Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase

We sought to evaluate the mechanisms by which mechanical perturbation elevates intracellular calcium in endothelial cells. We report that the transient elevation in intracellular calcium in cultured bovine aortic endothelial cells (BAEC) in response to gentle perturbation with the side of a micropipette was not blocked by depolarization (external K⁺, 130 mmol/L), nifedipine (10 μmol/L), or Bay K 8644 R(+) (10 μmol/L). Thus, voltage-dependent calcium channels were not involved in the response. Also, amiloride (10 μmol/L) and tetraethylammonium (1 mmol/L) had no effect on calcium mobilization, indicating that Na⁺ and K⁺ transporters were not involved. Pretreatment of the cells with the phospholipase C and phospholipase A₂ inhibitor manoalide (10 μmol/L) for 10 minutes at 37°C completely abolished the calcium response, as did a 10-minute pretreatment with the inhibitor of actin polymerization, cytochalasin B (1 μmol/L). We observed an inhibitory effect of the phospholipase A₂ and phospholipase C inhibitor 4-bromophenacyl bromide (10 μmol/L) on the mechanical response of BAEC that was not as potent as that observed with manoalide. To examine the role of arachidonic acid (AA) and subsequent metabolites that may be released after a putatively mechanical activation of phospholipase A₂, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 μmol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A₂, the liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium. Because extracellular calcium was required for the elevation of intracellular calcium, we suggest that mechanical deformation activates a plasmalemma ion channel permeable to extracellular calcium, and provides an amount of calcium sufficient to trigger release of internal calcium stores. Actin filaments may be required or involved in either the transfer of forces to the channel or in the subsequent activation of Ca²⁺-dependent phospholipases.