is the conduit for low oxygen tension-induced ATP release from human erythrocytes. Am J Physiol Heart Circ Physiol 299: H1146 -H1152, 2010. First published July 9, 2010; doi:10.1152/ajpheart.00301.2010.-Erythrocytes release ATP in response to exposure to the physiological stimulus of lowered oxygen (O 2) tension as well as pharmacological activation of the prostacyclin receptor (IPR). ATP release in response to these stimuli requires activation of adenylyl cyclase, accumulation of cAMP, and activation of protein kinase A. The mechanism by which ATP, a highly charged anion, exits the erythrocyte in response to lowered O 2 tension or receptor-mediated IPR activation by iloprost is unknown. It was demonstrated previously that inhibiting pannexin 1 with carbenoxolone inhibits hypotonically induced ATP release from human erythrocytes. Here we demonstrate that three structurally dissimilar compounds known to inhibit pannexin 1 prevent ATP release in response to lowered O 2 tension but not to iloprost-induced ATP release. These results suggest that pannexin 1 is the conduit for ATP release from erythrocytes in response to lowered O 2 tension. However, the identity of the conduit for iloprost-induced ATP release remains unknown.iloprost; carbenoxolone; probenecid; red blood cell; cystic fibrosis transmembrane conductance regulator ERYTHROCYTES CONTRIBUTE to the regulation of vascular caliber by virtue of their ability to release adenosine 5=-triphosphate (ATP) (14 -17, 48, 50). ATP released from erythrocytes binds to purinergic receptors on the vascular endothelium, which leads to the local formation of endothelium-derived vasodilators such as nitric oxide, prostaglandins, and endotheliumderived hyperpolarizing factor (20,46,50).Erythrocytes release ATP in response to mechanical deformation and exposure to lowered oxygen (O 2 ) tension and in response to incubation with pharmacological agents such as the prostacyclin analog iloprost (Ilo) (5,44,45). ATP release in response to these various stimuli requires activation of adenylyl cyclase, accumulation of cAMP, and activation of PKA (3,43,47). Although several components of the signaling pathway for ATP release from erythrocytes have been investigated, the identity of the conduit by which ATP exits these cells is not fully characterized. In other cell types, several membrane channels, including connexin hemichannels, voltage-dependent anion channels, volume-regulated anion channels, and ATP-binding cassette proteins, have been implicated as conduits for ATP release (2,27,29,37,40,41). In addition, the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette protein required for ATP release from erythrocytes in response to mechanical deformation (25, 45), was once considered to be a possible ATP conduit in cells. However, more recent studies demonstrate that CFTR is not likely to serve as an ATP conduit itself but rather regulates other channels that serve that role (1,7,19,24,51).Recently, the protein family of pannexins, orthologs of the invert...

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.

Objectives The purpose of this study was to establish that the prostacyclin (PGI2) receptor (IP receptor) is present on rabbit and human erythrocytes and that its activation stimulates cAMP synthesis and ATP release. Methods The effect of incubation of erythrocytes with the active PGI2 analogues, iloprost or UT-15C, on cAMP levels and ATP release was determined in the absence and presence of the IP receptor antagonist, CAY10441. Western analysis was used to determine the presence of the IP receptor on isolated membranes. To establish that effects of PGI2 analogues were not due to prostaglandin E2 (PGE2) receptor activation, the effect of PGE2 on cAMP levels and ATP release was determined. Results Rabbit and human erythrocytes possess IP receptors. Iloprost and UT-15C stimulated increases in cAMP and ATP release that were prevented by the IP receptor antagonist, CAY10441. PGE2 did not stimulate cAMP accumulation or ATP release and did not inhibit iloprost-induced increases in cAMP. Conclusions This study establishes that the IP preceptor is present on rabbit and human erythrocytes and that its activation results in increases in cAMP and ATP release. These results suggest a novel mechanism by which PGI2 and its active analogues, when administered pharmacologically, could produce vasodilation.

Objective ATP released from human erythrocytes in response to reduced oxygen tension (pO2) participates in the matching of oxygen (O2) supply with need in skeletal muscle by stimulating increases in blood flow to areas with increased O2 demand. Here we investigated the hypothesis that hyperinsulinemia inhibits ATP release from erythrocytes and impairs their ability to stimulate dilation of isolated arterioles exposed to decreased extra-luminal pO2. Methods Erythrocyte ATP release was stimulated pharmacologically (mastoparan 7) and physiologically (reduced pO2) in the absence or presence of insulin. We also examined the ability of isolated skeletal muscle arterioles perfused with buffer containing erythrocytes treated with insulin or its vehicle (saline) to dilate in response to decreased extra-luminal pO2. Results Insulin significantly attenuated mastoparan 7– and reduced pO2–induced ATP release. In vessels perfused with untreated erythrocytes, low extra-luminal pO2 resulted in an increase in vessel diameter. In contrast, when erythrocytes were treated with insulin, no vasodilation occurred. Conclusions These studies demonstrate that insulin inhibits ATP release from erythrocytes in response to reduced pO2 and impairs their ability to stimulate dilation of skeletal muscle arterioles. These results suggest that hyperinsulinemia could hinder the matching of O2 supply with need in skeletal muscle.

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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Exposure of erythrocytes to reduced oxygen (O2) tension activates the heterotrimeric G protein Gi, resulting in accumulation of cAMP and release of ATP. The mechanism by which exposure of erythrocytes to reduced O2 tension activates Gi is not known. Here we investigate the hypothesis that, in rabbit erythrocytes, ATP release in response to exposure to reduced O2 tension requires membrane deformation. If this hypothesis is correct, then decreasing the deformability of the erythrocyte membrane should decrease the release of ATP in response to reduced O2 tension. We report that treating erythrocytes with diamide, a compound that decreases erythrocyte deformability, inhibits low O2 tension induced ATP release. Treating erythrocytes with diamide does not, however, interfere with cAMP accumulation or ATP release in response to a direct activator of Gi (mastoparan 7) or in response to receptor mediated activation of Gs (the prostacyclin analog, iloprost). These results demonstrate that diamide (100 μM) does not directly inhibit the signaling pathways for ATP release from rabbit erythrocytes and support the hypothesis that deformability of the erythrocyte membrane is required for low O2 tension-induced ATP release from these cells.

In skeletal muscle, oxygen (O 2 ) delivery to appropriately meet metabolic need requires mechanisms for detection of the magnitude of O 2 demand and the regulation of O 2 delivery. Erythrocytes, when exposed to decreases in O 2 tension, release both O 2 and the vasodilator, adenosine triphosphate (ATP). The aims of this study were to establish that erythrocytes release ATP in response to reduced O 2 tension and determine if erythrocytes are necessary for dilation of isolated skeletal muscle arterioles exposed to reduced extra-luminal O 2 tension. Rabbit erythrocytes exposed to reduced O 2 tension in a tonometer (n = 5, PO 2 = 27 ± 3, p<0.01) released ATP in response to reduced O 2 tension. ATP release increased proportional to the decrease in O 2 tension. The contribution of erythrocytes to the response of skeletal muscle arterioles to reduced extra-luminal O 2 tension was determined using isolated hamster cheek pouch retractor muscle arterioles perfused with buffer (n = 11, mean diameter 52 ± 3 μm) in the absence and presence of rabbit erythrocytes. Without erythrocytes, arterioles did not dilate when exposed to reduced extraluminal O 2 tension (PO 2 = 32 ± 4 mm Hg). In contrast, when rabbit erythrocytes were present in the perfusate (hematocrit 15%) the same decrease in O 2 tension resulted in a 20 ± 4% dilation (p<0.01). These results provide support for the hypothesis that erythrocytes, via their ability to release O 2 along with ATP in response to exposure to reduced O 2 tension, can participate in the matching of O 2 delivery with metabolic need in skeletal muscle.