+ signaling, a 30-min VA treatment strongly suppressed ATP-triggered intracellular Ca2+ release; however, such treatment did not affect Ca2+ release triggered by cyclopiazonic acid, an inhibitor of SERCA Ca2+ pump, suggesting there was no reduction in Ca2+ store size. VA-activated p38 signaling, and VA-induced inhibition of ATP-triggered Ca2+ release was prevented by SB203580, a p38 inhibitor, suggesting VA caused the inhibition by activating p38. Remarkably, VA treatment did not affect acetylcholine-triggered Ca2+ release, suggesting VA may not inhibit inositol 1,4,5-trisphosphate-induced Ca2+ release per se, and may not act directly on Gq or phospholipase C. Taken together, our results suggest VA treatment, via a p38-dependent mechanism, led to an inhibition of purinergic receptor-effector coupling.

Eicosapentaenoic acid triggers Ca2+ release and Ca2+ influx in mouse cerebral cortex endothelial bEND.3 cells.

Eicosapentaenoic acid (EPA), an omega-3 fatty acid abundant in fish oil, protects endothelial cells (EC) from lipotoxicity and triggers EC NO release. The latter is related to an elevation of cytosolic Ca2+. Although EPA has been shown to cause human EC cytosolic Ca2+ elevation, the mechanism is unclear. Microfluorimetric imaging was used here to measure free cytosolic Ca2+ concentration. EPA was shown to cause intracellular Ca2+ release in mouse cerebral cortex endothelial bEND.3 cells; interestingly, the EPA-sensitive intracellular Ca2+ pool(s) appeared to encompass and was larger than the Ca2+ pool mobilized by sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition by cyclopiazonic acid. EPA also opened a Ca2+ influx pathway pharmacologically distinct from store-operated Ca2+ influx. Surprisingly, EPA-triggered Ca2+ influx was Ni2+-insensitive; and EPA did not trigger Mn2+ influx. Further, EPA-triggered Ca2+ influx did not involve Na+-Ca2+ exchangers. Thus, our results suggest EPA triggered unusual mechanisms of Ca2+ release and Ca2+ influx in EC.