Mechanisms of C-peptide-mediated rescue of low O2-induced ATP release from erythrocytes of humans with type 2 diabetes.

The circulating erythrocyte, by virtue of the regulated release of ATP in response to reduced oxygen (O2) tension, plays a key role in maintaining appropriate perfusion distribution to meet tissue needs. Erythrocytes from individuals with Type 2 diabetes (DM2) fail to release ATP in response to this stimulus. However, the administration of C-peptide and insulin at a 1:1 ratio was shown to restore this important physiological response in humans with DM2. To begin to investigate the mechanisms by which C-peptide influences low O2-induced ATP release, erythrocytes from healthy humans and humans with DM2 were exposed to reduced O2 in a thin-film tonometer, and ATP release under these conditions was compared with release during normoxia. We determined that 1) low O2-induced ATP release from DM2 erythrocytes is rescued by C-peptide in the presence and absence of insulin, 2) the signaling pathway activated by C-peptide in human erythrocytes involves PKC, as well as soluble guanylyl cyclase (sGC) and 3) inhibitors of cGMP degradation rescue low O2-induced ATP release from DM2 erythrocytes. These results provide support for the hypothesis that both PKC and sGC are components of a signaling pathway activated by C-peptide in human erythrocytes. In addition, since both C-peptide and phosphodiesterase 5 inhibitors rescue low O2-induced ATP release from erythrocytes of humans with DM2, their administration to humans with DM2 could aid in the treatment and/or prevention of the vascular disease associated with this condition.

Low O2-induced ATP release from erythrocytes of humans with type 2 diabetes is restored by physiological ratios of C-peptide and insulin.

ATP release from erythrocytes in response to reduced oxygen (O2) tension stimulates local vasodilation, enabling these cells to direct perfusion to areas in skeletal muscle in need of O2. Erythrocytes of humans with type 2 diabetes do not release ATP in response to low O2. Both C-peptide and insulin individually inhibit low O2-induced ATP release from healthy human erythrocytes, yet when coadministered at physiological concentrations and ratios, no inhibition is seen. Here, we determined: that 1) erythrocytes of healthy humans and humans with type 2 diabetes possess a C-peptide receptor (GPR146), 2) the combination of C-peptide and insulin at physiological ratios rescues low O2-induced ATP release from erythrocytes of humans with type 2 diabetes, 3) residual C-peptide levels reported in humans with type 2 diabetes are not adequate to rescue low O2-induced ATP release in the presence of 1 nM insulin, and 4) the effects of C-peptide and insulin are neither altered by increased glucose levels nor explained by changes in erythrocyte deformability. These results suggest that the addition of C-peptide to the treatment regimen for type 2 diabetes could have beneficial effects on tissue oxygenation, which would help to ameliorate the concomitant peripheral vascular disease.

Synergistic effects of C-peptide and insulin on low O2-induced ATP release from human erythrocytes.

Erythrocytes participate in the matching of oxygen (O2) delivery with local need in skeletal muscle via the release of O2 and the vasodilator, ATP. It was reported that a concentration of insulin found in humans with insulin resistance inhibits low O2-induced ATP release. However, in vivo, insulin is coreleased with connecting peptide (C-peptide) at equimolar concentrations, but because of the shorter insulin half-life, the peptides circulate at ratios of C-peptide to insulin ranging from 1:1 to 6:1. Here, we investigate the hypothesis that C-peptide and insulin work synergistically to maintain low O2-induced ATP release from human erythrocytes. Using a thin-film tonometer to alter O2 tension, we determined that either C-peptide or insulin alone inhibits low O2-induced ATP release in a concentration-dependent manner; however, coadministration of the peptides at a 1:1 ratio does not (n = 5; P < 0.05). Because this ratio of C-peptide to insulin is not present in vivo for extended periods, we also investigated the effect of additional physiological ratios on ATP release. In the presence of insulin concentrations that would be found in fasting humans (0.05 nM), C-peptide to insulin ratios of 4:1 and 6:1 did not adversely affect low O2-induced ATP release. However, at a concentration of insulin found in the peripheral circulation of humans under postprandial conditions (0.5 nM), a ratio of C-peptide to insulin of 6:1 inhibited low O2-induced ATP release (n = 5). These findings demonstrate a heretofore unrecognized synergism between C-peptide and insulin that could have physiological importance in the regulation of perfusion distribution in skeletal muscle.

Regulation of blood flow distribution in skeletal muscle: role of erythrocyte-released ATP.

The maintenance of adequate tissue O(2) levels in skeletal muscle is vital for normal physiology and requires a well regulated and appropriately distributed convective O(2) supply. Inherent in this fundamental physiological process is the requirement for a mechanism which both senses tissue O(2) need and locally adjusts flow to appropriately meet that need. Over the past several years we and others have suggested that, in skeletal muscle, O(2) carrying erythrocytes participate in the regulation of total blood flow and its distribution by releasing ATP. Importantly, the release of this vasoactive molecule must be both rapid and well controlled if it is to serve an important physiological role. Here we provide insights into three distinct regulated signalling pathways within the erythrocyte that are activated by exposure to reduced O(2) tension or in response to binding of agonists to the prostacyclin or ß-adrenergic receptors. Although much has been learned about the role of the erythrocyte in perfusion of skeletal muscle, much remains to be understood. However, what is clear is that the long established passive carrier of O(2) also contributes to the regulation of the distribution of microvascular perfusion in skeletal muscle by virtue of its capacity to release ATP.

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 ß-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.

Erythrocyte-derived ATP and perfusion distribution: role of intracellular and intercellular communication.

In complex organisms, both intracellular and intercellular communication are critical for the appropriate regulation of the distribution of perfusion to assure optimal O(2) delivery and organ function. The mobile erythrocyte is in a unique position in the circulation as it both senses and responds to a reduction in O(2) tension in its environment. When erythrocytes enter a region of the microcirculation in which O(2) tension is reduced, they release both O(2) and the vasodilator, ATP, via activation of a specific and dedicated signaling pathway that requires increases in cAMP, which are regulated by PDE3B. The ATP released initiates a conducted vasodilation that results in alterations in the distribution of perfusion to meet the tissue's metabolic needs. This delivery mechanism is modulated by both positive and negative feedback regulators. Importantly, defects in low O(2) -induced ATP release from erythrocytes have been observed in several human disease states in which impaired vascular function is present. Understanding of the role of erythrocytes in controlling perfusion distribution and the signaling pathways that are responsible for ATP release from these cells makes the erythrocyte a novel therapeutic target for the development of new approaches for the treatment of vascular dysfunction.

A selective phosphodiesterase 3 inhibitor rescues low PO2-induced ATP release from erythrocytes of humans with type 2 diabetes: implication for vascular control.

Erythrocytes, via release of ATP in areas of low oxygen (O(2)) tension, are components of a regulatory system for the distribution of perfusion in skeletal muscle ensuring optimal O(2) delivery to meet tissue needs. In type 2 diabetes (DM2), there are defects in O(2) supply to muscle as well as a failure of erythrocytes to release ATP. The goal of this study was to ascertain if a phosphodiesterase 3 (PDE3) inhibitor, cilostazol, would rescue low O(2)-induced ATP release from DM2 erythrocytes and, thereby, enable these cells to dilate isolated erythrocyte-perfused skeletal muscle arterioles exposed to decreased extraluminal O(2). Erythrocytes were obtained from healthy humans (HH; n = 12) and humans with DM2 (n = 17). We determined that 1) PDE3B is similarly expressed in both groups, 2) mastoparan 7 (G(i) activation) stimulates increases in cAMP in HH but not in DM2 erythrocytes, and 3) pretreatment of DM2 erythrocytes with cilostazol resulted in mastoparan 7-induced increases in cAMP not different from those in HH cells. Most importantly, cilostazol restored the ability of DM2 erythrocytes to release ATP in response to low O(2). In contrast with perfusion with HH erythrocytes, isolated hamster retractor muscle arterioles perfused with DM2 erythrocytes constricted in response to low extraluminal PO(2). However, in the presence of cilostazol (100 µM), DM2 erythrocytes induced vessel dilation not different from that seen with HH erythrocytes. Thus rescue of low O(2)-induced ATP release from DM2 erythrocytes by cilostazol restored the ability of erythrocytes to participate in the regulation of perfusion distribution in skeletal muscle.

Inhibition of ATP release from erythrocytes: a role for EPACs and PKC.

OBJECTIVE: Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). METHODS: ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). RESULTS: Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. CONCLUSION: These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes.

Identification of cytosolic phosphodiesterases in the erythrocyte: a possible role for PDE5.

BACKGROUND: Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of ß-adrenergic receptors (ßARs) 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. MATERIAL/METHODS: 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 ßAR 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 ßAR-induced increases in cAMP. CONCLUSIONS: 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.

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.

Divergent effects of low-O(2) tension and iloprost on ATP release from erythrocytes of humans with type 2 diabetes: implications for O(2) supply to skeletal muscle.

Erythrocytes release both O(2) and a vasodilator, ATP, when exposed to reduced O(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(2) levels of increases in blood flow directed to areas of increased O(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(2) tension (Po(2) < 30 mmHg). In addition, isolated hamster skeletal muscle arterioles dilated in response to similar decreases in extraluminal O(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(2)-induced ATP release) provides appropriate levels of tissue oxygenation and that this level of the matching of O(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(2) tension could contribute to the peripheral vascular disease of DM2.

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.

Protein kinases A and C regulate receptor-mediated increases in cAMP in rabbit erythrocytes.

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. 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 microM), in the presence of the PDE4 inhibitor, rolipram (10 microM), augmented isoproterenol (1 microM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 microM), pretreatment of erythrocytes with either H89 (1 microM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 microM) or GFX109203X (1 microM), potentiated iloprost (1 microM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-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.

Vascular disease in pre-diabetes: new insights derived from systems biology.

In many cases vascular disease is present before the clinical onset of type 2 diabetes, that is, during the pre-diabetic period when insulin levels are markedly increased. In pre-diabetes, microvascular dysfunction correlates with plasma insulin levels and not blood glucose. Here we discuss the concept that insulin, at levels found in pre-diabetes, contributes to microvascular disease in skeletal muscle by inhibiting the release of the vasodilator, adenosine triphosphate (ATP), from erythrocytes.

Insulin inhibits low oxygen-induced ATP release from human erythrocytes: implication for vascular control.

OBJECTIVE: ATP released from human erythrocytes in response to reduced oxygen tension (pO(2)) participates in the matching of oxygen (O(2)) supply with need in skeletal muscle by stimulating increases in blood flow to areas with increased O(2) demand. Here, we investigated the hypothesis that hyperinsulinemia inhibits ATP release from erythrocytes and impairs their ability to stimulate dilation of isolated arterioles exposed to decreased extraluminal pO(2). MATERIALS AND METHODS: Erythrocyte ATP release was stimulated pharmacologically (mastoparan 7) and physiologically (reduced pO(2)) in the absence or presence of insulin. We also examined the ability of isolated skeletal muscle arterioles perfused with buffer containing erythrocytes treated with insulin or its vehicle (saline) to dilate in response to decreased extraluminal pO(2). RESULTS: Insulin significantly attenuated mastoparan 7- and reduced pO(2)-induced ATP release. In vessels perfused with untreated erythrocytes, low extraluminal pO(2) resulted in an increase in vessel diameter. In contrast, when erythrocytes were treated with insulin, no vasodilation occurred. CONCLUSIONS: These studies demonstrate that insulin inhibits ATP release from erythrocytes in response to reduced pO(2) and impairs their ability to stimulate dilation of skeletal muscle arterioles. These results suggest that hyperinsulinemia could hinder the matching of O(2) supply with need in skeletal muscle.

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 (PGI(2)) 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. METHODS: The effect of incubation of erythrocytes with the active PGI(2) analogs, iloprost or UT-15C, on cAMP levels and ATP release was determined in the absence and presence of the IP receptor antagonist, CAY10441. Western analysis was used to determine the presence of the IP receptor on isolated membranes. To establish that effects of PGI(2) analogs were not due to prostaglandin E(2)(PGE(2)) receptor activation, the effect of PGE(2) on cAMP levels and ATP release was determined. RESULTS: Rabbit and human erythrocytes possess IP receptors. Iloprost and UT-15C stimulated increases in cAMP and ATP release that were prevented by the IP receptor antagonist, CAY10441. PGE(2) did not stimulate cAMP accumulation or ATP release and did not inhibit iloprost-induced increases in cAMP. CONCLUSIONS: This study establishes that the IP receptor is present on rabbit and human erythrocytes and that its activation results in increases in cAMP and ATP release. These results suggest a novel mechanism by which PGI(2) and its active analogs, when administered pharmacologically, could produce vasodilation.

Red not dead: signaling in and from erythrocytes.

The oxygen required to meet metabolic needs of all tissues is delivered by the erythrocyte, a small, flexible cell which, in mammals, is devoid of a nucleus and mitochondria. Despite its simple appearance, this 'bag of hemoglobin' has an important role in its own distribution, enabling the delivery of oxygen to precisely meet localized metabolic need. When an erythrocyte enters an area in which tissue oxygen demand exceeds supply, a signaling pathway is activated resulting in the release of adenosine 5'-triphosphate (ATP). This ATP acts in a paracrine fashion to increase vascular caliber resulting in increased oxygen delivery. Defects in this pathway are found in erythrocytes of humans with type 2 diabetes (DM2) and could contribute to the perfusion abnormalities in skeletal muscle associated with this disease.

Phosphodiesterase 5 inhibitors augment UT-15C-stimulated ATP release from erythrocytes of humans with pulmonary arterial hypertension.

Both prostacyclin analogs and phosphodiesterase 5 (PDE5) inhibitors are effective treatments for pulmonary arterial hypertension (PAH). In addition to direct effects on vascular smooth muscle, prostacyclin analogs increase cAMP levels and ATP release from healthy human erythrocytes. We hypothesized that UT-15C, an orally available form of the prostacyclin analog, treprostinil, would stimulate ATP release from erythrocytes of humans with PAH and that this release would be augmented by PDE5 inhibitors. Erythrocytes were isolated and the effect of UT-15C on cAMP levels and ATP release were measured in the presence and absence of the PDE5 inhibitors, zaprinast or tadalafil. In addition, the ability of a soluble guanylyl cyclase inhibitor to prevent the effects of tadalafil was determined. Erythrocytes of healthy humans and humans with PAH respond to UT-15C with increases in cAMP levels and ATP release. In both groups, UT-15C-induced ATP release was potentiated by zaprinast and tadalafil. The effect of tadalafil was prevented by pre-treatment with an inhibitor of soluble guanylyl cyclase in healthy human erythrocytes. Importantly, UT-15C-induced ATP release was greater in PAH erythrocytes than in healthy human erythrocytes in both the presence and the absence of PDE5 inhibitors. The finding that prostacyclin analogs and PDE5 inhibitors work synergistically to enhance release of the potent vasodilator ATP from PAH erythrocytes provides a new rationale for the co-administration of these drugs in this disease. Moreover, these results suggest that the erythrocyte is a novel target for future drug development for the treatment of PAH.

Liposomal delivery of a phosphodiesterase 3 inhibitor rescues low oxygen-induced ATP release from erythrocytes of humans with type 2 diabetes.

ATP release from erythrocytes in response to low oxygen tension requires an increase in cAMP, the level of which is regulated by phosphodiesterase 3 (PDE3). Such release is defective in erythrocytes of humans with type 2 diabetes (DM2). This study tested a hypothesis that direct delivery of the clinically useful PDE3 inhibitor, cilostazol, to erythrocytes of humans with type 2 diabetes using liposomes would restore low-oxygen tension-induced ATP release. Cilostazol was incorporated into liposomes prepared from dimyristoylphosphatidylcholine (DMPC). Liposome-delivery of cilostazol restored ATP release from DM2 erythrocytes to levels which were not different from that released from non-cilostazol treated healthy erythrocytes under the same conditions. There were no observed adverse effects of the liposomes on either healthy or DM2 erythrocytes. The directed liposomal delivery of PDE inhibitors to erythrocytes may help prevent or slow the development of peripheral vascular disease in individuals with DM2 by restoring an important physiological controller of microvascular perfusion while minimizing side effects associated with systemic delivery of some of these inhibitors.