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BackgroundInvestigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration.Methodology/Principal FindingsWe analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10–15 V/cm) and weak (≤5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation.Conclusion/SignificanceTaken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.

BackgroundIon transport proteins generate small electric fields that can induce directional cell motility; however, little is known about their mechanisms that lead to directedness. We investigated Na, K-ATPase (NaKA) and Na+/H+ exchanger isoforms (NHE1 and 3) in SaOS-2 and Calvarial osteoblasts, which present anode- and cathode- directed motility, during electrotaxis.ResultsSignificant colocalizations of NaKA with vinculin and pNHE3 with ß-actin were observed to occur at the leading edges of cells. The directedness were attenuated when NaKA or NHE3 was inhibited, confirming their implication in directional sensing. Depending on the perceived direction, a divergent regulation in PIP2 levels as a function of NHE3 and NaKA levels was observed, suggesting that PIP2 may act as a spatiotemporal regulator of the cell membrane during electrotaxis. Moreover, at the same places where pNHE3 accumulates, bubble-shaped H+ clouds were observed, suggesting a physio-mechanical role for NHE3. The cell membrane becomes hyperpolarized at the front and depolarized at the back, which confirms NaKA activity at the leading edge.ConclusionWe suggest a novel role for both NaKA and NHE3 that extends beyond ion translocation and conclude that they can act as directional sensors and Vmem as a regulatory cue which maintain the persistent direction in electrotaxis.

The ganglioside GD3 (Neu5Aca8Neu5Aca3Galb4GlcCer) is an intracellular lipid messenger that induces apoptosis by targeting mitochondria in various cell types. GD3 can also promote apoptosis when externally added to cells. Previous studies showed that the proapoptotic effects of GD3 can be counteracted by 9-O-acetylation. To determine whether 9-O-acetyl GD3 (acGD3) has a general antiapoptotic potential, the apoptosis-sensitive Jurkat cell line and an apoptosis-sensitive variant of the cell line Molt-4 were preincubated with micromolar concentrations of acGD3 and then treated with inducers of apoptosis. A reduced apoptotic index and an increased cell viability were observed. On the other hand, when the Jurkat cells were treated with GD3 for extended periods of time, a population was selected that was resistant to apoptosis induction by N-acetyl sphingosine as well as by the anti-leukemic drug daunorubicin. Comparative analysis of gangliosides revealed the formation of acGD3 in the resistant Jurkat cells that was not found in the apoptosis-sensitive cells. Conversely, exposing the acGD3 positive and apoptosis-resistant cell line Molt-4 to the Odeacetylating activity of salicylate resulted in a complete disappearance of acGD3 and an enhanced sensitivity to N-acetyl sphingosine-mediated apoptosis. Formation of acGD3 might thus represent a new mechanism how tumor cells can escape apoptosis. ' 2006 Wiley-Liss, Inc.Key words: apoptosis; 9-O-acetyl GD3; gangliosideThe intracellular ganglioside GD3 (Neu5Aca8Neu5Aca3Galb 4GlcCer) has been shown to serve as a second lipid messenger in the apoptotic pathway induced by FAS/FASL, TNF-a or b-amyloid in diverse lymphoid and myeloid cell types.1-3 Various effects have been ascribed to GD3, such as production of reactive oxygen species (ROS), 4 opening of the mitochondrial permeability transition pore (PTP), 4 release of cytochrome C, 1,4,5 activation of caspases 6 and inhibition of the translocation of NFjB to the nucleus. 7 The importance of ROS and PTP in the GD3-mediated apoptotic pathway is stressed by the finding that GD3-induced mitochondrial changes are prevented by antioxidants such as butylated hydroxytoluene (BHT) 2 and by preincubation of the cells with cyclosporin A, an inhibitor of the PTP.8 Despite the obvious involvement of mitochondria, the exact mechanism how GD3 promotes apoptosis still remains elusive.Accumulation of GD3 has been reported in a variety of tumors.9-11 The concomitant presence of an acetylated modification of GD3, 9-O-acetyl GD3 (acGD3), was observed in some tumors such as melanoma 12,13 and breast cancer, 14,15 as well as in tumor cell lines like MOLT 4 16 and SKMel28. 17In a previous report, we have shown that the proapoptotic activity of GD3 is suppressed by acetylation. 18 However, it remained unclear whether the observed resistance to apoptosis is due to inactivation of GD3 by acetylation or, rather, to an antiapoptotic potential of acGD3 on its own. To approach this question, more information is needed about the effects of acGD3 when applied ...

Several studies have reported that endogenous ion currents are involved in a wide range of biological processes from single cell and tissue behavior to regeneration. Various methods are used to assess intracellular and local ion dynamics in biological systems, e.g., patch clamping and vibrating probes. Here, we introduce an approach to detect ion kinetics in vivo using a noninvasive method that can electrophysiologically characterize an entire experimental tissue region or organism. Ion-specific vital dyes have been successfully used for live imaging of intracellular ion dynamics in vitro. Here, we demonstrate that cellular pH, cell membrane potential, calcium, sodium and potassium can be monitored in vivo during tail regeneration in the axolotl (Ambystoma mexicanum) using ion-specific vital dyes. Thus, we suggest that ion-specific vital dyes can be a powerful tool to obtain electrophysiological data during crucial biological events in vivo.

Dorzolamide reduced the damage inflicted on retinal neural cells by agents that induced apoptosis and, therefore, can be considered a neuroprotectant.

BackgroundThe disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown.MethodsNeuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24 h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the identification of isolated somas in phase-contrast images using image processing. Vmem was measured using voltage-sensitive dyes and whole-cell patch clamping. Immunocytochemistry and Western blotting were used to detect changes in the distribution and production of the proteins.ResultsData show that Vmem regulates cortical tissue shape and connectivity. Automated cluster analysis methods revealed that the degree of neural aggregation was significantly increased (0.26 clustering factor vs. 0.21 in controls, P ≤ 0.01). The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm. Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures. Glia cell densities increased (33.3%, P ≤ 0.05) under depolarizing conditions.ConclusionVmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

The aim of this study was to develop and characterize novel metal-polymer constructs to improve the biocompatibility of flexible but hydrophobic polyurethane (PUR) implants. Using a physical vapor deposition (PVD) technique, thin films (< or =100 nm) of zirconium (Zr) or titanium (Ti) were deposited on the polyurethane surface. Both coatings displayed good stability when subjected to cross-cutting test and especially Zr showed only minor and superficial cracks in the scanning electron microscopy analysis. PVD coating resulted in significantly lowered contact angles and the standard surface free energy of wetting (Delta(wet)G degrees ) turned to more favorable negative values (Ti: -40; Zr: -30; untreated PUR (uPUR): +10.1 mN/m). This may lead to the highly enhanced adhesion and proliferation properties observed with human umbilical vein endothelial cells (HUVECs). In addition, the novel coatings had no toxic effect and even drastically reduced apoptosis rates of HUVECs. Cell morphology, nitric oxide production, and mitochondrial membrane potential--both at static and flow conditions--were superior compared with uPUR, thus demonstrating intact physiological functions. Therefore, we suggest that combining PUR as a flexible material with a thin coating of Zr or Ti as the improved biocompatible surface may have advantages for use, for example, vascular graft material.