Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Suppression of Ca2+ influx by unfractionated heparin in non-excitable intact cells via multiple mechanisms

Effect of unfractionated heparin (UFH), described as a cell-impermeant IP3 receptor antagonist, was studied on the capacitive Ca(2+) entry in non-permeabilized, intact cells, measuring the intracellular Ca(2+) levels using fluorescence microplate technique. Ca(2+) influx induced via Ca(2+) mobilization by histamine in Hela cells or evoked by store depletion with thapsigargin in RBL-2H3 cells was dose-dependently suppressed by UFH added either before or after the stimuli. UFH also prevented the spontaneous Ba(2+) entry indicating that the non-capacitive Ca(2+) channels may also be affected. In addition, UFH caused a significant and dose-dependent delay in Ca(2+), and other bivalent cation inflow after treatment of the cells with Triton X-100, but it did not diminish the amount of these cations indicating that UFH did not act simply as a cation chelator, but modulated the capacitive Ca(2+) entry possibly via store operated Ca(2+) channels (SOCCs). Inhibitory activities of UFH and 2-aminoethyl diphenyl borate on the capacitive Ca(2+) influx was found reversible, but the time courses of their actions were dissimilar suggesting distinct modes of action. It was also demonstrated using a fluorescence potentiometric dye that UFH had a considerable hyperpolarizing effect and could alter the changes of membrane potential during Ca(2+) influx after store depletion by thapsigargin. We presume that the hyperpolarizing property of this agent might contribute to the suppression of Ca(2+) influx. We concluded that UFH can negatively modulate SOCCs and also other non-capacitive Ca(2+) channels and these activities might also account for its multiple biological effects.     

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.     

STIM1 but not STIM2 is an essential regulator of Ca influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

Extracellular Ca²⁺ entry, primarily mediated through store-operated Ca²⁺ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca²⁺ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca²⁺ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca²⁺ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca²⁺ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca²⁺ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca²⁺ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.     

BAFF activates Erk1/2 promoting cell proliferation and survival by Ca-CaMKII-dependent inhibition of PP2A in normal and neoplastic B-lymphoid cells

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca²⁺-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca²⁺-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca²⁺ may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.     

Distinct effects of N-ethylmaleimide on formyl peptide- and cyclopiazonic acid-induced Ca2+ signals through thiol modification in neutrophils

In this study, we demonstrate that N-ethylmaleimide (NEM), a cell permeable thiol-alkylating agent, enhanced the [Ca2+]i rise caused by stimulation with cyclopiazonic acid (CPA), a sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibitor, in rat neutrophils. In addition, NEM attenuated the formyl-Met-Leu-Phe (fMLP)-induced [Ca2+]i rise whether NEM was added to cells prior to or after fMLP stimulation. Moreover, application of NEM after fMLP activation in the absence of external Ca2+ inhibited the Ca2+ signal upon addition of Ca2+ to the medium. Similar patterns were also obtained by using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a cell impermeable dithiol-oxidizing agent, which replaced NEM in the CPA- and fMLP-induced [Ca2+]i rise experiments. Treatment with dithiothreitol (DTT), a cell permeable dithiol-reducing agent, N-acetyl-l-cysteine (NAC), a cell permeable monothiol-reducing agent, and tris-(2-carboxyethyl)phosphine (TCEP), a cell impermeable reductant without a thiol group, all rescued the fMLP-induced Ca2+ signal from NEM. Rat neutrophils express the mRNA encoding for transient receptor potential (TRP) C6, inositol trisphosphate receptor (IP3R) 2 and IP3R3. NEM had no effect on the mitochondrial membrane potential. NEM could restore the polarization and F-actin accumulation of fMLP-treated cells to those of the control. In the absence of external Ca2+, NEM rendered the CPA-induced [Ca2+]i elevation persistently but inhibited the fMLP-induced Ca2+ spike, which was reversed by tris-(2-cyanoethyl)phosphine (TCP), a cell permeable reductant without a thiol group. DTNB did not affect the Ca2+ spike caused by fMLP. These results indicate that through protein thiol oxidation, NEM affects the receptor-activated and the store depletion-derived Ca2+ signals in an opposing manner.     

Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB₁, CB₂) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ⁹-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids.     

TRPM7 channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3K and ERK pathways

TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl₄-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increase of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.     

Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1

Although cannabinoids exhibit a broad variety of anticarcinogenic effects, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel invasion assays we found a cannabidiol-driven impaired invasion of human cervical cancer (HeLa, C33A) and human lung cancer cells (A549) that was reversed by antagonists to both CB₁ and CB₂ receptors as well as to transient receptor potential vanilloid 1 (TRPV1). The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Knockdown of cannabidiol-induced TIMP-1 expression by siRNA led to a reversal of the cannabidiol-elicited decrease in tumor cell invasiveness, implying a causal link between the TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and p42/44 mitogen-activated protein kinases were identified as upstream targets conferring TIMP-1 induction and subsequent decreased invasiveness. Additionally, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls. Altogether, these findings provide a novel mechanism underlying the anti-invasive action of cannabidiol and imply its use as a therapeutic option for the treatment of highly invasive cancers.     

Characterization of endogenous calcium responses in neuronal cell lines

An increasing number of putative therapeutic targets have been identified in recent years for the treatment of neuronal pathophysiologies including pain, epilepsy, stroke and schizophrenia. Many of these targets signal through calcium (Ca²⁺), either by directly facilitating Ca²⁺ influx through an ion channel, or through activation of G proteins that couple to intracellular Ca²⁺ stores or voltage-gated Ca²⁺ channels. Immortalized neuronal cell lines are widely used models to study neuropharmacology. However, systematic pharmacological characterization of the receptors and ion channels expressed in these cell lines is lacking. In this study, we systematically assessed endogenous Ca²⁺ signaling in response to addition of agonists at potential therapeutic targets in a range of cell lines of neuronal origin (ND7/23, SH-SY5Y, 50B11, F11 and Neuro2A cells) as well as HEK293 cells, a cell line commonly used for over-expression of receptors and ion channels. This study revealed a remarkable diversity of endogenous Ca²⁺ responses in these cell lines, with one or more cell lines responding to addition of trypsin, bradykinin, ATP, nicotine, acetylcholine, histamine and neurotensin. Subtype specificity of these responses was inferred from agonist potency and the effect of receptor subtype specific antagonist. Surprisingly, HEK293 and SH-SY5Y cells responded to the largest number of agonists with potential roles in neuronal signaling. These findings have implications for the heterologous expression of neuronal receptors and ion channels in these cell lines, and highlight the potential of neuron-derived cell lines for the study of a range of endogenously expressed receptors and ion channels that signal through Ca²⁺.     

Increase of mesenchymal stem cell migration by cannabidiol via activation of p42/44 MAPK

Migration and differentiation of mesenchymal stem cells (MSCs) are known to be involved in various regenerative processes such as bone healing. However, little is known about the pharmacotherapeutical options aiming at the mobilization and differentiation of MSCs. The present study therefore focussed on cannabinoids which have been demonstrated to exhibit tissue healing properties. Using Boyden chamber assays, the non-psychoactive phytocannabinoid cannabidiol (CBD) was found to increase the migration of adipose-derived MSCs in a time- and concentration-dependent manner. CBD-induced migration was inhibited by AM-630 (CB₂ receptor antagonist) and O-1602 (G protein-coupled receptor 55 [GRP55] agonist). Moreover, the promigratory effect of CBD was antagonized by inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway which became activated upon CBD treatment. In line with this data, AM-630 and O-1602 attenuated CBD-induced p42/44 MAPK phosphorylation. A p42/44 MAPK-dependent promigratory effect was likewise demonstrated for the GPR55 antagonist O-1918 and the selective CB₂ receptor agonist JWH-133. Additional evidence for a functional effect of CBD on MSCs was provided by experiments demonstrating long-term stimulation with CBD to induce differentiation of MSCs into the osteoblastic lineage as evidenced by increased mineralization assessed by cresolphthalein complexone assay and enhanced activity of alkaline phosphatase. Collectively, this study demonstrates CBD to promote the migration of MSCs via activation of the CB₂ receptor and inhibition of GPR55 and to induce osteoblastic differentiation. CBD may therefore recruit MSCs to sites of calcifying tissue regeneration and subsequently support bone regeneration via an osteoanabolic action on MSCs.     

R-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone (WIN-2) ameliorates experimental autoimmune encephalomyelitis and induces encephalitogenic T cell apoptosis: partial involvement of the CB(2) receptor

Many reports have shown that cannabinoids might be beneficial in the symptomatic treatment of multiple sclerosis (MS). We have investigated the therapeutic properties of the non-selective cannabinoid receptor agonist WIN-2 as a suppressive drug in the experimental autoimmune encephalomyelitis (EAE) model of MS. In the passive variety of EAE, induced in Lewis rats by adoptive transfer of myelin-reactive T cells, WIN-2 ameliorates the clinical signs and diminishes the cell infiltration of the spinal cord. Due to the involvement of cannabinoids in the regulation of cell death and survival, we investigated the effects of WIN-2 on the encephalitogenic T cell population. WIN-2 induced a profound increase of apoptosis in a dose- and time-dependent manner. The potential involvement of cannabinoid receptors (CB) was investigated by encephalitogenic T cell stimulation in the presence of the CB(1) (SR141716A) and CB(2) (SR144528) antagonists, pertussis toxin (PTX) and the inactive enantiomer WIN-3. WIN-2-induced apoptosis was partially blocked by SR144528 and PTX, whereas, WIN-3 only exerted a mild effect on cell viability. These results point to the partial involvement of CB(2) receptor together with other receptor-independent mechanism or by yet unknown cannabinoid receptors. Moreover, WIN-2 induced the extrinsic pathway of apoptosis, as shown by caspase-10 and -3 activation. These results suggest that cannabinoid-induced apoptosis of encephalitogenic T cells may cooperate in their anti-inflammatory action in EAE models. The partial involvement of CB(2) receptors in WIN-2 action may open new therapeutic doors in the management of MS by non-psychoactive selective cannabinoid agonists.     

Role of endocannabinoid and glutamatergic systems in DOI-induced head-twitch response in mice

We previously reported that systemic administration of the endocannabinoid anandamide inhibited the head-twitches induced by the hallucinogenic drug 2,5-dimethoxy-4-iodoamphetamine (DOI) in mice, which is mediated via the activation of 5-HT_2A receptors. Endocannabinoid and glutamatergic systems have been suggested to modulate the function of 5-HT_2A receptors. In the present study, we further investigated the role of endocannabinoid and glutamatergic systems in DOI-induced head-twitch response in mice. An anandamide transport inhibitor AM404 (0.3-3 mg/kg, i.p.), a fatty acid amide hydrolase inhibitor URB597 (0.1-10 mg/kg, i.p.), a glutamate release inhibitor riluzole (0.3 and 1 mg/kg, i.p.), a natural glutamate analog l-glutamylethylamide (theanine, 1 and 3 mg/kg, p.o.) and an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist NBQX (0.01-0.3 mg/kg, i.p.) significantly inhibited DOI-induced head-twitch response. The AMPA receptor positive modulator aniracetam (30 or 100 mg/kg, p.o.) reversed inhibition of head-twitch response by NBQX and URB597. These findings indicated that endocannabinoid and glutamatergic systems participate in the mechanism of action of DOI to induce head-twitch response.