Search

Ca(2+) signalling is involved in virtually all cellular processes: among the others, it controls cell survival, proliferation and death regulating a plethora of intracellular enzymes located in the cytoplasm, nucleus and organelles. Changes in the cytosolic free Ca(2+) concentration may be due either to release from the intracellular Ca(2+) stores or to influx from the extracellular medium, through the opening of plasma membrane calcium-permeable channels. In particular, Ca(2+) entry from the extracellular space is a mechanism able to sustain long lasting intracellular Ca(2+) elevations: this signal, activated by many growth factors and mitogens in normal and tumoral tissues, is linked to DNA transcription and duplication, finally leading to cell proliferation. In the last years many informations have been provided about the transduction mechanisms related to Ca(2+) entry induced by mitogenic factors, mostly binding to tyrosine kinase receptors, but also to G-protein coupled ones. Nevertheless, some key points remain to be fully clarified: among them, the molecular structure of the Ca(2+) channels involved, their regulation by intracellular messengers, and the modes through which specificity is achieved. The increasing knowledge on Ca(2+) entry-dependent control of proliferation may provide a more satisfactory understanding of pathological alterations, including cancer progression and angiogenesis. A detailed description of the mechanisms that trigger Ca(2+) entry, and in particular the definition of calcium-permeable channels and their modulators at the molecular levels, will greatly improve our possibility to take advantage of Ca(2+) entry regulation as a therapeutic approach for the control of cell proliferation, designing antibodies or molecules with low side effects and specific channel blocker functions. The review will focus on this topic.

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation.

We have analyzed the expression and localization of bovine transient receptor potential-C1 (bTRPC1) in bovine aortic endothelial cells, and its possible involvement in the store-independent calcium influx induced by basic fibroblast growth factor (bFGF). RT-PCR experiments confirmed the existence of two btrpc1 mRNA isoforms; conversely, the btrpc3 gene was not transcribed. Anti-TRPC1 antibody revealed the presence of the protein in the membrane-rich compartment only. Application of anti-TRPC1 during the response to bFGF caused a partial but significant reduction of calcium entry. This is the first evidence of TRP channel involvement in a non-capacitative calcium influx induced by a biologically relevant agonist such as the angiogenic factor bFGF in native endothelial cells.

Basic fibroblast growth factor (bFGF), a peptide acting as a mitogen in different cell types, is able to induce a long lasting non capacitative calcium influx from the extracellular medium in Balb-c 3T3 mouse fibroblasts. This effect is mediated by the tyrosine kinase activity of bFGF receptors and the opening of voltage independent, agonist activated calcium channels. In this paper we investigate the signal transduction steps involved in this process using single cell calcium fluorimetry and electrophysiological techniques. One of the pathways initiated by the binding of growth factors to their tyrosine kinase receptors is the activation of cytosolic phospholipase A2 (cPLA2) and the release of arachidonic acid (AA) from the plasma membrane with the subsequent production of eicosanoids. We show here that, in our preparation, this pathway is involved in the opening of the bFGF-activated calcium permeable channels, through the activation of mitogen activated protein kinase (MAPK) and cPLA2. Evidence for direct involvement of AA is given by the finding that: (i) bFGF induces AA release from Balb-c 3T3 cells; (ii) blockers of AA metabolism are not effective; and (iii) the application of either arachidonic acid or its non metabolizable analogue 5,8,11,14-eicosatetraynoic acid (ETYA) reproduces the responses described for bFGF. Finally, single channel analysis indicates that bFGF, AA and ETYA can activate the same calcium permeable channel.

In order to investigate the action of basic fibroblast growth factor (bFGF) in the nervous system, we have studied the ionic signals elicited by this peptide in cultured quail mesencephalic neural crest neurons using patch-clamp and cytofluorimetric techniques. In this preparation stimulation with bFGF induced, with a delay of some tens of seconds, an inward cationic current. Single-channel experiments provided evidence for the activation of a calcium-permeable channel. In single-cell cytofluorimetric measurements, a sustained rise in [Ca2+]i was observed, which was dependent on the presence of external calcium. These events may play a role in the developmental effects of bFGF.

In quiescent Balb-c mouse 3T3 fibroblasts, the application of whole or dialyzed 10% foetal calf serum elicits a biphasic electrical response, consisting of a transient outward current, flowing through Ca(2+)-activated K+ channels, followed by an inward one, lasting up to 15 min. On the basis of experiments with ion substitutions and blockers, the inward current can be attributed to the opening of cationic channels permeable to Na+ and Ca2+ ions. This current could mediate the calcium influx involved in the sustained elevation of [Ca2+]i that has been observed in many preparations in response to mitogen stimulation and that is involved in triggering cell proliferation.

Abstract: Expression of the nerve cell phenotype is orchestrated by the REST/NRSF transcription repressor, working on hundreds of genes recognized at a specific regulatory binding sequence. Most PC12 clones, the most frequently employed neuronal model, maintain low levels of REST; however a few, defective of neurosecretion, express high levels. To investigate the role of REST in Ca2+ signalling we studied the [Ca2+]i changes in single cells of four clones, two wild-type and two defective, pre-treated for 5 days with NGF. We focused on Ca2+ influxes induced by depolarization and ATP. Only a subpopulation (∼15%) of the defective, high REST cells responded to depolarization (CaV expression ∼10%). The ATP-induced intracellular Ca2+ release was little changed, whereas influx via ionotropic P2X receptors was decreased, in agreement with the decreased expression of P2X2 receptors. The percentage of defective cells expressing store-operated calcium entry (SOCE) following ATP stimulation was also lower. The responses of the defective clones were little affected by their differentiated state. In conclusion, our results revealed important new aspects of REST control of Ca2+ homeostasis, of potential physiological importance. The mechanisms of this control remain to be investigated. [Copyright &y& Elsevier]

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels (VGCCs) and estrogen receptors are important cellular proteins that have been shown to interact with each other across varied cells and tissues. Estrogen hormone, the ligand for estrogen receptors, can also exert its effects independent of estrogen receptors that collectively constitute non-genomic mechanisms. Here, we provide insights into the VGCC regulation by estrogen and the possible mechanisms involved therein across several cell types. Notably, most of the interaction is described in neuronal and cardiovascular tissues given the importance of VGCCs in these electrically excitable tissues. We describe the modulation of various VGCCs by estrogen known so far in physiological conditions and pathological conditions. We observed that in most in vitro studies higher concentrations of estrogen were used while a handful of in vivo studies used meager concentrations resulting in inhibition or upregulation of VGCCs, respectively. There is a need for more relevant physiological assays to study the regulation of VGCCs by estrogen. Additionally, other interacting receptors and partners need to be identified that may be involved in exerting estrogen receptor-independent effects of estrogen. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels (VGCCs) are important mediators of pain hypersensitivity during inflammatory states, but their role in sensory nerve growth remains underexplored. Here, we assess the role of the N-type calcium channel Cav2.2 in the complete Freund's adjuvant (CFA) model of inflammatory pain. We demonstrate with in situ hybridization and immunoblotting, an increase in Cav2.2 expression after hind paw CFA injection in sensory neurons that respond to thermal stimuli, but not in two different mechanosensitive neuronal populations. Further, Cav2.2 upregulation post-CFA correlates with thermal but not mechanical hyperalgesia in behaving mice, and this hypersensitivity is blocked with a specific Cav2.2 inhibitor. Voltage clamp recordings reveal a significant increase in Cav2.2 currents post-CFA, while current clamp analyses demonstrate a significant increase in action potential frequency. Moreover, CFA-induced sensory nerve growth, which involves the extracellular signal-related kinase (ERK1/2) signaling pathway and likely contributes to inflammation-induced hyperalgesia, was blocked with the Cav2.2 inhibitor. Together, this work uncovers a role for Cav2.2 during inflammation, demonstrating that VGCC activity can promote thermal hyperalgesia through both changes in firing rates of sensory neurons as well as promotion of new neurite outgrowth. [ABSTRACT FROM AUTHOR]

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 24 h. Specific PGRMC1 inhibition by AG205 (1 μ m) showed this rise to be PGRMC1-dependent, primarily utilizing calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μ m) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for si RNA 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. [ABSTRACT FROM AUTHOR]

Axonal guidance and synaptic specification depends on specific signaling mechanisms that occur in growth cones. While several signaling pathways implicated in cone navigation have been identified, membrane dynamics in growth cones remains largely unknown. We took advantage of SynaptopHluorin and high-speed optical recordings to monitor the patterns of membrane dynamics in rat hippocampal growth cones. We show that exocytosis occurs both at the peripheral and central domains, including filopodia, and that SynaptopHluorin signals occur as spontaneous patterned peaks. Such transients average approximately two per minute and last ~30 s. We also demonstrate that the chemoattractant Netrin-1 elicits increases in the frequency and slopes of these transients, with peaks averaging up to six per minute in the peripheral domain. Netrin-1-dependent regulation of exocytotic events requires the activation of the Erk1/2 and SFK pathways. Furthermore, we show that domains with high SynaptopHluorin signals correlate with high local calcium concentrations and that local, spontaneous calcium increases are associated with higher SynaptopHluorin signals. These findings demonstrate highly stereotyped, spontaneous transients of local exocytosis in growth cones and that these transients are positively regulated by chemoattractant molecules such as Netrin-1. [ABSTRACT FROM AUTHOR]

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca2+ signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca2+ entry (SOCE) mediated by Ca2+ release-activated Ca2+ (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La3+, BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and inknock-inmice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca2+ signaling by Ca2+ microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca2+ entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis. [ABSTRACT FROM AUTHOR]

Ca2+ entry is essential for regulating vital physiological functions in all neuronal cells. Although neurons are engaged in multiple modes of Ca2+ entry that regulates variety of neuronal functions, we will only discuss a subset of specialized Ca2+-permeable non-selective Transient Receptor Potential Canonical (TRPC) channels and summarize their physiological and pathological role in these excitable cells. Depletion of endoplasmic reticulum (ER) Ca2+ stores, due to G-protein coupled receptor activation, has been shown to activate TRPC channels in both excitable and non-excitable cells. While all seven members of TRPC channels are predominately expressed in neuronal cells, the ion channel properties, mode of activation, and their physiological responses are quite distinct. Moreover, many of these TRPC channels have also been suggested to be associated with neuronal development, proliferation and differentiation. In addition, TRPCs also regulate neurosecretion, long-term potentiation and synaptic plasticity. Similarly, perturbations in Ca2+ entry via the TRPC channels have been also suggested in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of TRPCs in neuronal function and in neurodegenerative conditions would presumably unveil avenues for plausible therapeutic interventions for these devastating neuronal diseases. [ABSTRACT FROM AUTHOR]

Calcium signals play a major role in the control of all key stages of neuronal development, and in particular in the growth and orientation of neuritic processes. These signals are characterized by high spatial compartmentalization, a property which has a strong relevance in the different roles of specific neuronal regions in information coding. In this context it is therefore important to understand the structural and functional basis of this spatial compartmentalization, and in particular whether the behavior at each compartment is merely a consequence of its specific geometry or the result of the spatial segregation of specific calcium influx/efflux mechanisms. Here we have developed a novel approach to separate geometrical from functional differences, regardless on the assumptions on the actual mechanisms involved in the generation of calcium signals. First, spatial indices are derived with a wavelet-theoretic approach which define a measure of the oscillations of cytosolic calcium concentration in specific regions of interests (ROIs) along a cell, in our case developing chick ciliary ganglion neurons. The resulting spatial profile demonstrates clearly that different ROIs along the neuron are characterized by specific patterns of calcium oscillations. Next we have investigated whether this inhomogeneity is due just to geometrical factors, namely the surface to volume ratio in the different subcompartments (e.g. soma vs. growth cone) or it depends on their specific biophysical properties. To this aim correlation functions are computed between the activity indices and the surface/volume ratio along the cell: the data thus obtained are validated by a statistical analysis on a dataset of different cells. This analysis shows that whereas in the soma calcium dynamics is highly correlated to the surface/volume ratio, correlations drop in the growth cone-neurite region, suggesting that in this latter case the key factor is the expression of specific mechanisms controlling calcium influx/efflux. [ABSTRACT FROM AUTHOR]

The growth of neuritic processes in developing neurons is tightly controlled by a wide set of extracellular cues that act by initiating downstream signaling cascades, where calcium signals play a major role. Here we analyze the calcium dependence of the neurite growth promoted by basic fibroblast growth factor (bFGF or FGF-2) in chick embryonic ciliary ganglion neurons, taking advantage of dissociated, organotypic, and compartmentalized cultures. We report that signals at both the growth cone and the soma are involved in the promotion of neurite growth by the factor. Blocking calcium influx through L- and N-type voltage-dependent calcium channels and transient receptor potential canonical (TRPC) channels reduces, while release from intracellular stores does not significantly affect, the growth of neuritic processes. Simultaneous recordings of calcium signals elicited by FGF-2 at the soma and at the growth cone show that the factor activates different patterns of responses in the two compartments: steady and sustained responses at the former, oscillations at the latter. At the soma, both voltage-dependent channel and TRPC blockers strongly affect steady-state levels. At the growth cone, the changes in the oscillatory pattern are more complex; therefore, we used a tool based on wavelet analysis to obtain a quantitative evaluation of the effects of the two classes of blockers. We report that the oscillatory behavior at the growth cone is dramatically affected by all the blockers, pointing to a role for calcium influx through the two classes of channels in the generation of signals at the leading edge of the elongating neurites. [ABSTRACT FROM AUTHOR]

Aims Restenosis is an undesirable consequence following percutaneous vascular interventions. However, the current strategy for preventing restenosis is inadequate. The aim of this study was to investigate the role of low-voltage gated T-type calcium channels in regulating vascular smooth muscle cell (VSMC) proliferation during neointimal formation. Methods and results Wire injury of mice carotid arteries resulted in neointimal formation in the wild-type and Cav3.2−/− but not Cav3.1−/− mice, indicating a critical role of Cav3.1 in neointimal formation. In addition, we found a significant increase of Cav3.1 mRNA and protein in injured arteries. Cav3.1 knockout or knockdown (shCav3.1) reduced VSMC proliferation. Since T-channels are expressed predominantly in the G1 and S phases in VSMCs, we examined whether an abnormal G1/S transition was the cause of the reduced cell proliferation in shCav3.1 VSMCs. We found a disrupted expression of cyclin E in shCav3.1 VSMCs, and calmodulin agonist CALP1 partially rescued the defective cell proliferation. Furthermore, we demonstrated that infusion of NNC55-0396, a selective T-channel blocker, inhibited neointimal formation in wild-type mice. Conclusion Cav3.1 is required for VSMC proliferation during neointimal formation, and blocking of Cav3.1 may be beneficial for preventing restenosis. [ABSTRACT FROM PUBLISHER]

Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor. [ABSTRACT FROM AUTHOR]

Cytosolic calcium machinery is one of the principal signaling mechanisms by which endothelial cells (ECs) respond to external stimuli during several biological processes, including vascular progression in both physiological and pathological conditions. Low concentrations of angiogenic factors (such as VEGF) activate in fact complex pathways involving, among others, second messengers arachidonic acid (AA) and nitric oxide (NO), which in turn control the activity of plasma membrane calcium channels. The subsequent increase in the intracellular level of the ion regulates fundamental biophysical properties of ECs (such as elasticity, intrinsic motility, and chemical strength), enhancing their migratory capacity. Previously, a number of continuous models have represented cytosolic calcium dynamics, while EC migration in angiogenesis has been separately approached with discrete, lattice-based techniques. These two components are here integrated and interfaced to provide a multiscale and hybrid Cellular Potts Model (CPM), where the phenomenology of a motile EC is realistically mediated by its calcium-dependent subcellular events. The model, based on a realistic 3-D cell morphology with a nuclear and a cytosolic region, is set with known biochemical and electrophysiological data. In particular, the resulting simulations are able to reproduce and describe the polarization process, typical of stimulated vascular cells, in various experimental conditions. Moreover, by analyzing the mutual interactions between multilevel biochemical and biomechanical aspects, our study investigates ways to inhibit cell migration: such strategies have in fact the potential to result in pharmacological interventions useful to disrupt malignant vascular progression. [ABSTRACT FROM AUTHOR]

Aim Despite the fact that angiotensin (Ang) II is a critical regulator of the proliferation and migration of vascular smooth muscle cells (VSMCs), the effect of Ang II on VSMC proliferation has remained unclear. In this study, we determined whether Stim1- and Orai1-mediated store-operated calcium (Ca2+) entry (SOCE) plays a critical role in Ang II-induced VSMC proliferation and Ang II-accelerated neointimal growth after balloon injury of rat carotid arteries. Methods and results Knockdown of Stim1 and Orai1, putative calcium sensors/modulators, suppressed Ang II-mediated Ca2+ entry and cell proliferation in synthetic VSMCs. Stim1 and Orai1 short interfering RNAs (siRNAs) decreased neointimal growth induced by Ang II in balloon-injured rat carotid arteries. Ang II significantly increased the expression of Stim1 and Orai1 in neointima. In addition, our results showed that receptor subtype-1 (AT1) significantly contributed to Ang II-induced Ca2+ entry and proliferation of synthetic VSMCs. However, we found that transient receptor potential canonical 1 (Trpc1) had no effect on Ang II-induced SOCE or cell proliferation of synthetic VSMCs. Conclusions We show for the first time that Stim1- and Orai1-mediated SOCE may be critical for Ang II-induced VSMC proliferation. This provides important information with respect to targeting cardiovascular diseases under the enhanced renin–Ang system. [ABSTRACT FROM PUBLISHER]

Aim: The T-type calcium channel is expressed in vascular endothelial cells, but its role in endothelial cell function is yet to be elucidated. We analysed the endothelial functional role of T-type calcium channel-dependent calcium under angiotensin II (Ang II) stimulation. Methods: Human umbilical vein endothelial cells were co-incubated with hormone at 10−7 m and either Efonidipine 10−5 m or Verapamil 10−5 m or Mibefradil 10−5 m or Wortmannin 10−6 m. The contribution of Ang II receptors was evaluated using PD123319 10−7 m and ZD 7155 10−7 m. The calcium ion concentration was observed using Fluo-3 acetossimetil ester. The cells were observed after 3, 6, 9 and 12 h. Results: The microfluorescence method points out that Ang II induces intracellular calcium modulation in time by distinct mechanisms. AT2 receptor blockade is necessary to observe significant increase in [Ca2+]i levels. Pre-treatment with Mibefradil abolishes Ang II -induced cell migration. Conclusions: Our data show that Ang II, via AT1 receptor, modulates calcium concentration involving T-type calcium channel and L-type calcium channel but only the calcium influx via T-type calcium channels regulates endothelial cell migration which is essential for angiogenesis. [ABSTRACT FROM AUTHOR]

Background: N-4-Tert-Butyl benzyl haloperidol chloride (C3) was a novel calcium antagonist synthesized in our laboratory. The present study is to explore the effect of C3 on vascular smooth muscle cell proliferation and the mechanism involved. Methods: The effects of C3 on Ang II-induced cytosolic free Ca2+ concentration change, VSMC proliferation, the key early growth response factor 1 (Egr-1) were evaluated by laser scanning confocal microscopy, microtiter tetrazolium (MTT) proliferation assay, flow cytometry analysis, Western blot and RT-PCR analysis, respectively. An extracellular Ca2+ chelator EGTA and antisense Egr-1 oligodeoxyribonucleotides (ODNs) were used to establish the relation between Ca2+-dependent Egr-1 expression induced by Ang II and VSMC proliferation. Results: C3 attenuated the Ang II-induced extracellular Ca2+ influx, inhibited VSMCs proliferation and arrested VSMCs in G1-phase. C3 also triggered a significant reduction in PDGF-A and cyclin D1, Cdk2 along with an overexpression of p21Cip1. Antisense Egr-1 ODNs inhibited VSMCs proliferation, which was related to G1-phase arrest, due to inhibiting the expression of Egr-1 and C3 inhibited the overexpression of Egr-1. Conclusion: Egr-1 may play a key role in Ang II-induced proliferation of VSMCs. C3 inhibits vascular smooth muscle cell proliferation and the mechanism is involved with the inhibition of over-expression of Egr-1. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The existence of a mammalian family of TRPC ion channels, direct homologues of TRP, the visual transduction channel of flies, was discovered during 1995-1996 as a consequence of research into the mechanism by which the stimulation of the receptor- Gq-phospholipase Cβ signaling pathway leads to sustained increases in intracellular calcium. Mammalian TRPs, TRPCs, turned out to be nonselective, calcium-permeable cation channels, which cause both a collapse of the cell's membrane potential and entry of calcium. The family comprises 7 members and is widely expressed. Many cells and tissues express between 3 and 4 of the 7 TRPCs. Despite their recent discovery, a wealth of information has accumulated, showing that TRPCs have widespread roles in almost all cells studied, including cells from excitable and nonexcitable tissues, such as the nervous and cardiovascular systems, the kidney and the liver, and cells from endothelia, epithelia, and the bone marrow compartment. Disruption of TRPC function is at the root of some familial diseases. More often, TRPCs are contributing risk factors in complex diseases. The present article reviews what has been uncovered about physiological roles of mammalian TRPC channels since the time of their discovery. This analysis reveals TRPCs as major and unsuspected gates of Ca2+ entry that contribute, depending on context, to activation of transcription factors, apoptosis, vascular contractility, platelet activation, and cardiac hypertrophy, as well as to normal and abnormal cell proliferation. TRPCs emerge as targets for a thus far nonexistent field of pharmacological intervention that may ameliorate complex diseases. [ABSTRACT FROM AUTHOR]

In synthetic phenotype vascular smooth muscle cells (VSMC), activation of epidermal growth factor (EGF) receptor (EGFR) induces a sustained increase in intermediate conductance KCa (int-KCa; KCa3.1) channels that is essential for proliferation. However, a comparable mechanism has not been identified in native contractile phenotype VSMC, which express large conductance KCa (maxi-KCa; KCa1.1) channels, not int-KCa channels. Using patch clamp of freshly isolated contractile VSMC from rat basilar artery, we found that EGF (100 ng ml-1) caused hyperpolarization (7.9 ± 3.9 mV) due to activation of iberiotoxin-sensitive, maxi-KCa channels. The EGFR ligands EGF (100 ng ml-1), transforming growth factor α (0.4 ng ml-1) and heparin-binding EGF (100 ng ml-1) all caused a 20% increase in maxi-KCa channel current that was blocked by AG-1478 or by knock-down of EGFR expression using cisterna magna infusion of antisense oligodeoxynucleotide (AS-ODN). In controls, EGFR knock-down, and EGFR gain-of-expression (angiotensin II hypertension), the increase in maxi-KCa current correlated with the abundance of EGFR protein expressed. The EGFR-mediated increase in maxi-KCa channel activity was blocked by inhibiting cAMP-dependent protein kinase (cAK) using KT-5720 or Rp-cAMP, or by inhibiting adenylate cyclase type 5 (AC-5) using 2′,5′-dideoxyadenosine or knock-down of AC-5 expression by intracisternal AS-ODN. Direct infusion of EGF into cisterna magna caused up-regulation of proliferating cell nuclear antigen (PCNA) in VSMC that was prevented by coinfusion of iberiotoxin or of AG-1478. Our data, which are consistent with the hypothesis that hyperpolarization is critical for a proliferative response, are the first to implicate AC-5 and maxi-KCa channels in gene activation related to EGFR signalling in native contractile VSMC. [ABSTRACT FROM AUTHOR]

Unloading of endoplasmic reticulum (ER) Ca2+ stores activates influx of extracellular Ca2+ through‘store-operated’ Ca2+ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca2+ stores. Here, using high resolution imaging with the membrane-associated Ca2+ indicator, FFP-18, it is shown that store-operated Ca2+ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane–ER junctions. In the absence of extracellular Ca2+, depletion of ER Ca2+ stores using cyclopiazonic acid, an ER Ca2+-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca2+ concentration ([Ca2+]SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca2+ causes localized Ca2+ influx that first increases[Ca2+]SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs,markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying‘junctional’ ER. Thus, Ca2+ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca2+ stores. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) is known to play an important role in modulating renal transport functions. Thus, we investigated the effect of EGF on Ca2+ uptake and its related signals in the primary cultured rabbit renal proximal tubule cells. EGF (50 ng/ml, 1 h) stimulated Ca2+ uptake. Its effect was blocked by AG 1478 (an EGF receptor antagonist), genistein or herbimycin A (tyrosine kinase inhibitors). EGF increased intracellular cAMP level and SQ 22536 (an adenylate cyclase inhibitor), Rp-cAMP (a cAMP analogue), or PKI (a protein kinase A inhibitor) blocked the EGF-induced stimulation of Ca2+ uptake. EGF-induced stimulation of Ca2+ uptake was also blocked by neomycin or U-73122 (phospholipase C inhibitors), staurosporine, H-7, or bisindolylmaleimide I (protein kinase C inhibitors), nifedipine or methoxyverapamil (L-type Ca2+ channel blockers). It increased IPs formation by 167 ± 5% compare to control within 90 s. On the other hand, EGF increased [³H]-arachidonic acid release, which was significantly blocked by PKC inhibitors. In addition, PGE2, one of cyclooxygenase metabolites, and 5,6-EET, one of cytochrome P-450 metabolites, increased Ca2+ uptake. These results suggest that cAMP, PLC/PKC, and PLA2 are involved in EGFinduced stimulation of Ca2+ uptake. [ABSTRACT FROM AUTHOR]

Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factorregulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular pools under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel. [ABSTRACT FROM AUTHOR]

It has long been known that endothelial Ca2+ signals drive angiogenesis by recruiting multiple Ca2+-sensitive decoders in response to pro-angiogenic cues, such as vascular endothelial growth factor, basic fibroblast growth factor, stromal derived factor-1α and angiopoietins. Recently, it was shown that intracellular Ca2+ signaling also drives vasculogenesis by stimulation proliferation, tube formation and neovessel formation in endothelial progenitor cells. Herein, we survey how growth factors, chemokines and angiogenic modulators use endothelial Ca2+ signaling to regulate angiogenesis and vasculogenesis. The endothelial Ca2+ response to pro-angiogenic cues may adopt different waveforms, ranging from Ca2+ transients or biphasic Ca2+ signals to repetitive Ca2+ oscillations, and is mainly driven by endogenous Ca2+ release through inositol-1,4,5-trisphosphate receptors and by store-operated Ca2+ entry through Orai1 channels. Lysosomal Ca2+ release through nicotinic acid adenine dinucleotide phosphate-gated two-pore channels is, however, emerging as a crucial pro-angiogenic pathway, which sustains intracellular Ca2+ mobilization. Understanding how endothelial Ca2+ signaling regulates angiogenesis and vasculogenesis could shed light on alternative strategies to induce therapeutic angiogenesis or interfere with the aberrant vascularization featuring cancer and intraocular disorders. [ABSTRACT FROM AUTHOR]