Carla Distasi

ARACHIDONIC ACID MEDIATES CALCIUM INFLUX INDUCED BY BASIC FIBROBLAST GROWTH FACTOR IN BALB-C 3T3 FIBROBLASTS

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.

Neuronal survival and calcium influx induced by basic fibroblast growth factor in chick ciliary ganglion neurons.

Basic fibroblast growth factor (bFGF/FGF2) exhibits widespread biological activities in the nervous system. However, little is known about the cascade of intracellular events that links the activation of its tyrosine kinase receptors to these effects. Here we report that, in ciliary ganglion neurons from chick embryo, this trophic factor significantly enhanced neuronal survival. The percentage of surviving neurons was reduced when intracellular calcium was chelated by adding a membrane‐permeable BAPTA ester to the culture medium, while antagonists of L‐ and N‐type voltage‐dependent calcium channels were ineffective. The ionic signals in response to bFGF stimulation have been studied using cytofluorimetric and patch‐clamp techniques. In single‐cell Fura‐2 measurements, bFGF elicited a long lasting rise of the cytosolic calcium concentration that was dependent on [Ca2+]o. In whole‐cell experiments, we observed a reversible depolarization of the membrane resting potential and an inward cationic current. Single channel experiments, performed in the cell‐attached configuration, provide evidence for the activation of two families of Ca2+‐permeable cationic channels. Moreover, inositol 1,4,5‐trisphosphate opens channels with similar properties, suggesting that this cytosolic messenger can be responsible for the calcium influx induced by bFGF.

Activation of TRPV4 channels reduces migration of immortalized neuroendocrine cells.

J. Neurochem. (2011) 116, 606–615.

In vitro analysis of neuron-glial cell interactions during cellular migration.

Abstract. We used time-lapse microscopy to study the in vitro migration of neuronal cells from developing chick ciliary ganglion. These cells, when dissociated and cultured in a chemically defined medium, are able to migrate and to associate into clusters. We focused our attention on the study of the distribution of neuronal velocity components. Quantitative analysis of cell trajectories allowed us to demonstrate that, in many cells, velocities are well described by the Langevin equation, when deterministic components of the forces acting on the cells are taken into account. We also have shown that the majority of neurons whose movement is not purely random migrate in association with glial cells. We conclude that glial cells, by guiding neurons during migration, play an important role in the cell organization in vitro.

Calcium signals activated by ghrelin and D-Lys3-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys(3)-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca(2+)](i) followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca(2+) entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys(3)-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca(2+) activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys(3)-GHRP-6 ghrelin antagonist features ghrelin independent activities.

Basic Fibroblast Growth Factor Opens Calcium-Permeable Channels in Quail Mesencephalic Neural Crest Neurons

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+Ii was observed, which was dependent on the presence of external calcium. These events may play a role in the developmental effects of bFGF.

In vitro identification of dividing neuronal precursors from chick embryonic ciliary ganglion.

In chick parasympathetic ciliary ganglion the neuronal birthdate is well defined, between 2.5 and 5.5 days of embryonic development, and neuronal precursor cells that are able to differentiate into neurons in vitro can be isolated from E4.5 ganglia. In this report, using bromodeoxyuridine incorporation and Map1b immunostaining, we demonstrate that these cells can be isolated from E7-E8 chick embryos as well, suggesting that neuronal precursor cells are still present in the ciliary ganglion after the end of the in vivo neurogenesis. These precursor cells retain the ability to divide and generate newly differentiated neurons in vitro when cultured in a chemically defined medium. Such a capacity is highly stimulated by bFGF but not by CNTF.

Nanosized TiO2 is internalized by dorsal root ganglion cells and causes damage via apoptosis

UNLABELLED Titanium dioxide (TiO2) is widely used as ingredient in several products in the nanoform. TiO2-nanoparticles (NPs) are also currently studied for different medical applications. A large debate exists on possible adverse health effects related to their exposure. While there is some evidence of TiO2-NP central nervous system toxicity, their effects on peripheral neurons have been poorly explored. In this study we investigated the effects of TiO2-NPs on dorsal root ganglion (DRG) sensory neurons and satellite glial cells that may be reached by nanoparticles from the bloodstream. We found that TiO2-NPs are internalized in DRG cells and induce apoptosis in a dose dependent manner in both types of cells, ROS production and changes in expression of proinflammatory cytokine IL-1β. Furthermore, we found that the axonal retrograde transport is altered in neurons upon exposure to TiO2-NPs. Overall, the results indicate a potential neurotoxic effect of TiO2-NPs on DRG cells. FROM THE CLINICAL EDITOR Exposure to titanium dioxide nanoparticles is increasing in medical practice. Little is known about their potential toxic effects on the peripheral nervous system. The authors studied this aspect and showed that titanium nanoparticles might potentially cause toxicity over long term.

A simple method to study cellular migration

We describe here a simple and fast method for the characterisation of cell motion. By projecting on a single plane different positions of the cell a ribbon is generated, whose characteristics can be related to the type of motion. The proposed method allows both to determine, very quickly, the motility of a population of cells and to investigate and characterise properties of a single cells motion. The methodology presented here can be applied to a large range of cell movement and also adapted and extended to other problems involving biological motion.

Calcium Signals Activated by Arachidonic Acid in Embryonic Chick Ciliary Ganglion Neurons

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 µM), 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 ω-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.