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

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.

Independent signaling of grp78 gene transcription and phosphorylation of eukaryotic initiator factor 2 alpha by the stressed endoplasmic reticulum.

Perturbation of endoplasmic reticular (ER) function signals increased expression of the gene encoding the ER resident chaperone Grp78/BiP and rapid suppression of translational initiation accompanied by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 alpha phosphorylation and grp78 mRNA induction were measured in GH3 pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2 alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2 alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca2+ and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2 alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2 alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2 alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2 alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2 alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2 alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2 alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2 alpha phosphorylation than for grp78 mRNA induction.

Release of Ca2+ from intracellular organelles by peptide analogues: evidence against involvement of metalloendoproteases in Ca2+ sequestration by the endoplasmic reticulum.

N-Benzyloxycarbonyl-Gly-Phe-amide (Z-Gly-Phe-NH2), a competitive substrate for metalloendoproteases, mobilizes intracellular Ca2+ and suppresses protein synthesis and processing in a Ca(2+)-dependent, reversible manner. To ascertain whether Z-Gly-Phe-NH2 acts as Ca(2+)-storing organelles, effects of the dipeptide on Ca2+ sequestration by saponin-porated GH3 pituitary cells were examined. Porated preparations sequestered Ca2+ into two compartments with different Ca2+ affinities. Ca2+ accumulation at nM concentrations of free Ca2+ was inhibited by thapsigargin and inositol 1,4,5-triphosphate [Ins(1,4,5)P3], enhanced by oxalate and unaffected by oligomycin. Cation accumulation at microM concentrations of free Ca2+ was sensitive to oligomycin but not to thapsigargin. Z-Gly-Phe-NH2 reduced Ca2+ sequestration by both compartments. The dipeptide mobilized Ca2+ from the high-affinity compartment within 1-2 min without affecting Ca2+ uptake. Ca2+ was mobilized more rapidly by Z-Gly-Phe-NH2 and thapsigargin together than by either agent alone. The presence of a thiol-reducing agent was required for Ca2+ mobilization by Z-Gly-Phe-NH2 but not by thapsigargin or Ins(1,4,5)P3. Ca2+ mobilization by Z-Gly-Phe-NH2 could not be attributed to effects on anion-permeability or to actions at Ins(1,4,5)P3 or ryanodine receptors. Results with assorted peptide analogues did not favour suppression of metalloendoprotease activity in the Ca(2+)-mobilizing action of Z-Gly-Phe-NH2. The more hydrophobic analogue Z-L-Tyr-p-nitrophenyl ester was 60-80-fold more potent in mobilizing Ca2+ from intact and porated cells and perturbed the high-affinity Ca(2+)-sequestering compartment selectively. Z-Gly-Phe-NH2 and Z-L-Tyr-p-nitrophenyl ester are proposed to release Ca2+ from the endoplasmic reticulum through an ion pore with affinity for hydrophobic molecules containing internal peptide bonds.

Loss of a calcium requirement for protein synthesis in pituitary cells following thermal or chemical stress.

Ca2+ is required for the maintenance of high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). Following thermal stress at 46 degrees C or chemical stress from exposure to sodium arsenite or 8-hydroxyquinoline, rates of amino acid incorporation in Ca2+-restored GH3 cells were reduced acutely to those of unstressed, Ca2+-depleted control preparations. Sodium arsenite treatment resulted in loss of ability to accumulate polysomes in response to Ca2+. Stressed cells allowed to recover for 2-8 h either with or without Ca2+ in the medium exhibited comparable, increasing rates of amino acid incorporation and the induction of heat shock proteins (hsp). Abolition of the Ca2+-dependent component of translation was proportional to the intensity of the stress. Mild thermal stress (41 degrees C) resulted in the induction of hsp 68 and the retention of Ca2+-dependent protein synthesis; hsp 68 was synthesized in a Ca2+-dependent manner. After arsenite stress, restoration of the Ca2+ requirement for protein synthesis occurred by 24 h, and was preceded by a transitional period during which polysomes accumulated in response to Ca2+ without concomitant increased rates of incorporation. Responses to stress are proposed to include an acute inhibition of normal protein synthesis involving the destruction of Ca2+-stimulated initiation and a protracted period of recovery involving synthesis of the hsp accompanied by Ca2+-independent amino acid incorporation and slowed peptide chain elongation.

Stimulation of protein synthesis in pituitary cells by phorbol esters and cyclic AMP. Evidence for rapid induction of a component of translational initiation.

Phorbol esters such as phorbol myristate acetate (PMA) were employed to examine the involvement of protein kinase C in the regulation of protein synthesis in intact GH3 pituitary tumor cells. Amino acid incorporation increased as a function of time of pretreatment with these agents; 4-8- and 2-3-fold stimulations were observed for Ca2+-depleted and -restored preparations, respectively, following 2 h of exposure. PMA enhanced incorporation of amino acid into all detectable polypeptide species. Lysates of PMA-treated cells incorporated amino acid more efficiently than did lysates of untreated controls. Cells slowed at initiation by Ca2+ depletion responded to treatment with PMA with the production of low molecular weight polysomes and a concomitant decrease in 80 S monomers. In Ca2+-restored preparations, which form large polysomes, PMA treatment resulted in a decrease in 80 S monomers and a shift in average polysomal size from smaller to larger molecular weight. Ribosomal transit times, however, were not altered. PMA-stimulated amino acid incorporation and polysome formation were either eliminated or reduced significantly by actinomycin D and could not be ascribed to increased amino acid uptake or methionylation of tRNA. Substances which elevate cAMP in GH3 cells mimicked phorbol ester in its actions on protein synthesis. It is proposed that GH3 cells, in response to various stimuli, rapidly synthesize an mRNA that subsequently increases the synthesis of a rate-limiting component of translational initiation. Evidence that this pathway for translational control may function in alternative cell types is also presented.