Nerve growth factor and brain-derived neurotrophic factor but not granulocyte colony-stimulating factor, nimodipine and dizocilpine, require ATP for neuroprotective activity after oxygen-glucose deprivation of primary neurons.

In previous work, we have demonstrated by radiolabeling, mass spectrometry and site-directed mutagenesis that nerve growth factor (NGF) as well as brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) are capable of ATP-binding and that this binding appears to be essential for their neuroprotective activity. In this study, we attempted to shed some light on the question whether ATP is a general prerequisite for neuroprotection. Therefore, we used the non-ATP-binding granulocyte colony-stimulating factor (GCSF), the calcium antagonist nimodipine and the NMDA antagonist dizocilpine to find out whether they need ATP for neuroprotection comparable to NGF and BDNF. However, ATP was not necessary for the neuroprotective effects of GCSF, nimodipine and dizocilpine on primary cultures of rat cortical neurons damaged by oxygen-glucose deprivation whereas neuroprotection was demonstrable for NGF and BDNF only when ATP was present in the culture medium at a concentration higher than ca. 0.4nmol/l. In circular dichroism studies ATP caused changes of the secondary structure of NGF but not of GCSF. Taken together, we suggest that ATP is not a general prerequisite for neuroprotectivity but some growth factors like NGF and BDNF can stimulate their receptors only if they have bound ATP.

Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells.

BACKGROUND: ATP binding is essential for the bioactivity of several growth factors including nerve growth factor, fibroblast growth factor-2 and brain-derived neurotrophic factor. Vascular endothelial growth factor isoform 165 (VEGF-A(165)) induces the proliferation of human umbilical vein endothelial cells, however a dependence on ATP-binding is currently unknown. The aim of the present study was to determine if ATP binding is essential for the bioactivity of VEGF-A(165). RESULTS: We found evidence that ATP binding to VEGF-A(165) induced a conformational change in the secondary structure of the growth factor. This binding appears to be significant at the biological level, as we found evidence that nanomolar levels of ATP (4-8 nm) are required for the VEGF-A(165)-induced proliferation of human umbilical vein endothelial cells. At these levels, purinergic signaling by ATP via P2 receptors can be excluded. Addition of alkaline phosphate to cell culture lowered the ATP concentration in the cell culture medium to 1.8 nM and inhibited cell proliferation. CONCLUSIONS: We propose that proliferation of endothelial cells is induced by a VEGF-A(165)-ATP complex, rather than VEGF-A(165) alone.

ATP-dependent stabilization and protection of fibroblast growth factor 2.

Fibroblast growth factor 2 (FGF2) plays a pivotal role in cell proliferation, angiogenesis and neuroprotection. Several clinical trials using this growth factor in bone regeneration, wound healing and cardioprotection are initiated but the inadequate stability of FGF2 after application is one major problem. Binding of ATP to FGF2 and other growth factors has been demonstrated recently. Here we report that ATP, other nucleoside triphosphates and sodium triphosphate protect FGF2 from trypsin, plasmin and neutrophile elastase digestion in vitro. A molar ratio of 2:1 (ligand/FGF2) is sufficient for these protective effects. ADP shows only little, AMP no stabilizing effect on FGF2 indicating that the number of phosphate residues is important. Protection of FGF2 by ATP can be abolished by the addition of alkaline phosphatase hydrolyzing free and FGF2-bound ATP. The mutant FGF2 (K128A/R129A/K134A/K144A) with strongly reduced ATP-binding capacity revealed no detectable protease resistance after incubation with ATP. Furthermore, a stabilizing effect of ATP on FGF2 could also be demonstrated in cell culture experiments. ATP bound to FGF2 increased FGF2-dependent human umbilical vein endothelial cells proliferation when the growth factor was treated with neutrophile elastase or heat. For the first time these data demonstrate protection of FGF2 by bound ATP, other nucleoside triphosphates or sodium triphosphate from rapid protease digestion. Our data provide new evidence that nucleoside triphosphates are capable of protecting FGF2 and favours such stabilization for various, especially medical applications.