Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium

Abstract

Intracellular calcium ([Ca2+]i) was measured in FURA 2-loaded endothelial cells plated on fibronectin or vitronectin. Average values for [Ca2+]i increased to ∼twofold above basal levels by ∼1 h after plating, and then declined. The increase in [Ca2+]i required extracellular calcium. Substituting potassium for sodium in the medium reduced the elevation of [Ca2+]i, a result that rules out the involvement of Na Ca exchangers or voltage-dependent calcium channels, but that is consistent with the involvement of voltage-independent calcium channels. Plating cells on an anti-integrin β1 subunit antibody gave a similar [Ca2+]i response, but clustering β1 integrins with the same antibody, or occupying integrins with RGD (arggly-asp) peptides had no effect. Time course measurements on single cells revealed that in each cell [Ca2+]i rose abruptly at some point during spreading, from the basal level to a higher steady-state level that was maintained for some time. The elevated [Ca2+]i was unrelated to previously observed changes in intracellular pH, because chelating the Ca2+ in the medium failed to inhibit the elevation of pHi that occurred during cell spreading. In conclusion, these results show that integrin-mediated cell spreading can regulate [Ca2+]i, and the pathways involved are distinct from those that regulate intracellular pH.