Pulsatile and steady flow-induced calcium oscillations in single cultured endothelial cells

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.