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. 2012 Feb 1;302(3):H553-9.
doi: 10.1152/ajpheart.00998.2011. Epub 2011 Dec 9.

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Meera Sridharan  1 , Elizabeth A Bowles, Jennifer P Richards, Medina Krantic, Katie L Davis, Kristine A Dietrich, Alan H Stephenson, Mary L Ellsworth, Randy S Sprague
Affiliations

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Meera Sridharan et al. Am J Physiol Heart Circ Physiol. .

Abstract

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.

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Figures

Fig. 1.
Fig. 1.
Identification of voltage-dependent anion channel (VDAC) in human and rabbit erythrocyte membranes. Solubilized erythrocyte membranes of humans (H) and rabbits (R) were resolved on precast gradient (4–20%) gels and probed with antibodies generated against human or H. virescens VDAC. Crude H. virescens larva preparations (L) and isolated mouse cardiac mitochondria (M) were used as positive controls. The gels pictured are representative of 5 human and 5 rabbit membrane preparations. Gel A (A), larval antibody; gel B (B), AB1; gel C (C), AB4.
Fig. 2.
Fig. 2.
Effect of Bcl-xL BH44–23 (BCL; 5 × 10−2 μg/ml) on ATP release from human erythrocytes incubated with iloprost (1 μM, n = 7; A) or UT-15C (1 μM, n = 6; B). Erythrocytes were incubated with BCL or its vehicle, DMF, for 25 min before addition of the prostacyclin analog. Values are means ± SE. †Different from respective baseline and iloprost or UT-15C in the presence of BCL (P < 0.01); n, the number of different individuals studied. RBCs, red blood cells.
Fig. 3.
Fig. 3.
Effect of Bcl-xL BH44–23 (BCL; 5 × 10−2 μg/ml) on iloprost-induced increases in cAMP levels in human erythrocytes. Erythrocytes were incubated with BCL or its vehicle, DMF, for 30 min before addition of iloprost (1 μM, n = 6). The reaction was stopped 15 min after the addition of iloprost. Values are means ± SE. †Different from respective control (P < 0.01); n, the number of different individuals studied.
Fig. 4.
Fig. 4.
Effect of TRO19622 (Tro; 10 μM) on ATP release from human erythrocytes incubated with iloprost (1 μM, n = 7; A) or UT-15C (1 μM, n = 6; B). Erythrocytes were incubated with Tro or its vehicle, saline, for 30 min before addition of the prostacyclin analog. Values are means ± SE. *Different from respective baseline (P < 0.05); †different from respective baseline and UT15C in the presence of Tro (P < 0.01); n, the number of different individuals studied.
Fig. 5.
Fig. 5.
Effect of Bcl-xL Bh44–23 (BCL; 5 × 10−2 μg/ml, n = 6; A) or carbenoxolone (100 μM, n = 5; B) on ATP release from human erythrocytes incubated with isoproterenol. Erythrocytes were incubated with either the inhibitor or its respective vehicle, DMF or saline, for 25 min before addition of isoproterenol (1 μM). Values are means ± SE. *Different from respective baseline (P < 0.05); †different from respective baseline (P < 0.01); n, the number of different individuals studied.
Fig. 6.
Fig. 6.
Proposed signaling pathway for ATP release from erythrocytes in response to activation of the prostacyclin receptor. Exposure of human erythrocytes to prostacyclin (PGI2) analogs results in activation of Gs, leading to increases in cAMP that are regulated by phosphodiesterase 3 (PDE3) activity. Increases in cAMP activate PKA and, subsequently, CFTR. The final conduit for ATP release is VDAC. AC, adenylyl cyclase.
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