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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

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. adenylyl cyclase; cilostazol; milrinone; red blood cell; cyclic adenosine 3',5'-monophosphate

Erythrocytes (red blood cells) of either rabbits or healthy humans are required to demonstrate the participation of nitric oxide (NO) in the regulation of pulmonary vascular resistance in the isolated rabbit lung. The property of the erythrocyte that is responsible for the stimulation of NO synthesis was reported to be the ability to release ATP in response to physiological stimuli, including deformation. Moreover, a signal transduction pathway that relates mechanical deformation of erythrocytes to ATP release has been described, and the cystic fibrosis (CF) trans-membrane conductance regulator (CFTR) is a component, i.e., erythrocytes of individuals with CF do not release ATP in response to deformation. Here, we investigated the hypothesis that, in contrast to those of healthy humans, erythrocytes of humans with CF fail to stimulate endogenous NO synthesis in the isolated rabbit lung. We report that CFTR is a component of the membranes of both rabbit and human erythrocytes. The addition of the NO synthase inhibitor [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME, 100 [micro]M) produced increases in vascular resistance in isolated rabbit lungs perfused with physiological salt solution (PSS) containing erythrocytes of healthy humans, but L-NAME was without effect when the lungs were perfused with PSS alone or PSS containing erythrocytes of CF patients. These results provide support for the hypothesis that, in CF, a defect in ATP release from erythrocytes could lead to decreased endogenous pulmonary NO synthesis and contribute to pulmonary hypertension. adenosine triphosphate: red blood cells: pulmonary circulation

The release of ATP from erythrocytes involves a signal transduction pathway of which cystic fibrosis transmembrane conductance regulator, PKA, adenylyl cyclase, and the heterotrimeric G proteins [G.sub.s] and [G.sub.i] are components. In the pulmonary circulation, ATP released from the erythrocyte stimulates nitric oxide (NO) synthesis, thereby regulating vascular resistance. We reported that NO liberated from an NO donor inhibited ATP release from erythrocytes in response to decreased P[O.sub.2] or mechanical deformation. Here, we investigated the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of [G.sub.i]. Washed rabbit erythrocytes were incubated in the presence or absence of the NO donor N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate; 100 nM, 20 min), followed by treatment with agents that activate specific components of the signal transduction pathway promoting ATP release. Neither ATP release nor cAMP accumulation induced by either forskolin (100 [micro]M, n = 7) or iloprost (100 nM, n = 6) was inhibited by spermine NONOate. These experiments suggest that the inhibitory action of NO is not the result of inactivation of adenylyl cyclase or [G.sub.s], respectively. However, spermine NONOate completely inhibited ATP release in response to mastoparan (10 [micro]m, P < 0.05, n = 5), a specific activator of [G.sub.i]. Spermine (100 nM, 20 min), the polyamine remaining after liberation of NO from spermine NONOate, had no affect on mastoparan-induced ATP release (n = 4). These results support the hypothesis that NO inhibits ATP release from erythrocytes via inactivation of the heterotrimeric G protein [G.sub.i]. red blood cell; vascular control; adenine nucleotides; pulmonary circulation

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.sub.s] resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the [G.sub.i] subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], but not were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples [G.sub.i/o] from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 [micro]M) or Mas-7 (1 [micro]M), which are compounds that directly activate [G.sub.i] proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 [micro]M), which is an inactive Mas analog. In separate experiments, Mas (10 [micro]M) but not Mas-17 (10 [micro]M) 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.sub.i] is a component of a signal transduction pathway for ATP release from erythrocytes. pulmonary circulation; red blood cells; mechanical deformation; adenine nucleotides

Previously, it was reported that red blood cells (RBCs) are required to demonstrate participation of nitric oxide (NO) in the regulation of rabbit pulmonary vascular resistance (PVR). RBCs do not synthesize NO; hence, we postulated that ATP, present in millimolar amounts in RBCs, was the mediator, which evoked NO synthesis in the vascular endothelium. First, we found that deformation of RBCs, as occurs on passage across the pulmonary circulation with increasing flow rate, evoked increments in ATP release. Here, ATP (300 nM), administered to isolated, salt solution-perfused (PSS) rabbit lungs, decreased total and upstream (arterial) PVR, a response inhibited by [N.sup.G]-nitro-L-arginine methyl ester (L-NAME, 100 [micro]M). In lungs perfused with PSS containing RBCs, L-NAME increased total and upstream PVR. In lungs perfused with PSS containing glibenclamide-treated RBCs, which inhibits ATP release, L-NAME was without effect. Apyrase grade VII (8 U/ml), which degrades ATP to AMP, was without effect on PVR in PSS-perfused lungs. These results are consistent with the hypothesis that ATP, released from RBCs as they traverse the pulmonary circulation, evokes endogenous NO synthesis. adenosine-5' triphosphate; red blood cell

In the rabbit, 5,6-epoxyeicosatrienoic acid (EET) was reported both to dilate and to constrict pulmonary blood vessels. We propose that these seemingly contradictory results could be explained by differences in responses to 5,6-EET in large-conductance pulmonary arteries (PA) compared with smaller PA and resistance vessels. Thus we found that in rings of extralobar PA [>2-mm outside diameter (OD)], in which active tension had been increased with [PGF.sub.2[alpha]], 5,6-EET produced relaxation in a concentration- and cyclooxygenase (COX)-dependent manner. In contrast, 5,6-EET increased tension in intralobar (1- to 2-mm OD) PA. Small extralobar PA (2- to 2.5-mm OD) exhibited intermediate responses. In the intact lung, the net effect of 5,6-EET (1 x [10.sup.-8]-1 x [10.sup.-5] M) was an increase in pulmonary vascular resistance (PVR) from 13.0 [+ or -] 0.5 to 47.8 [+ or -] 4.6 mmHg*100 [ml.sup.-1]*[min.sup.-1] ([EC.sub.50] 5.9 [+ or -] 1.7 x [10.sup.-7] M). The increase in PVR was accompanied by a 10-fold increase in perfusate thromboxane (TX)[B.sub.2] concentration. The 5,6-EET-induced increase in PVR was prevented with indomethacin (100 [micro]M), a cyclooxygenase inhibitor, or ONO-3708 (20 [micro]M), a TX/[PGH.sub.2] (TP) receptor antagonist, but not with OKY-046 (700 [micro]M), a TX synthase inhibitor. These results demonstrate that although 5,6-EET dilates large extralobar PA segments in a COX-dependent manner, in the intact rabbit lung 5,6-EET produces constriction that requires synthesis of a COX-dependent agonist of the TP receptor other than TX. cytochrome P-450; pulmonary vascular resistance; arachidonic acid; prostaglandin; endoperoxide; 5,6-epoxy-eicosa-trienoic acid

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.

Isolated, perfused canine lungs were used in the investigation of the effect of 5,6-epoxyeicosatrienoic acid (5,6-EET) on the pulmonary vasculature. Different treatment protocols were designed to test the activity of 5,6-EET on the different segments of the pulmonary vasculature as well as the effects of various vasoconstrictors on 5,6-EET-induced relaxation. Results suggest that the vasodilator activity of 5,6-EET is dependent on the vasoconstrictor agent, segmental resistance and the activity of cyclooxigenase.

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.

Cytochrome P450 contributes to lung damage caused by ethchlorvynol by using arachidonic acid for the production of metabolites. Ethchlorvynol was used to induce acute lung injury in the dog, which led to an increase in the mixture of venous blood and oxygen. Cytochrome P-450 promoted venous admixture by producing 5,6-epoxyeicosatrienoic acid which is a vasodilator. Its relaxing effect on lung vessels may lead to hypoxemia.

Isolated blood-perfused rabbit lungs were used in a study to determine the role of nitric oxide (NO) in modulating pulmonary vascular resistance. The results show that NO lowers pulmonary vascular resistance only in the presence of blood. Furthermore, it was discovered that a component of red blood cells, rather than perfusate viscosity, is involved in mediating the effects of NO on pulmonary vascular resistance.

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

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