An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture

1. A regenerative calcium wave is an increase in cytosolic free calcium con centration ([Ca2+](i)) which extends beyond the stimulated cells without de crement of amplitude, kinetics of [Ca2+](i) increase and speed of propagati on. 2. The aim of the present study was to test the hypothesis that such a wave could be evoked by bradykinin stimulation and by scraping cultured endothe lial cells from porcine coronary arteries. 3. Calcium imaging was performed using the calcium-sensitive dye fura-2. A wound or a delivery of bradykinin to two to three cells on growing clusters of similar to 300 cells caused an increase in [Ca2+], which was propagated throughout the cluster in a regenerative manner over distances up to 400 m u m. This wave spread through gap junctions since it was inhibited by the c ell uncoupler palmitoleic acid. 4. The same experiments performed in confluent cultures caused a rise in [C a2+](i) which failed to propagate in a regenerative way. The wave propagati on probably failed because the confluent cells were less dye coupled than t he growing cells. This was confirmed by immunohistology which detected a dr amatic decrease in the number of connexin 40 gap junctions in the confluent cultures. 5. The regenerative propagation of the wave was blocked by inhibitors of ca lcium-induced calcium release (CICR) and phospholipase C (PLC), and by supp ression of extracellular calcium, but not by clamping the membrane potentia l with high-potassium solution. 6. We conclude that regenerative intercellular calcium waves exist in cultu red islets but not in confluent cultures of endothelial cells. An increase in [Ca2+](i), is not sufficient to trigger a regenerative propagation. The PLC pathway, CICR and extracellular calcium are all necessary for a fully r egenerated propagation.