The vascular endothelium, positioned between the flowing blood and the vessel wall, is uniquely exposed to hemodynamic shear stress forces. To study the effect of shear stress forces on vascular endothelial pHi and cytosolic [Ca2+] ([Ca2+]i), cells were cultured in 1 mm2 glass capillary tubes, loaded with the fluorescent indicator carboxy- seminaphtharhodafluor-1 (SNARF-1 for pHi) or indo-1 ([Ca2+]i) and studied on the stage of a modified inverted fluorescence microscope. These capillary tubes facilitate pHi or [Ca2+]i measurements in a closed system which does not allow gas diffusion when using CO2/HCO3-- buffered solutions; small changes in flow rate result in relatively large changes in shear stress forces. We have recently reported that flow-dependent intracellular acidification occurs in endothelial cells during brief exposures to continuous laminar shear stress forces in a physiologic buffer with bicarbonate due to parallel activation of Na+o- independent Cl-/HCO3- exchange and Na+/H+ exchange (Science 258: 656- 659, 1992). This change in intracellular pH (pHi) is sustained during a 30 minute exposure to shear stress forces of 13.4 dyne cm-2, although partial recovery from the acidification occurs during a 30 minute exposure to shear stress forces of 2.7 dyne cm-2 or less. To determine the mechanism of the partial recovery of pHi during a 30 minute exposure to shear stress forces of 2.7 dyne cm-2 or less, cells were exposed to ethylisopropylamiloride (EIPA), a Na+/H+ exchange inhibitor or to Na+-free buffer to inhibit Na+-dependent exchange mechanisms. While EIPA had no effect, removal of buffer Na+ significantly inhibited the pHi recovery. These results suggest that while Na+o-dependent Cl-/HCO3- exchange is also activated by hemodynamic shear stress exposure. Studies were also performed to characterize the pHi response following a 30 minute exposure to shear stress forces. After return to control conditions, a slowly-developing increase in endothelial pHi of approximately 0.20 pH units has been noted on return to control conditions. Following this alkalinization, pHi recovers to control values over 15-20 minutes. Thus, pHi appears to play a significant role in the response of the vascular endothelium to shear stress forces.
