An examination of the role of increased cytosolic free Ca²⁺ concentrations in the inhibition of mRNA translation

Mobilization of Ca²⁺ sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca²⁺ ([Ca²⁺](i)) due to plasmalemmal Ca²⁺ influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER C^a2+ stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca²⁺](i) per se causes slowing of peptide chain elongation is lacking. L- type Ca²⁺ channel activity of GH₃ pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca²⁺](i) on eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca²⁺, resting [Ca²⁺](i) values were high (154-255 nM) and eEF-2 was phosphorylated. The Ca²⁺ channel antagonist, nisoldipine, lowered [Ca₂₊](i) and reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca²⁺ channel agonist, Bay K 8644, produced sustained elevations of [Ca²⁺](i) that were associated with 25-50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca²⁺ influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50100% above control) and inhibited leucine incorporation by 20-70% but ATP content was reduced by 2550% and total cell-associated Ca²⁺ contents rose by 3-to 13-fold. elF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca²⁺](i) that resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF2-α, not eEF-2. It is concluded that changes in [Ca²⁺](i) occurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.