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Inhibition of protein synthesis in intact mammalian cells by arachidonic acid.

ABSTRACT: Optimal translation initiation in intact mammalian cells requires sequestered intracellular Ca2+. Arachidonic acid, which releases sequestered Ca2+ from cells and isolated organelles, was studied to assess its potential role in the regulation of protein synthesis via Ca2+ mobilization. Unsaturated fatty acids at microM concentrations inhibited protein synthesis in intact GH2 pituitary, C6 glial tumour and HeLa cells in a manner dependent on degree of unsaturation and cell number. Arachidonate was generally the most, and the fully saturated arachidic acid the least, potent of the fatty acids tested. At 2 x 10(6) GH3 cells/ml, amino incorporation into a broad spectrum of polypeptides was inhibited by 80-90% by 10-20 microM fatty acid. Inhibition was maximal at 4-8 min and was attenuated by 1-2 h and more pronounced at lower pH. Protein synthesis was maximally inhibited when arachidonate mobilized approx. 40% of cell-associated Ca2+. At lower concentrations (10 microM) arachidonate suppressed translational initiation, with the inhibition being reversed as extracellular Ca2+ concentrations were increased to supraphysiological values. At higher concentrations (20 microM) arachidonate inhibited peptide-chain elongation in a Ca(2+)-independent manner. Arachidonate also blocked elongation in reticulocyte lysates. The effects of arachidonate in intact cells were reversible with time via its metabolism or by washes containing BSA. Sufficient arachidonate appears to be synthesized during ischaemic stress to inhibit translation by either mechanism.

SUBMITTER: Rotman EI

PROVIDER: S-EPMC1130806 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Project description:Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca(2+) channels by an unknown mechanism at an unknown site. The Ca(2+) channel pore-forming subunit (Ca(V)alpha(1)) is a candidate for the site of AA inhibition because T-type Ca(2+) channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca(V)beta subunits on the inhibition of Ca(V)1.3b L-type (L-) current by AA. Whole cell Ba(2+) currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of -10 mV from a holding potential of -90 mV. A one-minute exposure to 10 microM AA inhibited currents with beta(1b), beta(3), or beta(4) 58, 51, or 44%, respectively, but with beta(2a) only 31%. At a more depolarized holding potential of -60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca(V)1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential-dependent inactivation or on recovery from inactivation regardless of Ca(V)beta subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca(V)2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of beta(2a) interfere with inhibition by AA. Our novel findings that the Ca(V)beta subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca(V)beta expression may regulate how AA modulates Ca(2+)-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.

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Inhibition of protein synthesis in intact mammalian cells by arachidonic acid.

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