LDL INCREASES (CA-CELLS AND AUGMENTS THROMBIN-INDUCED CELL SIGNALING(+)(I) IN HUMAN ENDOTHELIAL)

Low-density lipoproteins (LDLs) stimulate cytosolic calcium ([Ca++](i) ) in endothelial cells. To elucidate the mechanisms of this response, we compared the effects of low-density lipoprotein (LDL) with those of thrombin, a known endothelial cell agonist. [Ca++](i) was measured in cultured endothelial cells from human umbilical veins. Both spectrofl uorometry of single cells with fura-2 and confocal microscopy were use d. LDL (100 mu g/ml) led to a rapid increase in [Ca++](i) (143 +/- 46 nmol/L to 426 +/- 69 nmd/L; p < 0.05) followed by a sustained plateau phase. Higher concentrations did not increase this response further. R emoval of extracellular calcium resulted in a significant decrease of the plateau phase, which remained significantly elevated as compared w ith baseline values. On the other hand, the initial peak was only slig htly altered. Incubation of endothelial cells with thapsigargin (10(-6 ) mol/L) reduced the initial calcium peak, white the incubation of the cells with pertussis toxin (10(-6) mol/L) for 24 hours abolished the LDL-induced [Ca++](i) response together. Down-regulation of LDL recept ors by exposing the endothelial cells to high LDL concentrations (500 mu g/ml) for 24 hours abolished the LDL-induced calcium signal, while preincubation of the cells with acetylated LDL (500 mu g/ml) did not a lter the cellular response to LDL. Visualization of the calcium signal showed a rapid increase in [Ca++](i) followed by an increase in the n uclear calcium concentration. The LDL calcium signalling was shorter t han that observed with thrombin (0.1 U/ml). Administration of thrombin and LDL together resulted in an increased [Ca++](i) response as compa red with either substance alone. Our results show that (1) LDL reads t o both a release of calcium from intracellular stores and a transmembr anous calcium influx, (2) the effect of LDL is dependent on binding to a specific G-protein-coupled receptor, and (3) LDL enhances the activ ation induced by other agonists.