Wall shear stress in blood vessels appears to be a major determinant for the development of restenosis after angioplasty, coronary artery bypass graft occlusion and atherosclerosis. The cellular mechanisms for these effects are unknown. The purpose of this project is to characterize, in endothelial cells (EC), the effect of acute changes in shear stress forces on cytosolic pH (pH i) and [Ca 2+] (cA i) which are major determinants of cell function and growth. EC are obtained from the aorta of adult rats and grown in culture to passage 3 to 5. At this stage EC are seeded into glass capillary tubes and allowed additional 1 to 2 days for attachment and growth. Subsequently, EC are loaded either with the Ca 2+ probe indo-1 or the pH probe SNARF-1 and are suitable to monitor Cai or pHi with fluorescence microscopy. Cells in glass capillaries are perfused with continuous laminar flow. Shear stress forces are varied by changing flow rate and are calculated from the tube diameter, fluid viscosity and flow rate. Our initial results indicate that when cells are superfused with a bicarbonate-containing buffer an increase in shear stress forces from 0 to 7-30 dynes/squared cm decreases pHi. In contrast, when EC are superfused with a bicarbonate-free buffer a similar increase in shear stress forces is associated with an increase in pHi which is abolished by ethylisopropilamiloride, a Na+/H+ exchange blocker. This indicates that under our experimental conditions shear stress forces activate both Cl-/HCO 3- and Na+/H+ exchange and that the former mechanism predominates over the latter. In indo-1 loaded EC studied under similar conditions shear stress forces have no effect on Cai. However in the presence of 2 microM ATP EC respond to an increase in shear stress from 0 to 7-30 dynes/squared cm with an increase in Cai.
