| dc.description.abstract | Endothelial cells form a permeability barrier to macromolecules that can be modulated in post-capillary venules through the activation of Ca2+-dependent signaling pathways by pro-inflammatory agents, such as platelet-activating factor (PAF). This Ca2+ signaling depends on Ca2+ release from the endoplasmic reticulum and Ca2+ influx from the extracellular space, in which may be involved membrane channels formed by connexins (i.e. hemichannels) or pannexins. We analyzed the participation of these channels in the PAF-elicited intracellular Ca2+ concentration ([Ca2+]i) increase. To this end, we used the intact mesenteric vascular bed and primary cultures of mesenteric endothelial cells (EC) of resistance arteries (EC-A) and venules (EC-V). Changes in [Ca2+]i were recorded using the fluorescent Ca2+ indicator Fluo-4 and activity of connexin hemichannels or pannexin channels was evaluated by assessing ethidium uptake in EC and intact vessels. As expected, 1 μM acetylcholine induced an increase of [Ca2+]i in EC-A, but not in EC-V and, in contrast, 10 nM PAF evoked a response only in EC-V. ATP (5 μM) activated a Ca2+ response in both EC-A and EC-V. In addition to activate a Ca2+ signal in EC-V, PAF also induced an increase in ethidium uptake in these cells and in venules of intact mesenteric bed, without affecting the ethidium signal in mesenteric arteries. Treatment for 15 min with the connexin blocking peptide 37, 43Gap27 or the pannexin-1 blocking peptide 10Panx abolished the increase in [Ca2+]i and ethidium uptake induced by PAF, suggesting that connexin hemichannels and pannexin-1 channels contribute to the PAF-activated Ca2+ signal. Cx37, Cx40, Cx43 and pannexin-1 were found to be expressed in intact venules, intact resistance arteries and in EC-V and EC-A by immunofluorescence analysis and western blot. Connexin and pannexin channels are permeable to Ca2+, but they may also trigger Ca2+ signals through ATP release and, consistent with this, blockade of purinergic receptors with PPADS blunted the PAF-induced [Ca2+]i increase. These results suggest that the intracellular Ca2+ signaling activated by PAF in endothelial cells of post-capillary venules is mainly mediated by ATP release through connexin hemichannels and/or pannexin-1 channels, which, in turn, leads to activation of purinergic receptors. However, direct Ca2+ influx via connexin hemichannels or pannexin-1 channels may also contribute to the response. Support or Funding Information. FONDECYT 1150530 | |
