CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 µM) and 6-OH-BDE-47 (0.2 µM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.

Thrombin-induced ATP release from human umbilical vein endothelial cells.

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 µM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

Ammonium-evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity.

The clearance of extracellular glutamate is mainly mediated by pH- and sodium-dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH(4) (+)/NH(3)). Here, we examined the effects of NH(4) (+)/NH(3) on steady-state intracellular pH (pH(i)) and sodium concentration ([Na(+)](i)) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH(4) (+)/NH(3) on glutamate transporter activity by measuring D-aspartate-induced pH(i) and [Na(+)](i) transients. In 20-34 days in vitro (DIV) astrocytes, NH(4) (+)/NH(3) decreased steady-state pH(i) by 0.19 pH units and increased [Na(+)](i) by 21 mM. D-Aspartate-induced pH(i) and [Na(+)](i) transients were reduced by 80-90% in the presence of NH(4) (+)/NH(3), indicating a dramatic reduction of glutamate uptake activity. In 9-16 DIV astrocytes, in contrast, pH(i) and [Na(+)](i) were minimally affected by NH(4) (+)/NH(3), and D-aspartate-induced pH(i) and [Na(+)](i) transients were reduced by only 30-40%. Next we determined the contribution of Na(+), K(+), Cl(-)-cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20-34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH(4) (+)/NH(3)-evoked changes in steady-state pH(i) and [Na(+)](i) and attenuated the reduction of D-aspartate-induced pH(i) and [Na(+)](i) transients by NH(4) (+)/NH(3) to 30% in 20-34 DIV astrocytes. Our results suggest that NH(4) (+)/NH(3) decreases steady-state pH(i) and increases steady-state [Na(+)](i) in astrocytes by an age-dependent activation of NKCC. These NH(4) (+)/NH(3)-evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE.