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ATP and adenosine prevent via different pathways the activation of caspases in apoptotic AKR-2B fibroblasts
1Department of Physiological Chemistry, Biozentrum, University of Würzburg Am Hubland, 97074 Würzburg, Germany.
Abstract:
Confluent AKR-2B fibroblasts rapidly disintegrate after serum deprivation.27 ATP or adenosine added immediately after serum removal afforded substantial protection against cell death even for a long period of 24 h. ED50 values were 14 and 110 microM for ATP and adenosine, respectively. In the presence of 5 microg/ml cycloheximide the protective effect of both substances was suppressed, indicating that protein synthesis is required. The protective effect of ATP was highly specific since among numerous tested derivatives only ATP-[gamma-S] exhibited a substantial protective effect. The ability of ATP and adenosine to modulate cell division was analyzed. Both substances did not exhibit any mitogenic effect. Adenosine completely blocked PDGF-BB induced cell division, whereas ATP had no effect. Unlike adenosine, ATP strongly stimulated Ca2+-release from intracellular stores. On the other hand, adenosine stimulated an increase in the intracellular concentration of cAMP from 0.4 - 1.5 microM, whereas ATP decreased the content below 0.1 microM. ATP stimulated the phosphorylation of MAP-kinase, RSK and p70S6-kinase; adenosine was inactive. After complexation of [Ca2+]i the protective effect of ATP was greatly lost while adenosine was still active. Surprisingly neither ATP nor adenosine caused an activation of PKC-isoforms. After incubation with pertussis toxin, the protection by ATP was reduced indicating an involvement of Gi-proteins in the signal transduction induced by ATP. Our results indicate that ATP as well as adenosine are potent inhibitors of cell death caused by serum deprivation and that this protective effect apparently occurs via distinct pathways. However, both pathways must converge at the point of caspase activation, since the stimulation of DEVDase- and VEIDase-activities, respectively, are suppressed by either ATP or adenosine.
Insights
Adenosine triphosphate (ATP) and adenosine protect fibroblasts from serum deprivation-induced cell death via distinct pathways. Both molecules inhibit caspase activation, but their signaling mechanisms, including calcium and cAMP levels, differ significantly.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Fibroblast survival is crucial for tissue homeostasis.
- Serum deprivation triggers rapid fibroblast disintegration.
- Understanding protective mechanisms against cell death is vital.
Purpose of the Study:
- To investigate the protective effects of ATP and adenosine against serum deprivation-induced cell death in AKR-2B fibroblasts.
- To elucidate the distinct signaling pathways involved in the protective mechanisms of ATP and adenosine.
- To determine the role of protein synthesis, calcium, cAMP, and kinase activation in these processes.
Main Methods:
- Serum deprivation of AKR-2B fibroblasts.
- Treatment with ATP and adenosine and their derivatives.
- Assessment of cell viability and protein synthesis inhibition (cycloheximide).
- Measurement of intracellular calcium and cAMP levels.
- Analysis of MAP-kinase, RSK, and p70S6-kinase phosphorylation.
- Investigation of protein kinase C (PKC) and Gi-protein involvement (pertussis toxin).
- Assay of caspase activation (DEVDase and VEIDase).
Main Results:
- ATP and adenosine significantly protected fibroblasts from serum deprivation-induced death.
- Protein synthesis is required for the protective effects.
- ATP and adenosine trigger distinct intracellular signaling cascades.
- ATP stimulates Ca2+ release and MAP-kinase pathway activation.
- Adenosine increases cAMP levels and blocks PDGF-BB-induced cell division.
- Both ATP and adenosine inhibit caspase activation, converging on a common downstream pathway.
Conclusions:
- ATP and adenosine are potent inhibitors of serum deprivation-induced cell death in fibroblasts.
- Their protective effects are mediated through distinct, yet converging, signaling pathways.
- These findings highlight novel therapeutic targets for preventing cell death in various physiological and pathological conditions.