Dynamics of shear-induced ATP release from red blood cells

Abstract

Adenosine triphosphate (ATP) is a regulatory molecule for many cell functions, both for intracellular and, perhaps less well known, extracellular functions. An important example of the latter involves red blood cells (RBCs), which help regulate blood pressure by releasing ATP as a vasodilatory signaling molecule in response to the increased shear stress inside arterial constrictions. Although shear-induced ATP release has been observed widely and is believed to be triggered by deformation of the cell membrane, the underlying mechanosensing mechanism inside RBCs is still controversial. Here, we use an in vitro microfluidic approach to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolution. We demonstrate that there is a sizable delay time between the onset of increased shear stress and the release of ATP. This response time decreases with shear stress, but surprisingly does not depend significantly on membrane rigidity. Furthermore, we show that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is commensurate with a characteristic membrane relaxation time determined from observations of the cell deformation by using high-speed video. Taken together our results suggest a model wherein the retraction of the spectrin-actin cytoskeleton network triggers the mechanosensitive ATP release and a shear-dependent membrane viscosity controls the rate of release. mechanotransduction ͉ microfluidic ͉ RBCsA s the central component of the human circulatory system, RBCs have evolved highly specific mechanisms for responding to variations in the local environment. One key but poorly understood response mechanism involves the release of ATP to the extracellular space, which occurs in response to small changes in pH (1), oxygen concentration (2) or osmotic pressure (3). Although ATP is well known as the energy source for intracellular functions, extracellular ATP plays an important role as a signaling molecule in a variety of physiological processes. For example, the ATP released from RBCs helps regulate vascular tone by binding with purigenic receptors on endothelial cells, which then respond by releasing nitric oxide, a potent vasodilator, into the surrounding smooth muscle cells (1, 4-6). In addition, a variety of diseases are linked to impaired ATP release from RBCs, including cystic fibrosis (7), pulmonary hypertension (8), and diabetes (9, 10). Furthermore, extracellular ATP is known to inhibit growth of breast and lung tumors (11,12) and plays a role in the inflammation response to wounds (13). Knowledge of the circumstances under which RBCs release ATP is crucial for designing effective therapeutic strategies.A fundamental characteristic of RBCs is that they regularly encounter variations in hydrodynamic shear stress, especially on entering or exiting arterioles and capillaries (14). A mounting body of evidence suggests that RBCs respond to these variations in shear stress b...

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