ATP Release from Red Blood Cells Is Regulated by a Negative Feedback Pathway where ADP Acts on P2Y₁₃ Receptors.

Background: Red blood cells regulate tissue circulation and O₂ delivery by releasing the vasodilator adenosine triphosphate (ATP) in response to hypoxia. When released extracellularly, ATP is rapidly degraded to adenosine diphosphate (ADP) in the circulation by ectonucleotidases. ATP and ADP activate subtypes of the large P2 receptor family (15 subtypes). Here we show that ADP acting on P2Y₁₃ receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release.

Methods: mRNA was quantified with real-time PCR. Western blot was used to detect P2 receptors with available antibodies. cAMP levels were determined with an enzyme immunoassay. ATP release was measured in incubated red blood cells using microdialysis and a luciferase assay. In a pig model, catheters were inserted through the carotid artery to place a catheter in the left coronary artery, and through the jugular vein to place a microdialysis probe in the coronary vein. 2-MeSADP was injected in the artery and ATP levels were measured in the coronary vein.

Results: mRNA of the ADP receptor P2Y₁₃ was highly expressed in human red blood cells and reticulocytes, whilst other ADP receptors were not (Fig.1).

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The stable ADP analogue 2-MeSADP decreased ATP release from red blood cells by inhibition of cAMP. The P2Y₁₂ and P2Y₁₃ receptor antagonist AR-C67085 (30 mM), but not the P2Y₁ blocker MRS2179, inhibited the effects of 2-MeSADP. At doses where AR-C67085 only blocks P2Y₁₂ (100 nM), it had no effect. AR-C67085 and the nucleotidase apyrase increased cAMP per se, indicating a constant cAMP inhibitory effect of endogenous extracellular ADP. 2-MeSADP reduced plasma ATP concentrations in an in vivo pig model. Furthermore, a missense polymorphism in the coding region of P2Y₁₃ has been found that is in total disequilibrium with 5 polymorphisms in P2Y₁₂ (the important ADP receptor in platelets) forming a haplotype that could contribute to vascular disease.

Conclusion: Our results show that P2Y₁₃ is selectively expressed in human red blood cells. The ATP degradation product ADP inhibited ATP release by acting on this receptor. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation. Because blood consists of approximately 40% red blood cells, containing a 1000-fold higher ATP concentration than plasma (mM vs. uM), even a minor release of ATP from the high intracellular concentrations could have major circulatory effects. A negative system may therefore be of great physiological importance to mitigate ATP release. In addition, this finding could be of interest for efforts to preserve intracellular ATP in red blood cells during storage.