Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers

摘要

Previous investigation has demonstrated that adenosine 5'-triphosphate (ATP) appears in the vascular effluent following skeletal and cardiac muscle contraction. By cannulating the frog heart and perfusing through it hypoxic Ringer's solution, this author observed elevated levels of ATP. The presence of blood elements, however, made it necessary to ascertain if the frog red blood cells (RBC's) were a source of ATP. It was noted that RBC's up to 150/microliter were present in the clear effluent samples of the frog heart. To study cell suspensions diluted to this level, whole blood was diluted with frog Ringer's solution. ATP was assayed by firefly lantern extract + D-luciferin (threshold 1 nM). Well mixed suspensions produced ATP concentrations in proportion to the number of cells present. When cells were allowed to settle for one hour, nanomolar ATP concentrations the supernatant fluid were observed, regardless of cell count. Stirred cells did not increase the concentration of ATP in the medium, whereas cells allowed to settle in layers showed enhanced release of ATP. Anion channel blockers, probenecid and furosemide, diminished this release. Evidenced that cell lysis was not the source of the ATP was based on two observations: 1) time course of the recordings to the light signals were identical for cell samples and ATP standards, and 2) digitonin lysed the cells in the assay solution, verifying their presence after the assay.