Abstract

Well recognized for its role as the energy currency of cells, ATP plays an altogether different role in the mammalian vascular system. Erythrocyte-derived extracellular ATP is fast gaining recognition as a vital vasodilator in blood; stimulating a rapid widening of blood vessels when released into the circulation. Currently, the primary technique employed to determine the concentration of ATP released from erythrocytes is the luciferin-luciferase chemiluminescent assay, which is both robust in signal and specific for ATP. However, the assay is subject to interferences inherent to working with erythrocytes, such as the presence of hemoglobin, which filters the emitted chemiluminescence signal.
At the outset, the current work examined the luciferin-luciferase chemiluminescent assay in the microplate format and evaluated the parameters necessary to determine ATP in suspensions of intact erythrocytes. Based on the methodological considerations which arose from the examination of the assay, the concentration of ATP in suspensions of intact erythrocytes was compared to that of their supernatants. It was determined that the ATP released into the supernatants of erythrocyte suspensions represents but a fraction of the ATP which is externalized and accessible for enzymatic reactions. Known stimuli of ATP release, including CO2 and hypo-osmotic stress, were also used to compare the sensitivity of erythrocytes obtained from humans to those from rats, a common pharmacological model organism: human erythrocytes proved to be more sensitive to stimuli than rat erythrocytes.
Ultimately, a method for measuring ATP in suspensions of intact erythrocytes was developed, and the possibility of two modes of erythrocyte release was discovered. The erythrocyte has long been held as a sensor within the vasculature, and this work underscores the growing importance being placed on the erythrocyte’s role as an effector through the regulation of the release of the vasodilator ATP