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 th e transient elevation in intracellular calcium in cultured bovine aort ic endothelial cells (BAEC) in response to gentle perturbation with th e side of a micropipette was not blocked by depolarization (external K +, 130 mmol/L), nifedipine (10 mu mol/L), or Bay K 8644 R(+) (10 mu mo l/L). Thus, voltage-dependent calcium channels were not involved in th e response. Also, amiloride (10 mu mol/L) and tetraethylammonium (1 mm ol/L) had no effect on calcium mobilization, indicating that Na+ and K + transporters were not involved. Pretreatment of the cells with the p hospholipase C and phospholipase A, inhibitor manoalide (10 mu mol/L) for 10 minutes at 37 degrees C completely abolished the calcium respon se, as did a 10-minute pretreatment with the inhibitor of actin polyme rization, cytochalasin B (1 mu mol/L). We observed an inhibitory effec t of the phospholipase A, and phospholipase C inhibitor 4-bromophenacy l bromide (10 mu mol/L) on the mechanical response of BAEC that was no t as potent as that observed with manoalide. To examine the role of ar achidonic acid (AA) and subsequent metabolites that may be released af ter a putatively mechanical activation of phospholipase A,, we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 mu mol/L AA caused no elevation of intracel lular calcium. If mechanical stimulation activates phospholipase A,, t he liberated AA and subsequent metabolites do not appear to have much effect on BAEC intracellular calcium. Because extracellular calcium wa s 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.