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We investigated the expression pattern of gap junctional proteins (connexins, Cx) in situ and in vitro and their functional characteristics in cultured human umbilical vein endothelial cells (HUVEC) and cultured human umbilical artery endothelial cells (HUAEC). In both arteries and veins, Cx37, Cx40, and Cx43 could be detected in situ and in vitro (passages 2-4). Distribution patterns of Cx40 and Cx43 were homogeneous in situ but more heterogeneous in vitro. Cx37 is heterogeneously expressed both in situ and in vitro. Among most cells, no Cx37 staining could be detected; when present, it was found as bright spots between some clusters of cells. Cx40 was more abundant in cultured arterial endothelium than in cultured venous endothelium. Dye-coupling experiments with Lucifer yellow CH revealed extensive dye spread in HUVEC (15.2 +/- 0.4, mean +/- SE, n = 110) but was significantly restricted in HUAEC (9.8 +/- 0.3, n = 110). Electrophysiological gap junctional characteristics were determined in cultured HUVEC and HUAEC pairs by use of the dual voltage-clamp technique. In contrast to the dye-coupling experiments, mean macroscopic electrical conductance was significantly larger for HUAEC pairs (31.4 +/- 6.0 nS, n = 12) than for HUVEC pairs (16.6 +/- 2.8, n = 18). In HUVEC, we measured multiple single gap junctional channel conductances in the range of 19-75 pS. Interestingly, additional conductances of 80-200 pS were measured in HUAEC, possibly partially reflecting activity of channels formed of Cx40, which are more abundant in the cultured arterial endothelial cells.

Short-term (10 min) effects of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein kinase C (PKC) activator, on cardiac macroscopic (gj) and single channel (gamma j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell (WC) or perforated patch (PP) voltage-clamp, gj increased by 15.5 +/- 7.2% (mean +/- SD, n = 9) and by 46.3 +/- 17.0% (n = 5), respectively. The latter difference is not related to intracellular calcium concentration, because raising the Ca2+ concentration in the electrode solution did not change the TPA-induced increase in gj observed under WC conditions. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (alpha PDD), did not affect gj. Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two gamma j sizes of 20 and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas alpha PDD did not change this distribution, TPA shifted the gamma j distribution to the lower sizes. Diffusion of Lucifer Yellow (LY) and 6-carboxyfluorescein (6-CF), gap junction permeant tracers, was studied on small clusters of cardiomyocytes. Under control conditions, LY labeled 19.4 +/- 7.2 cells (mean +/- SD, n = 18) and 6-CF labeled 8.4 +/- 2.2 cells (n = 20). Whereas alpha PDD did not change the extent of dye transfer, TPA restricted the diffusion of LY to 2.8 +/- 1.3 cells (n = 11) and of 6-CF to 2.4 +/- 1.4 cells (n = 20). This suggests that permeability and single channel conductance of connexin 43 channels are parallely related. Altogether, these results point to the opposite modulation of electrical and metabolic coupling of cardiac cells evoked by TPA.