LDL increases (CA++)i in human endothelial cells and augments thrombin-induced cell signalling.

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 spectrofluorometry of single cells with fura-2 and confocal microscopy were used. LDL (100 micrograms/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. Removal of extracellular calcium resulted in a significant decrease of the plateau phase, which remained significantly elevated as compared with baseline values. On the other hand, the initial peak was only slightly altered. Incubation of endothelial cells with thapsigargin (10(-6) mol/L) reduced the initial calcium peak, while 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 receptors by exposing the endothelial cells to high LDL concentrations (500 micrograms/ml) for 24 hours abolished the LDL-induced calcium signal, while preincubation of the cells with acetylated LDL (500 micrograms/ml) did not alter the cellular response to LDL. Visualization of the calcium signal showed a rapid increase in [Ca++]i followed by an increase in the nuclear calcium concentration. The LDL calcium signalling was shorter than that observed with thrombin (0.1 U/ml). Administration of thrombin and LDL together resulted in an increased [Ca++]i response as compared with either substance alone. Our results show that (1) LDL leads to both a release of calcium from intracellular stores and a transmembranous calcium influx, (2) the effect of LDL is dependent on binding to a specific G-protein-coupled receptor, and (3) LDL enhances the activation induced by other agonists.