Autocrine P2X4 receptor activation in RBCs drives oxygen-dependent hyperemic responses in mouse skeletal muscle capillaries.

Microvascular blood flow is tightly coupled to tissue function and when disturbed, organ insult ensues. Changes in red blood cell (RBC) oxygenation are thought to be key to the initiation of robust microvascular responses, a process mechanistically linked to ATP release from RBCs and conducted signalling. The RBC signaling cascade governing ATP release was the focus of this study, and of note is the role of ATP-activated P2X4 receptors. Using an in-vivo mouse muscle preparation (extensor digitorum longus, EDL) and a focally delivered oxygen stimulus via a rapid gas exchange chamber, we probed capillary hemodynamics, prior to and following purinergic receptor antagonism. Dropping oxygen tension from 7% to 2% reduced RBC oxygen saturation in surface capillaries while increasing RBC supply rate. Incubating the EDL with PPADS (non-selective purinergic receptor antagonist, 30 µM) or 5-BDBD (selective P2X4 antagonist, 20 µM) reduced baseline RBC supply rate by ≍ 40% and 47%, respectively, and attenuated the capillary responses to the oxygen stimulus by ≍ 5 and 3 RBC/s, respectively. This blunting predictably enhanced the drop in capillary RBC oxygen saturation, as more oxygen was drawn per RBC to meet tissue demand. Immunofluorescence labeling revealed robust expression of P2X4 receptors in RBCs but not endothelial cells. Together, this data begins to elucidate a role for RBC P2X4 receptors, potentially in a positive feedback loop with pannexin channels, to dynamically regulate demand-supply coupling in skeletal muscle.