Human endometrium-derived mesenchymal stem cells (hMESCs) enter the premature senescence under sublethal oxidative stress, however underlying mechanism remains unknown. Here, we showed that exogenous H2O2 induces a rapid phosphorylation and co-localization of ATM, H2A.X, 53BP1 leading to DNA damage response (DDR) activation. DDR was accompanied with nuclear translocation of p-p53 followed by up-regulation of p21Waf1 and the permanent hypophosphorylation of pRb. Additionally, the increased p38MAPK/MAPKAPK-2 activation persisted in H2O2-treated cells. We suggest that both p53/p21/pRb and p38MAPK/MAPKAPK-2 pathways are responsible for establishing an irreversible cell cycle arrest that is typical of senescence. The process of further stabilization of senescence required prolonged DDR signaling activation that was provided by the permanent ROS production which in turn was regulated by both p38MAPK and the increased functional mitochondria. To reverse senescence, the pharmacological inhibition of p38MAPK was performed. Cell treatment with SB203580 was sufficient to recover partially senescence phenotype, to block the ROS elevation, to decrease the mitochondrial function, and finally to rescue proliferation. Thus, suppression of the p38MAPK pathway resulted in a partial prevention of H2O2-induced senescence of hMESCs. The current study is the first to reveal the molecular mechanism of the premature senescence of hMESCs in response to oxidative stress.

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pronociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca 2+ transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pronociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca 2ϩ imaging analysis, we report that ouabain, a specific ligand of the Na ϩ ,K ϩ -ATPase cardiac glycoside binding site, can prevent glutamate receptor agonistinduced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 M N-methyl-Daspartate (NMDA) or kainate caused apoptosis in ϳ50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic current's frequency and the intracellular Ca 2ϩ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid or inhibition of the plasma membrane Na ϩ ,Ca 2ϩ -exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA-or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca 2ϩ extrusion from neurons via the Na ϩ ,Ca 2ϩ exchanger. Because intracellular Ca 2ϩ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca 2ϩ extrusion abolishes its development. These antiapoptotic effects are independent of Na ϩ ,K ϩ -ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na ϩ ,K ϩ -ATPase in neuroprotection.