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Erythrocytes release ATP in response to exposure to the physiological stimulus of lowered oxygen ([O.sub.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.sub.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.sub.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.sub.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 doi: 10.1152/ajpheart.00301.2010.

Erythrocytes release both [O.sub.2] and a vasodilator, ATP, when exposed to reduced [O.sub.2] tension. We investigated the hypothesis that ATP release is impaired in erythrocytes of humans with type 2 diabetes (DM2) and that this defect compromises the ability of these cells to stimulate dilation of resistance vessels. We also determined whether a general vasodilator, the prostacyclin analog iloprost (ILO), stimulates ATP release from healthy human (HH) and DM2 erythrocytes. Finally, we used a computational model to compare the effect on tissue [O.sub.2] levels of increases in blood flow directed to areas of increased [O.sub.2] demand (erythrocyte ATP release) with nondirected increases in flow (ILO). HH erythrocytes, but not DM2 cells, released increased amounts of ATP when exposed to reduced [O.sub.2] tension ([Po.sub.2] < 30 mmHg). In addition, isolated hamster skeletal muscle arterioles dilated in response to similar decreases in extraluminal [O.sub.2] when perfused with HH erythrocytes, but not when perfused with DM2 erythrocytes. In contrast, both HH and DM2 erythrocytes released ATP in response to ILO. In the case of DM2 erythrocytes, amounts of ATP released correlated inversely with glycemic control. Modeling revealed that a functional regulatory system that directs blood flow to areas of need (low [O.sub.2]-induced ATP release) provides appropriate levels of tissue oxygenation and that this level of the matching of [O.sub.2] delivery with demand in skeletal muscle cannot be achieved with a general vasodilator. These results suggest that the inability of erythrocytes to release ATP in response to exposure to low-[O.sub.2] tension could contribute to the peripheral vascular disease of DM2. muscle blood flow; red blood cells; oxygen delivery; prostacyclin doi: 10.1152/ajpheart.00430.2010.

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.sub.2] delivery in skeletal muscle. Using in vivo video microscopy, we determined that the total [O.sub.2] supply to capillaries in the extensor digitorum longus muscle of prediabetic rats was reduced to 64% of controls with a lower [O.sub.2] supply rate per capillary and higher [O.sub.2] extraction resulting in a decreased [O.sub.2] saturation at the venous end of the capillary network. These findings suggest a lower average tissue [Po.sub.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.sub.2]-dependent release of ATP from rat red blood cells (RBCs). This inability to release ATP could contribute to the impaired [O.sub.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.sub.2] as well as an increase in the heterogeneity of tissue [Po.sub.2], implicating a failure of a regulatory system for [O.sub.2] supply. The finding that insulin attenuates the [O.sub.2]-dependent release of ATP from RBCs suggests that this defect in RBC physiology could contribute to a failure in the regulation of [O.sub.2] supply to meet the demand in skeletal muscle in prediabetes. adenosine 5'-triphosphate; erythrocyte; oxygen regulation; microvasculature; systems biology; computational modeling doi: 10.1152/ajpheart.01239.2009.

Adderley SP, Sridharan M, Bowles EA, Stephenson AH, Ellsworth ML, Sprague RS. Protein kinases A and C regulate receptormediated increases in cAMP in rabbit erythrocytes. Am J Physiol Heart Circ Physiol 298: H587-H593, 2010. First published December 11, 2009; doi: 10.1152/ajpheart.00975.2009.--Activation of the [beta]-adrenergic receptor ([beta]-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes, cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the [beta]-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs (1). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the [beta]-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 [micro]M), in the presence of the PDE4 inhibitor, rolipram (10 [micro]M), augmented isoproterenol (1 [micro]M)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 [micro]M), pretreatment of erythrocytes with either H89 (1 [micro]M) or two chemically dissimilar inhibitors of PKC, calphostin C (1 [micro]M) or GFX109203X (1 [micro]M), potentiated iloprost (1 [micro]M)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFXI09203X in the presence of cilostazol augmented the rioprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway. erythrocyte; isoproterenol; iloprost; phosphodiesterases doi: 10.1152/ajpheart.00975.2009

Activation of the G protein [G.sub.s] results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (I1o), a PG[I.sub.2] analog, and isoproterenol (Iso), a [beta]-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways. red blood cell; adenosine 3', 5'-cyclic monophosphate; adenosine 5'-trisphophate; rolipram: [beta]-adrenergic receptors

Upon exposure to low P[O.sub.2], the red blood cells of most species, including humans, release increased amounts of ATP that ultimately serves as a regulator of vascular tone matching oxygen supply with demand. In pathological conditions such as malaria and sepsis, a maldistribution of perfusion exists with its severity often correlated with the extent of elevation of serum lactate frequently in the absence of an alteration in pH. We hypothesized that the increased levels of lactate might impair the ability of red blood cells to appropriately respond to conditions of low P[O.sub.2], thus preventing its important blood flow regulatory role. Using an in vitro system and rabbit red blood cells, we evaluated the capacity of cells incubated with lactate to release increased amounts of ATP in response to acute exposure to low P[O.sub.2]. We found that in the presence of lactate, the red blood cells did not release ATP. However, when sodium dichloroacetate, a drug used clinically to lower blood lactate levels, was added, ATP release was restored to levels that were not different from that of control cells (no lactate), even though intracellular levels of ATP were not. These results support the presence of a distinct flow regulatory pool of ATP within the red blood cell that can he independently regulated, and that lactate interferes with the ATP production within this pool, thereby diminishing the amount of ATP available for release on exposure to low P[O.sub.2]. Therefore, if lactate levels can be reduced, the vascular regulatory capacity of the red blood cell should be restored, thus enabling the appropriate matching of oxygen supply with oxygen demand. vascular control; microcirculation; adenosine 5'-triphosphate; erythrocytes; lactate dichloroacetate doi:10.1152/ajpheart.01238.2006.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the release of ATP from red blood cells induced by mechanical deformation was investigated. Pharmacological inhibitors of CFTR activity and red blood cells from cystic fibrosis patients were used to determine whether CFTR is required for ATP release from red blood cells. Results confirmed the hypothesis that red blood cells of healthy humans and chronic obstructive lung disease patients release ATP in response to deformation. In contrast, there is no ATP release from red blood cells of cystic fibrosis patients.

The responses of hamster rectractor muscle arterioles to adenosine 3',5'-triphosphate (ATP) were analyzed by in vivo video microscopy. Vasodilator responses where initiated by the intraluminal administration of ATP in the arterioles of Saran-covered hamster cheek pouch retractor muscles. ATP was also released by circulating red blood cells during the exposure of cremaster muscles to low hydrogen-ion concentrations.

The role of mechanical deformation in the release of adenosine triphosphate (ATP) by red blood cells (RBCs) and the role of RBC-mediated release of ATP in the synthesis of NO were analyzed in human, rabbit and dog RBC. Mechanical deformation induced the release of ATP by human and rabbit RBCs which enhanced the synthesis of endogenous NO in the pulmonary circulation. However, mechanical deformation did not induce the release of ATP in dog RBCs.

Erythrocytes isolated from hamsters and exposed to low oxygen concentration were used in a study to test the hypothesis that erythrocytes also serve as oxygen sensors that communicate changes in tissue oxygen demand to the microvasculature to effect adjustments in microvascular blood flow. The results show that erythrocytes participate in the regulation of blood vessel caliber by releasing ATP at low oxygen concentration. ATP induces vasodilation by binding to the P2y receptor in the vascular endothelium of arterioles and venules.

The relationship of systemic venous PO2 and end-capillary PO2 at three rates of inspired oxygen in the hamster retractor muscle was studied. Measurements of capillary oxygen saturation (SO2), venular PO2 and systemic arterial and venous blood gases were jointly employed with capillary homodynamic measurements to prove that animals had to be made hypoxic to reveal a link between systemic venous and end-capillary PO2.

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 [micro] M), with IBMX (100 [micro] M), resulted in ATP release and increases in intracellular cAMP. In addition, epinephrine (1 [micro] M), 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 [micro] M)] resulted in ATP release. In contrast, forskolin and Sp-cAMP were without effect on dog RBCs, cells known not to re]ease 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 [micro] M), 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. pulmonary circulation; red blood cells; cystic fibrosis transmembrane conductance regulator; nitric oxide

Erythrocyte deformability has been recognized as a determinant of microvascular perfusion. Because nitric oxide (NO) is implicated in the modulation of red blood cell (RBC) deformability and NO levels increase during sepsis, we tested the hypothesis that a NO-mediated decrease in RBC deformability contributes to decreased functional capillary density (CD) in remote organs. With the use of a peritonitis model of sepsis in the rat [cecal ligation and perforation (CLP)] and aminoguanidine (AG) to prevent increases in NO, we measured CD in skeletal muscle (intravital microscopy), mean erythrocyte membrane deformability ([bar][Delta]; micropipette aspiration), systemic NO production [plasma nitrite/nitrate ([NO.sub.x]) chemiluminescence], and NO accumulation in RBC [NO bound to hemoglobin (HbNO) detected by electron paramagnetic resonance spectroscopy]. In untreated CLP animals relative to sham, [NO.sub.x] increased 254% (P [is less than] 0.05), stopped flow capillaries increased 149% (P [is less than] 0.05), and [bar][Delta] decreased 12.7% (P [is less than] 0.05), with a subpopulation (5%) of RBC with deformabilities below the normal range. AG prevented increases in [NO.sub.x], accumulation of HbNO, and decreases in both [bar][Delta] and functional CD. We found no evidence of leukocyte plugging postcapillary venules. Our findings suggest that decreased functional CD during sepsis resulted from a NO-mediated decrease in erythrocyte deformability. microcirculation; abnormalities; septicemia; functional capillary density

The release of ATP from red blood cells (RBC) in response to low [O.sub.2] levels is linked to ATP production and the oxygenation state of hemoglobin. Because [O.sub.2] is unloaded from the RBC, the concentration of deoxygenated hemoglobin increases, displacing phosphofructokinase from the cytoplasmic domain of band 3. We hypothesize that the ATP molecules produced through this glycolytic stimulation at the membrane surface result in the release of ATP from the RBC. Rat whole blood exposed to 5 min of low [Po.sub.2] in vitro increased plasma [ATP] by 1.0 [micro]M (+45%). This increase was reduced to 0.1 [micro]M (+ 12%, P [is less than] 0.05) after citrate incubation and reversed after fluoride treatment (both glycolytic inhibitors) by -0.2 [micro]M (-23%, P [is less than] 0.05). Plasma [ATP] of control RBC decreased -0.3 [micro]M (-12%) when 8% CO (P [is less than] 0.05) was added to the chamber. Because CO and [O.sub.2] bind competitively to heme, these results support our hypothesis that the release of ATP from RBC is linked to ATP production through the oxygenation state of the hemoglobin molecule. adenosine triphosphate; [O.sub.2] regulation; glycolysis; microcirculation; membrane transport

Dietrich, Hans H., Mary L. Ellsworth, Randy S. Sprague, and Ralph G. Dacey, Jr. Red blood cell regulation of microvascular tone through adenosine triphosphate. Am J Physiol Heart Circ Physiol 278: H1294-1298, 2000.--The matching of blood flow with metabolic need requires a mechanism for sensing the needs of the tissue and communicating that need to the arterioles, the ultimate controllers of tissue perfusion. Despite significant strides in our understanding of blood flow regulation, the identity of the [O.sub.2] sensor has remained elusive. Recently, the red blood cell, the Hb-containing [O.sub.2] carrier, has been implicated as a potential [O.sub.2] sensor and contributor to this vascular control by virtue of its concomitant carriage of millimolar amounts of ATP, which it is able to release when exposed to a low-[O.sub.2] environment. To evaluate this possibility, we exposed perfused cerebral arterioles to low extraluminal [O.sub.2] in the absence and presence of red blood cells or 6% dextran and determined both vessel diameter and ATP in the vessel effluent. Only when the vessels were perfused with red blood cells did the vessels dilate in response to low extraluminal [O.sub.2]. In addition, this response was accompanied by a significant increase in vessel effluent ATP. These findings support the hypothesis that the red blood cell itself serves a role in determining [O.sub.2] supply to tissue. rat; cerebral arterioles; microvascular regulatory mechanism; oxygen tension