MECHANISM FOR SIGNAL TRANSDUCTION OF SHEAR STRESS FORCES IN ENDOTHELIAL CELLS

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Wall shear stress in blood vessels plays a role in the development of restenosis after angioplasty, coronary artery bypass graft occlusion and atherosclerosis. In addition these processes are also modulated by the interaction of vascular cells with the endothelium. The purpose of this project is: 1) To characterize, in endothelial cells (EC), the effect of acute changes in shear stress forces on cytosolic pH (pH)i and [Ca2+](Cai). 2) To determine the effects of changes in Cai on EC's pHi and 3) to determine how the interaction between the endothelium and other cells affects Cai homeostasis in EC. The effect of laminar shear stress on EC's pHi was studied by examining EC cultured in glass capillary tubes. Shear stress forces led to a rapid decrease in EC's pHi in the presence of HCO3-. This response was markedly blunted by the anion exchange inhibitor 4-acetamido,4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS) but unaffected by Na+ removal. In the absence of HCO3-, shear stress forces caused a small increase in pHi which was abolished by ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor. The effect of acidification on Cai was examined in the absence of significant shear stress forces either by removal of NH4Cl, changing from a bicarbonate- free to a 5% CO2/HCO3--buffered solution at constant buffer pH, or changing from a 5% CO2/HCO3- to a 20% CO2/HCO3- solution. Regardless of the method employed, intracellular acidification resulted in an increase in Cai indexed by the fluorescent Ca2+ indicator indo-1. The increase in the indo-1 fluorescence ratio induced by changing from a 5% CO2/HCO3- to a 20% CO2/HCO3- solution was not significantly altered by removal of buffer Ca2+ either before or after depletion of bradykinin-sensitive intracellular Ca2+ stores. In other experiments we examined the effect of leukocyte adhesion on EC's Cai. Upon contact between granulocytes or monocytes with EC there was a rapid increase in EC's Cai which exhibited a partial recovery toward control. This response was abolished after depletion of EC's endoplasmic reticulum Ca2+ with thapsigargin while it was not affected in a Ca2+-free buffer. Similar results were obtained when melanoma cells were used in place of leukocytes. EC contact with 8 fm inert beads did not elicit an increase in Cai. Thus, in vascular endothelial cells, shear stress forces activate both an alkali extruder, Na+-independent Cl-/HCO3- exchange, and to a smaller extent an acid extruder, Na+/H+ exchange; the net effect in a physiologic bicarbonate buffer is a decrease in pHi