An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell-cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM-mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras-mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)-Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.     

Ca2+- and protein kinase C-dependent signaling pathway for nuclear factor-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha production in lipopolysaccharide-stimulated rat peritoneal macrophages

Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factor-kappaB (NF-kappaB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2+ concentration ([Ca2+]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2+- and PKC-dependent NF-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha (TNF-alpha) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2+]i increase is due to Ca2+ release and influx. Extracellular and intracellular Ca2+ chelators inhibited phosphorylation of PKCalpha and PKCbeta. A PKCbeta-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitorykappaB kinase and NF-kappaB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) gamma. Furthermore, a PLC inhibitor eliminated the transient [Ca2+]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2+ and PKC in the signaling pathway for NF-kappaB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCgamma1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCbeta regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory kappaB kinase activation. Sequentially, NF-kappaB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-alpha production is promoted.     

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.     

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

The neural cell adhesion molecule NCAM regulates neuritogenesis by multiple mechanisms of interaction

The neural cell adhesion molecule NCAM and its glycosylation with polysialic acid (polySia) are crucially involved in proliferation, migration and differentiation of neural progenitors. Modification with polySia, homophilic and heterophilic interactions set the function of NCAM, but little is known on their interplay. We have shown recently that removal of polySia induces neuronal differentiation via heterophilic NCAM interactions at cell contacts between SH-SY5Y neuroblastoma cells. Here we analyze the additional impact of NCAM-positive fibroblasts as a ligand-presenting cellular environment, a model often used to demonstrate the neuritogenic effect of homophilic NCAM interactions. Native SH-SY5Y cells did not respond to interactions with fibroblast NCAM. However, after induction of neuronal differentiation by retinoic acid the previously ineffective NCAM signals activated extracellular signal-regulated kinase (ERK) and promoted neuritogenesis. Removal of polySia increased neuritogenesis in retinoic acid-treated cells additive to the NCAM substrate effect. The change in responsiveness to substrate NCAM was associated with a rearrangement of polysialylated NCAM away from its enrichment at homotypic cell-cell contacts and with the appearance of non-polysialylated NCAM, i.e. changes facilitating NCAM interactions with the substrate. Thus, heterophilic and homophilic NCAM interactions are integrated into the cell's response yet they have the capacity to independently trigger neuritogenesis. The actual occurrence of each of these interactions, however, depends on the cellular context, targeted cell surface presentation of NCAM and the dynamic regulation of its modification by polysialic acid. In summary, this study reveals how the complex interplay of NCAM interactions and polysialylation provides an elaborate system to regulate neuritogenesis.     

Structure and interactions of NCAM Ig1-2-3 suggest a novel zipper mechanism for homophilic adhesion

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.     

A novel collagen-binding peptide promotes osteogenic differentiation via Ca2+/calmodulin-dependent protein kinase II/ERK/AP-1 signaling pathway in human bone marrow-derived mesenchymal stem cells

The intracellular signaling events controlling human mesenchymal stem cell (hMSC) differentiation into osteoblasts are poorly understood. Collagen-binding domain is considered an essential component of bone mineralization. In the present study, we investigated the regulatory mechanism of osteoblastic differentiation of hMSC by the peptide with a novel collagen-binding motif derived from osteopontin. The peptide induced influx of extracellular Ca2+ via calcium channels and increased intracellular Ca2+ concentration ([Ca2+]i) independent of both pertussis toxin and phospholipase C, and activated ERK, which was inhibited by Ca2+/calmodulin-dependent protein kinase (CaMKII) antagonist, KN93. The peptide-induced increase of [Ca2+]i is correlated with ERK activation in a various cell types. The peptide stimulated the migration of hMSC but suppressed cell proliferation. Furthermore, the peptide increased the phosphorylation of cAMP-response element-binding protein, leading to a significant increase in the transactivation of cAMP-response element and serum response element. Ultimately, the peptide increased AP-1 transactivation, c-jun expression, and bone mineralization, which are suppressed by KN93. Taken together, these results indicate that the novel collagen-binding peptide promotes osteogenic differentiation via Ca2+/CaMKII/ERK/AP-1 signaling pathway in hMSC, suggesting the potential application in cell therapy for bone regeneration.     

Effects of multiple metal binding sites on calcium and magnesium-dependent activation of BK channels

BK channels are activated by physiological concentrations of intracellular Ca2+ and Mg2+ in a variety of cells. Previous studies have identified two sites important for high-affinity Ca2+ sensing between [Ca2+]i of 0.1-100 microM and a site important for Mg2+ sensing between [Mg2+]i of 0.1-10 mM. BK channels can be also activated by Ca2+ and Mg2+ at concentrations>10 mM so that the steady-state conductance and voltage (G-V) relation continuously shifts to more negative voltage ranges when [Mg2+]i increases from 0.1-100 mM. We demonstrate that a novel site is responsible for metal sensing at concentrations>=10 mM, and all four sites affect channel activation independently. As a result, the contributions of these sites to channel activation are complex, depending on the combination of Ca2+ and Mg2+ concentrations. Here we examined the effects of each of these sites on Ca2+ and Mg2+-dependent activation and the data are consistent with the suggestion that these sites are responsible for metal binding. We provide an allosteric model for quantitative estimation of the contributions that each of these putative binding sites makes to channel activation at any [Ca2+]i and [Mg2+]i.     

Cellular mechanisms of thromboxane A2-mediated contraction in pulmonary veins

Our objectives were to identify the relative contributions of [Ca2+]i and myofilament Ca2+ sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane A2 mimetic U-46619 and to assess the roles of PKC, tyrosine kinases (TK), and Rho-kinase (ROK) in that response. We tested the hypothesis that U-46619-induced contraction in PVSM is mediated by both increases in [Ca2+]i and myofilament Ca2+ sensitivity and that the PKC, TK, and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E-) canine pulmonary venous (PV) rings. In addition, [Ca2+]i and tension were simultaneously measured in fura-2-loaded E- PVSM strips. U-46619 (0.1 nM-1 microM) caused dose-dependent (P < 0.001) contraction in PV rings. U-46619 contraction was attenuated by inhibitors of L-type voltage-operated Ca2+ channels (nifedipine, P < 0.001), inositol 1,4,5-trisphosphate-mediated Ca2+ release (2-aminoethoxydiphenylborate, P < 0.001), PKC (bisindolylmaleimide I, P < 0.001), TK (tyrphostin A-47, P = 0.014), and ROK (Y-27632, P = 0.008). In PV strips, U-46619 contraction was associated with increases in [Ca2+]i and myofilament Ca2+ sensitivity. Both Ca2+ influx and release mediated the early transient increase in [Ca2+]i, whereas the late sustained increase in [Ca2+]i only involved Ca2+ influx. Inhibition of both PKC and ROK (P = 0.006 and P = 0.002, respectively), but not TK, attenuated the U-46619-induced increase in myofilament Ca2+ sensitivity. These results suggest that U-46619 contraction is mediated by Ca2+ influx, Ca2+ release, and increased myofilament Ca2+ sensitivity. The PKC, TK, and ROK signaling pathways are involved in U-46619 contraction.     

Role of PKC,tyrosine kinases,and Rho kinase in alpha-adrenoreceptor-mediated PASM contraction

Our objectives were to identify the relative contributions of intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in the pulmonary artery smooth muscle (PASM) contractile response to the alpha-adrenoreceptor agonist phenylephrine (PE) and to assess the role of PKC, tyrosine kinases (TK), and Rho kinase (ROK) in that response. Our hypothesis was that multiple signaling pathways are involved in the regulation of [Ca2+]i, myofilament Ca2+ sensitization, and vasomotor tone in response to alpha-adrenoreceptor stimulation of PASM. Simultaneous measurement of [Ca2+]i and isometric tension was performed in isolated canine pulmonary arterial strips loaded with fura 2-AM. PE-induced tension development was due to sarcolemmal Ca2+ influx, Ca2+ release from inositol 1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2+ stores, and myofilament Ca2+ sensitization. Inhibition of either PKC or TK partially attenuated the sarcolemmal Ca2+ influx component and the myofilament Ca2+ sensitizing effect of PE. Combined inhibition of PKC and TK did not have an additive attenuating effect on PE-induced Ca2+ sensitization. ROK inhibition slightly decreased [Ca2+]i but completely inhibited myofilament Ca2+ sensitization. These results indicate that PKC and TK activation positively regulate sarcolemmal Ca2+ influx in response to alpha-adrenoreceptor stimulation in PASM but have relatively minor effects on myofilament Ca2+ sensitivity. ROK is the predominant pathway mediating PE-induced myofilament Ca2+ sensitization.     

Glial cell line-derived neurotrophic factor increases intracellular calcium concentration. Role of calcium/calmodulin in the activation of the phosphatidylinositol 3-kinase pathway

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.     

Co-expression of mCysLT1 receptors and IK channels in Xenopus laevis oocytes elicits LTD4-stimulated IK current, independent of an increase in [Ca2+]i

Addition of LTD4 (10 nM) to Xenopus laevis oocytes expressing the mCysLT1 receptor together with hBK or hIK channels resulted in the activation of both channels secondary to an LTD4-induced increase in [Ca2+]i. In addition, the hIK channel is activated by low concentrations of LTD4 (<0.1 nM), which did not result in any increase in [Ca2+]i. Even though activation of hIK by low concentrations of LTD4 was independent of an increase in [Ca2+]i, a certain "permissive" level of [Ca2+]i was required for its activation, since buffering of intracellular Ca2+ by EGTA completely abolished the response to LTD4. Neither hTBAK1 nor hTASK2 was activated following stimulations with LTD4 (0.1 and 100 nM).     

Brevetoxin-induced autocrine excitotoxicity is associated with manifold routes of Ca2+ influx

Real-time alterations in intracellular Ca2+ ([Ca2+]i) were monitored in fluo-3-loaded cerebellar granule neurons (CGNs) exposed to the brevetoxin PbTx-1. [Ca2+]i was measured using a fluorescent plate reader (FLIPR), which measures simultaneously the mean intracellular Ca2+ change in a population of cultured cells in each well of a 96-well plate. PbTx-1 produced rapid and concentration-dependent increases in neuronal [Ca2+]i with a potency nearly identical to that determined previously for PbTx-1-induced neurotoxicity. The NMDA receptor antagonists MK-801, dextrorphan, and D(-)-2-amino-5-phosphonopentanoic acid, and tetanus toxin, an inhibitor of Ca2+-dependent exocytotic neurotransmitter release, effected significant reductions in both the integrated fluo-3 fluorescence response and excitatory amino acid release and protected CGNs against PbTx-1 neurotoxicity. The L-type Ca2+ channel antagonist nifedipine produced a modest reduction in the fluo-3 response but reduced substantially the plateau phase of the PbTx-1 increment in [Ca2+]i when combined with MK-801. When nifedipine and MK-801 were combined with the Na+/Ca2+ exchanger (reversed mode) inhibitor KB-R7943, the PbTx-1 increment in [Ca2+]i was nearly completely attenuated. These data show that Ca2+ entry into PbTx-1-exposed CGNs occurs through three primary routes: NMDA receptor ion channels, L-type Ca2+ channels, and reversal of the Na+/Ca2+ exchanger. There was a close correlation between reduction of the integrated fluo-3 fluorescence response and the level of neuroprotection afforded by blockers of each Ca2+ entry pathway; however, simultaneous blockade of L-type Ca2+ channels and the Na+/Ca2+ exchanger, although reducing the integrated [Ca2+]i response to a level below that provided by NMDA receptor blockade alone, failed to completely attenuate PbTx-1 neurotoxicity. This finding suggests that in addition to total [Ca2+]i load, neuronal vulnerability is governed principally by the NMDA receptor Ca2+ influx pathway.     

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.     

Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

Regulation of Transmembrane Signaling by Ganglioside GM1 :

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.