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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]

Abstract Background A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence. Results RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca2+ channels characterized by low Mg2+-sensitivity and low Ca2+-permeability, generating small, long-lasting currents. Ca2+-imaging experiments showed slow [Ca2+]i increases in 45% of the cells following 8 μM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca2+ in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca2+]i, in 99% of the cells. These intracellular Ca2+ signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals. Conclusions In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca2+]i increase in ST14A neural progenitors; NRG1-induced migration is Ca2+-dependent and can be positively modulated by activation of the NMDA receptor.

Forty years ago, the introduction of a new electrophysiological technique, the patch clamp, revolutionized the fields of Cellular Physiology and Biophysics, providing for the first time the possibility of describing the behavior of a single protein, an ion-permeable channel of the cell plasma membrane, in its physiological environment. The new approach was actually much more potent and versatile than initially envisaged, and it has evolved into several different modalities that have radically changed our knowledge of how cells (not only the classical "electrically excitable "ones, such as nerves and muscles) use electrical signaling to modulate and organize their activity. This review aims at telling the history of the background from which the new technique evolved and at analyzing some of its more recent developments., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Bisphenol A (BPA) is one of the most widely used chemicals worldwide, e.g., as a component of plastic containers for food and water. It is considered to exert an estrogenic effect, by mimicking estradiol (E2) action. Because of this widespread presence, it has attracted the interest and concern of researchers and regulators. Despite the vast amount of related literature, the potential adverse effects of environmentally significant doses of BPA are still object of controversy, and the mechanisms by which it can perturb endocrine functions, and particularly the neuroendocrine axis, are not adequately understood. One of the ways by which endocrine disruptors (EDCs) can exert their effects is the perturbation of calcium signaling mechanisms. In this study, we addressed the issue of the impact of BPA on the neuroendocrine system with an in vitro approach, using a consolidated model of immortalized Gonadotropin-Releasing Hormone (GnRH) expressing neurons, the GT1-7 cell line, focusing on the calcium signals activated by the endocrine disruptor. The investigation was limited to biologically relevant doses (nM-µM range). We found that BPA induced moderate increases in intracellular calcium concentration, comparable with those induced by nanomolar doses of E2, without affecting cell survival and with only a minor effect on proliferation.

Aim: To clarify the mechanisms of interaction between SiO 2 nanoparticles (NPs) and the plasma membrane of GT1-7 neuroendocrine cells, with focus on the activation of calcium-permeable channels, responsible for the long lasting calcium influx and modulation of the electrical activity in these cells., Materials & Methods: Nontoxic doses of SiO 2 NPs were administered to the cells. Calcium imaging and patch clamp techniques were combined with a pharmacological approach., Results: TRPV4, Cx and Panx-like channels are the major components of the NP-induced inward currents. Preincubation with the antioxidant N-acetyl-L-cysteine strongly reduced the [Ca 2+ ] i increase., Conclusion: These findings suggest that SiO 2 NPs directly activate a complex set of calcium-permeable channels, possibly by catalyzing free radical production.

Engineered silica nanoparticles (NPs) have attracted increasing interest in several applications, and particularly in the field of nanomedicine, thanks to the high biocompatibility of this material. For their optimal and controlled use, the understanding of the mechanisms elicited by their interaction with the biological target is a prerequisite, especially when dealing with cells particularly vulnerable to environmental stimuli like neurons. Here we have combined different electrophysiological approaches (both at the single cell and at the population level) with a genomic screening in order to analyze, in GT1-7 neuroendocrine cells, the impact of SiO 2 NPs (50 ± 3 nm in diameter) on electrical activity and gene expression, providing a detailed analysis of the impact of a nanoparticle on neuronal excitability. We find that 20 µg mL -1 NPs induce depolarization of the membrane potential, with a modulation of the firing of action potentials. Recordings of electrical activity with multielectrode arrays provide further evidence that the NPs evoke a temporary increase in firing frequency, without affecting the functional behavior on a time scale of hours. Finally, NPs incubation up to 24 hours does not induce any change in gene expression.

The repressor element-1 silencing transcription factor (REST) has emerged as a key controller of neuronal differentiation and has been shown to play a critical role in the expression of the neuronal phenotype; however, much has still to be learned about its role at specific developmental stages and about the functional targets affected. Among these targets, calcium signaling mechanisms are critically dependent on the developmental stage and their full expression is a hallmark of the mature, functional neuron. We have analyzed the role played by REST in GN11 cells, an immortalized cell line derived from gonadotropin hormone releasing hormone (GnRH) neurons at an early developmental stage, electrically non-excitable and with a strong migratory activity. We show for the first time that functional voltage-dependent calcium channels are expressed in wild type GN11 cells; down-regulation of REST by a silencing approach shifts these cells towards a more differentiated phenotype, increasing the functional expression of P/Q-type channels and reducing their migratory potential., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

SiO2 nanoparticles (NPs), in addition to their widespread utilization in consumer goods, are also being engineered for clinical use. They are considered to exert low toxicity both in vivo and in vitro, but the mechanisms involved in the cellular responses activated by these nanoobjects, even at non-toxic doses, have not been characterized in detail. This is of particular relevance for their interaction with the nervous system: silica NPs are good candidates for nanoneuromedicine applications. Here, by using two neuronal cell lines (GT1-7 and GN11 cells), derived from gonadotropin hormone releasing hormone (GnRH) neurons, we describe the mechanisms involved in the perturbation of calcium signaling, a key controller of neuronal function. At the non-toxic dose of 20μgmL(-1), 50nm SiO2 NPs induce long lasting but reversible calcium signals, that in most cases show a complex oscillatory behavior. Using fluorescent NPs, we show that these signals do not depend on NPs internalization, are totally ascribable to calcium influx and are dependent in a complex way from size and surface charge. We provide evidence of the involvement of voltage-dependent and transient receptor potential-vanilloid 4 (TRPV4) channels., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nanosized objects are increasingly present in everyday life and in specialized technological applications. In recent years, as a consequence of concern about their potential adverse effects, intense research effort has led to a better understanding of the physicochemical properties that underlie their biocompatibility or potential toxicity, setting the basis for a rational approach to their use in the different fields of application. Among the functional parameters that can be perturbed by interaction between nanoparticles (NPs) and living structures, calcium homeostasis is one of the key players and has been actively investigated. One of the most relevant biological targets is represented by the nervous system (NS), since it has been shown that these objects can access the NS through several pathways; moreover, engineered nanoparticles are increasingly developed to be used for imaging and drug delivery in the NS. In neurons, calcium homeostasis is tightly regulated through a complex set of mechanisms controlling both calcium increases and recovery to the basal levels, and even minor perturbations can have severe consequences on neuronal viability and function, such as excitability and synaptic transmission. In this review, we will focus on the available knowledge about the effects of NPs on the mechanisms controlling calcium signalling and homeostasis in neurons. We have taken into account the data related to environmental NPs, and, in more detail, studies employing engineered NPs, since their more strictly controlled chemical and physical properties allow a better understanding of the relevant parameters that determine the biological responses they elicit. The literature on this specific subject is all quite recent, and we have based the review on the data present in papers dealing strictly with nanoparticles and calcium signals in neuronal cells; while they presently amount to about 20 papers, and no related review is available, the field is rapidly growing and some relevant information is already available. A few general findings can be summarized: most NPs interfere with neuronal calcium homeostasis by interactions at the plasmamembrane, and not following their internalization; influx from the extracellular medium is the main mechanism involved; the effects are dependent in a complex way from concentration, size and surface properties.

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 [Formula: see text] 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.

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.

CdSe Quantum Dots (QDs) are increasingly being employed in both industrial applications and biological imaging, thanks to their numerous advantages over conventional organic and proteic fluorescent markers. On the other hand a growing concern has emerged that toxic elements from the QDs core would render the nanoparticles harmful to cell cultures, animals and humans. The interaction between QDs and neuronal cells in particular needs to be carefully evaluated, since nanoparticles could access the nervous system by several pathways, including the olfactory epithelium, even if no data are presently available about QDs. The pH of the environment to which the nanoparticles are exposed may play a crucial role in the stability of QDs coating. For this reason we investigated the release of metal ions from CdSe/ZnS QDs in artificial media reproducing the cytosolic and lysosomal cellular compartments characterized respectively by a neutral and an acidic pH. In the latter significant amounts of both Cd(2+) and Zn(2+) were released. We provide evidence that these QDs are internalized in the GT1-7 neuronal cell line and located in the lysosomal compartment. These findings can be related to a slight but significant reduction in cell survival and proliferation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Basic Fibroblast Growth Factor, or FGF-2, has been shown to promote neuronal survival and neurite outgrowth in dissociated neurons from the embryonic chick ciliary ganglion; in these effects the three main signal transduction pathways downstream the activated FGFR receptor, i.e. the MAPK, the PI3-K and the PLCγ ones, are differentially involved. While it has been shown that FGF-2 can elicit long lasting elevations in intracellular calcium concentration, [Ca(2+)](i), the role of the three pathways in this process has not been elucidated. Here we show, by means of pharmacological inhibitors, that all three are involved, at a different extent, in the generation of the [Ca(2+)](i) increase induced by FGF-2; in particular, inhibition of the PLCγ pathway, in addition to reducing the number of responsive cells, induces, in a significant population of cells, basal calcium oscillations in the absence of the growth factor and interferes with calcium signals elicited by depolarization. We propose that this complex behaviour can be due to a perturbation in PIP(2) levels at the plasmamembrane., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Gonadotropin-releasing hormone (GnRH)-secreting neurons are key regulators of the reproductive behaviour in vertebrates. These neurons show a peculiar migratory pattern during embryonic development, and its perturbations have profound impact on fertility and other related functional aspects. Changes in the intracellular calcium concentration, [Ca(2+)](i), induced by different extracellular signals, play a central role in the control of neuronal migration, but the available knowledge regarding GnRH neurons is still limited. Our goal was to investigate mechanisms that may be involved in the Ca(2+) dependence of the migratory behaviour in these neurons. We focused on the "classical" Transient Receptor Potential (TRPC) subfamily of Ca(2+)-permeable cation channels, recently shown to be involved in other aspects of neuronal development. Using GN11 cells, immortalized early stage GnRH neurons, we set to investigate Ca(2+) signals under basal conditions and in the presence of a well-established motogen, fetal calf serum (FCS), and the effect of pharmacological TRPC agonists and antagonists on Ca(2+) oscillations, cell motility and proliferation. We have found that a subpopulation of GN11 cells shows spontaneous Ca(2+) transients and that this activity is increased in the presence of serum. Quantitative real-time PCR showed that transcripts of some TRPC members are expressed in GN11 cells. Interestingly, pharmacological experiments with inhibitors, SKF-96365, lanthanum, anti-TRPC1 antibody, and activators, 1-oleil 2-acetyl-sn-glycerol, of TRPCs suggested that the activation of these channels can account for both the basal Ca(2+) oscillations and the increased activity in the presence of FCS. Moreover, functional studies using the same pharmacological tools supported their involvement in the control of motility and proliferation. Thus, our data provide evidence for the involvement of Ca(2+)-permeable channels of the TRPC subfamily in the control of functional properties of neurosecretory cells and neuronal motility., (2011 Elsevier Ltd. All rights reserved.)

The effects of Stöber silica nanoparticles on neuronal survival, proliferation, and on the underlying perturbations in calcium homeostasis are investigated on the well-differentiated neuronal cell line GT1-7. The responses to nanoparticles 50 and 200 nm in diameter are compared. The 50-nm silica affects neuronal survival/proliferation in a dose-dependent way, by stimulating apoptotic processes. In contrast, the 200-nm silica does not show any toxic effect even at relatively high concentrations (292 μg mL−1). To identify the mechanisms underlying these effects, the changes in intracellular calcium concentration elicited by acute and chronic administration of the two silica nanoparticles are analyzed. The 50-nm silica at toxic concentrations generates huge and long-lasting increases in intracellular calcium, whereas the 200-nm silica only induces transient signals of much lower amplitude. These findings provide the first evidence that silica nanoparticles can induce toxic effects on neuronal cells in a size-dependent way, and that these effects are related to the degree of perturbation of calcium homeostasis.

Basic fibroblast growth factor (bFGF) exerts multiple neurotrophic actions on cultured neurons from the ciliary ganglion of chick embryo, among them promotion of neuronal survival and of neurite outgrowth. To understand the specificity of the signal transduction cascades involved in the control of these processes, we used pharmacological inhibitors of the three main effectors known to act downstream of the bFGF receptor (FGFR): phospholipase Cgamma (PLCgamma), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Neuronal survival was assessed at 24 and 48 hr; neurite growth was analyzed both on dissociated neurons and on explants of whole ganglia. Our data show that only the PI3-K pathway is involved in the survival-promoting effect of bFGF; on the other hand, all three effectors converge on the enhancement of neurite outgrowth, both on isolated neurons and in whole ganglia., (2009 Wiley-Liss, Inc.)

We have developed a device for recording the extracellular electrical activity of cultured neuronal networks based on a hydrogen terminated (H-terminated) conductive diamond. GT1-7 cells, a neuronal cell line showing spontaneous action potentials firing, could maintain their functional properties for days in culture when plated on the H-terminated diamond surface. The recorded extracellular electrical activity appeared in the form of well-resolved bursts of fast and slow biphasic signals with a mean duration of about 8ms for the fast and 60ms for the slow events. The time courses of these signals were in good agreement with those recorded by means of conventional microelectrode array (MEAs) and with the negative derivative of the action potentials intracellularly recorded with the patch clamp technique from single cells. Thus, although hydrophobic in nature, the conductive H-terminated diamond surface is able to reveal the spontaneous electrical activity of neurons mainly by capacitative coupling to the cell membrane. Having previously shown that the optical properties of H-terminated diamond allow to record cellular activity by means of fluorescent probes (Ariano, P., Baldelli, P., Carbone, E., Giardino, A., Lo Giudice, A., Lovisolo, D., Manfredotti, C., Novara, M., Sternschulte, H., Vittone, E., 2005. Diam. Relat. Mater. 14, 669-674), we now provide evidence for the feasibility of using diamond-based cellular biosensors for multiparametrical recordings of electrical activity from living cells.

Calcium-permeable cation channels of the transient receptor potential (TRP) superfamily are involved in agonist-induced calcium influx in several cell types. In this work we evaluated expression and localisation of classical TRP (TRPC) channels in two immortalised cell lines derived from the gonadotrophin releasing hormone (GnRH) neuroendocrine system, at different developmental stages: GT1-7 cells display many characteristics of mature hypothalamic GnRH neurons and are a suitable model to study neuritogenesis and neurosecretion, whereas GN11 cells retain a more immature phenotype with migratory activity. Immunoblotting analysis demonstrates that GN11 and GT1-7 cells differentially express several members of the TRPC family: TRPC1 and TRPC5 are expressed at high levels in GN11 cells, and TRPC4 is expressed at higher levels in GT1-7 cells. Immunocytochemical experiments show a widespread localisation for TRPC proteins in GN11 cells and a characteristic staining along neurites in GT1-7 cells. These data suggest that different TRPC proteins could play specific functional roles at different developmental stages of the GnRH system.

Basic fibroblast growth factor (bFGF) is a potent and multifunctional neurotrophic factor that can influence neuronal survival and differentiation. It has been shown to modulate growth and orientation of neuritic processes both in intact organs and in neuronal cultures, with a wide spectrum of effects on different preparations. Here we report that it promotes neurite growth in developing parasympathetic neurons from the chick ciliary ganglion. We have used both organotypic cultures and dissociated neurons, and we have combined assessment of global neurite growth by immunocytochemical techniques with evaluation of dynamic parameters of single neurites via time-lapse microscopy. We show that laminin, a molecule of the extracellular matrix that has been associated with stimulation of neurite extension, has only a limited and short-lived effect on neurite outgrowth. In contrast, bFGF can promote global growth of the neuritic network both in whole ganglia and in dissociated cultures for times up to 48 hr, and this effect is related to an increase in the growth rate of single neurites. Moreover, the effect can be observed even in enriched neuronal cultures, pointing to a direct action of bFGF on neurons.

We have studied calcium signals and their role in the migration of neuronal and nonneuronal cells of embryonic chick ciliary ganglion (CG). In vitro, neurons migrate in association with nonneuronal cells to form cellular aggregates. Changes in the modulus of the velocity of the neuron-nonneuronal cell complex were observed in response to treatments that increased or decreased intracellular calcium concentration. In addition, both cell types generated spontaneous calcium activity that was abolished by removal of extracellular calcium. Calcium signals in neurons could be characterized as either spikes or waves. Neuronal spikes were found to be related to action potential generation whereas neuronal waves were due to voltage-independent calcium influx. Nonneuronal cells generated calcium oscillations that were dependent on calcium release from intracellular stores and on voltage-independent calcium influx. Application of thimerosal, a compound that stimulates calcium mobilization from internal stores, increased: (1) the amplitude of spontaneous nonneuronal oscillations; (2) the area of migrating nonneuronal cells; and (3) the velocity of the neuronal-nonneuronal cell complex. We conclude that CG cell migration is a calcium dependent process and that nonneuronal cell calcium oscillations play a key role in the modulation of velocity.

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.

Several peptides, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), activate the release of arachidonic acid (AA) and nitric oxide (NO) in endothelial cells (ECs). Both messengers are involved in EC proliferation and vascular permeability and control calcium homeostasis in different ways. Interestingly, it has been recently suggested that NO acts as a downstream mediator of AA-induced calcium entry in smooth muscle cells and isolated mouse parotid cells. In this paper, we have investigated the complex relationships that link intracellular calcium, AA, and NO in cultured endothelial cells. Using different experimental approaches, mainly simultaneous Ca2+ and NO fluorimetric confocal imaging, we provide evidence for a complex pathway leading to noncapacitative calcium entry (NCCE) in bovine aortic endothelial cells (BAECs). In particular, AA is able to induce NCCE through two different pathways: one dependent on eNOS recruitment and NO release, the other NO-independent. Finally, we show that NO increase is involved in the control of BAEC proliferation.

Arachidonic acid (AA, 20:4) has been reported to modulate a variety of calcium-permeable ionic channels, both in the plasma membrane and in the endoplasmic reticulum. We have studied the effects of AA on calcium signaling in a well-characterized model of developing peripheral neurons, embryonic chick ciliary ganglion neurons in culture. When given at low non-micellar concentrations (5 microM), in the majority of cells AA directly activated a delayed and long-lasting increase in [Ca2+]i, involving both the cytoplasm and the nucleoplasm, that was completely reversed by abolition of extracellular calcium. Other fatty acids (FAs), either saturated like arachidic acid (20:0), or unsaturated like linoleic (18:2) and docosahexaenoic acid (22:6), shared its ability to activate calcium influx. This entry was not suppressed by voltage-dependent calcium channel inhibitors omega-conotoxin and nifedipine, by the voltage-independent calcium channel antagonist LOE-908, by pre-treatment with blockers of AA metabolic pathways or with pertussis toxin. The arachidonate-activated calcium pathway was permeable to Mn2+ and blocked by La3+, Gd3+ and Ni2+. In a neuronal subpopulation, AA at the same concentration was also able to elicit calcium release from thapsigargin-sensitive intracellular stores; we provide evidence that cytochrome P450 epoxygenase is involved in this process., (Copyright 2005 S. Karger AG, Basel)

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.

We have shown that the neurotrophic factors glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) exert different effects on glial cells in cultures from chick embryo ciliary ganglia. bFGF acts as a mitogen on glial cells, and induces their aggregation to neuronal bodies; after 48 h of culture no glial cells could be observed along neurites. GDNF has no proliferative role; in contrast, it promotes the expression of the differentiative marker O4 and the association of glial cell bodies to neurites to form robust bundles.

In physiological conditions, endothelial cell proliferation is strictly controlled by several growth factors, among which bFGF and VEGF are the most effective. Both bind to specific tyrosine kinase receptors and trigger intracellular signal cascades. In particular, bFGF stimulates the release of arachidonic acid (AA) and its metabolites in many types of endothelial cells in culture. In bovine aortic endothelial cells, it has been suggested that AA is released by the recruitment of cytosolic phospholipase A2 (cPLA2). AA metabolites are involved in the control of both endothelial cell motility (mostly via the cyclooxygenase pathway) and proliferation (via the lipoxygenase (LOX) cascade). On the other hand, evidence has been provided for a proliferative role of AA-induced calcium influx. By using a pharmacological approach, we have tried to elucidate the contribution to bovine aortic endothelial proliferation of the different pathways leading to production of AA and its metabolites. Two main informations were obtained by our experiments: first, AA release is not entirely due to cPLA2 involvement, but also to DAG lipase recruitment; second, cyclooxygenase derivatives play a role in the control of cell proliferation, and not only of motility. Moreover, by combining proliferation assays and single cell calcium measurements, we show that the blocking effect of carboxyamido-triazole (CAI), an inhibitor of tumor growth and angiogenesis acting on calcium influx-dependent pathways, including AA metabolism, is at least in part due to a direct effect on AA-induced calcium influx.

Embryonic chick ciliary ganglion (CG) neurons obtained from E7-E8 ganglia maintained in serum-free medium were stimulated with 50 microM muscarine. A fast hyperpolarization of the membrane potential was observed in 25% of the cells tested, that in some cases was associated with a slower depolarization. Accordingly, in voltage clamp experiments, either an outward current or a biphasic current response could be observed. Single-channel experiments provide evidence that these signals can be associated to the activation of a K(+) channel whose conductance is 20 pS.

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.

We have shown that neurons from embryonic chick ciliary ganglia in primary culture possess receptors for insulin-like growth factor I (IGF-I). When added to serum- and insulin-free culture medium, the factor potently enhanced neuronal survival as observed after 24 and 48 h of culture. The effect saturated at 5 ng/ml. Laminin was not necessary for the trophic effects of IGF-I; in the absence of the factor, it had no effect on neuronal survival. Insulin exerted a trophic effect similar to that observed with IGF-I, but at higher doses. The trophic effect of IGF-I was sharply and specifically reduced when either a membrane-permeable calcium chelating agent or blockers of voltage-dependent calcium channels were added to the medium.