Role of Protein‐Phosphorylation Events in the Anoxia Signal‐Transduction Pathway Leading to the Inhibition of Total Protein Synthesis in Isolated Hepatocytes

Abstract

Incubation of isolated hepatocytes under N,/COZ (no 0,) produced a rapid and strong inhibition of overall polypeptide biosynthesis, which was neither related to cell death nor to the appearance of specific stress proteins. Treatment of the cells with the tyrosine-kinase inhibitor genistein or with the serine/ threonine-protein-kinaqe inhibitor H7 did not modify the impairment of protein synthesis induced by oxygen deprivation, indicating that such signal-transduction pathways are probably not involved in the anoxia-mediated effect. Okadaic acid (100 nM) and Na,VO, (1 mM) reduced the incorporation of[I4C]Leu into proteins of hepatocytes maintained under aerobic conditions (93.3 kPa OJ. The effects of oxygen deprivation and okadaic acid were additive, whereas sodium vanadate did not enhance the impairment of protein synthesis induced by anoxia. This observation suggests that a common mechanism, involving the net phosphorylation of protein tyrosine residues, that is insensitive to genistein might participate in the negative control of the translation induced by oxygen deprivation. The effect of anoxia on the synthesis of proteins was fully and rapidly reversible upon the restoration of oxygen supply, thus indicating that hepatocytes are able to sense 0,. Although high concentrations of cobalt chloride partially mimic the effect of oxygen deprivation on protein biosynthesis, the nature of such an oxygen sensor remains unknown, and appears unlikely to be a part of a classic haem protein.Keywords: anoxia; protein synthesis ; protein phosphorylation ; isolated hepatocytes.Hypoxia produces a variety of physiological and biochemical adaptations so that energy can be supplied to the tissues to maintain cellular activities [l -41. A decrease in the production of almost all polypeptides appears to be an important feature of oxygen deprivation in numerous cell types [5-71. Thus, protein synthesis, a major function of the liver, is inhibited as soon as hepatocytes are kept under hypoxic conditions, indicating that cells react to a change in PO, levels rather than to a change in either ATP content or ATP/ADP ratio [8, 91. Moreover, such an inhibition was not related to a decreased amino acid uptake or to enhanced proteolytic activities, but also occurred in cold-preserved hepatocytes [lo] and in acute ischemic rat liver [ll].In mammalian cells, reduced 0, tension modulates genes for proteins such as erythropoietin [ 12, 131, glycolytic enzymes, which increase anaerobic ATP synthesis [14-161, vascular endothelial growth factor [16, 171, and transcription factors, such as hypoxia inducible factor-I [IS], nuclear factor KB and activator protein 1 [19]. Such responses indicate that cells are able to sense oxygen, but the nature of the oxygen sensor and the mechanisms involved in the hypoxia signal-transduction pathway are not fully understood.Correspondence to P.

Physiological and Pathological Responses to Hypoxia

“…Studies on hepatocytes have shown that protein synthesis is largely inhibited in response to hypoxia. 16 Buttgereit and Brand 17 demonstrated that, in fact, the ATP consuming processes are arranged in a hierarchy, with protein synthesis and RNA/DNA synthesis being the first to be inhibited as energy becomes limiting and with Na ϩ /K ϩ pumping and Ca 2ϩ cycling taking the greatest priority. This phenomenon, known as oxygen conformance, involves precise regulatory mechanisms notably at the level of translation initiation.…”

Regulation of Transcription and Translation by Hypoxia

“…43,54,55 For example, anoxia activates AMPK and this leads to the phosphorylation of eEF2 and inhibition of translation elongation (Fig. 2).…”

The role of eukaryotic initiation factor 2α during the metabolic depression associated with estivation

“…This mechanism and the sequence of the phosphorylation site in eIF2α are conserved between yeast and man. An increase in the extent of eIF2α phosphorylation occurs concomitantly with a downregulation of the rate of protein synthesis in normal tissues responding to a diverse array of stresses, including heat shock (Hu et al, 1993), amino acid deprivation (Kimball et al, 1991), reperfusion following ischemia (Martin de la Vega et al, 2001) and anoxia (Tinton et al, 1997).…”