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.

Recently, we reported that rabbit red blood cells (RBCs) were required for the expression of nitric oxide (NO) activity on pulmonary vascular resistance (PVR) in rabbit lungs. Here, we investigate the hypothesis that RBCs participate in the regulation of PVR via release of ATP in response to mechanical deformation that, in turn, evokes vascular NO synthesis. We found that rabbit and human RBCs, but not dog RBCs, release ATP in response to mechanical deformation. To determine the contribution of this ATP to NO synthesis and PVR, we compared the effects of human and dog RBCs on pressure-flow relationships in isolated rabbit lungs. In the presence of human RBCs, NG-nitro-L-arginine methyl ester (100 microM) produced a shift in the pressure-flow relationship consistent with a reduction in vascular caliber. NG-nitro-L-arginine methyl ester had no effect in lungs perfused with dog RBCs. These results suggest a unique mechanism for the control of PVR in rabbits and humans whereby release of ATP by RBCs in response to mechanical deformation leads to stimulation of NO synthesis that, in turn, modulates the PVR.

Through oxygen-dependent release of the vasodilator ATP, the mobile erythrocyte plays a fundamental role in matching microvascular oxygen supply with local tissue oxygen demand. Signal transduction within the erythrocyte and microvessels as well as feedback mechanisms controlling ATP release have been described. Our understanding of the impact of this novel control mechanism will rely on the integration of in vivo experiments and computational models.1548-9213/09 8.00

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...

Previously, we reported that red blood cells (RBCs) of rabbits and humans release ATP in response to mechanical deformation and that this release of ATP requires the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). It was reported that cAMP, acting through a cAMP-dependent protein kinase, PKA, is an activator of CFTR. Here we investigate the hypothesis that cAMP stimulates ATP release from RBCs. Incubation of human and rabbit RBCs with the direct activator of adenylyl cyclase, forskolin (10 or 100 microM), with IBMX (100 microM), resulted in ATP release and increases in intracellular cAMP. In addition, epinephrine (1 microM), a receptor-mediated activator of adenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover, incubation of human and rabbit RBCs with an active cAMP analog [adenosine 3'5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 microM)] resulted in ATP release. In contrast, forskolin and Sp-cAMP were without effect on dog RBCs, cells known not to release ATP in response to deformation. When rabbit RBCs were incubated with the inactive cAMP analog and inhibitor of PKA activity, adenosine 3',5'-cyclic monophosphorothioate Rp-isomer (100 microM), deformation-induced ATP release was attenuated. These results are consistent with the hypothesis that adenylyl cyclase and cAMP are components of a signal-transduction pathway relating RBC deformation to ATP release from human and rabbit RBCs.

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein G(s) resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the G(i) subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins G(alphai1), G(alphai2), and G(alphai3) but not G(alphao) were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples G(i/o) from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 microM) or Mas-7 (1 microM), which are compounds that directly activate G(i) proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 microM), which is an inactive Mas analog. In separate experiments, Mas (10 microM) but not Mas-17 (10 microM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein G(i) is a component of a signal transduction pathway for ATP release from erythrocytes.

Human erythrocytes, by virtue of their ability to release ATP in response to physiological stimuli, have been proposed to participate in the regulation of local blood flow. A signal transduction pathway that relates these stimuli to ATP release has been described and includes the heterotrimeric G protein G i and adenylyl cyclase (AC). In this cell, G i activation results in increases in cAMP and, ultimately, ATP release. It has been reported that G i expression is decreased in animal models of diabetes and in platelets of humans with type 2 diabetes. Here, we report that G i2 expression is selectively decreased in erythrocytes of humans with type 2 diabetes and that this defect is associated with reductions in cAMP accumulation and ATP release in response to incubation of erythrocytes with mastoparan 7 (10 mol/l), an activator of G i . Importantly, this defect in ATP release correlates inversely with the adequacy of glycemic control as determined by levels of HbA 1c (A1C). These results demonstrate that in erythrocytes of humans with type 2 diabetes, both G i expression and ATP release in response to mastoparan 7 are impaired, which is consistent with the hypothesis that this defect in erythrocyte physiology could contribute to the vascular disease associated with this clinical condition. Diabetes 55: 3588 -3593, 2006

Previously, we reported that In the Isolated perfused rabbit lung, red blood cells (RBCs) obtained from either rabbits or healthy humans were a required component of the perfusate to unmask evidence of nitric oxide (NO) participation in regulation of the pulmonary circulation. In addition, we found that mechanical deformation of rabbit and healthy human RBCs released ATP, a known agonist for enhanced NO synthesis. In contrast, RBCs obtained from patients with cystic fibrosis (CF) did not release ATP In response to mechanical deformation. The coexistence of airway disease and alveolar hypoxia In patients with CF precluded the drawing of conclusions relating a defect In RBC ATP release with the pulmonary hypertension associated with CF. Airway disease and alveolar hypoxia are not, however, features of primary pulmonary hypertension (PPH), a human condition of unknown etiology. We postulated that a defect In NO generation might contribute to the Increased pulmonary vascular resistance In PPH, and as a first step, we hypothesized that RBCs obtained from patients with PPH would not release ATP. In contrast to RBCs of healthy humans, when RBCs of PPH patients were passed through filters (average pore size 12, 8, or 5 101m), ATP was not released and the RBCs exhibited reduced deformabllity. Moreover, when incubated with the active cAMP analogue, Sp-cAMP (100 101M), an activator of the CF transmembrane conductance regulator, ATP was not released. These results demonstrate that RBCs obtained from patients with PPH fail to release ATP whether the stimulus Is mechanical or pharmacological. Thus, failure of RBCs to release ATP in patients with PPH might be a major pathogenetic factor that accounts for the heretofore unknown etiology of their pUlmonary hypertension.