Detection of the Initial Step of Mesenchymal Differentiation of Teratocarcinoma Cells Using the Monoclonal Antibody Eccd-11

The monoclonal antibody ECCD-1 recognizes the Ca²⁺-dependent cell-cell adhesion molecule of teratocarcinoma stem cells (EC cells) and of a certain class of differentiated epithelial cells. It actively disrupts cell-cell adhesion when added to monolayer cultures of these cells, but does not affect adhesion of mesenchymal or neuronal cells. When ECCD-1 was added to clonal cultures of EC cells (PCC3/A/1 line), all the cells were initially sensitive to the antibody, but after 5 to 6 days of culture a fraction of the cells in certain colonies no longer reacted with the antibody although they expressed alkaline phosphatase activity, which is a marker of undifferentiated EC cells. We isolated these ECCD-1-resistant cells by recloning and examined their differentiation by clonal culture. Most of them differentiated into fibroblastic cells and a few into skeletal muscle-like cells, but none differentiated into any other cell types. From these observations, we suppose that the ECCD-1-resistant population of EC cells are committed to mesenchymal differentiation. The use of ECCD-1, thus, permitted us to detect EC cells at the initial stage of a particular differentiation pathway.     

Calmodulin Mediates the Ca-Dependent Regulation of Cx44 Gap Junctions

We have shown previously that the Ca²⁺-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca²⁺]_i in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca²⁺-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca²⁺-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca²⁺ release from the complex. Our data suggest a common mechanism by which the Ca²⁺-dependent inhibition of the α-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca²⁺-CaM.     

Immunological detection of cell surface components related with aggregation of Chinese hamster and chick embryonic cells.

Previous studies suggested that Chinese hamster V79 cells possess two mechanisms for their mutual adhesion, Ca²⁺-dependent and Ca²⁺-independent ones. We could prepare cells with only the Ca²⁺-dependent mechanism intact by dispersing cell monolayers with trypsin (0.01%) containing Ca²⁺. In the present study, we found that cells dispersed with a very low concentration of trypsin (0.0001%) in the absence of Ca²⁺ retain only the Ca²⁺-independent mechanism intact. Fab fragments of antibodies directed against surface antigens of V79 cells inhibited the aggregation of V79 cells by the Ca²⁺-independent mechanism, but did not inhibit the aggregation of these cells by the Ca²⁺-dependent mechanism. These results suggest that the two mechanisms of cell adhesion are based on different cellular components. Molecules responsible for the Ca²⁺-independent adhesion mechanism are probably cell surface components, because they were released from cells by the treatment with 0.01% trypsin without losing their specific antigenicity. The presence of adhesion mechanisms similar to those in V79 cells was shown in neural retinal cells of chick embryos. It was assumed, therefore, that these mechanisms of cell adhesion are generally present among a variety of cell types.     

Characterisation of the Egeria densa Planch. leaf symplast

The hydrophyllic dyes fluorescein glutamic acid, fluorescein glutamylglutamic acid (F(Glu)₂), fluorescein hexaglycine, fluorescein leucyldiglutamyl-leucine and 6-carboxyfluorescein are unable to pass the plasmalemma in leaves of E. densa. However, when injected into single cells the dye conjugates of molecular weight 665 dalton or less move freely from cell-to-cell. This intercellular movement presumably occurs via the plant symplast. Movement of F(Glu)₂ from the injected cell occurs with greatly reduced frequency when Ca²⁺, Mg²⁺ or Sr²⁺ are injected into the cell immediately prior to the dye. The fraction of dye injections leading to movement declines with increasing group II ion concentration in the electrode tip, up to 10 mM. Sodium and K ions do not affect dye movement. When dye injection is delayed 30 min after Ca²⁺ injection, dye movement is no longer inhibited. Thus the cells recover from the Ca²⁺ injection, indicating that the ion does not cause major cell damage. Recovery from Mg²⁺ injection is not complete within 60 min. Treatment of leaves with chemicals expected to raise the concentration of free intracellular group II ions, notably the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, the inhibitor of mitochondrial Ca²⁺ uptake trifluralin, or the ionophore A23187 also inhibits dye movement, while the calmodulin inhibitor trifluoperazine does not. Cytoplasmic streaming is inhibited by Ca²⁺ or Mg²⁺ injection and by the metabolic inhibitors. However when streaming is stopped by cytochalasin B, dye movement is not inhibited. Hence steaming is not necessary for dye movement. Thus the cytoplasmic concentration of free group II ions may directly regulate the permeability of the plant symplast.     

Molecular nature of the calcium-dependent cell-cell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody

The molecular nature of the Ca2+-dependent cell-cell adhesion system in mouse teratocarcinoma (t-CDS) was studied using a monoclonal antibody recognizing t-CDS. We isolated a hybridoma clone producing a monoclonal antibody (ECCD-1) able to disrupt cell-cell adhesion when added to monolayer cultures of teratocarcinoma cells. This antibody bound to the cells with intact t-CDS, resulting in an inhibition of their aggregation, but did not bind to cells from which t-CDS was removed by trypsin treatment in the absence of Ca2+. The binding of ECCD-1 to cell surfaces required Ca2+ but not other ions. Western blot analysis showed that ECCD-1 recognizes multiple cell surface proteins, the major one of which is a component with a molecular weight of 124,000. The binding of ECCD-1 to these antigens was Ca2+-dependent even in cell-free systems, suggesting that the molecules involved in t-CDS undergo conformational changes by binding with Ca2+, leading to conversion of their molecular structure into an active form. ECCD-1 also reacted with 8-cell stage mouse embryos and with certain types of epithelial cells (excluding fibroblastic cells) in various differentiated tissues collected from mouse fetuses, again affecting their cell-cell adhesion. We also showed that a monoclonal antibody (DE1) raised against gp84 (F. Hyafil et al., 1981, Cell 26, 447-454) recognizes the same antigens as ECCD-1.     

Calcium-dependent cell-cell adhesion molecules common to hepatocytes and teratocarcinoma stem cells

The molecules involved in Ca2+-dependent cell-cell adhesion systems (CDS) in mouse hepatocytes were characterized and compared with those in teratocarcinoma cells. Fab fragments of antibody raised against liver tissues (anti-liver) inhibited Ca2+-dependent aggregation of both liver and teratocarcinoma cells. A monoclonal antibody raised against teratocarcinoma CDS (ECCD-1) also inhibited the Ca2+-dependent aggregation of these two cell types equally. These antibodies induced disruption of cell-cell adhesion in monolayers of hepatocytes. Thus, CDS in these two cell types are not immunologically distinctive. Immunochemical analyses with these antibodies showed that CDS in both hepatocytes and teratocarcinoma cells involved at least two classes of cell surface proteins with molecular weights of 124,000 and 104,000. ECCD-1 selectively bound to hepatocytes but not to fibroblastic cells in liver cell cultures. Thus, the molecular constitution of CDS in hepatocytes and teratocarcinoma stem cells is identical. As ECCD-1 reacts with other classes of embryonic and fetal cells, the molecules identified here could have a major role in cell-cell adhesion in various tissues at any developmental stage of animals.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. V. Evidence that manganese inhibits a calcium-dependent step in programming for lysis

The mechanism of T-lymphocyte-mediated cytolysis consists of three successive steps: adhesion formation, programming for lysis, and killer-cell-independent lysis. Mg²⁺, but not Ca²⁺, is required for adhesion formation, whereas programming for lysis is strongly Ca²⁺ dependent. We have previously reported that the transition metal manganese can substitute for Mg²⁺ in supporting adhesion formation. In the present paper, we demonstrate that manganese inhibits programming for lysis. The inhibitory effect of Mn²⁺ on cytolysis can be reduced by increasing the concentration of Ca²⁺. Furthermore, inhibitor sequencing experiments were unable to distinguish the step blocked by Mn²⁺ from the Ca²⁺-dependent step. These results suggest that Mn²⁺ blocks a Ca²⁺-dependent step(s) in programming for lysis. Present evidence does not distinguish whether the action of Ca²⁺ in programming for lysis is via a Ca²⁺ influx (as a “second messenger?”) or whether Ca²⁺ simply serves as a cofactor at the cell exterior.     

Experimental manipulation of cell surface to affect cellular recognition mechanisms.

The mechanism of selective cell adhesion was studied using Chinese hamster V79 and chick embryonic neural retinal cells. Both of these cell types have been shown to have two experimentally separable mechanisms of adhesion; Ca²⁺-dependent and Ca²⁺-independent. Cells can be dispersed so that either or both of the mechanisms remain intact by use of different treatments. A method of labeling cells with FITC was devised to identify one of the two types of cells in a binary cell population. When cells with one of the two adhesion mechanisms were mixed with cells with the other mechanism, they segregated completely, forming independent aggregates, not only in the heterotypic combination of these cell types but also in the homotypic combination of each cell type. In contrast, when cells were mixed with others with the same adhesion mechanism, either Ca²⁺-dependent or -independent, they formed chimeric aggregates, even in the heterotypic cell combination. These results suggest that the specificity in each of those two mechanisms of cell adhesion plays an important role in cellular recognition processes.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca²⁺ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP₃) concentration changes using a fluorescent IP₃ sensor in single HeLa cells showing different patterns of histamine-induced Ca²⁺ oscillations in terms of the time constant of Ca²⁺ spike amplitude decay and the Ca²⁺ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca²⁺ signals and we found that there were cell-specific IP₃ dynamics depending on the patterns of Ca²⁺ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP₃ increases in HeLa cells. Modulation of IP₃ dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca²⁺ oscillations, such as the time constant of the temporal changes in the Ca²⁺ spike amplitude and the Ca²⁺ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP₃ production, rather than IP₃-induced Ca²⁺ release, can cause cell-to-cell variability in the patterns of Ca²⁺ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca²⁺ signals evoked by G protein-coupled receptor stimulation.     

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract

Resveratrol is a natural compound that affects cellular Ca²⁺ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i and WST-1 was used to measure viability. Resveratrol-evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Resveratrol-evoked Ca²⁺ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca²⁺]_i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca²⁺]_i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca²⁺]_i by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca²⁺-dependent cell proliferation.     

Maintenance of compaction and adherent-type junctions in mouse morula-stage embryos

Timed morulae of different stages of development were exposed to cytochalasin B causing depolymerisation of microfilaments and to ECCD-1 antibodies interacting with Ca²⁺-dependent adhesion molecules or cultured in the absence of calcium. All three treatments decompacted mid-morula-stage embryos within one hour. Late morulae were resistant to ECCD-1 antibody treatment and relatively resistant to calcium-free cultivation, but not to cytochalasin B treatment. Scanning electron microscopy revealed that the decompacting treatments not only loosened the interblastomere contacts but also resulted in rearrangement of the cell surface microvilli. Transmission electron microscopy showed that normal, untreated embryos had specialized membrane junctions in the most apical regions of the interblastomere contacts. Immunoelectron microscopy revealed that these apical junction areas contained vinculin, a protein typical of adherent junctions. Upon decompaction the apical junctions disappeared completely. When transferred back to the normal medium, the embryos rapidly started to recompact. Simultaneously the apical junctions and cell surface microvilli reassumed the organization characteristic of the morula stage. Late morulae that were resistant to treatment had normal apical junctional areas. During subcultivation in the normal medium, the treated morulae developed into morphologically normal blastocysts. These data indicate that adherent-type junctions and cell surface microvilli participate in the initiation and maintenance of compaction of morula-stage embryos.     

Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca²⁺ signal generated in cells by different effectors, where the Ca²⁺-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca²⁺-dependent and Ca²⁺-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca²⁺/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca²⁺-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca²⁺/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca²⁺ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.     

Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP₃) and intracellular free calcium (Ca_i²⁺) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Ca_i²⁺ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Ca_i²⁺ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Ca_i²⁺ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM × 1 h) and H-7 (30 μM × 18 h) potentiated the effects of 1 μg/ml bPTH(1–84) on Ca_i²⁺ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 μg/ml) was without significant effect on adherent cell Ca_i²⁺ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (∼ λ510 nm) detected and optimized at λ348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Ca_i²⁺ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Ca_i²⁺ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 μM × 30 min). PTH-induced Ca_i²⁺ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP₃ increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP₃/Ca_i²⁺). J. Cell. Biochem. 65:276–285. © 1997 Wiley-Liss, Inc.     

Binding of cadmium (Cd2+) to E-CAD1, a calcium-binding polypeptide analog of E-cadherin

Recent studies have shown that Cd²⁺ can damage the Ca²⁺-dependent junctions between renal epithelial cells in culture, and preliminary evidence suggests that this effect may involve the interaction of Cd²⁺ with E-cadherin, a Ca²⁺-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To determine whether or not Cd²⁺ might bind directly to the E-cadherin molecule, we studied the binding of Cd²⁺ to E-CAD1, a recombinant, 145-residue polypeptide that corresponds to one of the extracellular Ca²⁺-binding regions of mouse E-cadherin. By using an equilibrium microdialysis technique, we were able to show that Cd²⁺ could, in fact, bind to E-CAD1. The binding was saturable, with a maximum of one Cd²⁺ binding site per E-CAD1 molecule. The apparent dissociation constant (K_D) for the binding was about 20 μM, a concentration similar to that which has been shown to disrupt the junctions between epithelial cells. Other results showed that the binding of Cd²⁺ was greatly reduced when excess Ca²⁺ was included in the dialysis solution. These results suggest that Cd²⁺ can interact with the Ca²⁺ binding regions on the E-CAD1 molecule, and they provide additional support for the hypothesis that E-cadherin might be a molecular target for Cd²⁺ toxicity.     

Subcloning the RBL-2H3 mucosal mast cell line reduces Ca2+ response heterogeneity at the single-cell level

Ca²⁺ imaging experiments have revealed that for a wide variety of cell types, including RBL-2H3 mucosal mast cells, there are considerable cell-to-cell differences of the Ca²⁺ responses of individual cells. This heterogeneity is evident in both the shape and latency of the responses. Mast cells within a single microscopic field of view, which have experienced identical culture conditions and experimental preparation, display a wide variety of responses upon antigen stimulation. We have subcloned the RBL-2H3 mucosal mast cell line to test the hypothesis that genetic heterogeneity within the population is the cause of the Ca²⁺ response heterogeneity. We found that cell-to-cell variability was significantly reduced in four of five clonal lines. The response heterogeneity remaining within the clones was not an experimental artifact caused by differences in the amount of fura-2 loaded by individual cells. Factors other than genetic heterogeneity must partly account for Ca²⁺ response heterogeneity. It is possible that the complex shapes and variability of the Ca²⁺ responses are reflections of the fact that there are multiple factors underlying the Ca²⁺ response to antigen stimulation. Small differences from cell to cell in one or more of these factors could be a cause of the remaining Ca²⁺ response heterogeneity. © 1996 Wiley-Liss, Inc.     

Effects of ruthenium red on Ca2+ uptake and ATPase of sarcoplasmic reticulum of rabbit skeletal muscle

Ruthenium red, a powerful inhibitor of Ca²⁺ transport by mitochondria, does not inhibit the active Ca²⁺ uptake by sarcoplasmic reticulum isolated from rabbit skeletal muscle promoted by 5 mM ATP-Mg in the presence or absence of potassium oxalate. Although concentrations of ruthenium red up to 100 μM do not affect the active uptake of Ca²⁺, 25 μM of the inorganic dye inhibit the passive binding of Ca²⁺ by about 50%. This inhibitory effect is observed in sarcoplasmic reticulum even after its lipid fraction is extracted with acetone.Although active Ca²⁺ uptake by sarcoplasmic reticulum is not inhibited by ruthenium red, in the absence of oxalate it inhibits significantly the Ca²⁺-dependent ATPase activity but not the Mg²⁺-ATPase. However, if potassium oxalate is present, the Ca²⁺-stimulated ATPase is not sensitive to the dye. It is not clear how oxalate functions to protect the Ca²⁺-ATPase against the inhibitor effect of ruthenium red.The high sensitivity to ruthenium red of the Ca²⁺ transport mechanism in mitochondria as compared to the Ca²⁺ transport in sarcoplasmic reticulum may be useful in determining the extent to which each organelle functions in the cell to regulate intracellular free Ca²⁺.