Calcium influx inhibits MT1-MMP processing and blocks MMP-2 activation

We have previously reported that concanavalin A (ConA)-induced MMP-2 activation involves both transcriptional and non-transcriptional mechanisms. Here we examined the effects of calcium influx on MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. The calcium ionophore ionomycin caused a dose-dependent inhibition of ConA-induced MMP-2 activation, but had no effect on MT1-MMP mRNA levels. However, Western analysis revealed an accumulation of pro-MT1-MMP (63 kDa), indicating that ionomycin blocked the conversion of pro-MT1-MMP protein to the active 60 kDa form. This suggests that increased calcium levels inhibit the processing of MT1-MMP. This finding may help to elucidate the mechanism(s) which regulates MT1-MMP activation.

Role of calcium influx through voltage-operated calcium channels and of calcium mobilization in the physiology of Schistosoma mansoni muscle contractions

We tested the hypothesis that voltage-operated Ca2+ channels mediate an extracellular Ca2+ influx in muscle fibres from the human parasite Schistosoma mansoni and, along with Ca2+ mobilization from the sarcoplasmic reticulum, contribute to Muscle contraction. Indeed, whole-cell voltage clamp revealed voltage-gated inward currents carried by divalent ions with a peak current elicited by steps to + 20 mV (from a holding potential of -70 mV). Depolarization of the fibres by elevated extracellular K+ elicited contractions that were completely dependent on extracellular Ca2+ and inhibited by nicardipine (half inhibition at 4(.)1 mu M). However these contractions were not very sensitive to other classical blockers of voltage-gated Ca2+ channels, indicating that the schistosome Muscle channels have an atypical pharmacology when compared to their mammalian counterparts. Furthermore, the contraction induced by 5 mM caffeine was inhibited after depletion of the sarcoplasmic reticulum either with thapsigargin (10 mu M) or ryanodine (10 mu M). These data suggest that voltage-operated Ca2+ channels docontribute to S. mansoni contraction as does the mobilization of stored Ca2+, despite the small volume of sarcoplasmic reticulum in schistosome smooth muscles.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurriagomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Glutamate-induced potentiation of calcium influx in primary hippocampal culture neurons

ABSTRACT This works aim was to test whether LTP-like features can also be measured in cell culture and by methods that allow to analyse a alrger number of cells. A suitable method for this purpose is calcium imaging. The rationale for this approach lies in the fact that LTP/LTD are dependent on changes in intracellular calcium concentrations. Calcium levels have been measured using the calcium sensitive dye fura-2, whose fluorescence spectrum changes upon formation of the [fura-2-Ca2+] complex. Our LTP-inducing protocol comprised of two glutamate stimuli of identical size and duration (50 mM, 30 s) which were separated by 35 min. We could demonstrate that such a stimulation pattern gives rise to approx. 25% larger calcium influx at the second stimulus. It has been shown than such a stimulation pattern gives rise to an average of 25% augmentation (potentiation) of the second response, with 69% of potentiated cells. This experimental paradigm shows the pharmacological properties of LTP, established by previous electrophysiological studies:- blocking of NMDARs and mGluRs eliminates LTP induction;- blocking of AMPARs and L-type VGCCs does not eliminate LTP induction. Having obtained a system for induction and following of LTP-like changes, a preliminary application example was performed. Its purpose was to investigate possible influence of nicotine and galanthamine on our potentiation effect. Nicotine (100 mM) was shown both to increase and to eliminate glutamate-induced potentiation. Galanthamine coapplication (0.5 mM) with nicotine and glutamate exerted no effect on nicotinic modulation. However, galanthamine coapplied with glutamate alone seems to augment glutamate-induced potentiation. An LTP model system presented here could be additionally refined, by variation of glutamate application times, and testing for dependence on various forms of protein kinases. Galanthamine effect would probably be better addressed by cell-to-cell measurements instead of statistical approach, with subsequent identification of the cell type. Alternatively, combined calcium imaging – electrophysiological experiments could be performed. Spatial and temporal properties of intracellular ion dynamics could be utilised as diagnostic tools of the physiological state of the cells, thereby finding its application in functional proteomics.

Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors

During illumination, Ca2+ enters fly photoreceptor cells through light-activated channels that are located in the rhabdomere, the compartment specialized for phototransduction. From the rhabdomere, Ca2+ diffuses into the cell body. We visualize this process by rapidly imaging the fluorescence in a cross section of a photoreceptor cell injected with a fluorescent Ca2+ indicator in vivo. The free Ca2+ concentration in the rhabdomere shows a very fast and large transient shortly after light onset. The free Ca2+ concentration in the cell body rises more slowly and displays a much smaller transient. After ≈400 ms of light stimulation, the Ca2+ concentration in both compartments reaches a steady state, indicating that thereafter an amount of Ca2+, equivalent to the amount of Ca2+ flowing into the cell, is extruded. Quantitative analysis demonstrates that during the steady state, the free Ca2+ concentration in the rhabdomere and throughout the cell body is the same. This shows that Ca2+ extrusion takes place very close to the location of Ca2+ influx, the rhabdomere, because otherwise gradients in the steady-state distribution of Ca2+ should be measured. The close colocalization of Ca2+ influx and Ca2+ extrusion ensures that, after turning off the light, Ca2+ removal from the rhabdomere is faster than from the cell body. This is functionally significant because it ensures rapid dark adaptation.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca2+ entry through voltage-sensitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca2+. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca2+ response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca2+-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants

In this paper, we report direct measurement of an influx of extracellular Ca2+ induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca2+-selective vibrating probe. Ca2+ influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca2+ influx in the vicinity of the sperm entry site with a delay of 1.8 ± 0.6 sec. The Ca2+ influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 μm⋅sec−1. Once established, Ca2+ influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol⋅cm−2⋅sec−1 and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca2+ (c[Ca2+]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca2+ ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca2+ influx. In turn, c[Ca2+] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.

Increased calcium influx mediates increased cardiac stiffness in hyperthyroid rats

Cardiac remodeling (hypertrophy and fibrosis) and an increased left ventricular diastolic stiffness characterize models of hypertension such as the SHR and DOCA-salt hypertensive rats. By contrast, hyperthyroidism induces hypertrophy and hypertension, yet collagen expression and deposition is unchanged or decreased, whereas diastolic stiffness is increased. We determined the possible role of increased calcium influx in the development of increased diastolic stiffness in hyperthyroidism by administering verapamil (15 mg/[kg(.)d] orally) to rats given triiodothyronine (T-3) (0.5 mg/[kg.d] subcutaneously for 14 d). Administration of T3 significantly increased body temperature (control: 36.7 +/- 0.2 degrees C; T-3: 39.6 +/- 0.2 degrees C), left ventricular wet weight (control: 2.09 +/- 0.02 mg/kg; T-3 3.07 +/- 0.07 mg/kg), systolic blood pressure (control: 128 +/- 5 mmHg; T-3: 156 +/- 4 mmHg), and left ventricular diastolic stiffness (control: 20.6 +/- 2.0; T-3: 28.8 +/- 1.4). Collagen content of the left ventricle was unchanged. Contractile response to noradrenaline in thoracic aortic rings was reduced. Relaxation in response to acetylcholine (ACh) was also reduced in T-3-treated rats, whereas sodium nitroprusside response was unchanged. Verapamil treatment of hyperthyroid rats completely prevented the increased diastolic stiffness and systolic blood pressure while attenuating the increased body temperature and left ventricular weight; collagen content remained unchanged. ACh response in thoracic aortic rings was restored by verapamil. Thus, in hyperthyroid rats, an increased calcium influx is a potential mediator of the increased diastolic stiffness independent of changes in collagen.

The effect of cis-5,8,11,14,17-eicosapentaenoic acid upon the contractile mechanisms linked to calcium influx and the mobilisation of intracellular calcium in aortic smooth muscle

Epidemiological studies previously identified cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as the biologically active component of fish oil of benefit to the cardiovascular system. Although clinical investigations demonstrated its usefulness in surgical procedures, its mechanism of action still remained unclear. It was shown in this thesis, that EPA partially blocked the contraction of aortic smooth muscle cells to the vasoactive agents KCl and noradrenaline. The latter effect was likely caused by reducing calcium influx through receptor-operated channels, supporting a recent suggestion by Asano et al (1997). Consistently, EPA decreased noradrenaline-induced contractures in aortic tissue, in support of previous reports (Engler, 1992b). The observed effect of EPA on cell contractions to KCl was not simple due to blocking calcium influx through L-type channels, consistent with a previous suggestion by Hallaq et al (1992). Moreover, EPA caused a transient increase in [Ca2+]i in the absence of extracellular calcium. To resolve this it was shown that EPA increased inositol phosphate formation which, it is suggested, caused the release of calcium from an inositol phosphate-dependent internal binding site, possibly that of an intracellular membrane or superficial sarcoplasmic reticulum, producing the transient increase in [Ca2+]i. As it was shown that the cellular contractile filaments were not desensitised to calcium by EPA, it is suggested that the transient increase in [Ca2+]i subsequently blocks further cell contraction to KCl by activating membrane-associated potassium channels. Activation of potassium channels induces the cellular efflux of potassium ions, thereby hyperpolarising the plasma membrane and moving the membrane potential farther from the activation range for calcium channels. This would prevent calcium influx in the longer term and could explain the initial observed effect of EPA to block cell contraction to KCl.

Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.

Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone ‘Norgestrel’ as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24h. Specific PGRMC1 inhibition by AG205 (1 μM) showed this rise to be PGRMC1-dependent, primarily utilising calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μM) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells in vitro.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

A Calcium-Dependent Plasticity Rule for HCN Channels Maintains Activity Homeostasis and Stable Synaptic Learning

Theoretical and computational frameworks for synaptic plasticity and learning have a long and cherished history, with few parallels within the well-established literature for plasticity of voltage-gated ion channels. In this study, we derive rules for plasticity in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and assess the synergy between synaptic and HCN channel plasticity in establishing stability during synaptic learning. To do this, we employ a conductance-based model for the hippocampal pyramidal neuron, and incorporate synaptic plasticity through the well-established Bienenstock-Cooper-Munro (BCM)-like rule for synaptic plasticity, wherein the direction and strength of the plasticity is dependent on the concentration of calcium influx. Under this framework, we derive a rule for HCN channel plasticity to establish homeostasis in synaptically-driven firing rate, and incorporate such plasticity into our model. In demonstrating that this rule for HCN channel plasticity helps maintain firing rate homeostasis after bidirectional synaptic plasticity, we observe a linear relationship between synaptic plasticity and HCN channel plasticity for maintaining firing rate homeostasis. Motivated by this linear relationship, we derive a calcium-dependent rule for HCN-channel plasticity, and demonstrate that firing rate homeostasis is maintained in the face of synaptic plasticity when moderate and high levels of cytosolic calcium influx induced depression and potentiation of the HCN-channel conductance, respectively. Additionally, we show that such synergy between synaptic and HCN-channel plasticity enhances the stability of synaptic learning through metaplasticity in the BCM-like synaptic plasticity profile. Finally, we demonstrate that the synergistic interaction between synaptic and HCN-channel plasticity preserves robustness of information transfer across the neuron under a rate-coding schema. Our results establish specific physiological roles for experimentally observed plasticity in HCN channels accompanying synaptic plasticity in hippocampal neurons, and uncover potential links between HCN-channel plasticity and calcium influx, dynamic gain control and stable synaptic learning.

Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.