Pulsatile and Steady Flow-Induced Calcium Oscillations in Single Cultured Endothelial Cells

Abstract

The influence of flow-imposed shear stress on the intracellular calcium concentration ([Ca²⁺]i) of cultured endothelial cells (ECs) remains incompletely understood. In the present study, we measured [Ca²⁺]i in single bovine aortic ECs, using fluorescence ratiometric image analysis. The effects of several flow patterns were analysed: steady shear stress (5–70 dyn/cm²), 1-Hz pulsatile shear stress (nonreversing 40 ± 20 dyn/cm², reversing 20 ± 40 dyn/cm², or purely oscillatory 0 ± 20 dyn/cm²), or changing shear stress levels. Under all flow conditions, single-cell analyses revealed flow-induced asynchronous [Ca²⁺]i oscillations, which occurred randomly over the monolayer and which were not seen in the average [Ca²⁺]i signal corresponding to the monolayer response. The number of single-cell [Ca²⁺]i oscillations and the corresponding oscillation frequency rose as the shear stress associated with the steady flow increased: 0.06 ± 0.02 min^–1 at 5 dyn/cm², 0.19 ± 0.03 min^–1 at 20 dyn/cm², and 0.28 ± 0.02 min^–1 at 70 dyn/cm² (means ± SD). Also, the number of oscillations was greater for any type of pulsatile flow (0.53 ± 0.07 min^–1 at 40 ± 20 dyn/cm²,0.54 ± 0.08 min^–1 at 20 ± 40 dyn/cm², and 0.39 ± 0.07 min^–1 at 0 ± 20 dyn/cm²), as compared to any level of steady flow. The most dramatic finding was that purely oscillatory flow induced numerous single-cell [Ca²⁺]i oscillations, yet the average [Ca²⁺]i response for the monolayer did not change. Furthermore, an EC monolayer switched from low to high (or from high to low) steady flow consistently showed an increase (or a decrease) in the number of single-cell [Ca²⁺]i oscillations. These experiments show that ECs respond to different flow conditions by varying single-cell [Ca²⁺]i oscillatory activity. This may have important implications in the endothelium-dependent control of vascular physiology, such as the release of vasoactive substances.