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

S L Diamond, F Sachs, W J Sigurdson
1994 Arteriosclerosis and Thrombosis A Journal of Vascular Biology  
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 junol/L), or Bay K 8644 R(+) (10 mol/L). Thus, voltage-dependent calcium channels were not involved in the response.
more » ... Also, amiloride (10 /xmol/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 A 2 inhibitor manoalide (10 jimol/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 £imol/L). We observed an inhibitory effect of the phospholipase A 2 and phospholipase C inhibitor 4-bromophenacyl bromide (10 /xmol/L) on the mechanical response of BAEC that was not as potent as that A the interface between the vessel wall and flowing blood, the endothelium must function in a complex fluid mechanical and biomechanical environment. Endothelial cell adjustments to physical stimuli may have roles in various types of vessel physiology and pathology. The fastest endothelial responses to chemical stimuli occur on a time scale of milliseconds to seconds and include ion channel regulation and receptor-mediated signal transduction. These pathways are fairly well characterized in endothelial cells (for review, see Reference 1). The endothelial response to the onset of steady laminar fluid shear stress is also rapid. Flow-activated hyperpolarization of bovine aortic endothelial cells (BAEC) by means of the activation of a K + -selective whole cell current requires about 10 seconds and is half maxima] at a shear stress of 0.07 N/m 2 (0.7 dyne/cm 2 ). 2 For cultured rat aorta endothelial cells subjected to flow, the net intracellular acidification brought about by activation of both Na + -independent Cr/HCO 3 " exchangers and Na + /H + exchangers requires about 30 seconds to reach a full response and is half maximal at a shear stress of about 0.1 N/m 2 . 3 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 A 2 , we exposed BAEC to exogenous AA. We found that continued exposure of BAEC for 5 minutes to 10 nmol/L to 10 fimol/L AA caused no elevation of intracellular calcium. If mechanical stimulation activates phospholipase A 2 , 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 Ca 2+ -dependent phospholipases.
doi:10.1161/01.atv.14.12.2000 fatcat:dxo7nqqvvbbypf2cuxlvybgr3m