Alan H. Stephenson

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

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.

Erythrocytes : Oxygen Sensors and Modulators of Vascular Tone

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.

Pannexin 1 is the conduit for low oxygen tension-induced ATP release from human erythrocytes.

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.

Reduced Expression of Gi in Erythrocytes of Humans With Type 2 Diabetes Is Associated With Impairment of Both cAMP Generation and ATP Release

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 Gi and adenylyl cyclase (AC). In this cell, Gi activation results in increases in cAMP and, ultimately, ATP release. It has been reported that Gi expression is decreased in animal models of diabetes and in platelets of humans with type 2 diabetes. Here, we report that Gi2 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 μmol/l), an activator of Gi. Importantly, this defect in ATP release correlates inversely with the adequacy of glycemic control as determined by levels of HbA1c (A1C). These results demonstrate that in erythrocytes of humans with type 2 diabetes, both Gi 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.

Impaired Release of ATP from Red Blood Cells of Humans with Primary Pulmonary Hypertension

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 μm), ATP was not released and the RBCs exhibited reduced deformability. Moreover, when incubated with the active cAMP analogue, Sp-cAMP (100 μM), 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.

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

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.

Rabbit erythrocytes release ATP and dilate skeletal muscle arterioles in the presence of reduced oxygen tension

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 a decrease 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 the dilation of isolated skeletal muscle arterioles exposed to reduced extraluminal 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 in proportion to the decrease in O(2) tension. The contribution of erythrocytes to the response of skeletal muscle arterioles to reduced extraluminal O(2) tension was determined using isolated hamster cheek pouch retractor muscle arterioles perfused with buffer (n = 11, mean diameter 52 +/- 3 mum) 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 mmHg). 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.

Prostacyclin analogs stimulate receptor-mediated cAMP synthesis and ATP release from rabbit and human erythrocytes.

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 cyclic adenosine monophosphate (cAMP) synthesis and adenosine triphosphate (ATP) release.

Defects in oxygen supply to skeletal muscle of prediabetic ZDF rats

In humans, prediabetes is characterized by marked increases in plasma insulin and near normal blood glucose levels as well as microvascular dysfunction of unknown origin. Using the extensor digitorum longus muscle of 7-wk inbred male Zucker diabetic fatty rats fed a high-fat diet as a model of prediabetes, we tested the hypothesis that hyperinsulinemia contributes to impaired O(2) delivery in skeletal muscle. Using in vivo video microscopy, we determined that the total O(2) supply to capillaries in the extensor digitorum longus muscle of prediabetic rats was reduced to 64% of controls with a lower O(2) supply rate per capillary and higher O(2) extraction resulting in a decreased O(2) saturation at the venous end of the capillary network. These findings suggest a lower average tissue Po(2) in prediabetic animals. In addition, we determined that insulin, at concentrations measured in humans and Zucker diabetic fatty rats with prediabetes, inhibited the O(2)-dependent release of ATP from rat red blood cells (RBCs). This inability to release ATP could contribute to the impaired O(2) delivery observed in rats with prediabetes, especially in light of the finding that the endothelium-dependent relaxation of resistance arteries from these animals is not different from controls and is not altered by insulin. Computational modeling confirmed a significant 8.3-mmHg decrease in average tissue Po(2) as well as an increase in the heterogeneity of tissue Po(2), implicating a failure of a regulatory system for O(2) supply. The finding that insulin attenuates the O(2)-dependent release of ATP from RBCs suggests that this defect in RBC physiology could contribute to a failure in the regulation of O(2) supply to meet the demand in skeletal muscle in prediabetes.

Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP

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.