Bioluminescent Monitoring of ATP Release from Human Red Blood Cells Treated with Nonionic Detergent

Abstract 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). Figure Figure 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.

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Deformation-induced ATP release from red blood cells requires CFTR activity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.5.h1726 ◽

1998 ◽

Vol 275 (5) ◽

pp. H1726-H1732 ◽

Cited By ~ 94

Author(s):

Randy S. Sprague ◽

Mary L. Ellsworth ◽

Alan H. Stephenson ◽

Mary E. Kleinhenz ◽

Andrew J. Lonigro

Keyword(s):

Cystic Fibrosis ◽

Red Blood Cells ◽

Blood Cells ◽

Atp Release ◽

Chronic Obstructive Lung Disease ◽

Mechanical Deformation ◽

Chronic Obstructive ◽

Healthy Humans ◽

Human Red Blood Cells ◽

Human Rbcs

Recently, it was reported that rabbit and human red blood cells (RBCs) release ATP in response to mechanical deformation. Here we investigate the hypothesis that the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP binding cassette, is required for deformation-induced ATP release from RBCs. Incubation of rabbit RBCs with either of two inhibitors of CFTR activity, glibenclamide (10 μM) or niflumic acid (20 μM), resulted in inhibition of deformation-induced ATP release. To demonstrate the contribution of CFTR to deformation-induced ATP release from human RBCs, cells from healthy humans, patients with cystic fibrosis (CF), or patients with chronic obstructive lung disease (COPD) unrelated to CF were studied. RBCs of healthy humans and COPD patients released ATP in response to mechanical deformation. In contrast, deformation of RBCs from patients with CF did not result in ATP release. We conclude that deformation-induced ATP release from rabbit and human RBCs requires CFTR activity, suggesting a previously unrecognized role for CFTR in the regulation of vascular resistance.

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Localization of calcium in red blood cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/31.9.6411806 ◽

1983 ◽

Vol 31 (9) ◽

pp. 1109-1116 ◽

Cited By ~ 16

Author(s):

M Borgers ◽

F J Thone ◽

B J Xhonneux ◽

F F De Clerck

Keyword(s):

Plasma Membrane ◽

Red Blood Cells ◽

Blood Cells ◽

Potassium Phosphate ◽

Human Red Blood Cells ◽

A 23187 ◽

Nonionic Detergent ◽

Capillary Endothelial Cells ◽

Membrane Bound ◽

Triton X

The distribution of calcium is demonstrated in human red blood cells (RBC) with a combined phosphate-pyroantimonate technique (PPA). Freshly collected blood and tissue biopsies were initially fixed in potassium phosphate-glutaraldehyde and the complexed calcium was subsequently visualized on Vibratome sections with potassium pyroantimonate. The majority of cells, both in isolated as well as "in situ" preparations, show a fine granular precipitate located at the inner leaflet of the plasma membrane. A minority of cells lack these membrane-associated deposits, exhibiting instead a random distribution of very fine precipitate in their cytoplasm. Capillary endothelial cells and pericytes are devoid of plasma membrane-bound precipitate. When irreversible crenation of RBC is induced by exposure to ionophore A 23187 and calcium, the sphero-echinocytes loose their membrane-bound precipitate, whereas the cells that retain their discocyte shape demonstrate the usual pattern of membrane-bound deposits. Contrarily, cells showing reversible shape changes induced by either A 23187-Ca2+ challenge, by adenosine triphosphate depletion during aging, or contact with lysolecithin, retain or regain the membrane-bound calcium. This cytochemical demonstrable calcium at the inner leaflet of the plasma membrane is probably bound to acidic phospholipids, since it is readily extractable with the nonionic detergent Triton X-100.

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