Influx Signaling Research Articles

Involvement of protein kinase C and E2F-5 in euxanthone-induced neurite differentiation of neuroblastoma

Euxanthone, a neuritogenic agent isolated from the medicinal herb Polygala caudata, has been shown to induce morphological differentiation and neurite outgrowth in murine neuroblastoma Neuro 2a cells (BU-1 subclone). In order to elucidate the underlying mechanisms of euxanthone-induced neurite outgrowth, a proteomic approach was employed. In the present study, two dimensional (2-D) gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry were performed to investigate the alterations in protein expression profile of euxanthone-treated BU-1 cells. Fourteen identified proteins were changed in expression levels after induction of neurite growth. These proteins included participants in transcription and cell cycle regulation, calcium influx and calcium signaling, fatty acid metabolism, cytoskeleton reorganization, casein kinase signal transduction, putative transbilayer amphipath transport and protein biosynthesis. Among the 14 identified proteins, E2F transcription factor 5 (E2F-5) was significantly up-regulated after euxanthone treatment. Go6976, a protein kinase C (PKC) α/βI inhibitor, was found to inhibit neuritogenesis and expression of E2F-5 in the euxanthone-treated BU-1 cells, while SH-6, the Akt/PKB inhibitor, had no inhibitory effect. The gene silencing of E2F-5 by small interfering RNA (siRNA) was found to abolish the euxanthone-induced neurite outgrowth. In conclusion, these results indicated that the transcription factor E2F-5 was actively involved in the regulation of euxanthone-induced neurite outgrowth via PKC pathway.

A Novel, Selective, and Orally Available Antagonist for CC Chemokine Receptor 3

CC chemokine ligand 11 (CCL11/eotaxin) and other CC chemokine receptor 3 (CCR3) ligands (CCL24/eotaxin-2, CCL26/eotaxin-3, CCL13/monocyte chemotactic protein-4, etc.) play important roles in the chemotaxis and activation of eosinophils and other CCR3-expressing cells (basophils, mast cells, and CD4(+) T helper 2 cells) in allergic inflammation incidents, including asthma and rhinitis. A newly synthesized compound, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-{1-[(5-hydroxy-3-methylpyridin-2-yl)carbonyl]piperidin-4-ylidene}-acetamide hemifumarate (YM-355179), inhibited the binding of CCL11 and CCL5/regulated on activation normal T cell expressed and secreted to CCR3-expressing B300-19 cells with IC(50) values of 7.6 and 24 nM, respectively. In contrast, YM-355179 did not affect the binding of CCL5 to CCR1 or CCR5. In functional assays, YM-355179 inhibited CCL11-induced, intracellular Ca(2+) influx, chemotaxis, and eosinophil degranulation with IC(50) values of 8.0, 24, and 29 nM, respectively. YM-355179 did not, however, affect any CC chemokine receptor (CCR1, CCR2, CCR4, or CCR5)-mediated Ca(2+) influx signals. Furthermore, oral administration of YM-355179 (1 mg/kg) inhibited CCL11-induced shape change of whole blood eosinophils in cynomolgus monkeys. Intravenous injection of YM-355179 (1 mg/kg) also inhibited eosinophil infiltration into airways of cynomolgus monkeys after segmental bronchoprovocation with CCL11. These results indicate that YM-355179 is a novel, selective, and orally available CCR3 antagonist with therapeutic potential for treating eosinophil-related allergic inflammatory diseases.

Phospholipase Cγ1 Is Required for Activation of Store-Operated Channels in Human Keratinocytes

Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R.

Hybridization signatures during thymus ontogeny reveals modulation of genes coding for T-cell signaling proteins

Non-manipulated inbred mouse strains constitutes an interesting model-system for in vivo studies on thymus ontogeny due to the possibility to observe the molecular events of the thymocyte maturation. In previous studies, using RT-PCR method, we have found that several immune system genes such as interleukins and MHC are differentially expressed during ontogeny of the thymus whose genes act as modulators of T-cell differentiation. To determine which other genes are modulated on a large-scale basis, we measured the levels of mRNA expression in mouse fetal thymus (14–17 days of gestation) by hybridization with cDNA microarrays containing 1,576 cDNA sequences derived from the IMAGE MTB library. T-cell maturation was monitored by detection of the T-cell receptor beta TRBV8.1-BD2.1 rearranged DNA segment. Each developmental phase of thymus, displayed a characteristic expression profile, as evaluated by the Cluster and Tree-View softwares. Genes differentially and significantly expressed were selected on the basis of significance analysis of the microarray data (SAM program). With the reclustering of only significantly expressed genes, it was possible to characterize the phases of thymus ontogeny, based on the differential profile of expression. Our method provided the detection of genes implicated in the cell signaling, such as the hematopoietic cell signal transducer gene, genes implicated in T-cell calcium influx (tyrosine phosphatase) and calcium signaling proteins (vesicle transport binding protein 3, proline rich Gla, casein kinase alpha 1 and Down syndrome homolog protein 1) and a gene important for the protein transport, including T-cell receptors chains, towards the cell membrane (Golgi SNAP receptor complex member 2). The results demonstrate that the cDNA microarray used to explore the gene expression was useful for understanding the modulation of several cell-signaling genes, including the calcium cascade pathway, which is important for individual stages of T-cell maturation and control of anergy during thymus ontogeny.

Elicitation of reactive oxygen species in Chlamydia pneumoniae-stimulated macrophages: a Ca2+-dependent process involving simultaneous activation of NADPH oxidase and cytochrome oxidase genes.

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected with C. pneumoniae and to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated with C. pneumoniae exhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose of C. pneumoniae led to an increase in the expression of these enzymes gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding of C. pneumoniae to CD14 receptors of macrophages. The data reported here imply that macrophages infected with C. pneumoniae produce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals.

Expression of iNOS gene in macrophages stimulated with 17beta-estradiol is regulated by free intracellular Ca2+.

17Beta-estradiol has potent Ca2+ ionophore capability and its signaling in macrophages is mediated through binding to surface and genomic receptors, resulting in transient nitric oxide (NO) elaboration. We decided to examine if the transient release of NO is due to Ca2+ influx pattern or the quenching effect of superoxide (*O2-) through peroxynitrite formation. Differential chelation of intracellular Ca2+ ([Ca2+]i) showed that NO generation was favored by [Ca2+]i concentration of 237 nM. Application of an estrogen receptor antagonist ICI 182 780 resulted in attenuation of estradiol mediated NO release. Studies directed at identifying the possible role of *O2-; in the attenuation of NO showed no supportive evidence. Inhibition of extracellular Ca2+ channel or extracellular and intracellular Ca2+ channels showed data consistent with a case for optimum Ca2+ influx signal favoring iNOS gene expression, accompanied by an elevation in iNOS protein. These data show that Ca2+ influx pattern determines macrophage NO elaboration.

Induction of lipoprotein lipase gene expression in Chlamydia pneumoniae-infected macrophages is dependent on Ca2+ signaling events.

Unregulated uptake of low density lipoprotein (LDL) in macrophages is the hallmark of early atherogenic lesions, and Chlamydia pneumoniae infection of macrophages induces this process by an unknown mechanism. It was therefore aimed in this study to investigate (i) the role of C. pneumoniae in macrophage expression of the lipoprotein lipase (LpL) gene, (ii) the probable role of Ca2+ influx signals and (iii) the effect of the process on LDL uptake. Lipoprotein lipase mRNA expression and LpL activity in infected RAW-264.7 cells were significantly upregulated. A biphasic Ca2+ influx signal was observed in infected cells with a moderate influx (303 nM Ca2+) favoring optimal LpL gene expression. Also, the antagonists of L-type Ca2+ channel in macrophages significantly down-regulated LpL gene expression and the biomolecular content of C. pneumoniae responsible for the observed events was in part found to be Chlamydia lipopolysaccharide (cLPS). Investigations aimed at determining the specific relevance of Ca(2+)-dependent lipoprotein lipase gene expression in C. pneumoniae-infected macrophages showed that the condition caused enhanced uptake of LDL which was abrogated by Calphostin-C-mediated down-regulation of LpL. This discovery of a specialized Ca2+ influx signal-mediated LpL upregulation in C. pneumoniae-infected macrophages provides a mechanistic insight into early events involving C. pneumoniae in macrophage foam cell formation resulting from LDL uptake.

Lack of evidence for functional ADP-activated human P2X1 receptors supports a role for ATP during hemostasis and thrombosis

AbstractPurine nucleotides acting through P2 receptors play key roles in platelet signaling. The P2X1 receptor is an adenosine triphosphate (ATP)-gated ion channel that mediates a rapid calcium influx signal, but can also synergize with subsequent adenosine diphosphate (ADP)-evoked P2Y1 receptor-mediated responses and thus may contribute to platelet activation during hemostasis. Recent studies have shown that P2X1 receptors contribute to the formation of platelet thrombi, particularly under conditions of high shear stress. Based on intracellular Ca2+ measurements a previous report has suggested that a splice variant of the P2X1 receptor, P2X1del, is expressed in platelets and, in contrast to the full-length P2X1WT receptor, is activated by ADP. In the present study we show that the P2X1del receptor fails to form functional ion channels and is below the limit of detection in human platelets. Furthermore, ADP does not contribute to the rapid ionotropic P2X receptor-mediated response in platelets. These results support the notion that ATP is the principal physiologic agonist at P2X1 receptors and that it plays a role in the activation of platelets. (Blood. 2003;102:3646-3651)

Decoding of Short-lived Ca2+ Influx Signals into Long Term Substrate Phosphorylation through Activation of Two Distinct Classes of Protein Kinase C

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca(2+) channels eliciting Ca(2+) influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca(2+) influx alone can activate PKC. The present study was conducted to investigate the Ca(2+) influx-induced activation mechanisms for two classes of PKC, conventional PKC (cPKC; PKCalpha) and novel PKC (nPKC; PKCtheta), in insulin-secreting cells. We have demonstrated simultaneous translocation of both DsRed-tagged PKCalpha to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate to the cytosol as a dual marker of PKC activity in response to depolarization-evoked Ca(2+) influx in the DsRed-tagged PKCalpha and GFP-tagged myristoylated alanine-rich C kinase substrate co-expressing cells. The result indicates that Ca(2+) influx can generate diacylglycerol (DAG), because cPKC is activated by Ca(2+) and DAG. We showed this in three different ways by demonstrating: 1) Ca(2+) influx-induced translocation of GFP-tagged C1 domain of PKCgamma, 2) Ca(2+) influx-induced translocation of GFP-tagged pleckstrin homology domain, and 3) Ca(2+) influx-induced translocation of GFP-tagged PKCtheta, as a marker of DAG production and/or nPKC activity. Thus, Ca(2+) influx alone via voltage-dependent Ca(2+) channels can generate DAG, thereby activating cPKC and nPKC, whose activation is structurally independent of Ca(2+).

Effective macrophage redox defense against Chlamydia pneumoniae depends on L-type Ca2+ channel activation.

Macrophage immune capability depends on their efficient redox potential expressed in the effective release of reactive oxygen species (ROS) and nitric oxide. In this study the effect of the activation of a specialized Ca(2+) channel on macrophage redox function during Chlamydia pneumoniae infection was explored. C. pneumoniae exhibited a profound and sustained Ca(2+) influx capacity, with evidence of activity attributable to their lipopolysaccharide (cLPS) content. Also the organism showed an additional Ca(2+) influx signal in macrophages exposed to thapsigargin, and there was evidence for the operation of a single ion channel of the L type as demonstrated by the effect of L-type channel antagonists (methoxyverapamil and nimodipine) despite exposure to Ca(2+)-rich medium. C. pneumoniae or cLPS induced intracellular ROS and NO generation in a manner consistent with dependence on intracellular calcium. L-type Ca(2+) channel blocking significantly prompted C. pneumoniae inclusion formation. These findings suggest that Ca(2+) influx signal and redox function in C. pneumoniae-infected macrophages depend on L-type Ca(2+) channel activation.

TEMPERATURE EFFECT ON ENTRAINMENT, PHASE SHIFTING, AND AMPLITUDE OF CIRCADIAN CLOCKS AND ITS MOLECULAR BASES

Effects of temperature and temperature changes on circadian clocks in cyanobacteria, unicellular algae, and plants, as well as fungi, arthropods, and vertebrates are reviewed. Periodic temperature with periods around 24 h even in the low range of 1–2°C (strong Zeitgeber effect) can entrain all ectothermic (poikilothermic) organisms. This is also reflected by the phase shifts—recorded by phase response curves (PRCs)—that are elicited by step- or pulsewise changes in the temperature. The amount of phase shift (weak or strong type of PRC) depends on the amplitude of the temperature change and on its duration when applied as a pulse. Form and position of the PRC to temperature pulses are similar to those of the PRC to light pulses. A combined high/low temperature and light/dark cycle leads to a stabile phase and maximal amplitude of the circadian rhythm—when applied in phase (i.e., warm/light and cold/dark). When the two Zeitgeber cycles are phase-shifted against each other the phase of the circadian rhythm is determined by either Zeitgeber or by both, depending on the relative strength (amplitude) of both Zeitgeber signals and the sensitivity of the species/individual toward them. A phase jump of the circadian rhythm has been observed in several organisms at a certain phase relationship of the two Zeitgeber cycles.Ectothermic organisms show inter- and intraspecies plus seasonal variations in the temperature limits for the expression of the clock, either of the basic molecular mechanism, and/or the dependent variables. A step-down from higher temperatures or a step-up from lower temperatures to moderate temperatures often results in initiation of oscillations from phase positions that are about 180° different. This may be explained by holding the clock at different phase positions (maximum or minimum of a clock component) or by significantly different levels of clock components at the higher or lower temperatures. Different permissive temperatures result in different circadian amplitudes, that usually show a species-specific optimum.In endothermic (homeothermic) organisms periodic temperature changes of about 24 h often cause entrainment, although with considerable individual differences, only if they are of rather high amplitudes (weak Zeitgeber effects). The same applies to the phase-shifting effects of temperature pulses. Isolated bird pineals and rat suprachiasmatic nuclei tissues on the other hand, respond to medium high temperature pulses and reveal PRCs similar to that of light signals. Therefore, one may speculate that the self-selected circadian rhythm of body temperature in reptiles or the endogenously controlled body temperature in homeotherms (some of which show temperature differences of more than 2°C) may, in itself, serve as an internal entraining system. The so-called heterothermic mammals (undergoing low body temperature states in a daily or seasonal pattern) may be more sensitive to temperature changes.Effects of temperature elevation on the molecular clock mechanisms have been shown in Neurospora (induction of the frequency (FRQ) protein) and in Drosophila (degradation of the period (PER) and timeless (TIM) protein) and can explain observed phase shifts of rhythms in conidiation and locomotor activity, respectively.Temperature changes probably act directly on all processes of the clock mechanism some being more sensitive than the others. Temperature changes affect membrane properties, ion homeostasis, calcium influx, and other signal cascades (cAMP, cGMP, and the protein kinases A and C) (indirect effects) and may thus influence, in particular, protein phosphorylation processes of the clock mechanism. The temperature effects resemble to some degree those induced by light or by light-transducing neurons and their transmitters. In ectothermic vertebrates temperature changes significantly affect the melatonin rhythm, which in turn exerts entraining (phase shifting) functions.

A ratiometric imaging method for mapping ion flux densities

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiom etric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the loc al influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratio ing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux dens ity distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution pat terns of ion channels active during a given stimulus may be derived.

Gonadotropin subunit transcriptional responses to calcium signals in the rat: evidence for regulation by pulse frequency.

Alterations in the frequency of calcium influx signals to rat pituitary cells can regulate the expression of gonadotropin subunit mRNAs in a differential manner, producing effects that are similar to those previously found for GnRH. The present study was conducted to investigate whether this reflects a transcriptional response to calcium pulse frequency, as determined by alterations in primary transcript (PT) expression. Perifused rat pituitary cells were given pulses of the calcium channel-activator Bay K 8644 (BK; with 10 mM KCl in the injectate) for 6 h. The response to alterations in pulse dose was examined by giving pulses of 1, 3, or 10 microM BK at 60-min intervals. Maximal increases in LHbeta and FSHbeta PTs were obtained with the 3-microM BK pulse dose and with the 10-microM dose for alpha. To investigate the effect of calcium pulse frequency, 3-microM BK pulses were given at intervals of 15, 60, or 180 min. Alpha PT was selectively stimulated by 15-min pulses and LHbeta by 15- and 60-min pulses of BK. In contrast, FSHbeta PT was maximally stimulated by the slower, 180-min pulse interval. These findings reveal that pulsatile increases in intracellular calcium stimulate alpha, LHbeta, and FSHbeta transcription in a differential manner. Thus, intermittent changes in intracellular calcium appear to be important in the transmission of GnRH pulse signals from the plasma membrane to the gene, and they may mediate the differential actions of pulse frequency on gonadotropin subunit gene expression.

Immunogold-labeled L-type Calcium Channels are Clustered in the Surface Plasma Membrane Overlying Junctional Sarcoplasmic Reticulum in Guinea-pig Myocytes—Implications for Excitation–contraction Coupling in Cardiac Muscle

Ca2+release through ryanodine receptors, located in the membrane of the junctional sarcoplasmic reticulum (SR), initiates contraction of cardiac muscle. Ca2+influx through plasma membrane L-type Ca2+channels is thought to be an important trigger for opening ryanodine receptors (««Ca2+-induced Ca2+-release»»). Optimal transmission of the transmembrane Ca2+influx signal to SR release is predicted to involve spatial juxtaposition of L-type Ca2+channels to the ryanodine receptors of the junctional SR. Although such spatial coupling has often been implicitly assumed, and data from immunofluorescence microscopy are consistent with its existence, the definitive demonstration of such a structural organization in mammalian tissue is lacking at the electron-microscopic level. To determine the spatial distribution of plasma membrane L-type Ca2+channels and their location in relation to underlying junctional SR, we applied two high-resolution immunogold-labeling techniques, label-fracture and cryothin-sectioning, combined with quantitative analysis, to guinea-pig ventricular myocytes. Label-fracture enabled visualization of colloidal gold-labeled L-type Ca2+channels in planar freeze-fracture electron-microscopic views of the plasma membrane. Mathematical analysis of the gold label distribution (by nearest-neighbor distance distribution and the radial distribution function) demonstrated genuine clustering of the labeled channels. Gold-labeled cryosections showed that labeled L-type Ca2+channels quantitatively predominated in domains of the plasma membrane overlying junctional SR. These findings provide an ultrastructural basis for functional coupling between L-type Ca2+channels and junctional SR and for excitation–contraction coupling in guinea-pig cardiac muscle.

L-type Ca2+ current as the predominant pathway of Ca2+ entry during I(Na) activation in beta-stimulated cardiac myocytes.

In the present study Ca2+ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca2+ indicator before and after beta-adrenergic stimulation. To clearly distinguish between Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum the Ca2+ store was blocked with 0.1 microM thapsigargin and 10 microM ryanodine. Omitting Na+ from the pipette filling solution minimized Ca2+ entry via Na+-Ca2+ exchange. Individual guinea-pig ventricular myocytes were voltage clamped in the whole-cell configuration of the patch-clamp technique and different membrane currents were activated using specific voltage protocols. The intracellular Ca2+ concentration was simultaneously recorded with a laser-scanning confocal microscope using fluo-3 as a Ca2+ indicator. Ca2+ entry pathways were discriminated using pharmacological blockers under control conditions and during beta-adrenergic stimulation with 1 microM isoproterenol (isoprenaline) in the bathing solution or 100 microM cAMP in the patch-clamp pipette. Isoproterenol or cAMP potentiated the Ca2+ influx signals recorded during L-type Ca2+ current activation but, more interestingly, also during Na+ current (INa) activation. The Ca2+ influx signal arising from L-type Ca2+ current activation was usually blocked by 50 microM Cd2+. However, the Ca2+ influx signal elicited by the Na+ current activation protocol was only curtailed to 56.4 +/- 28.2 % by 100 microM Ni2+ but was reduced to 17.9 +/- 15.1 % by 50 microM Cd2+ and consistently eliminated by 5 mM Ni2+. The pronounced Cd2+ and moderate Ni2+ sensitivity of the Ca2+ influx signals suggested that the predominant source of Ca2+ influx during the Na+ current activation - before and during beta-adrenergic stimulation - was a spurious activation of the L-type Ca2+ current, presumably due to voltage escape during Na+ current activation. Calculations based on the relationship between Ca2+ current and fluorescence change revealed that, on average, we could reliably detect rapid Ca2+ concentration changes as small as 5.4 +/- 0.7 nM. Thus, we can estimate an upper limit for the Ca2+ permeability of the phosphorylated TTX-sensitive Na+ channels which is less than 0.04:1 for Ca2+ ions flowing through Na+ channels via the proposed 'slip-mode' Ca2+ conductance. Therefore the slip-mode Ca2+ conductance of Na+ channels does not contribute noticeably to the Ca2+ signals observed in our experiments.

Psoriasis and Altered Calcium Metabolism: Downregulated Capacitative Calcium Influx and Defective Calcium-Mediated Cell Signaling in Cultured Psoriatic Keratinocytes1

Intracellular calcium plays an important part in the regulation of proliferation and differentiation of keratinocytes. Detached from their in vivo environment, cultured psoriatic keratinocytes were investigated by monitoring free intracellular calcium concentration, which was measured using fura-2/AM as a calcium-sensitive probe. The mean increase in intracellular calcium of psoriatic keratinocytes was significantly reduced compared with control keratinocytes when intracellular calcium stores were mobilized from endoplasmic reticulum with thapsigargin. This finding suggests defective capacitative calcium influx of psoriatic cells. Intracellular calcium stores were similar in psoriatic and control keratinocytes, when extracellular calcium was chelated with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid and intracellular calcium was depleted with thapsigargin. Mechanical wounding of keratinocyte monolayer resulted in a significantly reduced rise in intracellular calcium of psoriatic cells in low (< 0.1 mM) and high (1.8 mM) extracellular calcium suggesting defective intercellular coupling of psoriatic keratinocytes. Blocking of gap-junctions with heptanol in wounded keratinocytes did not affect the intracellular calcium response in psoriatic keratinocytes in contrast to healthy keratinocytes. Adding adenosine triphosphate to culture medium resulted in a more pronounced intracellular calcium increase than thapsigargin in psoriatic keratinocytes, suggesting that inositol triphosphate-mediated, P2-purinergic signaling was enhanced in these cells. Moreover, psoriatic keratinocytes maintained their defective responses up to at least fifth passage suggesting that psoriatic keratinocytes have an inborn error in calcium metabolism, rather than a localized defect in response to altered extracellular calcium gradient observed in vivo.

TAS-301 Blocks Receptor-Operated Calcium Influx and Inhibits Rat Vascular Smooth Muscle Cell Proliferation Induced by Basic Fibroblast Growth Factor and Platelet-Derived Growth Factor

The purpose of this study was to determine the effect of a recently synthesized drug, TAS-301 [3-bis(4-methoxyphenyl)methylene-2-indolinone], on vascular smooth muscle cell (VSMC) proliferation and the intracellular signal transduction pathways involved in VSMC proliferation. In an in vitro assay, TAS-301 inhibited the proliferation of rat VSMCs stimulated by platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor, or 2% fetal bovine serum in a concentration-dependent manner. TAS-301 dose-dependently inhibited the PDGF-induced Ca2+ influx; the concentration for the inhibition of Ca2+ influx was nearly identical to that for inhibition of VSMC proliferation. The Ca2+ influx induced by PDGF was also attenuated by NiCl2 but not by nifedipine, suggesting that PDGF-induced Ca2+ influx would be mediated by some non-voltage-dependent mechanisms. Furthermore, TAS-301 inhibited PDGF-induced activation of protein kinase C (PKC) and the phorbol 12-myristate 13-acetate-mediated induction of activator protein 1 (AP-1) in a concentration-dependent manner. These findings indicate that TAS-301 inhibited the proliferation of VSMCs by blocking voltage-independent Ca2+ influx and downstream signals such as the Ca2+/PKC signaling pathway, leading to AP-1 induction.

Function of Glycoprotein VI and Integrin α2β1 in the Procoagulant Response of Single, Collagen-Adherent Platelets

Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI.

Co-localization of Calcium-modulating Cyclophilin Ligand with Intracellular Calcium Pools

The calcium-modulating cyclophilin ligand (CAML) protein activates Ca2+ influx signaling when overexpressed in Jurkat T cells. Although CAML appears to directly participate in Ca2+-dependent signaling initiated by the transmembrane activator and CAML interactor cell surface receptor, its mechanism of action is unknown. To address this issue, we have determined its membrane topology, subcellular localization, and ability to mobilize intracellular Ca2+ pools. Fractionation of cell extracts on discontinuous sucrose gradients and indirect immunofluorescence indicate that CAML co-localizes with sarcoplasmic/endoplasmic reticulum calcium/ATPase-2 and calreticulin at membrane-bound cytosolic vesicles. Limited trypsin digests indicate that the hydrophilic NH2-terminal domain of CAML is directed toward the cytoplasm. Functionally, CAML overexpression was shown to deplete thapsigargin-sensitive intracellular Ca2+ pools. These data suggest that CAML may initiate Ca2+ signaling through activation of a capacitative Ca2+ influx pathway.

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways.

To investigate the mechanism of the regulation of human red blood cell deformability, we examined the deformability under mechanical stress. Washed human red blood cells were rapidly injected through a fine needle, and their filterability was measured using a nickel mesh filter. The decrease in filterability showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at approximately 50 muM. The decreased filterability was accompanied by no change in cell morphology and cell volume, indicating that the decrease in filterability can be ascribed to alterations of the membrane properties. Ca2+ entry blockers (nifedipine and felodipine) inhibited the impairment of filterability under mechanical stress. Prostaglandins E1 and E2, epinephrine, and pentoxifylline, which are thought to modulate the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level of red blood cells, improved or worsened the impaired filterability according to their expected actions on the cAMP level of the cells. These results strongly suggest that the membrane properties regulating red blood cell deformability are affected by the signal transduction system, including Ca(2+)-dependent and cAMP-mediated signaling pathways.