The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation

Challenge of protease-activated receptors induces cytosolic Ca2+ concentration ([Ca2+]c) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca2+]c rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca2+]c rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca2+]c response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca2+amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O2 center dot(-)-release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca2+]c increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration.