An increase in intracellular [Ca2+] is important in mediating many response of vascular endothelial cells, which are positioned between the blood and the vessel wall. Although considerable information has been learned about endothelial Ca2+ regulation, the identity and sensitivity of the intracellular compartments are not completely known. In addition, endothelial cells are uniquely exposed in vivo to hemodynamic shear stress forces, and the intracellular signalling mechanisms involved in the respons to these forces have been the subject of considerable recent attention. To study endothelial intracellular cytosolic pH and [Ca2+], cells were culture in 1 mm2 glass capillary tubes and loaded with either the fluorescent indicator carboxy-seminaphtharhodafluor-1 (SNARF-1 for pH) or indo-1 ([Ca2+]) and studied on the stage of a modified inverted fluorescence microscope. These capillary tubes facilitate measurements of cytosolic pH or [Ca2+] in a closed system which does not allow gas diffusion when using CO2/HCO3--buffered solutions. In addition, the effect of shear stress is readily examined using these tubes as relatively small changes in flow rate produce rather large changes in shear stress. We recently demonstrated that a functional ryanodine-sensitive intracellular Ca2+ store exists in vascula endothelial cells (Circ Res 74:151-6, 1994) which may be involved in the regulation of Ca2+ storage and release from agonist-sensitive intracellular compartments. We have also shown that cytosolic [Ca2+] is an important intracellular signal in mediating firm adhesion of cancer cells (J Clin Invest 92:3017-22, 1993) and leukocytes (Circulation 1994, in press) to the vascular endothelium. Recently we demonstrated that flow-dependent intracellular acidification occurs in endothelial cells during brief exposures to continuous laminar shear stress forces in bicarbonate buffer due to activation of both Na+o-independent Cl-/HCO3- exchange and Na+/H+ exchange (Science 258: 656-9, 1992). Partial recovery from this acidification occurs during a 30 min exposure to shear stress forces of 2.7 dyne cm-2 or less. Studies were also performed to characterize the pH response following a 30 min shear stress exposure. After return to control conditions, a slowly-developing increase in endothelial pH of approximately 0.20 pH units was noted on return to control conditions. Following this alkalinization, pH recovers to control values over 15-20 min. Thus, cytosolic pH appears to play a significant role in the response of the vascular endothelium to shear stress.
