Formation of immature cell-cell contacts in proliferating HMEC-1 enables TRPC4-dependent Ca 2+ entry. A , Ca 2+ entry into thrombin or EGF-stimulated single HMEC-1 cells. Left , representative recordings of fluo-4 fluorescence intensity in single cells transfected with pcDNA3-vector (control; open circles ), TRPC4 ( closed circles ), or a functional dominant negative TRPC4 fragment (DNTRPC; squares ). Time courses during acute stimulation with 0.5 unit/ml thrombin ( arrow ) and Ca 2+ readdition (2 m m ) are shown. Center , mean values of peak fluo-4 intensity ( n > 6; open columns , control; hatched columns , TRPC4; filled columns , DNTRPC). Right , mean increases in fura-2 fluorescence ratios ( n ≥ 6) obtained during Ca 2+ readdition after stimulation with EGF (100 ng/ml). B , left , representative recordings of fura-2 fluorescence ratios in the subconfluent, contact-forming state is shown for controls (sham-transfected; open circles ) and DNTRPC-transfected ( closed circles ) cells, HMEC-1 cells were challenged with EGF (100 ng/ml; arrow ), as indicated. Right , increases in fura-2 fluorescence ratios (mean ± S.E.; n ≥ 8). C , left , representative recordings of fura-2 fluorescence ratios in the confluent, barrier-forming state is shown for controls (sham-transfected; open circles ) and DNTRPC-transfected ( closed circles ) cells, and HMEC-1 cells were challenged with EGF (100 ng/ml; arrow ), as indicated. Right , increases in fura-2 fluorescence ratios (mean ± S.E.; n ≥ 8).
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