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.

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.

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.

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.

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.

Synergistic effects of prostacyclin analogs and phosphodiesterase inhibitors on cyclic adenosine 3',5' monophosphate accumulation and adenosine 3'5' triphosphate release from human erythrocytes.

Prostacyclin (PGI2) and phosphodiesterase 5 (PDE5) inhibitors are potent vasodilators that are used alone and in combination for the treatment of pulmonary arterial hypertension (PAH). Although these vasodilators are known to stimulate relaxation of vascular smooth muscle directly, other cells in circulation, including erythrocytes, express prostacyclin receptor (IPR) and contain PDE5. The binding of PGI2 analogs to the erythrocyte IPR results in activation of a signaling pathway that increases cyclic adenosine 3',5' monophosphate (cAMP), a requirement for adenosine 3'5' triphosphate (ATP) release.

Toward a multiscale description of microvascular flow regulation: o(2)-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks.

Integration of the numerous mechanisms that have been suggested to contribute to optimization of O(2) supply to meet O(2) need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O(2) tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP.

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.

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.

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.

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).

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 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.

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).

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.

Diamide decreases deformability of rabbit erythrocytes and attenuates low oxygen tension-induced ATP release.

Exposure of erythrocytes to reduced oxygen (O(2)) tension activates the heterotrimeric G-protein Gi, resulting in the accumulation of cyclic AMP (cAMP) and release of ATP. The mechanism by which exposure of erythrocytes to reduced O(2) tension activates Gi is not known. Here we investigate the hypothesis that, in rabbit erythrocytes, ATP release in response to exposure to reduced O(2) tension is linked to erythrocyte membrane deformability.

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.

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.

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.

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.

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.

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.

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.

Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans.

Activation of the G protein G(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 (Ilo), a PGI(2) analog, and isoproterenol (Iso), a beta-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes.

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.

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).