Biomedical subjects

Davide Lovisolo

Publications and source records attributed to Davide Lovisolo.

11 recordsLinked to original sources

PubMed2006

Temporal dynamics of neurite outgrowth promoted by basic fibroblast growth factor in chick ciliary ganglia.

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.

Animals↗

PubMed2006

Calcium signals and the in vitro migration of chick ciliary ganglion cells.

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.

Animals↗

PubMed2006

Interaction between TRPC channel subunits in endothelial cells.

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.

Animals↗

PubMed2005

Regulation of noncapacitative calcium entry by arachidonic acid and nitric oxide in endothelial cells.

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.

Analysis of Variance↗

PubMed2005

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 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.

Animals↗

PubMed2004

Blocking Ca2+entry: a way to control cell proliferation.

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.

Animals↗

PubMed2003

GDNF and bFGF are differentially involved in glial cell differentiation and neurite bundle formation in cultures from chick embryonic ciliary ganglia.

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.

Animals↗

PubMed2003

A K(+) channel activated by cholinergic muscarinic receptors in chick ciliary ganglion neurons at early developmental stage.

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.

Animals↗

PubMed2003

Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach.

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.

Animals↗

PubMed2002

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons.

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.

Animals↗

PubMed2002

Expression and functional role of bTRPC1 channels in native endothelial cells.

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.

Alternative Splicing↗