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[structure: see text] The total synthesis of the polyether antibiotic ionomycin, a calcium ionophore, is described. The synthesis demonstrates the utility of ring-opening methodologies as applied to the synthesis of polypropionate and deoxypolypropionate subunits, which are found in two of the four fragments in the synthesis.
A Zn-initiated triepoxide cascade cyclization reaction was employed for the synthesis of the bistetrahydrofuran core segment of the polyether ionophore antibiotic ionomycin.
Two associated calcium-binding proteins (CaBPs) have recently been identified specifically in cells of myeloid origin. These proteins have relative molecular masses (Mr) of 8,000 and 14,000 and are variously referred to as the cystic fibrosis antigen, the L1 light chain, MRP-8 or p8, and the L1 heavy chain, MRP14 or p14, respectively. The expression of p8 and p14 seems to be confined to a specific stage of myeloid cell differentiation, because both proteins are expressed in circulating neutrophils and monocytes but not in normal tissue macrophages. In chronic inflammatory conditions, however, such as rheumatoid arthritis, macrophages in affected tissues express both p8 and p14. These proteins are members of a family of CaBPs of low Mr, which include S-100 alpha and beta proteins, calcyclin (2A9), intestinal CaBP and p11. All the proteins have an Mr of approximately 10,000 with the exception of p14 which has a longer C-terminal sequence after the second calcium-binding domain. Little is known about their function, although by analogy with calmodulin they could be molecules involved in intracellular signalling that are activated by an increase in the intracellular Ca2+ concentration ([Ca2+]). Here we report that p14 is phosphorylated in both monocytes and neutrophils. The level of p14 phosphorylation can be increased by elevating the [Ca2+]i using the ionophore ionomycin, but is not affected by activation of protein kinase C using phorbol 12,13-dibutyrate. The phosphorylated residue is threonine at position 113, which is the penultimate amino acid in p14 and contained in the longer 'tail' sequence. Part of this sequence is identical to the neutrophil immobilizing factors NIF-1 and NIF-2, indicating that the phosphorylation event could have a role in the generation of NIF activity in the p14 protein.
Key steps in the synthesis of the C1-C16 polyketide fragment of ionomycin were the nucleophilic addition of an organocuprate to a neutral (eta3-allyl)iron complex and the construction of a beta-diketone moiety by the Rh-catalysed rearrangement of an alpha-diazo-beta-hydroxyketone.
A system for the study of the regulation of the release of triacylglycerols by mammary gland slices was developed. By prelabelling the triacylglycerol pool with [3H]oleate measurements of release of both mass of triacylglycerol and of newly synthesized triacylglycerol have been made. Oxytocin and ovine prolactin stimulated release of triacylglycerol and protein, but the former was 40-fold more effective. Recombinant bovine prolactin was even less active than ovine prolactin, suggesting that contamination of the latter with oxytocin and/or vasopressin was partly responsible for its stimulatory effect on release. The findings support the view that the major effect of oxytocin is to stimulate contraction of myoepithelial cells and thus release secreted lipid stored in the lumen of the mammary gland alveoli. Ionomycin, a Ca2+ ionophore, also stimulated lipid release, but probably not by the usual apocrine route. Parathyroid hormone-related protein, a peptide produced by the mammary gland, did not stimulate release or antagonize the effects of oxytocin. Release of lipid was also measured in mammary gland slices from late-pregnant, early- and mid-lactating rats and lactating rats made prolactin-deficient. Hormonal stimulation in vitro showed the maturation of response seen in vivo on transition from late pregnancy to peak lactation. Prolactin deficiency resulted in decreased release of newly synthesized lipid in response to oxytocin.
PBMC from tuberculoid (BT/TT) and lepromatous leprosy (BL/LL) leprosy patients showed spontaneous apoptosis when cultured in the absence of mitogen for 24 h, which was inhibited by anti-tumour necrosis factor-alpha (TNF-alpha) antibodies. Apoptosis was also inhibited by ionomycin and zinc, which also increased IL-2 and decreased TNF-alpha production. The increase in IL-2 production suggests a mechanism whereby dietary supplements with zinc might alter the cell-mediated immunity response in leprosy patients.
3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (Acrp30), which is also designated as AdipoQ. In order to study the expression and secretion of the human homologue of this protein, apM1 (adipose Most abundant gene transcript 1, also named gelatin-binding protein of 28 kD [GBP28] or adiponectin), a polyclonal antibody was produced. Both expression and secretion can be detected beginning with day 4 after induction of differentiation. The amount of expressed apM1 correlates with the specific activity of the differentiation marker glycerol-3-phosphate dehydrogenase. Secretion of apM1 is increased by the addition of ionomycin. Both the nonhydrolysable dibutyryl-cycloAMP and tumour necrosis factor alpha reduce the expression and secretion of apM1.
Fertilization of mature mouse oocytes triggered highly repetitive Ca2+ oscillations lasting 2-3 h. However, immature oocytes generated only two or three oscillations, which ceased within 1 h. Development of repetitive Ca2+ transients to sperm occurred late in oocyte maturation and was dependent on cytoplasmic modifications that were independent of cell cycle progression from metaphase I to metaphase II. Immature oocytes released significantly less Ca2+ from stores than mature oocytes in response to ionomycin and thapsigargin. Ryanodine had no effect on intracellular Ca2+ in maturing oocytes but stimulated an increase in Ca2+ in mature oocytes. The ability of ryanodine to increase Ca2+ levels was, however, strain-dependent. Preincubation of oocytes with thapsigargin or ryanodine significantly attenuated the normal fertilization Ca2+ response, causing a decrease in the number and the rate of rise of the transients. The inhibition of sperm-induced Ca2+ transients by ryanodine was independent of its ability to cause an immediate Ca2+ increase. Low concentrations of ryanodine had no effect on resting Ca2+ levels but inhibited Ca2+ oscillations at fertilization. Similarly Ca2+ oscillations were blocked in oocytes from a strain of mouse that showed no immediate Ca2+ increase with ryanodine. These results suggest that modifications in Ca2+ stores and ryanodine-sensitive Ca2+ release mechanisms during oocyte maturation play an important role in Ca2+ oscillations at fertilization.
Lymphotoxin-beta (LT- beta) is a tumor necrosis factor (TNF)-related membrane-bound cytokine that forms a heterotrimeric surface lymphotoxin (LT) complex with LT-alpha on the surface of lymphoid cells. Although knockout studies have revealed a role in lymph node biogenesis during development, the regulation and function of surface LT in mature cell types are poorly understood. The present study aims to understand the physiologic signals that regulate the components of surface LT in Jurkat T cells. We show that the previously observed upregulation of surface LT by phorbol myristate acetate (PMA) is markedly abrogated by cotreatment with ionomycin through posttranscriptional mechanisms. In addition, the observation of striking similarities between the mRNA accumulation kinetics of LT-alpha and LT-beta during these treatments indicates tight coupling of expression under certain conditions. In investigating the reported upregulation of LT-beta during inflammation, we tested the effects of various proinflammatory and anti-inflammatory cytokines on LT-beta expression. Our data demonstrate an upregulation of LT-beta mRNA by the inflammatory cytokines TNF and LT-alpha.
Secretion of IgM and IgG in vitro by B cells from patients with common variable immunodeficiency (CVI) has been used to classify the disease into three groups. On stimulation with anti-IgM and IL-2, group A patients' cells fail to secrete IgM or IgG, group B patients' cells secrete no IgG and significantly lower levels of IgM than normal cells, and group C patients' cells produce normal levels of both isotypes. Direct activation of protein kinase C using 12,13-phorbol dibutyrate and ionomycin followed by IL-2 or IL-4 has been reported to induce immunoglobulin secretion by normal human B cells. We therefore attempted to induce B cells from group A and group B CVI patients to secrete IgM and IgG after direct activation of protein kinase C together with IL-2 or IL-4. The data show that the failure of secretion of immunoglobulin by B cells from CVI patients could not be reversed using this approach. This finding suggests that the activation channel involving protein kinase C in B cells from CVI patients is not involved in the defect in cell differentiation.
Guinea-pig cerebral cortex synaptosomes steadily release H2O2 into the suspending medium, at the rate of 20-30 pmol min-1 mg protein-1. A transient increase of the H2O2 release is induced by the addition of 1 mM Ca2+, which declines within 60-90 s to a rate identical or slightly higher than that before Ca2+. The extra H2O2 following Ca2+ addition varies between 40-100 pmol/mg protein and is insensitive to verapamil. The H2O2 release increases strongly (up to 250 pmol min-1 mg-1) upon depletion of the synaptosomal glutathione by treatment with 1-chloro-2,4-dinitrobenzene, a substrate for glutathione transferase. This treatment however has no effect on the Ca2+-induced H2O2 transient. In these treated synaptosomes a further increase of the output of H2O2 is rapidly induced upon addition of the Ca2+ ionophore ionomycin. This increase (about 100 pmol min-1 mg-1) lasts several minutes and requires the presence of Ca2+. A similar, though less pronounced increased H2O2 release is obtained (also in the absence of Ca2+) upon depolarization of the synaptosomal plasma membrane with KCl or with veratridine.
In order to investigate signalling pathways involved in the control of granule cell differentiation, survival and other functions by depolarization or activation of NMDA receptors we have characterized protein phosphorylation in cerebellar granule cells. Cultures of cerebellar granule cells were incubated with 32P orthophosphate and then challenged with NMDA, K+ or the Ca2+ ionophore ionomycin, agents which raise [Ca2+]i and stimulate differentiation and survival. Upon separation of labelled phosphoproteins by two-dimensional gel electrophoresis three differences were found in response to all of these agents. These were an increase in acidity of two phosphoproteins of 87 and 48 kDa (p87 and p48) and increased 32P-incorporation into a phosphoprotein of 120 kDa (p120). Treatment with PMA which stimulates neurite outgrowth but not survival affected p87 (increased its acidity) but not p48. The acidic shift of p87, therefore, is not sufficient to stimulate granule cell survival. The identification of p87 as the actin-binding MARCKS protein and the demonstration of its presence in neurites and growth cones of granule cells suggests that it may be involved in NMDA-stimulated neurite outgrowth. The phosphoproteins p120 and p48 may potentially be involved in events linking the rise in [Ca2+]i to increased granule cell survival or other aspects of granule cell differentiation.
It has been hypothesized that modulation of epithelial paracellular permeability may be mediated by initiation of contraction of a band of actin and myosin located at the tight junction. Phosphorylation of myosin light chain (MLC) is an important determinant of actomyosin contraction. We asked if ionomycin (iono) and phorbol 12,13-dibutyrate (PDBU), which increase paracellular permeability of Madin-Darby canine kidney (MDCK) cell monolayers, increased MLC phosphorylation in MDCK cells. MDCK cell MLC was constitutively phosphorylated by myosin light chain kinase (MLCK), and after PDBU and iono > 99% of MLC continued to be phosphorylated by MLCK. Neither iono or PDBU, nor the combination of iono and PDBU, increased MLC phosphorylation. In contrast, the phosphatase inhibitor okadaic acid did increase MLC phosphorylation. Adenosine 3',5'-cyclic monophosphate (cAMP) and forskolin decreased MLC phosphorylation in control MDCK cells and in cells exposed to iono and PDBU. In contrast, cAMP and forskolin did not blunt the decrease in transepithelial resistance caused by iono and PDBU. Iono and PDBU increase MDCK monolayer permeability independently of an increase in MLC phosphorylation.
Recent studies have demonstrated that pulmonary surfactant protein (SP)-A plays a potential role in modifying inflammation and immune function. To see whether SP-A could modify IL-8 production and release by eosinophils stimulated with ionomycin, SP-A purified from surfactant recovered from patients with alveolar proteinosis was added to eosinophils isolated by the negative-selection method with immunomagnetic beads, and cultured for 24 h. The concentrations of IL-8 in the cell-free supernatants and cell lysates were then measured by ELISA. SP-A attenuated the production of IL-8 by eosinophils in a concentration-dependent manner. SP-A also attenuated the release of IL-8 from the eosinophils. The addition of SP-A antibody (PE10) reversed these effects of SP-A completely. These data suggest that SP-A may have the potential to modify allergic inflammation by inhibiting the release and production of IL-8 by eosinophils.
How 4 beta-phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io), a calcium ionophore, affect on the atrial natriuretic peptide (ANP) stimulated cyclic-3',5'-guanosine monophosphate (cGMP) production in cultured rat mesangial cells was examined. Cultured mesangial cells were prepared by isolated glomeruli from Sprague Dawley rats employing the sieving method and were used between the 3rd and 15th passage for experiments. cGMP and protein contents were measured by radioimmunoassay and Lowry method. Incubations with effectors were carried out either in the presence or absence of 0.5 mM 1-methyl-3-isobutyl-xanthine (MIX). The intracellular concentration of calcium ([Ca2+]i) was determined by using the Fura-2 method. Pretreatment with PMA, an activator of protein kinase C (PKC), attenuated ANP stimulated cGMP production in a time- and dose-dependent fashion, while alpha PDD (an inactive analog of PMA) did not inhibit cGMP production. PMA inhibition was reversed by addition of staurosporine, a protein kinase C inhibitor. Io attenuated ANP stimulated cGMP production in the absence but not in the presence of MIX. These findings suggested that PMA acts on ANP receptor or guanylate cyclase via activation of PKC in rat mesangial cells. Io may inhibit ANP stimulated cGMP production via activation of cyclic nucleotide phosphodiesterase.
In order to study the regulation of cathepsin B expression in the thyroid, cathepsin B mRNA concentrations were measured in rat thyroid cells (FRTL5) in culture. Northern blot analysis demonstrated that cathepsin B mRNA concentrations were increased in FRTL5 cells cultured for up to 6 days in TSH. The effect of TSH on cathepsin B mRNA concentrations was dose dependent over the range 25-150 mu units/ml. Cytoplasmic dot-blot analysis was used to characterize this effect further. The TSH-induced increase in cathepsin B mRNA concentrations (approximately fivefold over that in untreated cells) was partially mimicked by forskolin (approximately threefold) and ionomycin, while phorbol ester decreased cathepsin B mRNA concentrations. Similar changes were observed for thyroglobulin and actin mRNA concentrations. TSH had no effect on cathepsin B enzymatic activity or immunoreactive protein concentration. These results demonstrate (1) that cathepsin B expression in the thyroid is regulated in parallel with that of thyroglobulin and actin, and (2) that cyclic AMP- and Ca2+-dependent processes stimulate gene expression, while phorbol ester treatment inhibits gene expression in FRTL5 cells.
Vitamin D may regulate pituitary function, as there are selective effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on gene expression in clonal pituitary tumour cells, and on TRH-induced TSH release in normal rat pituitary cells in vitro. The role of Ca2+ in 1,25-(OH)2D3-enhanced TSH release from primary rat pituitary cell cultures was investigated. Pretreatment with 10 nmol 1,25-(OH)2D3/l for 24 h augmented KCl (3-60 mmol/l)-induced TSH release over 1 h at all KCl concentrations greater than 7.5 mmol/l (P less than 0.001), with a 76% enhancement of TSH release induced by 30 mmol KCl/l (P less than 0.001). The Ca2+ channel antagonist nifedipine (10 nmol/l-10 mumol/l) caused a concentration-dependent inhibition of KCl (60 mmol/l)-induced TSH secretion. Pretreatment with 1,25-(OH)2D3 enhanced KCl-induced release at all concentrations of nifedipine (P less than 0.001). The Ca2+ selective divalent cation ionophore ionomycin (1 nmol/l-1 mumol/l), and the Ca2+ channel agonist BAY K 8644 (10 nmol/l-1 mumol/l) increased prolactin secretion but did not increase TSH release, and 1,25-(OH)2D3 had no effect. At an extracellular Ca2+ concentration of less than 500 nmol/l, TRH-induced TSH release was observed only after treatment with 1,25-(OH)2D3 (P less than 0.01). As the extracellular Ca2+ concentration was increased, greater increments of TRH-induced TSH release were observed following pretreatment with 1,25-(OH)2D3 (P less than 0.01). However, the effect of 1,25-(OH)2D3 in the thyrotroph was independent of the pretreatment extracellular Ca2+ concentration. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in-vitro responsiveness to TRH and KCl. These data suggest that the action of 1,25-(OH)2D3 in the thyrotroph is to enhance intracellular signal transduction. They further support a permissive or regulatory role of vitamin D in the normal pituitary gland.
AIM: To compare the suppressions of kanglemycin C (Kan) with that of cyclosporine (Cyc) on lymphocyte proliferations induced by tetradecanoylphorbol acetate (TPA) with ionomycin (IM), and concanavalin A (Con A). METHODS: Cell proliferation was quantified with 3-(4, 5-dimetyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) or [3H]thymidine ([3H]TdR) incorporating method. Calcineurin (CN) activity was measured with molybdate dye colorimetry. RESULTS: Kan (8, 40, 80, and 400 nmol.L-1) strongly suppressed splenocyte proliferation induced by TPA and IM, and the suppressive effect of Kan gradually decreased along with the increasing concentrations of TPA (1-100 micrograms.L-1) and IM (125-500 micrograms.L-1). But, the suppression of Cyc on the splenocyte proliferation induced by TPA was mild. Cyc suppressed CN activity of mouse splenocytes stimulated by Con A stronger than Kan. Moreover, Kan and Cyc strongly suppressed spleen enriched T-cell proliferation induced by Con A and TPA + IM, and the suppression of Kan on proliferation was partly attenuated by exogenous IL-2. CONCLUSION: Kan competitively suppressed the proliferation induced by TPA and IM, and Cyc mainly suppressed IM part of the proliferation induced by TPA and IM.