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

Abstract

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07527.x Abstract 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•−‐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.

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

“…Hypoxia stimulates angiogenesis and thrombin is a key mediator of the angiogenic process (Dupuy et al, 2003; Green and Karpatkin, 2010; Zhou et al, 2012). Beyond its central role in the coagulation cascade, thrombin can directly affect cellular processes in a variety of cell types including endothelial cells, glia, and neurons (Lee da et al, 2006; Huang et al, 2008; Zundorf and Reiser, 2011; Alabanza and Bynoe, 2012). There is an extensive literature identifying the ability of hypoxia-stimulated peripheral endothelial cells to express a proangiogenic/inflammatory phenotype that is mediated by thrombin (Chen et al, 2010; Sabit et al, 2010).…”

Abruption-Induced Preterm Delivery Is Associated with Thrombin-Mediated Functional Progesterone Withdrawal in Decidual Cells

“…Thrombin augments the expression of several biochemical endpoints in different cell lines and primary cells via phosphorylated ERK1/2 or p38 MAP kinase or NF-B mediation. 27,31,38 However, a literature search revealed no examples in which thrombin directly affects the expression of steroid receptors including PR and GR.…”

“…36 Recent studies have demonstrated that thrombin activates p38 MAPK and ERK1/2 MAPK by increasing their phosphorylation in several cell types. 27,31,32,37,38 In the present study, with the goal of understanding the molecular mechanisms underlying these thrombin effects, in situ and in vitro observations were extended to assess the involvement of the ERK signaling pathway on thrombin-mediated PR expression in DCs.…”

Calcium-Signalling in Human Glaucoma Lamina Cribrosa Myofibroblasts

“…Oxidative stress can arise from Ca 2+ dysregulation through several mechanisms, including increasing metabolic rate [ 110 ] and the activation of ROS-producing enzymes such as nitric oxide synthase and nicotinamide adenine dinucleotide phosphate oxidase [ 97 , 98 , 99 , 111 ]. ROS formation can damage proteins, lipids, and nucleic acids.…”