Individual gap junction plaques contain multiple connexins in arterial endothelium

Gap-junctional intercellular communication in endothelial cells is implicat ed in the coordination of growth, migration, and vasomotor responses. Up to 3 connexin types, connexin40 (Cx40), Cx37, and Cx43 may be expressed in va scular endothelium according to vascular site, species, and physiological c onditions. To establish how these connexins are organized at the level of t he individual endothelial gap junction, we used affinity-purified connexin- specific antibodies raised in 3 different species to permit double and trip le immunolabeling, in combination with confocal and electron microscopy. Us ing HeLa cells transfected with Cx37 and Cx40 for characterization, the ant i-Cx37 antibody (raised in rabbit) and the anti-Cx40 antibody (raised in gu inea pig) were shown to recognize single bands of 37 and 40 kDa, respective ly, on Western blots and to give prominent punctate labeling at the cell bo rders, specifically in the corresponding transfectant. By applying these an tibodies together with mouse monoclonal anti-Cx43 for double and triple imm unofluorescence labeling at confocal microscopy, rat aortic and pulmonary a rterial endothelia were found to express all 3 connexin types, whereas coro nary artery endothelium expressed Cx40 and Cx37 but lacked Cx43. High-resol ution en face confocal viewing of the aortic endothelium after double label ing demonstrated frequent colocalization of connexins, with distinct variat ion in the expression pattern within a given cell, where it made contact wi th different neighbors. Triple immunogold labeling at the electron-microsco pic level revealed that aortic endothelial gap junctions commonly contain a ll 3 connexin types. This represents the first definitive demonstration of any cell type in vivo expressing 3 different connexins organized within the same gap-junctional plaque.