Translational suppression by Ca2+ ionophores: Reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion

Abstract

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca2+ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50-300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca2+ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca2+ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH3 pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca2+ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca2+. Ionomycin produced more Ca2+ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca2+ by ionomycin were readily reversed in GH3 cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca2+ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca2+ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca2+-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca2+ accumulation. Increased Ca2+ contents in response to Ca2+ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.

Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level

“…half that in ionophore-free control cells (results not shown). This result was not unexpected, since the transient inhibition of translation due to activation of protein kinase R and phosphorylation of the elongation initiation factor eIF2α in response to calcium ionophore has been described [2]. In a second experiment, we examined the relative expression level of the same proteins after a longer exposure to ionophore in cells loaded with BAPTA and maintained in the absence of extracellular calcium, and compared this with the corresponding level in BAPTA-free control cells.…”

“…The concentration of Ca# + in the cytosol of eukaryotic cells (typically 10-100 nM) is maintained in homoeostatic balance with millimolar Ca# + levels in both the extracellular environment and the lumen of the endoplasmic reticulum (ER), the major dynamic Ca# + storage compartment of non-muscle cells [1]. The rapidly exchanging Ca# + store in this organelle is essential for a variety of cellular functions, including correct folding and post-translational modification of newly synthesized secretory and membrane proteins, translation of newly transcribed mRNA and in maintaining the structural integrity of the ER [1,2]. The intracellular calcium store participates in the regulation of the cytosolic free Ca# + concentration ([Ca# + ] i ) through rapid Ca# + release and by controlling store-operated Ca# + influx from outside the cell [3][4][5].…”

Fluorescent Annexin A1 Reveals Dynamics of Ceramide Platforms in Living Cells

“…After its initial reduction, the ceramide concentration did not further decrease to the levels observed in non‐stimulated cells, presumably because of an incomplete removal of intracellular Ca 2+ (Figure 6C: time = 0, 2nd Ca 2+ challenge, Fluo‐3 AM). This can be ascribed to an ionomycin‐induced depletion of intracellular ATP and subsequent inhibition of Ca 2+ pumps (31). As [Ca 2+ ] i decreased, the annexins dissociated from the plasma membrane and assumed a cytoplasmic localization (Figure 6C: time = 0, 2nd Ca 2+ challenge).…”

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis

“…43 Because ionomycin depletes Ca 2ϩ from mitochondria and disrupts the mitochondrial transmembrane potential and adenosine triphosphate generation, it may deplete the cell of the energy needed for endocytosis. 44 Ionomycin is known to lead to a dramatic loss of clathrin puncta and dissociation of endocytic adaptors from the plasma membrane. 28 In fact, sublytic concentrations of ionomycin actually inhibit clathrin-mediated endocytosis.…”