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

Abstract

1. A regenerative calcium wave is an increase in cytosolic free calcium concentration ([Ca2+]i) which extends beyond the stimulated cells without decrement of amplitude, kinetics of [Ca2+]i increase and speed of propagation. 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 endothelial 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 approximately 300 cells caused an increase in [Ca2+]i which was propagated throughout the cluster in a regenerative manner over distances up to 400 micrometer. This wave spread through gap junctions since it was inhibited by the cell uncoupler palmitoleic acid. 4. The same experiments performed in confluent cultures caused a rise in [Ca2+]i which failed to propagate in a regenerative way. The wave propagation probably failed because the confluent cells were less dye coupled than the growing cells. This was confirmed by immunohistology which detected a dramatic 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 calcium-induced calcium release (CICR) and phospholipase C (PLC), and by suppression of extracellular calcium, but not by clamping the membrane potential with high-potassium solution. 6. We conclude that regenerative intercellular calcium waves exist in cultured 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 regenerated propagation.