Platelet agonists and calcium homeostasis in endothelial cells: possible role in the interaction of endothelium with hemostatic system.

BACKGROUND AND OBJECTIVE

The endothelium is complex tissue that modulates a vast array of biological functions and Ca++ transients are critically important to these endothelium-dependant functions. We addressed the hypothesis that some platelet agonists and products of activation of the hemostatic system could determine Ca++ transients in a bovine pulmonary artery endothelial cell line (CPA-47).

DESIGN AND METHODS

The effect of thrombin, collagen, ADP, PAF, PDGF, GRGDS, and the TxA2 mimetic U46619 on CPA-47-cytoplasmic Ca++ transients was evaluated using a Platelet ionized Calcium Aggregometer, after cells were loaded with the photoprotein aequorin.

RESULTS

ADP, GRGDS, PAF, U46619 and collagen were able to induce rapid Ca++ transients in CPA-47 endothelial cells and the response was stable after repeated additions, while thrombin acted slightly differently, as cells became refractory to this agonist after the first response, but they remained sensitive to the other inducers. Only PDGF was completely ineffective. Furthermore, calcium-channel blockers verapamil and flunarizine (but not nifedipine) caused a reduction only of thrombin-induced cytoplasmic Ca++ transients, while the addition of depolarizing concentration of KCl suggests the presence also of voltage-operated channels on endothelial cell membrane. Finally, EGTA caused the complete suppression of Ca++ transients induced by all the tested agonists but collagen.

INTERPRETATION AND CONCLUSIONS

Our study demonstrated that the different agents tested are able to induce Ca++ transients on bovine endothelial cultured cells, similarly to that observed in platelets and other non excitable cells, including tumor cells, and that calcium channel blockers had only a limited inhibitory effect on these changes; these results may help more thorough understanding of the biochemical basis of the interaction between endothelium and the hemostatic system.