ABSTRACT
The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca2+ ([Ca2+]c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca2+]c both in the presence and absence of extracellular Ca2+. In the absence of extracellular Ca2+, depletion of the intracellular Ca2+ stores with either ATP (1 microM) or thapsigargin (100 nM) prevented any further release of Ca2+ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca2+), a prior release of Ca2+ induced by okadaic acid reduced the magnitude of the response to ATP (1 microM). Taken together these observations indicate that okadaic acid induces Ca2+ release from the agonist-sensitive pool. The okadaic acid-induced Ca2+ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca2+]c oscillations, which at their peak resulted in 40-100% cells, at any one time, having an elevated [Ca2+]c. The response appeared to propagate between adjacent cells and the elevation of [Ca2+]c appeared initially in the cell periphery. In adherent cells, the release of Ca2+ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca2+ from the agonist- and thapsigargin-sensitive intracellular stores.
