Hypoxia increases the association of 4E-binding protein 1 with the initiation factor 4E in isolated rat hepatocytes.

Incubation of hepatocytes under hypoxia increases binding of translation initiation factor eIF-4E to its inhibitory regulator 4E-BP1, and this correlates with dephosphorylation of 4E-BP1. Rapamycin induced the same effect in aerobic cells but no additive effect was observed when hypoxic cells were treated with rapamycin. This enhanced association of 4E-BP1 with eIF-4E might be mediated by mTOR. Nevertheless, only hypoxia produces a rapid inhibition of protein synthesis. Although hypoxia might be signalling via the rapamycin-sensitive pathway by changing eIF-4E availability, such a pathway is unlikely to be responsible for the depression in overall protein synthesis under hypoxia.

Adenosine stimulates calcium influx in isolated rat hepatocytes.

The mechanism of stimulation of Ca2+ entry into hepatocytes by adenosine was investigated. When Fura-2-loaded hepatocytes were suspended in a nominally Ca(2+)-free buffer, adenosine produced only a small transient increase in the cytosolic free Ca2+ concentration ([Ca2+)i). However, on restoration of an extracellular Ca2+ concentration of 1.3 mM, a rapid increase in [Ca2+]i occurred, which indicates activation of a Ca(2+)-influx pathway. Adenosine augmented the rate of Ca2+ influx triggered by maximally effective concentrations of thapsigargin or cAMP, but was without effect on the rate of Ca2+ entry that resulted from phospholipase-C-linked-receptor activation by maximally effective concentrations of vasopressin or ATP. However, in contrast to vasopression and ATP, adenosine did not stimulate Mn2+ entry. The rate of Mn2+ influx after stimulation of the hepatocytes with vasopressin was not increased by adenosine treatment. The stimulation of hepatocytes with adenosine did not result in significant accumulation of inositol phosphates or cAMP. Furthermore, the rate of adenosine-induced Ca2+ entry in hepatocytes was only slightly reduced in the presence of the P1 purinoceptor antagonist 8-phenyltheophylline. In contrast, the receptor-mediated-Ca(2+)-entry antagonist SK&F 96365 nearly completely blocked the Ca(2+)-entry response without any effect on internal-Ca(2+)-pool mobilisation by adenosine. It is concluded that adenosine activates the internal-pool-regulated pathway of Ca2+ entry and an additional pathway that appears comparable to the previously reported receptor-dependent pathway, except that Mn2+ entry is not stimulated.

Adenosine inhibits protein synthesis in isolated rat hepatocytes. Evidence for a lack of involvement of intracellular calcium in the mechanism of inhibition.

Extracellularly added adenosine and ATP are potent inhibitors of protein synthesis in liver cells. In this study, the possible involvement of Ca2+ in the mechanism of inhibition of protein synthesis by adenosine was investigated. Stimulation of freshly isolated hepatocytes with adenosine or ATP, at concentrations that impaired protein synthesis, induced an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). However, there was no correlation between the increase in [Ca2+]i and inhibition of radiolabelled leucine incorporation into proteins. Thus, the stimulation of hepatocytes with the V1-receptor agonist, vasopressin, or with the nucleotide triphosphates, UTP and GTP, elicited changes in [Ca2+]i similar to those observed after ATP or adenosine addition, but did not affect protein synthesis. ATP produced near complete discharge of Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in isolated hepatocytes, whereas adenosine only had a partial effect. Depletion of the hormone-sensitive Ca2+ pool by adenosine was transient. In contrast, prolonged depletion of internal Ca2+ by thapsigargin resulted in the inhibition of protein synthesis in hepatocytes. However, the inhibition of radiolabelled leucine incorporation into proteins by thapsigargin was further augmented by the additional presence of adenosine. These results show that the inhibition of protein synthesis by adenosine in isolated hepatocytes is not mediated by an increase in [Ca2+]i or depletion of internal pool(s) sensitive to inositol 1,4,5-trisphosphate or thapsigargin.

Cytolytic effects and biochemical changes induced by extracellular ATP to isolated hepatocytes.

Cell death, as estimated by the release of lactate dehydrogenase (LDH), was induced by incubating isolated hepatocytes for 60 min in the presence of extracellular ATP (ecATP), while AMP, adenosine, GTP and UTP were without any significant effects, even when tested at 3 mM (final concentration). At such a concentration, the release of LDH induced by ecATP, but also by ecADP, reached almost 50% and 30%, respectively. Since UTP and GTP (which have no lytic effects) were able to activate phosphorylase a at the same rate as ATP, we excluded the possibility that an increase of free cytosolic Ca2+ triggers the onset of a process leading to cell lysis. Moreover, such a lytic ability of ecATP (1.7 mM) can not be the result of a previous complexation of ionic iron (making it catalytically available for a Fenton reaction), because Desferal, a strong iron chelator, did not modify the cytolytic effect of the ecATP observed after 60 min of incubation. A major cellular function such as protein synthesis was impaired in a dose-dependent way by incubating hepatocytes during 60 min in the presence of ecATP. The inhibition was already observed at 0.1 mM ecATP, a dose without any effect on cell viability. The biological relevance of such metabolic impairment, however, remains to be elucidated.