Low oxygen (O(2)) tension and mechanical deformation are stimuli for ATP release from erythrocytes. It has been shown previously that rabbit erythrocytes made less deformable with diamide, a thiol cross-linking agent, release less ATP in response to low O(2) tension, suggesting a link between these two stimuli. In nonerythroid cells, activation of the Rho/Rho kinase signaling pathway has been reported to decrease cell deformability by altering Rho kinase-dependent cytoskeleton-protein interactions. We investigated the hypothesis that the Rho kinase inhibitor Y-27632 would increase erythrocyte deformability and thereby increase low O(2) tension-induced ATP release from erythrocytes. Here we show that Y-27632 (1 μM) increases erythrocyte deformability (5%) and increases low O(2) tension-induced ATP release (203%) from healthy human erythrocytes. In addition, we found that, when erythrocytes were made less deformable by incubation with diamide (100 μM), Y-27632 restored both deformability and low O(2) tension-induced ATP release to levels similar to those measured in the absence of diamide. These findings suggest that the Rho kinase inhibitor Y-27632 is able to reverse the diamide-induced decrease in erythrocyte deformability and rescue low O(2) tension-induced ATP release. These results further support a link between erythrocyte deformability and ATP release in response to low O(2) tension.

SUMMARY Background Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP. Methods/Materials Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP). Results Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP. Conclusion PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.

Mechanical deformation and low oxygen (O2) tension were reported to stimulate ATP release from RBCs via a signaling pathway that includes the G protein Gi. Stiffening of the RBC with diamide, which promotes cross linking of cytoskeletal proteins, resulted in decreased low O2 tension‐induced ATP release. In the RBC, spectrin molecules must fold for deformation to occur. Phosphorylation of adducin and moesin by Rho kinase may stabilize cytoskeletal‐membrane interactions. Rho kinase signaling has been linked to increased stiffness of fibroblasts, ventricular myocytes, airway smooth muscle cells and endothelial cells. We hypothesized that Rho kinase inhibits low O2 tension‐induced ATP release from RBCs by stiffening the membrane cytoskeleton. We examined the effect of the Rho kinase inhibitor, Y27632 (1 uM), on low O2 tension‐induced ATP release from RBCs. In human RBCs, ATP release was increased in the presence of Y27632 (p<0.01, n=13). To support the hypothesis that ATP release is not the result of a direct effect of Y27632 to inhibit the activity of Gi, RBCs were incubated with Mastoporan 7, a direct Gi activator. Y27632 had no effect on Mastoporan 7‐ induced ATP release (n=3). These results are consistent with the hypothesis that Rho kinase inhibition increases RBC deformability resulting in augmented ATP release in response to exposure of these cells to low O2 tension. (NIH grant HL089094)

Expression of P2X7 purinergic receptors on red blood cell (RBC) membranes has been reported. ATP, released from RBCs has been proposed to stimulate the P2X7 receptor in an autocrine fashion to induce the production and release of epoxyeicosatrienoic acids (EETs). However, RBCs from humans with type 2 diabetes (DM2) have been reported to release less ATP than healthy humans in response to physiological stimuli. To examine whether DM2 also alters RBC expression of the P2X7 receptor we examined its expression in humans with DM2 and healthy humans. We also compared RBC P2X7 expression from an animal model of DM2, the Zucker Diabetic Fatty rat (ZDF) to its control, the lean ZDF rat. Blood was obtained from healthy humans, humans with type 2 diabetes and from 12 and 18 week control and DM2 ZDF rats. RBCs were separated and membranes were isolated. Western immunoblotting was conducted on the membranes and P2X7 expression was measured by densitometry using beta‐actin as a loading control. Expression of P2X7 was 3 fold higher in RBC membranes from humans with DM2 than in healthy humans (p<0.0002). RBC membranes of fatty ZDF rats at 12 and 18 weeks did not express greater P2X7 than lean ZDF rats. These results suggest that the greater P2X7 expression in humans with DM2 may act as a compensatory mechanism in the presence of reduced ATP release and that the ZDF rat may not accurately model this aspect of human DM2.