Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca2+ waves. Gap junction- deficient HeLa cells were transfected with plasmids encoding for green fluo rescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx 43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labele d gap junctions rendered the cells dye- and electrically coupled and were d etected at the plasma membranes at points of contact between adjacent cells . To correlate the distribution of gap junctions with the changes in [Ca2+] (i) associated with Ca2+ waves and the distribution of the endoplasmic reti culum (ER), cells were loaded with fluorescent Ca2+-sensitive (fluo-3 and f ura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca2+ waves were induced in these cells by mechanical stimulat ion with or without extracellular apyrase, an Am-degrading enzyme. In untra nsfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca2+](i) changes were characterized by initiating Ca2+ puffs associated w ith the perinuclear En By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca2+](i) changes were propagated without initiating p erinuclear Ca2+ puffs and were communicated between cells at the sites of t he Cx-GFP gap junctions. The efficiency of Cx expression determined the ext ent of Ca2+ wave propagation. These results demonstrate that intercellular Ca2+ waves may be propagated simultaneously via an extracellular pathway an d an intracellular pathway through gap junctions and that one form of commu nication may mask the other.