Merck

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

American journal of physiology. Cell physiology (2017-09-01)
Alexander S Keller, Lukas Diederich, Christina Panknin, Leon J DeLalio, Joshua C Drake, Robyn Sherman, Edwin Kerry Jackson, Zhen Yan, Malte Kelm, Miriam M Cortese-Krott, Brant E Isakson
RESUMEN

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Firefly Lantern Extract, for ATP determination
Sigma-Aldrich
Monoclonal Anti-Spectrin (α and β) antibody produced in mouse, clone SB-SP1, ascites fluid
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
Anti-Connexin-43 antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution