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At least 703 records · Page 39Linked to original sources
PubMed1993

Phosphorylation of CD20 in cells from a hairy cell leukemia cell line. Evidence for involvement of calcium/calmodulin-dependent protein kinase II.

Hairy cell leukemia is an uncommon B cell lymphoproliferative disease of unknown etiology. We previously observed that CD20, a membrane protein involved in B cell activation, is hyperphosphorylated on hairy cells and that these cells have unusually high levels of intracellular free Ca2+. Therefore, we used a hairy cell line, HCLL-7876, to study the potential involvement of Ca(2+)-activated protein kinases in CD20 phosphorylation. Addition of the Ca2+ ionophore, ionomycin, increased CD20 phosphorylation both in activated B cells and in cells from the hairy cell line; addition of EGTA to either cell type decreased basal levels of CD20 phosphorylation. Ionomycin treatment of these cells resulted in increased kinase activity of cytosolic extracts toward syntide-2, a synthetic peptide substrate for calcium/calmodulin-dependent kinase II (CaM-KII), with kinetics similar to those of CD20 phosphorylation in the cell line. CD20 isolated from the cell line was a substrate for purified CaM-KII in vitro. Phosphopeptide maps of CD20 from untreated hairy cells or ionomycin-treated HCLL-7876 cells were similar to maps of CD20 that had been phosphorylated in vitro by CaM-KII. These results suggest that the unusually high levels of intracytoplasmic Ca2+ in hairy cells may enhance the phosphorylation of key surface proteins.

Amino Acid Sequence↗
PubMed1994

Tepoxalin, a novel immunosuppressive agent with a different mechanism of action from cyclosporin A.

Tepoxalin, a compound previously identified as a dual cyclooxygenase/lipoxygenase (CO/LO) inhibitor, is a potent inhibitor of T cell proliferation. Comparing the suppressive effects of tepoxalin and cyclosporin A (CsA) on OKT3-, PMA-, IL-2-, and PMA+ionomycin-induced T cell proliferations revealed marked differences in the mechanism of action between the two compounds. Whereas CsA was most effective in suppressing OKT3-stimulated proliferation, tepoxalin was more potent in inhibiting PMA-, PMA+ionomycin-, and IL-2-induced proliferation. Quantitative PCR (QPCR) assays used to detect cytokine messages showed that tepoxalin blocked IL-2 mRNA transcription in PMA- and PMA+ionomycin-, but not OKT3-stimulated T cells whereas CsA was most potent in inhibiting OKT3-induced IL-2 mRNA induction in these cells. Both tepoxalin and CsA did not inhibit the expression of IL-2R; however, only tepoxalin, but not CsA, inhibited the proliferation of IL-2-dependent blasts and the transcription of IFN-gamma, an IL-2-dependent target gene. Moreover, addition of exogenous IL-2 restored OKT3-induced proliferation to CsA- but not tepoxalin-treated cells. These data suggest that tepoxalin, but not CsA, suppressed T cell proliferation by inhibiting IL-2-induced signal transduction. Consistent with these findings, tepoxalin, unlike CsA, which was most potent when added at the initiation of OKT3 stimulation, was equally active, regardless of whether it was added at the beginning or 48 h after culture initiation. The difference in mechanism of action between tepoxalin and CsA was confirmed further by the synergistic suppressive effects on T cell proliferation upon co-administration of the two compounds.

Base Sequence↗
PubMed1994

Cyclic AMP inhibits phosphatidylinositol-coupled and -uncoupled mitogenic signals in T lymphocytes. Evidence that cAMP alters PKC-induced transcription regulation of members of the jun and fos family of genes.

T lymphocyte stimulation via the Ag receptor results in activation of phospholipase C gamma 1 that catalyses the hydrolysis of phosphatidylinositol (PI). The hydrolysis generates inositol phosphate and diacylglycerol, which in turn, increase intracellular Ca2+ concentration and activates protein kinase C, respectively. Agonists operating via the adenylate cyclase pathway or cell permeable cAMP analogues inhibit T cell activation by interfering with the PI-turnover. We have shown that dbcAMP inhibits PI-independent mitogenic signals in T cells after stimulation with TPA plus ionomycin. dbcAMP inhibited the TPA plus ionomycin-induced transcription of IL-2 and IL-2R genes in EL4 cells, suggesting interference with biochemic events downstream to PI hydrolysis and upstream to transcription of early activation genes. Because many of the early genes operating in T cell mitogenesis possess a TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, AP-1. dbcAMP increased the binding activity of nuclear proteins consisting of Fos:Jun heterodimers to a TRE-containing oligonucleotide, but altered the composition of Jun proteins in the AP-1. Furthermore, the TPA plus ionomycin-induced transcription program of members of the jun and fos family of genes was altered by dbcAMP, suggesting that inhibition of T cell proliferation by dbcAMP is a consequence of intervention in transcriptional regulation by TRE-binding proteins.

Animals↗
PubMed1993

Calcium ionophore-induced transient down-regulation of c-myb mRNA levels in Friend erythroleukemia cells.

The effects of calcium ionophores A23187 and ionomycin on the c-myb and c-myc mRNA levels have been investigated in the Friend erythroleukemia cell line F4-6 using Northern blot analysis. Treatment of the cells with 0.5-4 microM A23187 or 1-4 microM ionomycin induced a concentration-dependent decrease in c-myb mRNA; this decrease was abolished by EGTA. c-myc mRNA levels were only moderately affected. After 12-24 h of calcium ionophore exposure, c-myb mRNA returned to pretreatment levels. No similar decrease in c-myb mRNA was seen with the sodium ionophore monensin (up to 16 microM). The dimethyl sulfoxide-induced suppression of c-myb and also of c-myc mRNA levels was not prevented in Ca(2+)-free medium and thus appeared Ca(2+)-independent. A23187 and ionomycin were capable of inducing beta-globin mRNA synthesis in F4-6 cells. Prolonged calcium ionophore exposure, however, strongly reduced cell viability and resulted only in a slight hemoglobin increase at lower concentrations. These results suggest that a rise in [Ca2+]i may be a signal leading to a transient decrease in c-myb mRNA and the initiation of erythroid differentiation in Friend cells. The transient suppression of c-myb mRNA levels represents a common feature of the action of dimethyl sulfoxide and calcium ionophores.

Actins↗
PubMed1995

Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.

Lactating mammary epithelial cells synthesize large quantities of milk proteins, which they secrete vectorially at the apical membrane into the alveolar lumen of the gland. Recent work suggested that mammary protein secretion is not wholly constitutive, but may also occur in part through a regulated secretory pathway. This study used mouse mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix to compare the proportions of basally and apically-directed proteins secreted constitutively or in a regulated manner. On EHS matrix, mammary cells formed mammospheres, multicellular structures enshrouded in matrix material, within which they became polarised, formed tight intercellular junctions, and secreted milk proteins vectorially. Protein secreted basolaterally was collected in culture medium, whereas apically-secreted milk proteins accumulated in a closed lumen within the mammosphere, and were recovered by EGTA treatment of the cells in situ. Protein secretion was measured by following the release of radiolabelled protein after pulse-labelling with [35S]-methionine. Basolateral and apical secretion of [35S]-protein appeared complete within 1 h of pulse-labelling, consistent with immediate secretion through constitutive secretory pathways. However, addition of the calcium ionophore ionomycin induced a second wave of secretion in both directions. Ca(2+)-stimulated secretion occurred within 15 min of ionomycin addition, doubled the extent of basolateral and apical secretion, but did not change the populations of proteins secreted. Ionomycin treatment did not affect mammosphere morphology or mammary cell ultrastructure. The results suggest that lactating mammary epithelial cells secrete proteins apically and basolaterally by two pathways, one a Ca(2+)-independent constitutive pathway, the other a regulated pathway stimulated by elevation of intracellular Ca2+.

Animals↗
PubMed1996

Deficient CD4+ T cell proliferation in the class 1 MHC-restricted 2C TCR-transgenic mouse.

A comparative study of immune function and marker expression of CD4+ T cells from MHC class 1-restricted 2C TCR-transgenic (2C+) and control transgene-negative littermate (2C-) mice was performed. While 2C+CD4+ T cells resembled memory T cells on the basis of CD44highCD45RBlow expression, the majority of 2C-CD4+ T cells were of the CD44lowCD45RBhigh naive phenotype. Slightly lower levels of TCR-beta and CD3 were found on 2C+CD4+ T cell than 2C-CD4+ T cells. Vigorous proliferation by 2C-CD4+ T cells was observed upon stimulation with 1) anti-CD3 mAb presented through the FcR of macrophages; 2) immobilized (plate-bound) anti-CD3 + anti-CD28 mAbs; and 3) PMA + ionomycin. In marked contrast, all three mitogenic stimuli stimulated highly deficient proliferative responses by 2C+CD4+ T cells. However, significant IL-2 production was detected both in anti-CD3 and in PMA + ionomycin-stimulated cultures of 2C+CD4+ T cells. While intracellular calcium in 2C-CD4+ T cells rapidly increased following anti-CD3 addition, no such increase was observed for similarly stimulated 2C+CD4+ T cells. Anti-CD28, PMA, and coculture with 2C-CD4+ T cells each failed to significantly correct the deficient 2C+CD4+ T cells proliferation as induced by anti-CD3. In addition, IL-2, IL-4, and IL-7 supplements also failed to reverse the deficient proliferation of 2C+CD4+ T cells despite expression of IL-2R component alpha-, beta-chains and the gamma-chain common also to IL-4R and IL-7R. Thymus CD4+8- T cells from the 2C-transgenic mouse were similarly deficient in proliferation as spleen CD4+ T cells. A small subpopulation of CD4+ T cell from the 2C-transgenic mouse expressed the transgenic TCR alpha:beta heterodimer as detected by the 1B2 anti-2C clonotypic mAb; both 1B2+ and 1B2- subpopulations proliferated poorly in response to anti-CD3 and to PMA + ionomycin. These results raise the possibility that TCR engagement with MHC class 1 molecules during early intrathymic development can result in the emergence of CD4+ T cells characterized by unusual marker expression and function.

Animals↗
PubMed1996

Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: relationship to the cell-cycle and role of the retinoblastoma protein.

Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.

Antibodies, Anti-Idiotypic↗
PubMedDate not supplied

Modulation of calcium signalling and proliferation in monoblastoid U-937 cells.

The effects of TPA (12-0-tetradecanoylphorbol-13-acetate) and G-protein modulators on the concentration of cytoplasmic Ca2+ ([Ca2+]i), cytoplasmic pH and cell growth were investigated in monoblastoid U-937 cells. The G-protein activator NaF causes a dose-dependent increase of [Ca2+]i, that is partially sensitive to inhibition by pertussis toxin. The [Ca2+]i rise appears to come mainly from extracellular sources, and the Ca2+ influx is mediated by channels insensitive to the Ca2+ blocker verapamil. The Ca2+ ionophore ionomycin causes a biphasic rise of [Ca2+]i, reaching steady state levels slightly higher than those attained with NaF. TPA per se has no effect on [Ca2+]i, but potently reverses the NaF or ionomycin induced [Ca2+]i rise. Also, TPA partially counteracted the acidification induced by NaF. Both NaF and ionomycin per se had no effect on cell growth but partially counteracted TPA induced growth inhibition. Interferon-gamma and tumor necrosis factor-alpha did not affect [Ca2+]i by themselves but lowered the [Ca2+]i of NaF stimulated cells. The cytokines had no effect on cytoplasmic pH. This study indicates that elevations of [Ca2+]i in themselves does not trigger proliferation, but alterations of [Ca2+]i modulates the regulation of U937-cell growth.

Calcium↗
PubMed1997

Mechanism of nitric oxide production induced by H2O2 in cultured endothelial cells.

This study examined the mechanism of stimulation of nitric oxide (NO) synthesis induced by hydrogen peroxide (H2O2). To determine the role of Ca2+ on H2O2-induced NO synthesis in bovine aortic endothelial cells, the increases in intracellular Ca2+ and NO production induced by H2O2 were compared with the effects of the Ca2+ ionophore ionomycin in the same batches of endothelial cells. NO production was assessed by formation of [3H]citrulline from [3H]arginine, and changes in intracellular Ca2+ were measured using fluorescent indicator fluo-3 with a confocal laser scanning system. Both H2O2 (1-10 mM) and ionomycin (10(-7)-10(-5) M) increased intracellular Ca2+ and stimulated the synthesis of L-citrulline from L-arginine. Although H2O2 induced only a small increase in intracellular Ca2+, it markedly increased L-citrulline formation compared with ionomycin. Thus, stimulation of NO synthesis induced by H2O2 may involve the mechanisms other than the increases in intracellular Ca2+ in endothelial cells. In the particulate fraction from cultured endothelial cells, addition of exogenous H2O2 (1 mM) or catalase (100 U/ml) did not affect L-citrulline formation. However, co-administration of H2O2 and catalase stimulated L-citrulline formation. These findings suggested that not only the increases in intracellular Ca2+ but also the products by the reaction with H2O2 and catalase are likely to be involved in the stimulation of NO synthesis induced by H2O2.

Aniline Compounds↗
PubMedDate not supplied

Enhanced expression of cyclin-dependent kinase inhibitor in apoptosis of androgen-independent prostatic cancer cell line induced by calcium ionophore.

In order to examine the relationship between apoptosis of androgen-independent prostatic cancer cells and cell cycle-associated proteins, TSU-pr1 human prostatic cancer cells were chronically exposed in vitro to the calcium ionophore ionomycin to sustain an elevation in their intracellular free calcium concentration. Temporal analysis demonstrated that the death of these cells does not require cell proliferation and involves fragmentation of genomic DNA into nucleosome sized pieces. Morphological analysis demonstrated that this death process is via apoptosis. During the apoptotic process induced by ionomycin, expression of cyclin-dependent kinase inhibitor p27Kip1 increased. Flow cytometric analysis showed that the treatment resulted in a block in G0/G1 of the cell cycle. These results demonstrate that even nonproliferating androgen-independent prostatic cancer cells can be induced to undergo apoptosis if a modest elevation in the intracellular free calcium is sustained for a sufficient time. p27Kip1 protein is a candidate for the cell cycle regulator in ionomycin-treated TSU-pr1 cells.

Androgens↗
PubMed1997

Cyclosporine-insensitive partial signaling and multiple roles of Ca2+ in Fas ligand-induced lysis.

The induction of Fas ligand (FasL) mRNA expression and FasL-mediated cytotoxicity in CD8+ CTL is a rapid and transient response to activation via the TCR. This response can also be initiated by pharmacologic activation of two major TCR signaling pathways using phorbol ester (PMA) and calcium ionophore (ionomycin). In these experiments using CD8+ alloreactive cell lines, we demonstrate that induction of FasL mRNA can occur in response to either PMA or ionomycin independently. However, only the ionomycin pathway is sensitive to inhibition by cyclosporine A. Both pathways are blocked by genistein, a general protein tyrosine kinase inhibitor. The magnitude of induction of FasL mRNA is not proportional to the manifested FasL-dependent cytotoxicity. We also found a calcium requirement for cytotoxicity that is unrelated to FasL mRNA induction. In addition to the positive effects of constitutive and induced calcium levels on FasL-mediated cytotoxicity, calcium may play a role in the rapid down-regulation of the response. We also present data suggesting that CD2 and LFA-1 contribute to FasL-mediated cytotoxicity. Together these results suggest pathways by which partial TCR activation could, among the many activation-induced functions of a T cell, selectively lead to the induction of FasL-mediated cytotoxicity that can be regulated by lineage/ activation-dependent accessory molecules on the target.

Animals↗
PubMed1998

Desensitization of formyl peptide receptors is abolished in calcium ionophore-primed neutrophils: an association of the ligand-receptor complex to the cytoskeleton is not required for a rapid termination of the NADPH-oxidase response.

Binding of ligands to N-formyl peptide chemoattractant receptors exposed on human neutrophils generates signals in the cells that induce an activation of the superoxide anion producing NADPH-oxidase. Ligand binding is followed by a rapid association of the ligand-receptor complex with the cytoskeleton, a process leading to desensitization of the cells with respect to NADPH-oxidase activation. We show that neutrophils that have experienced an intracellular calcium rise obtained through interaction with the calcium-specific ionophore ionomycin are "primed" with respect to the FMLP-induced production of superoxide anions. Mobilization of FMLP receptors from intracellular pools is one well-known mechanism behind the primed response. Based on our finding that ionomycin-treated neutrophils could not be desensitized, we suggest that the lack of association between the ligand-receptor complex and the cytoskeleton is an additional priming mechanism. Since in vivo-exudated neutrophils, which also had mobilized intracellular organelles, could be desensitized, we suggest that the abolished desensitization in ionomycin-treated neutrophils is not due to an inability of newly recruited receptors to couple to the cytoskeleton. We show that a rapid termination of FMLP-induced superoxide anion production is obtained in both desensitizable and nondesensitizable neutrophils, suggesting that the desensitization phenomenon is of limited importance in the oxidase termination process.

Adult↗
PubMed1998

Differences between responses of naive and activated T cells to anergy induction.

T cell unresponsiveness to Ag stimulation can be induced by several means. The precise mechanism by which this process occurs remains poorly understood. Preincubating T cells with either EDCI-fixed APC or ionomycin is a proven means of inducing T cell anergy with reduced IL-2 production in response to Ag stimulation. Using T cells from mice expressing the TCR transgene DO11.10, which is specific for a peptide (323-339) derived from hen egg OVA, we demonstrate that naive cells obtained directly from the host are resistant to the anergy induction by either fixed APC or ionomycin. TCR transgenic mice also deficient in the recombination-activating gene-2 (RAG-2(-/-)), preventing the formation of T cells with endogenous TCRs, were immunized with OVA, and in vivo activated T cells with low expression of CD62 were isolated. These primed cells possess the same sensitivity to ionomycin-induced anergy as in vitro activated cell lines. This unresponsive state most profoundly affects Ag-induced IL-2 production, with IFN-gamma and IL-3 affected to a lesser degree and no effect observed on IL-4 production. Thus, T cells in vivo can be distinguished phenotypically by their susceptibility to anergic stimuli. Anergy so induced affects selected T cell functions.

Animals↗
PubMed1999

MUC5AC mucin is a component of the human precorneal tear film.

PURPOSE: Mucins are important structural and functional components of the precorneal tear film, yet little is known of their composition and synthesis. The mRNAs of MUC1, MUC4, and MUC5AC have previously been identified in human conjunctiva. Of these, only MUC5AC mRNA appears to be associated with goblet cells. The purpose of the this study was to quantify MUC5AC transcript levels, to identify MUC5AC protein in conjunctiva, tears, and goblet cells and to determine whether this mucin is secreted in response to the calcium ionophore ionomycin. METHODS: MUC5AC mRNA from normal human conjunctiva was identified, quantified, and compared with beta2-microglobulin levels using a competitive reverse transcription-polymerase chain reaction (RT-PCR) method. An antibody to a MUC5AC peptide was used to localize this mucin in conjunctival sections by immunohistochemistry. Anti-MUC5AC antiserum was used to label western blot analysis of conjunctiva and tears. Conjunctival tissues were incubated with ionomycin, and secreted mucins were detected with Helix pomatia agglutinin conjugated to horseradish peroxidase and with anti-MUC5AC antiserum. RESULTS: MUC5AC and beta2-microglobulin transcripts were expressed at a ratio of approximately 1:500. Immunochemical labeling showed that MUC5AC was localized in conjunctival goblet cells and at the apical surface of the conjunctival epithelium. MUC5AC protein was present in conjunctiva and in the tear film. Ionomycin stimulation of conjunctival secretion resulted in a fourfold increase in total mucin secretion and in a corresponding increase in secreted MUC5AC. CONCLUSIONS: MUC5AC is synthesized by goblet cells of the normal human conjunctiva, and this mucin is a component of conjunctival secretions and of normal human tears.

Adult↗
PubMed1998

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C.

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC.

Alkaline Phosphatase↗
PubMed1996

Transmembrane potential responses during HL-60 promyelocyte differentiation.

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/ macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC2-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by KCa channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl- conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions.

Calcium↗
PubMed1995

Dynamics of triton-insoluble and triton-soluble F-actin pools in calcium-activated human polymorphonuclear leukocytes: evidence for regulation by gelsolin.

Gelsolin, a Ca++ activated, 90 kd actin binding protein, can regulate actin polymerization in polymorphonuclear leukocytes (PMNs) via severing of filaments to dissolve gels or by capping of filament ends to limit polymerization. In Triton-lysed PMNs, 30% of gelsolin is bound to the Triton-soluble F-actin (TSF) pool and none is bound to the Triton-insoluble F-actin (TIF) pool. Calcium-activated PMNs exhibit concurrent temporal and quantitative TIF growth and TSF and total F-actin loss. To determine if gelsolin plays a role in regulating TSF pool size, we monitored gelsolin-actin interactions and TIF, TSF and G-actin content at 5 second intervals in PMNs activated with the calcium ionophore, ionomycin. Actin pools were measured by NBDphallacidin binding and by gel scans and expressed relative to basal; gelsolin-actin interactions were measured as change in the amount of EGTA-resistant gelsolin:actin (G:A) complexes and by immunoblot quantification of gelsolin in actin pools. In basal PMNs, 33% of PMN gelsolin is bound in 1:1 EGTA-resistant G:A complexes and TSF and TIF retain 30% and 0% of PMN gelsolin, respectively. By 20 seconds after ionomycin addition, TSF decreases, TIF increases and a fraction of gelsolin repartitions from the TSF to the TIF pool. At maximum change (60 seconds), total F-actin (TIF + TSF) and TSF decrease and TIF increases by 25%; gelsolin is bound to both TSF and TIF (35% of total gelsolin in each pool), and 1:1 EGTA-resistant G:A complexes increase from 33% to 70%. No changes occur in cells activated by ionomycin in the absence of Ca++. The data show Ca++ activated TIF growth and TSF loss are temporally and quantitatively associated with an increase in the percent of gelsolin bound to actin and the translocation of gelsolin from TSF to TIF. This is unique, since no other PMN activator is known to repartition gelsolin into TIF actin. Further, the Ca++ activated initial increase in TIF concurrent with a fall in TSF without a change in total F-actin or G-actin content suggest that TIF grows initially only by TSF annealing/cross-linking to TIF. Gelsolin may regulate these events.

Actins↗
PubMed1986

Requirements for growth of immature thymocytes from fetal and adult mice in vitro.

We report here defined culture conditions that allow reproducibly the growth of the majority of immature thymocytes from both fetal (14-15 days of gestation) and adult mice. The combination of phorbol myristate acetate (PMA), ionomycin and recombinant interleukin 2 (IL2) is both sufficient and necessary to induce growth of about 1/6.2 (range 1/3-1/9) and 1/4.3 (range 1/2-1/7) immature thymocytes from adult and fetal mice, respectively, in serum-free cultures. Several other combinations tested (e.g. PMA + IL2, concanavalin A + IL2) were poorly or not active. None of the agents tested alone (PMA, ionomycin, concanavalin A, pokeweed mitogen, IL2) had any effect. We found no evidence for a role of IL1 and IL3 on growth of these cells. The growth of activated immature thymocytes from either fetal or adult mice was inhibited by a monoclonal antibody against mouse IL2 receptors. Under the same conditions that stimulated growth of most immature thymocytes, they did not mature into cells expressing Lyt-2, L3T4 or T cell antigen receptor (KJ16) after 7 to 15 days of continuous proliferation in culture. Nor did they give rise to cells with cytolytic activity after 7-9 days of culture. In some but not all experiments cultures of immature thymocytes from adult mice but not from fetal mice generated cells (1 out of 120-310) with helper function for B lymphocytes. While we confirmed here that approximately 50-70% freshly isolated immature thymocytes express receptors for IL2, our results indicate that these cells need to be activated (by e.g. PMA + ionomycin) to respond to IL2. A possible mechanism to account for the expression of nonfunctionally competent IL2 receptors is proposed and our results concerning the maturation of immature thymocytes in vitro are discussed.

Animals↗