Presence Of Ionomycin Research Articles

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development.

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.

Th1/Th2 shift in HIV lymph nodes: no contribution of CD6o cells.

An expansion of CD8+60+ cells with Th2 type helper function has been observed in HIV-infected individuals. A Th1/Th2 shift in late HIV infection might be related to the functional activity of this subset. Our objective was to test the ability of lymph node (LN) lymphocytes to produce cytokines of the Th1 and Th2 type. LN cells were stimulated with PMA in the presence of ionomycin and brefeldin A. After surface staining for CD4, CD8 and CD60 and intracellular staining for interferon gamma (IFNgamma), interleukin 2 (IL-2) or IL-4 and IL-10, the percentage of cytokine producing lymphocytes (CPL) was determined by flow cytometry. LN of nine individuals in the early stage of HIV infection were compared to late stage patients. CD4+ subset: in early stages of HIV infection the percentage of Th1 CPL was 1.9 times higher than that of Th2 CPL. In late stages of infection the Th1 responding cells were not found more frequently than Th2 ceLLs. CD8+ subset: no Th1/Th2 shift was detected during early or late stages of HIV infection. CD60+ subset: a maximum of 32.1 +/- 7.8% of double positive cells of the CD8+60+ type produced Th2 type cytokines. A small percentage (< 8%) of CD60+ cells also produced Th1 cytokines. No shift in the cytokine production was seen in early or late stages of infection. At single cell level a shift in cytokine production in LN cells can only be detected in the CD4+ subset. Thus, the CD60+ subset does not seem to contribute to the putative Th1/Th2 shift attributed to the immunopathogenesis of HIV-induced destruction of the immune system.

Hypoxia-Effects on Cai-Signaling and Ion Transport Activity of Lung Alveolar Epithelial Cells

In excitatory cells specific responses upon changes in PO₂ are mediated by changes in intracellular Ca (Ca_i). We wanted to know whether ion transport of lung alveolar epithelial cells is regulated by Ca_i and whether Ca_i and Ca_i -signaling are affected by hypoxia in a way that might explain hypoxic transport inhibition (Mairbäurl et al. AJP 273: L797, 1997). The activity of transport (Na/K-pump, Na/K/2Cl-cotransport) was measured as unidirectional ⁸⁶Rb-uptake after A549 cells were exposed to hypoxia (3% O₂). Ca_i of primary cultured rat alveolar type II cells was measured by fura-2 epifluorescence. Depletion of Ca_i by extracellular chelators in presence of ionomycin or with thapsigargin as well as PKC inhibition decreases ⁸⁶Rb-uptake of normoxic and hypoxic A549 cells, whereas an increased Ca_i activates transport. Neither immediate nor prolonged exposure to hypoxia changes Ca_i significantly. The increase in Ca_i upon stimulation with ATP, which is caused mainly by release from intracellular stores, is smaller in hypoxia than in normoxia. These results indicate that ion transport of alveolar epithelial cells is modulated by Ca_i. A change in Ca_i does not mediate hypoxic transport inhibition. The decreased Ca_i transients in hypoxia might indicate a blunted response to extracellular stimuli.

Evaluating the role of dibutyl-cAMP and Ca++ as an MT inducer in a clonal rat hepatoma cell

Metallothioneins (MT) is a low molecular weight, cysteine-rich, metal-binding protein that can be induced when cells and tissues are exposed to metal such as Cd, Zn, Cu and Hg. MT could also be induced by non-metallic stimuli including physiological, chemical and pathological stresses. It has been suggested that signalling molecules such as cAMP or Ca ++ might also induce MT. In the present study, the mode of induction of MT in the RH-35 cells was investigated with reference to the integrity of the cells. The degree of MT induction was measured by the Cd-saturation assay in which total Cd bound to the heat-stable MT was determined by the graphite furnace atomic absorption spectrometry. It was demonstrated that Zn and Cd could markedly increase cellular MT content. Alpha-amanitin, which blocks DNA transcription, can significantly decrease the Cd induced MT induction. MT induction was also lowered by cycloheximide, which blocks protein translation. However, dibutyl-cAMP, Ca ++ and Ca ++ in the presence of ionomycin were unable to induce cellular MT. Using this model, the results supported that notion that MT induction might not be mediated by cAMP or Ca ++ in this cell line.

Positive and Negative Regulation of Human T Cell Activation Mediated by the CTLA-4/CD28 Ligand CD80

CD28 and CTLA-4 are related receptors that differentially regulate T cell activation. Despite the fact that they bind the same ligands, CD28 is a classical costimulator enhancing proliferation whereas CTLA-4 appears to perform negative regulatory functions. In this study, we have utilized the natural ligand for CD28 and CTLA-4 (CD80) to determine under what circumstances positive and negative effects are operative. We show here that the stimulation of purified human T cells with phorbol ester and ionomycin is inhibited in the presence of Chinese hamster ovary (CHO) cells expressing CD80. This inhibition is reversed by blocking with both anti-CD80 or Fab fragments of anti-CTLA-4 but also requires CD28 engagement. Furthermore, we show that the inhibitory function of CD80 requires elevated intracellular calcium since inhibition was observed only in the presence of ionomycin. In the absence of intracellular calcium elevation, CTLA-4 was not expressed at the cell surface, and CD80 acted positively as a costimulator of T cells, via CD28. These results demonstrate that the natural ligand CD80 can either costimulate or inhibit T cell responses depending on the conditions of T cell stimulation.

Insulin inhibits vascular smooth muscle contraction at a site distal to intracellular Ca2+ concentration.

Several hypertensive states are associated with resistance to insulin-induced glucose disposal and insulin-induced vasodilation. Insulin can inhibit vascular smooth muscle (VSM) contraction at the level of the VSM cell, and resistance to insulin's inhibition of VSM cell contraction may be of pathophysiological importance. To understand the VSM cellular mechanisms by which insulin resistance leads to increased VSM contraction, we sought to determine how insulin inhibits contraction of normal VSM. It has been shown that insulin lowers the contractile agonist-stimulated intracellular Ca2+ (Ca2+i) transient in VSM cells. In this study, our goal was to see whether insulin inhibits VSM cell contraction at steps distal to Ca2+i and, if so, to determine whether the mechanism is dependent on nitric oxide synthase (NOS) and cGMP. Primary cultured VSM cells from canine femoral artery were bathed in a physiological concentration of extracellular Ca2+ and permeabilized to Ca2+ with a Ca2+ ionophore, either ionomycin or A-23187. The resultant increase in Ca2+i contracted individual cells, as measured by photomicroscopy. Preincubating cells with 1 nM insulin for 30 min did not affect basal Ca2+i or the ionomycin-induced increase in Ca2+i, as determined by fura 2 fluorescence measurements, but it did inhibit ionomycin- and A-23187-induced contractions by 47 and 51%, respectively (both P < 0.05). In the presence of 1.0 microM ionized Ca2+, ionomycin-induced contractions were inhibited by insulin in a dose-dependent manner. In the presence of ionomycin, insulin increased cGMP production by 43% (P < 0.05). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), a selective inhibitor of guanylate cyclase that blocked cGMP production in these cells, completely blocked the inhibition by insulin of ionomycin-induced contraction. It was found that the cells expressed the inducible isoform of NOS. NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester (0.1 mM), inhibitors of NOS, did not affect ionomycin-induced contraction but prevented insulin from inhibiting contraction. We conclude that insulin stimulates cGMP production and inhibits VSM contraction in the presence of elevated Ca2+i. This inhibition by insulin of VSM contraction at sites where Ca2+i could not be rate limiting is dependent on NOS and cGMP.

Autocrine proliferating B cells are susceptible to terminal differentiation and apoptosis

Lymphocytes that proliferate autonomously are thought to be arrested at certain steps in differentiation. Here we demonstrate that autocrine proliferating B cells can be induced to terminal differentiation in the presence of ionomycin plus phorbol dibutyrate. The mature CD23high/CD38low B cell phenotype converts to the CD23low/CD38high plasma cell phenotype associated with increased immunoglobulin secretion and PC1 expression and a loss in surface immunoglobulin. Simultaneously, the cells arrest in proliferation and enter apoptosis at day 10. The cytokines IL-1alpha, IL-6, TNFalpha and TNFbeta that are required to sustain continuous growth are secreted in substantially increased amounts mediating entry into apoptosis of the proliferating cells. Decrease in IL-10 secretion sustains this process. Our results draw the concept that once plasmacytoid differentiation is initiated, the growth sustaining network of autocrine cytokines is disturbed in a particular fashion depriving appropriate signals to suppress the differentiation associated apoptotic program.

In Situ Compositional Analysis of Acidocalcisomes in Trypanosoma cruzi

We measured the elemental content of different compartments in Trypanosoma cruzi epimastigotes using quick freezing, ultracryomicrotomy, and electron probe microanalysis. Vacuoles identified by high electron density contained (in units of mmol/kg dry weight +/- S.E.) large amounts of phosphorus (1390 +/- 13), magnesium (646 +/- 19), calcium (171 +/- 5), sodium (161 +/- 18), and zinc (148 +/- 6). No other compartment had appreciable calcium or zinc content. Iron (128 +/- 16 mmol/kg) was detected only in vacuoles distinct from the electron-dense vacuoles and other organelles. Incubation of cells for 70 min in culture medium in the presence of ionomycin plus nigericin led to a very significant 3- or 2-fold increase in potassium in the electron-dense vacuoles and the iron-rich vacuoles, respectively, with no significant change in the other elements investigated. This indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to what were described previously as acidocalcisomes, i.e. acidic compartments rich in Ca2+. The acidocalcisomes were investigated by separation of epimastigote fractions on Percoll gradients in combination with Triton WR-1339 treatment. This detergent caused a rapid vacuolation; these vacuoles were shown by electron microscopy to be largely transparent, with a diffuse matrix. Percoll gradient fractionation demonstrated decreases in the density of various organelle markers in detergent-treated cells compared with controls. Large decreases in the density of the acidocalcisome and the mitochondrion were seen, as well as smaller decreases in the density of the other markers. Conventional electron microscopy of epimastigotes loaded with gold-labeled transferrin indicated that the endosomal system was separate from vacuoles that probably corresponded to the calcium-containing organelles detected by electron probe microanalysis. The combined results provide evidence that acidocalcisomes are organelles different from lysosomes or other organelles previously described in these parasites.

Regulation of Mitogen-activated Protein Kinase Phosphatase-1 Expression by Extracellular Signal-related Kinase-dependent and Ca2+-dependent Signal Pathways in Rat-1 Cells

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.

Regulation of motility of cells from marine sponges by calcium ions

Sponges are known not to contain muscle and nerve cells. Since sponge cells are characterized by high motility we determined the effect of intracellular calcium ion concentration ([Ca 2+] i) on their motility. Addition of the Ca 2+ ionophore ionomycin to dissociated cells from the marine sponge Suberites domuncula caused in Ca 2+-containing artificial seawater (ASW) an increase in motility from 0.2 μm/min (absence of the ionophore) to 3.7 μm/min (presence of ionomycin). When the experiments were performed in Ca 2+-free medium, no effect of ionomycin could be observed. In parallel experiments the changes of [Ca 2+] i, using the dye Fura-2 were measured. The experiments revealed that ionomycin causes an influx of Ca 2+ into the cytosol of cells suspended in Ca 2+-containing artificial seawater. In contrast, if cells were suspended in Ca 2+-free artificial seawater, no increase of [Ca 2+] i occurred. Incubation of cells in the presence of inhibitors, specific for endoplasmatic Ca 2+-ATPase in mammals such as thapsigargin, cyclopiazonic acid, or 2,5 di-t-butylhydrochinone, did not influence the [Ca 2+] i if cells were suspended in Ca 2+-free artificial seawater. From these data we conclude that the [Ca 2+] i is primarily regulated through channels in the plasma membrane. In addition we summarize experimental evidence indicating that the [Ca 2+] i is involved in the control of cell motility. From the marine sponge Geodia cydonium a partial sequence of the myosin cDNA has been cloned. The deduced amino acid sequence comprises highest homology to nonmuscle myosin type II found in higher invertebrates and vertebrates. Taken together, these data show that the [Ca 2+] i level in sponge cells can be modulated by incubation with ionomycin. An increase of the Ca 2+ level parallels with higher motility of cells, suggesting an activation of Ca 2+-dependent protein kinases of myosin type II. Investigations on the ionomycin-activated influx of Ca 2+ into the cytosol revealed that predominantly the Ca 2+ channels in plasma membrane control the level of [Ca 2+] i.

Effect of Ionomycin on Cell pH in Isolated Renal Proximal Tubules

In isolated rabbit proximal tubules the addition of 2.0 μM but not 0.2 μM ionomycin induced a sustained increase in cell pH ([pH]i). This [pH]iresponse to 2.0 μM ionomycin was shown to be independent of several transporters such as Na+/H+exchanger, Na+-HCO−3cotransporter, Cl−/HCO−3exchanger, or H+-ATPase. On the other hand, the removal of extracellular Ca2+abolished the [pH]iincrease or even induced a transient [pH]idecrease in the presence of ionomycin. These results are consistent with the induction of Ca2+/H+exchange by ionomycin. Therefore Ca2+ionophores should be used with caution as probes to estimate renal tubule functions.

Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid

Abstract The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all- trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.

Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid

The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all-trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.

Cytotoxic action of tri-n-butyltin on dissociated rat cerebellar neurones: a flow-cytometric study.

The effects of tri-n-butyltin on cell viability and intracellular Ca2+ concentration ([CA2+]i) were examined by a flow cytometer in rat cerebellar neurones to reveal the contribution of tri-n-butyltin-induced increased in the [Ca2+]i to its cytotoxicity. Tri-n-Butyltin decreased the cell viability in association with increased [Ca2+]i at concentrations of 0.3 microM or more. Decrease or increase of the extracellular Ca2+ concentration ([Ca2+]o) respectively decreased or increased the cell viability. However, cell viability in the presence of ionomycin which increased [Ca2+]i more significantly than tri-n-butyltin was higher than that in the presence of tri-n-butyltin. Tri-n-butyltin also decreased cell viability under nominally [Ca2+]o-free conditions although the [Ca2+]i increase by tri-n-butyltin was still lower than the control [Ca2+]i under normal [Ca2+]o (2 mM). Therefore, it is unlikely that neuronal death induced by tri-n-butyltin is entirely dependent on the tri-n-butyltin-induced increase in [Ca2+]i.

Calcium-activated potassium current in cultured rabbit retinal pigment epithelial cells.

Calcium-activated potassium current was studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell and single channel patch-clamp recording techniques. When K+ was the principal cation in the electrode, depolarizing voltage steps from a holding potential of -60 mV activated outwardly rectifying current. Outward K+ current was increased by the Ca2+ ionophore ionomycin and reduced when the extracellular Ca2+ concentration was decreased from 2.5 mM to 100 nM in the presence of ionomycin. Outward K+ current recorded in the presence of ionomycin was blocked by iberiotoxin and by charybdotoxin. Single channel recording from cell-attached and excised membrane patches revealed a large conductance Ca2+-activated K+ (K(Ca)) channel. Identification of K(Ca) channels was based on: 1) the voltage-dependence of channel opening; 2) the large unitary conductance (> 200 pS with symmetrical 130 mM K+); 3) the dependence of the reversal potential on the K+ gradient; and 4) increased channel opening after exposure of the cytosolic surface of excised membrane patches to elevated Ca2+. These results demonstrate that Ca2+-activated K+ channels are present in rabbit RPE cells and may play an essential role in the regulation of membrane potential and ion transport.

Regulation of interferon-gamma mRNA in a cytolytic T cell clone: Ca(2+)-induced transcription followed by mRNA stabilization through activation of protein kinase C or increase in cAMP.

Activation pathways inducing the expression of the interferon (IFN)-gamma gene in a cytotoxic T lymphocyte (CTL) clone were studied for their effects on transcription and on mRNA stability. IFN-gamma was secreted by the CTL clone in response to the Ca2+ ionophore ionomycin when used in conjunction with either protein kinase C (PKC)-activating phorbol 12-myristate 13-acetate (PMA) or with agents increasing cAMP, including prostaglandin E2. We describe that ionomycin induced IFN-gamma gene transcription, which was totally inhibited in the presence of cyclosporin A (CSA), an immunosuppressant forming a calcineurin-inhibiting complex with cyclophilin. Ionomycin did not, however, permit accumulation of IFN-gamma mRNA. Activation of PKC by PMA or of cAMP-dependent protein kinase through increase in cAMP had no transcription-inducing effect, either alone or in conjunction with ionomycin, as measured in run on assays of the IFN-gamma gene. When transcription of the IFN-gamma gene, initiated in the presence of ionomycin and an agent increasing intracellular cAMP, was inhibited by CSA in the absence of PKC or cAMP-dependent protein kinase activation, the IFN-gamma mRNA was rapidly degraded (half-life = 30 min). When either PKC was activated or intracellular cAMP was increased at the time of inhibition with CSA, a stabilizing effect was observed on IFN-gamma mRNA, which led to an increase in secreted IFN-gamma. These effects were selective, they did not affect the rate of transcription of the actin gene, nor the accumulation of actin mRNA. These results show that (i) post-transcriptional events can be critical for IFN-gamma expression in activated lymphocytes, and (ii) specific stabilization of IFN-gamma mRNA can be mediated by activation of two different protein kinases involved in T cell activation.

Modulation of cytokine patterns of human autoreactive T cell clones by a single amino acid substitution of their peptide ligand

We demonstrate that cognate peptide ligands altered at T call receptor (TCR) contact residues and bound to class II major histocompetability complex can change the cytokine pattern of mature T cell clones. Myelin basic protein peptide 85–99-reactive Th0 T cell clones were stimulated with altered peptide ligands, which acted both as TCR antagonist and induced new mRNA synthesis and protein secretion of TGF-β1, while no longer inducing mRNA synthesis or protein secretion of IL-2, IL-4, IL-10, and IFNγ. The modified peptides failed to induce a detectable calcium flux, p58 lck activation, or thymidine Incorporation, yet triggered nearly equal amounts of IL-4 secretion in the presence of ion-omycin. Antigen-induced modulation of T cell cytokine secretion may be important in regulating the immune response.

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V alpha 1 gene.

Previous studies of the TCR alpha chain gene have located promoter elements 5' to the start of the various V alpha genes. The only fully characterized enhancer for the entire alpha chain gene (V, J and C genes) has been located approximately 3 kb from the 3' end of C alpha. We now report the existence of additional regulatory elements located in the introns of several murine V alpha genes (V alpha 1, V alpha 3 and V alpha B6.2.16). In the case of V alpha 1, this element appears to be a promoter with bidirectional activity that is not T cell specific. Interestingly, upstream of the promoter in the antisense strand, an open reading frame has been found that codes for a small molecular weight protein (approximately 60 amino acids) that contains a proline-rich region and a tyrosine-isoleucine motif that has homology to Ig beta (the B29 gene product). A rabbit antiserum made against this sequence has confirmed its existence by Western blot and immunoprecipitation. Thus this V alpha 1 intronic promoter has the potential not only to induce the formation of a truncated V alpha 1 gene product, but also regulates the expression of a small molecular weight protein that may be involved in lymphocyte antigen receptor signaling. The activity of this promoter is regulated by changes in intracellular calcium. In the presence of ionomycin the promoter is down-regulated in the sense direction and its activity is enhanced in the antisense direction. This result suggests that this promoter can act differentially to produce two very different gene products. The bidirectional V alpha 1 promoter appears to be the first in the Ig superfamily to induce potentially functional proteins in both directions.

Carbachol-stimulated phosphorylation of the Na-K-Cl cotransporter of avian salt gland. Requirement for Ca2+ and PKC Activation.

The Na-K-Cl cotransporter of avian salt gland is a membrane-bound 170-kDa protein that is phosphorylated in response to cAMP- and Ca(2+)-dependent secretogogues and is homologous to the Na-K-Cl cotransporter in another Cl-secreting epithelia; the shark rectal gland (Torchia, J., Lytle, C., Pon, D. J., Forbush, B., and Sen, A. K. (1992) J. Biol. Chem. 267, 25444-25450). In the present study we assess the role of Ca2+ and protein kinase C (PKC) activation on the phosphorylation of the Na-K-Cl cotransporter. Although the addition of ionomycin alone did not significantly stimulate cotransporter phosphorylation, concurrent addition of ionomycin plus the tumor promoter phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent increase in phosphorylation. Immunoprecipitation experiments, using a monoclonal antibody which specifically recognizes the cotransporter, suggested that the response to CCh or ionomycin plus PMA was quantitatively similar (5-fold) and was localized exclusively on serine residues. In contrast, when 4 alpha-phorbol was added in the presence of ionomycin, no stimulation was observed. To further assess the involvement of PKC on cotransporter phosphorylation the effects of protein kinase inhibitors were tested. Both staurosporine and calphostin C inhibited phosphorylation of the cotransporter at concentrations known to inhibit PKC, whereas the calmodulin antagonist W-7 had no significant effect. The requirement for Ca2+ was tested further by removing Ca2+ from the incubation medium and stimulating with CCh. Under these conditions, the CCh-stimulated phosphorylation was transient and, furthermore, could be completely inhibited by preloading the cells with the Ca2+ chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid) prior to stimulation. The involvement of protein phosphatases on the phosphorylation of the Na-K-Cl cotransporter was also tested. The addition of okadaic acid stimulated phosphorylation by approximately 3-fold. Taken together these results suggest that the phosphorylation state of the cotransporter involves a dynamic interplay between changes in intracellular Ca2+, PKC, and protein phosphatase activities.

Intracellular Ca 2+ transients in isolated perfused rat heart: measurement using the fluorescent indicator [formula omitted

We have investigated the nature of Fura-2 AM loading into isolated perfused rat heart and the temporal and kinetic relationship between left ventricular [Ca 2+] i dependent fluorescence and isovolumic pressure. The contribution of hydrolysed mitochondrial matrix Fura-2 fluorescence to that measured from the surface of the heart was estimated to be 43.9 ± 5.5% by the addition of 100 μM Mn 2+ to the perfusate. Maximum endothelial Fura-2 fluorescence ratio, estimated by the addition of 3 μM bradykinin to the perfusate, was found to constitute 33.6 ± 2.7% of the maximum myocardial Fura-2 fluorescence ratio. Approximately 11.2% of the 340 nm surface fluorescence was insensitive to 20 mM Mn 2+ in the presence of ionomycin (3 μM) and therefore indicates the degree of partial hydrolysis of Fura-2 AM . Thus, depending on the contribution of endothelial Fura-2 fluorescence at a physiological endothelial calcium concentration, cytosolic fluorescence may comprise between 11–45% of the total cellular fluorescence at 340 nm. Net tissue interference of the Fura-2 fluorescence ratio by NADH emission and myoglobin absorption remained unaltered, providing the oxygenation state of the tissue was unaltered throughout the experiment. The [Ca 2+] i dependent fluorescence decay from peak systole was best fitted to a biexponential decay with fast and slow rate constants of 18.08 ± 1.97 s −1 and 0.23 ± 0.02 s −1, respectively. In addition, a phase shift was observed between temporal and kinetic measurements of the left ventricular isovolumic pressure and calcium dependent fluorescence traces during a contraction-relaxation cycle. We conclude that despite imperfect Fura-2 AM loading, the temporal and kinetic characteristics of intracellular [Ca 2+] transients in normal isolated perfused rat heart are similar to those reported in more controlled preparations such as isolated myocytes and cardiac trabeculae.