Endothelial Ca²⁺signaling by Cx43 Hemichannels Interplay with eNOS-Caveolae Internalization During Inflammation-induced Hyperpermeability

Endothelial cells (EC) barrier maintain tissue homeostasis. In post-capillary venules, inflammatory agents such as Platelet-activating Factor (PAF) or Vascular Endothelial Growth Factor (VEGF) evoke an increase in endothelial permeability to macromolecules, a.k.a Hyperpermeability, which leads to edema. Hyperpermeability is mediated by an increase in cytosolic Nitric Oxide (NO) production. Then, the endothelial isoform of NO synthase (eNOS) translocates from the membrane to the cytosol via caveolae (Caveolin-1) leading to the NO-dependent S-nitrosylation of adherens junctions (i.e. Vascular endothelial (VE)-cadherin) and disrupting the endothelial barrier. Intracellular Ca 2+ concentration ([Ca 2+ ] i ) increases at the onset of hyperpermeability and is crucial for eNOS activation. First, Ca 2+ increases transiently by intracellular Ca 2+ store release and successive Ca 2+ influx results in a sustained Ca 2+ signal. In this context, Connexin 43 (Cx43) is a transmembrane protein that forms Hemichannels (HC) and is permeable to Ca 2+ . Additionally, Cx43 interacts with Caveolin-1 (Cav-1), can undergo caveolae-dependent internalization, and is regulated by NO-dependent S-nitrosylation. Thus, we will test the hypothesis that at the onset of Endothelial Hyperpermeability: NO produced by eNOS activation in the plasma membrane increases Cx43 HC activity and Ca 2+ influx leading to Cx43-eNOS-caveolae internalization. We used Human Microvascular Endothelial Cells (HMVEC) to determine spatial distribution of Cx43, eNOS and Cav-1 proteins. Immunofluorescence results showed that these proteins are localized in the plasma membrane and under inflammatory conditions (PAF, VEGF or extracellular Ca 2+ exposure), they redistribute to the cytosol, suggesting an endocytic process. We confirmed that co-localized by proximity ligation assay. Redistribution was prevented when we specifically blocked Cx43-HC with Gap19, caveolae lipid rafts with β-cyclodextrin (β-CD), or chelate intracellular Ca 2+ with BAPTA (1,2-bis (o-aminophenoxy) ethane-N, N, N′, N'-tetra-acetic acid). Moreover, Gap19 did not prevent S1177 phosphorylation or T495 dephosphorylation of eNOS, suggesting that eNOS activation is upstream to Cx43 hemichannel Ca 2+ signaling. We demonstrated in HMVEC that PAF, VEGF or Ca 2+ -induced Hyperpermeability in vitro was blunted when we pretreated with Gap19, β-CD or BAPTA. To further understand this concept, we used eNOS-deficient mouse mesenteric endothelial cells to discover that Cx43 and Cav-1 have a disorganized distribution after PAF, VEGF, or extracellular Ca 2+ stimulation, and the Cx43-HC Ca 2+ signal and Hyperpermeability response ( in vivo and in vitro ) is blunted. In summary, we demonstrate a novel endocytic crosstalk pathway between eNOS-caveolae endocytosis and Cx43-HC Ca 2+ signal during Endothelial Inflammation-stimulated Hyperpermeability. Support: American Heart Association (AHA) Career Development Award (# 932684) and Research Supplement to Promote Diversity in Science (#23DIVSUP1054931). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.