Endothelial Ca(2+) waves preferentially originate at specific loci in caveolin-rich cell edges

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). In a single bovine aortic EC, the [Ca(2+)](i) rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca(2+) wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca(2+) wave was unaffected by the removal of extracellular Ca(2+), demonstrating its dependence on intracellular Ca(2+) release. Microinjection of heparin into the cell inhibited the ATP-induced Ca(2+) responses, indicating that the Ca(2+) wave is at least partly mediated by the inositol 1,4,5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca(2+) waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca(2+)](i) increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca(2+) waves in ECs.