Lysophosphatidic acid positively regulates the fluid flow-induced local Ca2+ influx in bovine aortic endothelial cells

Using real-time confocal microscopy, we have demonstrated that lysophosphat idic acid (LPA), a bioactive phospholipid existing in plasma, positively re gulates fluid flow-induced [Ca2+](i) response in flue 4-loaded, cultured, b ovine aortic endothelial cells. The initial increase in [Ca2+](i) was local ized to a circular area with a diameter of <4 mum and spread concentrically , resulting in a mean global increase in [Ca2+](i) The local increase often occurred in a stepwise manner or repetitively during constant flow. The pe rcentage of cells that responded and the averaged level of increase in [Ca2 +](i) were dependent on both the concentration of LPA (0.1 to 10 mu mol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca2+ or by the application of Gd3+, an inhibitor of mechanose nsitive (MS) channels, but not by thapsigargin, an inhibitor of the endopla smic reticular Ca2+-ATPase. It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinas e G (PKG). These results suggest that the [Ca2+](i) response arises from Ca 2+ influx through Gd3+-sensitive MS channels, which are negatively regulate d by the activation of PKG. The spatiotemporal properties of the [Ca2+](i) response were completely different from those of a Ca2+ wave induced by ATP , a Ca2+-mobilizing agonist. Therefore, we called the phenomenon Ca2+ spots . We conclude that LPA positively regulates fluid flow-induced local and os cillatory [Ca2+](i) increase, ie, the Ca2+ spots, in endothelial cells via the activation of elementary Ca2+ influx through PKG-regulating MS channels . This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.