Increase In Intracellular Free Ca2 Research Articles

Endothelin-1 enhances proliferation of lung cancer cells by increasing intracellular free Ca 2+

Endothelin-1 (ET-1), the most potent vasoconstrictor, has been shown to be mitogenic in many tumor cells as well as in vascular cells. It was previously reported that the mRNA of ET-1 and endothelin receptors (ETRs) are expressed in lung cancer cells. However, their biological role in lung cancer remains to be explored. The purpose of this study was to determine whether ET-1 stimulates proliferation of the human lung adenocarcinoma cell SPC-A1 and probe its cellular mechanism. Reverse-transcription polymerase chain reaction and Western blot analysis showed that both the mRNA and protein of ET-1, ET AR and ET BR are expressed in SPC-A1 cells. Application of ET-1 at 10 − 15 –10 − 8 M caused a dose-dependent cell proliferation and an increase in intracellular free Ca 2+ concentration ([Ca 2+] i). This ET-1-induced cell proliferation and [Ca 2+] i increase were completely abolished by BQ123, a selective ET AR antagonist, but not by BQ788, a selective ET BR antagonist. Furthermore, it was significantly reduced by U73122, a specific inhibitor of phospholipase C (PLC), but not by U73433, the structural isomer of U73122. Chelating extracellular Ca 2+ or blocking voltage dependent calcium channels by nifedipine also significantly reduced the mitogenic effect of ET-1 and [Ca 2+] i increase in SPC-A1 cells. These results indicate that ET-1 acts as an autocrine growth factor and enhances proliferation of SPC-A1 cells via activation of ET AR. The phosphoinositol/Ca 2+ pathway and Ca 2+ influx through voltage dependent Ca 2+ channels activated by ET AR contribute to this process.

Development and Validation of a Cell-Based High-Throughput Screening Assay for TRPM2 Channel Modulators

TRPM2 is a member of the transient receptor potential melastatin (TRPM)-related ion channel family. The activation of TRPM2 induced by oxidative/nitrosative stress leads to an increase in intracellular free Ca(2+). Although further progress in understanding TRPM2's role in cell and organism physiology would be facilitated by isolation of compounds able to specifically modulate its function in primary cells or animal models, no cell-based assays for TRPM2 function well suited for high-throughput screening have yet been described. Here, a novel suspension B lymphocyte cell line stably expressing TRPM2 was used to develop a cell-based assay. The assay uses the Ca(2+)-sensitive fluorescence dye, Fluo-4 NW (no wash), to measure TRPM2-dependent Ca(2+) transients induced by H(2)O(2) and N-methyl-N'-nitrosoguanidine in a 96-well plate format. Assay performance was evaluated by statistical analysis of the Z' factor value and was consistently greater than 0.5 under optimal conditions, suggesting that the assay is very robust. For assay validation, the effects of known inhibitors of TRPM2 and TRPM2 gating secondary messenger production were determined. Overall, the authors have developed a cell-based assay that may be used to identify TRPM2 ion channel modulators from large compound libraries.

Abstract 674: Coupling Factor 6 Activates c-Src By Intracellular Acidosis And Enhances Calcium Signaling In Resistance Arteriole Smooth Muscle Cells

In vascular endothelial cells, coupling factor 6 (CF6) binds to the β subunit of plasma membrane ATP synthase and attenuates prostacyclin generation by inducing intracellular acidosis. However, intracellular signaling in resistance arterioles that is directly related to vascular tonus and hypertension has not been determined yet. We investigated the direct effect of CF6 on Ca 2+ signaling, involvement of c-Src in it, and the role of CF6 in the pathogenesis of hypertension. CF6 at 10 −7 M induced a monophasic increase in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in A7r5 cells (rat vascular smooth muscle cells (VSMC) expressing voltage-gated Ca 2+ channel). When CF6 at 10 −7 M was added after pretreatment with nifedipine at 10 −5 M, the sustained increase in [Ca 2+ ] i was abolished. In cultured resistance arteriole VSMC obtained from the mesenteric artery network of spontaneously hypertensive rats (SHR, n = 8) and Wistar Kyoto rats (WKY, n = 8), angiotensin II (Ang II) at 10 −7 M caused a spike increase in [Ca 2+ ] i followed by a sustained increase. When Ang II at 10 −7 M was added after pre-administration of CF6 at 10 −7 M, the spike phase of [Ca 2+ ] i was enhanced and it was greater in SHR than in WKY (412 ± 26 vs 162 ± 14 nM, p < 0.05). Pretreatment with PP1 at 50 μM, a c-Src inhibitor, blocked not only the CF6-induced Ca 2+ influx but the CF6-induced increase in the AngII-mediated spike phase of [Ca 2+ ] i . CF6 receptor of VSMC was the β-subunit of ATP synthase, and the affinity was higher in SHR than in WKY (Kd: 3.6 ± 0.3 vs 9.6 ± 0.3 nM, p < 0.05). The increase in ATPase activity by CF6 was greater in SHR than in WKY (p < 0.05), and the decrease in intracellular pH was greater in SHR than in WKY (0.26 ± 0.03 vs 0.13 ± 0.02, p < 0.05). CF6 activated c-Src at 15 minutes by 2 folds greater in SHR than in WKY, and it was blocked by efrapeptine at 10 −5 M, an ATPase inhibitor. In mesenteric arterioles obtained from CF6-transgenic mice (2 fold over-expressed), AngII caused greater vasoconstriction by 2 folds than in those from wild mice (p < 0.05), but it was abolished by pretreatment with PP1. These suggest that CF6 activates c-Src by inducing intracellular acidosis and enhances Ca 2+ signaling in resistance arteriole VSMC. Since the c-Src activation was enhanced in SHR, CF6 might be involved in the genesis of hypertension.

The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted

In pulmonary arterial hypertension (PAH), antiapoptotic, proliferative, and inflammatory diatheses converge to create an obstructive vasculopathy. A selective down-regulation of the Kv channel Kv1.5 has been described in human and animal PAH. The resultant increase in intracellular free Ca(2+) ([Ca(2+)](i)) and K(+) ([K(+)](i)) concentrations explains the pulmonary artery smooth muscle cell (PASMC) contraction, proliferation and resistance to apoptosis. The recently described PASMC hyperpolarized mitochondria and increased bcl-2 levels also contribute to apoptosis resistance in PAH. The cause of the Kv1.5, mitochondrial, and inflammatory abnormalities remains unknown. We hypothesized that these abnormalities can be explained in part by an activation of NFAT (nuclear factor of activated T cells), a Ca(2+)/calcineurin-sensitive transcription factor. We studied PASMC and lungs from six patients with and four without PAH and blood from 23 PAH patients and 10 healthy volunteers. Compared with normal, PAH PASMC had decreased Kv current and Kv1.5 expression and increased [Ca(2+)](i), [K(+)](i), mitochondrial potential (Delta Psi m), and bcl-2 levels. PAH but not normal PASMC and lungs showed activation of NFATc2. Inhibition of NFATc2 by VIVIT or cyclosporine restored Kv1.5 expression and current, decreased [Ca(2+)](i), [K(+)](i), bcl-2, and Delta Psi m, leading to decreased proliferation and increased apoptosis in vitro. In vivo, cyclosporine decreased established rat monocrotaline-PAH. NFATc2 levels were increased in circulating leukocytes in PAH versus healthy volunteers. CD3-positive lymphocytes with activated NFATc2 were seen in the arterial wall in PAH but not normal lungs. The generalized activation of NFAT in human and experimental PAH might regulate the ionic, mitochondrial, and inflammatory remodeling and be a therapeutic target and biomarker.

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca2+ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell-endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell-ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell-cell contact region, and thus inhibits cell-cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca2+ -CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell-cell contact regions might be essential for this process under hypoxic stress.

Proteolytic Degradation of SCOP in the Hippocampus Contributes to Activation of MAP Kinase and Memory

Because activation of ERK1/2 MAP kinase (MAPK) is critical for hippocampus-dependent memory, there is considerable interest in mechanisms for regulation of MAPK during memory formation. Here we report that MAPK and CREB-mediated transcription are negatively regulated by SCOP (suprachiasmatic nucleus [SCN] circadian oscillatory protein) and that SCOP is proteolyzed by calpain when hippocampal neurons are stimulated by brain-derived neurotrophic factor (BDNF), KCl depolarization, or NMDA. Moreover, training for novel object memory decreases SCOP in the hippocampus. To determine if hippocampus-dependent memory is influenced by SCOP in vivo, we generated a transgenic mouse strain for the inducible overexpression of SCOP in the forebrain. Overexpression of SCOP completely blocked memory for novel objects. We conclude that degradation of SCOP by calpain contributes to activation of MAPK during memory formation.

Na+-dependent Inactivation of the Retinal Cone/Brain Na+/Ca2+-K+ Exchanger NCKX2

The SLC24 gene family Na+/Ca2+-K+ exchangers (NCKX) are bidirectional plasma membrane transporters whose main function is the extrusion of Ca2+ from the cytosol. In this study, we used human embryonic kidney 293 cells expressing human retinal cone/brain NCKX2 to examine its Na+ affinity and kinetic parameters of Ca2+ transport. With the use of the ionophore gramicidin to control alkali cation concentrations across the plasma membrane, application of high intracellular Na+ promoted large NCKX2-mediated increases in intracellular free Ca2+ in the 15-20 microm range; this also resulted in inactivation of NCKX2 transport, the first description of this novel kinetic state. The affinity of NCKX2 for internal Na+ was found to be sigmoidal, with a Hill coefficient of 2.6 and Kd = 50 mm. The time-dependent (t(1/2) approximately 40s) inactivation of NCKX2 required high intracellular Na+ levels (Kd > 50 mm) as well as high occupancy of the extracellular Ca2+-binding site. Also reported are two residues whose substitution resulted in an increase in internal Na+ affinity to values of approximately 19 mm; these mutants also displayed enhanced inactivation, suggesting that inactivation requires binding of Na+ to its intracellular transport sites. These findings are the first report of a regulatory kinetic state of Ca2+ transport via NCKX2 Na+/Ca2+-K+ exchangers that may play a prominent role in regulation of Ca2+ extrusion in cellular environments such as neuronal synapses that experience frequent and dynamic Ca2+ fluxes.

Inhibitory Effects of Thunberginols A, B, and F on Degranulations and Releases of TNF-.ALPHA. and IL-4 in RBL-2H3 Cells

Thunberginols A, B, and F from the processed leaves of Hydrangea macrophylla var. thunbergii (Hydrangeae Dulcis Folium) substantially inhibited the degranulations by antigen and calcium ionophore A23187, and the releases of TNF-alpha and IL-4 by antigen in RBL-2H3 cells. Phyllodulcin and hydrangenol also showed significant inhibition for the antigen-induced degranulations, but their effects were weaker than those of thunberginols A, B, and F. Among them, thunberginol B showed the most potent activity. With regard to structural requirements of thunberginols for the activity, the 3,4-double bond was essential for the strong activity and the 6-hydroxyl group and lactone ring enhanced the activity. Thunberginols A, B, and F inhibited increase in intracellular free Ca2+ levels, which is an essential process for the degranulation and production of cytokines, in RBL-2H3 cells induced by antigen, but not by calcium ionophore A23187. These results suggested that these active compounds inhibited the degranulation processes both before and after increase in intracellular free Ca2+ levels.

Currently Evaluated Calpain and Caspase Inhibitors for Neuroprotection in Experimental Brain Ischemia

Currently available therapies for brain ischemia, with a few exceptions, provide only symptomatic relief in patients. Recent investigations in experimental models provided an understanding of the cellular and molecular mechanisms that lead to neurodegeneration in ischemic injury, and also indicate targets for prevention and amelioration of the devastating consequences of stroke. An enormous increase in intracellular free Ca(2+) levels following stroke activates Ca(2+)-dependent enzymes, contributing to neuronal death and dysfunction. Additionally, ischemic injury generates highly reactive free radicals and triggers release of cytotoxic cytokines for activation of cysteine proteases. A number of studies already indicated a prominent role for the cysteine proteases of the calpain and caspase families in the pathogenesis of brain ischemia. Proteolytic activities of these proteases degrade various cytoskeletal proteins and membrane proteins, destabilizing the structural integrity and forcing the neurons to delayed death in ischemic penumbra. Some current studies have unequivocally confirmed the neuronal apoptosis in ischemia and showed that administration of calpain and caspase inhibitors alone or in combination can provide functional neuroprotection in various animal models of cerebral ischemia. This article will discuss the molecular structures and activities of calpain and caspase inhibitors and their therapeutic efficacy in experimental brain ischemia. However, further investigations are necessary for improvements in the structural design of calpain and caspase inhibitors for their persistent therapeutic efficacy in animal models of stroke and for clinical trials in the future.

The Orexin OX1Receptor Regulates Ca2+Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells

We studied the cellular response to orexin type 1 receptor (OX1R) stimulation in differentiated IMR-32 neuroblastoma cells. In vitro differentiation of IMR-32 cells with 5-bromo-2'-deoxyuridine leads to a neuronal phenotype with long neurite extensions and an upregulation of mainly N-type voltage-gated calcium channels. Transduction of differentiated IMR-32 cells with baculovirus harboring an OX1R-green fluorescent protein cDNA fusion construct resulted in appearance of fluorescence that was confined mainly to the plasma membrane in the cell body and to neurites. Application of orexin-A to fluorescent cells led to an increase in intracellular free Ca2+ concentration, [Ca2+]i. At low nanomolar concentrations of orexin-A, the response was reversibly attenuated by removal of extracellular Ca2+, by application of a high concentration (10 mM) of Mg2+, and by the pharmacological channel blocker dextromethorphan. A diacylglycerol, dioctanoylglycerol, but not thapsigargin or depolarization with potassium, mimicked the OX1R response with regard to Mg2+ sensitivity. A reverse transcription-PCR screening identified mRNAs for all transient receptor potential canonical (TRPC) channels, including TRPC3, TRPC6, and TRPC7, which are known to be activated by diacylglycerol. Expression of a dominant-negative TRPC6 channel subunit blunted the responses to both dioctanoylglycerol and OX1R stimulation. The results suggest that the OX1R activates a Ca2+ entry pathway that involves diacylglycerol-activated TRPC channels in neuronal cells.

Ca 2+ microdomains and the control of insulin secretion

Nutrient-induced increases in intracellular free Ca 2+ concentrations are the key trigger for insulin release from pancreatic islet β-cells. These Ca 2+ changes are tightly regulated temporally, occurring as Ca 2+ influx-dependent baseline oscillations. We explore here the concept that locally high [Ca 2+] concentrations (i.e. Ca 2+ microdomains) may control exocytosis via the recruitment of key effector proteins to sites of exocytosis. Importantly, recent advances in the development of organelle- and membrane-targeted green fluorescent protein (GFP-) or aequorin-based Ca 2+ indicators, as well as in rapid imaging techniques, are providing new insights into the potential role of these Ca 2+ microdomains in β-cells. We summarise here some of the evidence indicating that Ca 2+ microdomains beneath the plasma membrane and at the surface of large dense core vesicles may be important in the normal regulation of insulin secretion, and may conceivably contribute to “ATP-sensitive K +-channel independent” effects of glucose. We also discuss evidence that, in contrast to certain non-excitable cells, direct transfer of Ca 2+ from the ER to mitochondria via localised physical contacts between these organelles is relatively less important for efficient mitochondrial Ca 2+ uptake in β-cells. Finally, we discuss evidence from single cell imaging that increases in cytosolic Ca 2+ are not required for the upstroke of oscillations in mitochondrial redox state, but may underlie the reoxidation process.

Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta

The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone, associated with capacitative Ca(2+) entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca(2+)-free solution. The incubation with estradiol (1-100 nM, 5 min) inhibited both the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 microM; inhibitor of protein synthesis) nor tamoxifen (1 microM; blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 microM) also blocked the contractile response to serotonin (10 microM) but not to caffeine (10 mM). In addition, estradiol (100 microM) inhibited the contractile responses to cyclopiazonic acid (1 microM; selective Ca(2+)-ATPase inhibitor) associated with capacitative Ca(2+) influx through non-L-type Ca(2+) channels. Finally, estradiol inhibited the Ca(2+)-induced increases in intracellular free Ca(2+) (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca(2+)-free solution. In conclusion, estradiol interfered in a concentration-dependent manner with Ca(2+)-dependent contractile effects mediated by the stimuli of alpha(1)-adrenergic and serotonergic receptors and inhibited the capacitative Ca(2+) influx through both L-type and non-L-type Ca(2+) channels. Such effects are in essence nongenomic and not mediated by the intracellular estrogenic receptor.

HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection

The chemokine receptors CCR5 and CXCR4 serve, in addition to CD4, as coreceptors for human immunodeficiency virus-1 (HIV-1), and infection with HIV-1 can cause dementia. In brain-derived cells, HIV-1 envelope glycoprotein gp120 initiates a signaling cascade that involves p38 mitogen-activated protein kinase and leads to neuronal cell death. Using mixed neuronal/glial cultures from rats and mice genetically deficient in one or both HIV coreceptors, we show here that CCR5, CXCR4 or both can mediate HIV/gp120 neurotoxicity depending on the viral strain. Paradoxically, we also found evidence for a CCR5-mediated neuroprotective pathway. We identify protein kinase Akt/PKB as an essential component of this pathway, which can be triggered by the CCR5 agonists macrophage inflammatory protein-1beta and regulated-and-normal-T-cell-expressed-and-secreted. Moreover, these CCR5 ligands prevent neuronal cell death induced by stromal cell-derived factor-1, a CXCR4 agonist. Both neurons and glia coexpress CXCR4 and CCR5. Ca2+ imaging experiments demonstrate that engagement of CCR5 prevents CXCR4-triggered increases in intracellular free Ca2+. This finding suggests that CCR5 ligands can protect neurons at least, in part, by modulating CXCR4-mediated toxicity through heterologous desensitization.

Mitochondrial Redox Control of Matrix Metalloproteinase Signaling in Resistance Arteries

The importance of free radicals in the regulation of vascular tone was recognized in the late 1980s, when endothelium-derived relaxing factor was identified to be NO.1,2 More recently, it has become clear that other free radicals, such as superoxide (O2−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-) also modulate vascular reactivity.3,4 However, the exact role of reactive oxygen species (ROS) in the regulation of vascular contraction and relaxation remains elusive because vascular effects of free radicals are heterogeneous. Responses may differ depending on the species studied, the vascular bed under investigation, whether the endothelium is intact or denuded, and if studies are conducted in vitro or in vivo.5,6 Furthermore, vascular actions of ROS may be direct or indirect and responses vary depending on the source of the free radicals and their site of compartmentalization. See page 819 Superoxide has been implicated both as a vasoconstrictor and as a vasodilator. In cerebral vessels, low concentrations of O2−, generated by NADPH oxidase or xanthine oxidase, induce vasodilation.7 These effects may be mediated directly by activating potassium channels or indirectly through interactions with NO.8 On the other hand, high concentrations of O2− are potent vasoconstrictors, probably induced by ROS-stimulated increase in intracellular free Ca2+ concentration ([Ca2+]i) or by increasing ONOO- production through interactions with NO.9,10 The inactivation of NO by superoxide results in the loss of the vasodilator effect of NO, which, together with the direct contractile actions of ONOO-, leads to vasoconstriction. Similar biphasic dose-dependent responses have been observed in aorta and resistance arteries11,12 and in cultured vascular smooth muscle cells.13 H2O2, which is lipid soluble and more stable than O2−, has been considered …

Caged Vanilloid Ligands for Activation of TRPV1 Receptors by 1- and 2-Photon Excitation

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.

Effect of PDBu on [alpha]1‐adrenoceptor‐mediated contraction and changes of intracellular free Ca2+ concentration in uterine artery of pregnant sheep

PKC activation by phorbol 12, 13-dibutyrate (PDBu) shows inhibitory effects on phenylephrine (PE)-induced contractions in pregnant uterine arteries. We hypothesize that PDBu inhibits PE-induced increases of intracellular free Ca2+ concentration ([Ca2+]i). Uterine arteries were isolated from near term pregnant ewes. Vascular smooth muscle [Ca2+]i was measured simultaneously with muscle contraction in the same tissue loaded with Fura-2 by using a CAF-110 intracellular Ca2+ analyzer. PE-induced contraction and simultaneously measured [Ca2+]i were compared between before and after PDBu treatment. Compared with before PDBu treatment, both 0.1 and 1 μM PDBu produced a significant decrease in PE-induced contraction and completely blocked PE-induced increases of [Ca2+]i as indicated by no changes of Rf340/380. These results suggested that PKC activation in pregnant uterine artery plays an inhibitory role in α1-adrenoceptor-mediated contraction through blocking the availability of free Ca2+ to the contractile proteins. (Supported in part by NIH grants HL57787 and HD 31226)

Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction

We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient (Cav-1(-/-)) mice and characterized these cells with respect to their proliferation, migration, and Ca(2+) response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2'-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in Cav-1(-/-) compared with wild-type (Cav-1(+/+)) AoSMCs. Cav-1(-/-) AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor p27(Kip1). The Ca(2+) response was examined in the presence of ET-1 and assessed by confocal microscopy with the Ca(2+)-sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-1(-/-) AoSMCs exhibited a faster and larger increase in free intracellular Ca(2+) than Cav-1(+/+) cells. The ET-1-induced response in Cav-1(-/-) cells was mediated by the ET(B) receptor, as shown using the ET(B) receptor antagonist BQ-788 and the ET(A) receptor antagonist BQ-123. In Cav-1(-/-) cells, ET(A) receptor expression was reduced and ET(B) receptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced Ca(2+) response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and Ca(2+)-mediated signaling.

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca 2+ was detected by fura-2 assay, suggesting activation of Ca 2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca 2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in ‘second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.’ Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.

Econazole induces increases in free intracellular Ca2+ concentrations in human osteosarcoma cells

Econazole is an antifungal drug with different in vitro effects. However, econazole's effect on osteoblast-like cells is unknown. In human MG63 osteosarcoma cells, the effect of econazole on intracellular Ca2+ concentrations ([Ca2+]i) was explored by using fura-2. At a concentration of 0.1 microM, econazole started to cause a rise in [Ca2+]i in a concentration-dependent manner. Econazole-induced [Ca2+]i rise was reduced by 74% by removal of extracellular Ca2+. The econazole-induced Ca2+ influx was mediated via a nimodipine-sensitive pathway. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca+ -ATPase, caused a [Ca2+]i rise, after which the increasing effect of econazole on [Ca2+]i was abolished. Pretreatment of cells with econazole to deplete Ca2+ stores totally prevented thapsigargin from releasing Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not econazole-induced, [Ca2+]i rise. Econazole inhibited 76% of thapsigargin-induced store-operated Ca2+ entry. These findings suggest that in MG63 osteosarcoma cells, econazole increases [Ca2+]i by stimulating Ca2+ influx and Ca2+ release from the endoplasmic reticulum via a phospholipase C-independent manner. In contrast, econazole acts as a potent blocker of store-operated Ca2+ entry.

Amyloid β‐peptide(1–42) and hydrogen peroxide‐induced toxicity are mediated by TRPM2 in rat primary striatal cultures

Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.