An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP

Abstract

A novel microflow technique is used to demonstrate that a weakened oxidant defense system found in diabetic erythrocytes leads to decreased levels of deformation-induced release of adenosine triphosphate (ATP) from erythrocytes. Addition of an oxidant to rabbit erythrocytes resulted in a 63% decrease in deformation-induced ATP release before eventually recovering to a value that was statistically equivalent to the initial value. Inhibition of glucose-6-phosphate dehydrogenase prevents recovery from the oxidant attack. Finally, results indicated that the ATP release from the erythrocytes of type II diabetics (91 nM ± 10 nM) was less than half of that measured from the erythrocytes of healthy controls (190 ± 10 nM). These data suggest that the antioxidant status of erythrocytes is a critical determinant in the ability of these cells to release ATP, a known nitric oxide stimulus.

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Introduction

A vast amount of research involving diabetes and the associated complications arising from this disease is focused on such areas including, but not limited to, pancreatic beta cells and insulin, blood glucose levels, and cytokines to name a few. However, there have also been reports concerning the physical characteristics of erythrocytes, or red blood cells (RBCs), from diabetic patients. Specifically, it has been reported that the deformability of RBCs from diabetic patients is lower than of those RBCs obtained from healthy non-diabetic controls.^1–3 Unfortunately, there have been no reports linking these physical characteristics to a physiological consequence.

RBCs, when traversing microvascular beds, are subjected to mechanical deformation. Previously, it has been reported in the case of the isolated perfused rabbit lung that in order to demonstrate flow-induced endogenous nitric oxide (NO) synthesis in the pulmonary circulation system, RBCs obtained from either rabbits or healthy humans were a required component of the perfusate.⁴ It was shown that the property of these RBCs responsible for the stimulation of NO synthesis was their ability to release adenosine triphosphate (ATP) in response to mechanical deformation.⁵ Moreover, it was reported that the release of ATP from the RBCs of rabbits and humans increased as the degree of deformation increased.⁵ Taken collectively, these reports suggest that as the RBC is increasingly deformed by increments in the velocity of blood flow through a vessel and/or by reductions in vascular diameter, it releases ATP that stimulates endothelial synthesis of NO. It has been reported that RBC-derived ATP contributes to the control of vascular resistance in both pulmonary^4–7 and systemic circulation.^8–12

The findings that ATP is released from RBCs in response to mechanical deformation,⁷ and that the levels of ATP released from RBCs increase as the RBC deformability increases,¹³ suggest that the RBC may be an important determinant of endothelium-derived NO as these cells traverse the intact circulatory system. Interestingly, it has been reported by more than one group^1–3,14 that the RBCs of patients suffering from type II diabetes are less deformable than the RBCs obtained from healthy patients and that this decrease in deformability may be related to a susceptibility to oxidant insults.^15–19

Reports pertaining to the weakened oxidant defense mechanism found in diabetic RBCs have been known for nearly two decades. However, there has never been an attempt to link all of these findings as a possible mechanism by which RBCs maintain their deformability and subsequent ability to stimulate NO production (via their ability to release deformation-induced ATP). Based on the aforementioned previous reports concerning RBC antioxidant metabolism, we demonstrate that RBC-derived ATP is affected by oxidant insults and that the recovery from this insult is dependent upon the pentose phosphate pathway. This pathway, which is catalyzed by glucose-6-phosphate dehydrogenase (G6PD), results in the production of the most abundant non-enzymatic antioxidant found in RBCs, the reduced form of glutathione (GSH). The relationship between the pentose phosphate pathway (in abbreviated form), RBC deformability, RBC-derived ATP, and subsequent endothelium-derived NO production is shown in Fig. 1. Deformation-induced ATP release from the erythrocytes of non-diabetic and diabetic patients is examined in order to demonstrate that long-term oxidative stress may result in decreased amounts of RBC-derived ATP, a known stimulus of the potent vasodilator, NO. In this construct, the RBC becomes a possible determinant in diabetic complications. Previous studies by Stamler et al. have shown the ability of RBCs to carry and deliver NO.^20–22 Interestingly, work by James et al. describes the inability of diabetic RBCs to carry and deliver NO as well as RBCs from healthy patients.²³ Here, we provide evidence that diabetic RBCs may not be able to stimulate endothelium-derived NO as well as RBCs from healthy controls. Moreover, using fluorescence microscopy, we demonstrate that NO production in bovine pulmonary artery endothelial cells (bPAECs), increases with increments of ATP concentration.

Fig. 1 Proposed mechanism for the maintenance of deformability in RBCs through antioxidant activities. Glucose-6-phosphate (G6P) is converted to 6-phosphogluconolactone (6GP) via enzymatic (glucose-6-phosphate dehydrogenase, G6PD) catalyzed reduction of NADP⁺ to NADPH. NADPH is a co-factor in the glutathione reductase (GR) catalyzed reduction of oxidized glutathione (GSSG) to the oxidant-fighting reduced form (GSH). The GSH is known to protect spectrin against oxidant attack. Spectrin is often viewed as a determinant of membrane deformability which, in turn, has been shown to be related to ATP release from the RBC. ATP is a known stimulus of nitric oxide (NO) production in endothelial cells.

Results and discussion

In RBCs the most prominent non-enzymatic antioxidant is the reduced form of GSH, which acts to protect important proteins such as spectrin from being oxidized.^18,24–26 It has been reported that oxidation of spectrin in RBCs leads to an overall increase in membrane stiffness.^19,27–29 In type I and type II diabetes, the levels of GSH are lower than non-diabetic patients while levels of the oxidized form of GSH (a dimer linked by a sulfide bond, GSSG) are increased.^{24–26,30–32} In connection with these lowered levels of GSH is the fact that the activity of glutathione reductase (GR) in the RBCs of type II diabetics is less than the activity in the RBCs of healthy patients.^32,33 GR maintains proper levels of GSH by enzymatically catalyzing the protonation of GSSG by oxidation of nicotinamide dinucleotide phosphate (NADPH).

NADPH, which is produced during the pentose phosphate pathway, is at lower than normal levels in the RBCs of diabetics.^24,25,33,34 Importantly, while other cells have multiple ways of producing NADPH, in RBCs, the lone method for the production of NADPH is through the pentose phosphate pathway. The starting material for the pentose phosphate pathway in RBCs is glucose-6-phosphate (G6P), the level of which in type II diabetics has not been shown to be abnormal. However, G6PD, the enzyme that converts G6P to 6-phosphogluconolactone (6GP) while converting NADP⁺ to NADPH in the process, has less activity in the RBCs of diabetics as compared to the RBCs of healthy patients.^24,35,36

It is well established that ATP is released from RBCs that are subjected to mechanical deformation.^{5,6,13,37–39} It has also been reported that the amount of ATP released from mechanically deformed RBCs is related to a cell's overall deformability.¹³ Thus, a determination of those factors governing the overall deformability of an RBC becomes important, especially because patients with pulmonary hypertension⁵ or diabetes^1–3,14 are known to have RBCs that are less deformable than those of healthy patients.

In order to demonstrate that oxidative stress is a determinant of ATP release from RBCs that are subjected to mechanical deformation, RBCs from rabbits were stiffened with diamide.⁴⁰ Diamide is known to stiffen cell membranes without damaging the cell cytosol and, as such, is a molecule with which to examine the effect of cell membrane stiffness on cell function.^41,42 This alteration in cell deformability decreases the overall amount of ATP released from the RBCs, possibly due to a decrease in G-protein activation as suggested elsewhere.⁴³ As shown in Fig. 2, an initial decrease in ATP release of approximately 28% is seen after 5 min of incubation with diamide. In fact, after 20 min, the ATP release has been decreased by 63% from its original value. However, due to the RBC's ability to maintain stasis, the RBC deformability recovers as evident by the increase in ATP release to a value that is statistically equivalent to the original. The concentrations of diamide employed in this study were such that the diamide is depleted while oxidizing GSH to GSSG in a 1 ∶ 2 ratio.⁴⁴ These data demonstrate that deformation-induced ATP release from RBCs is affected by oxidant stress to the cell.

Fig. 2 The effect of oxidant insult on the ability of mechanically-deformed RBCs to release ATP. The initial ATP release from the RBCs is determined in the absence of diamide (which is known to oxidatively stiffen RBC membranes). Upon addition of diamide, the RBC-derived ATP is determined at 5 min intervals for 30 min. Note the ability of the RBCs to recover from the oxidant insult to near-initial levels of ATP release. The RBC sample was a 7% hematocrit from rabbits flowing through microbore tubing having an inside diameter of 50 µm. The crosses indicate ATP values that are statistically equivalent to the initial value at t = 0 min (p > 0.02). Other details are found in the text.

The diamide that was used as the oxidant insult in the studies leading to the results in Fig. 2 is known to be rather specific for oxidation of reduced GSH to GSSG.⁴⁰Fig. 3 shows the changes in GSH over time after insult with diamide. Interestingly, the data in Fig 3 show the same trends as the ATP release data in Fig. 2. Specifically, there is an immediate decrease in ATP release in Fig. 2 (with a low level at 20 min) and a decrease in GSH levels in Fig. 3 (with a low occurring between 15 and 20 min, not unlike the data in Fig. 2). At its lowest point, the percent decrease in GSH levels decreased by 7.2%. However, after approximately 15–20 min, both the RBC-derived ATP levels and the GSH concentrations begin to increase. It is important to note that the small decrease in GSH levels is not surprising because, while the diamide is converting GSH to the oxidized dimer, the GR is also converting the GSSG back to the protective, reduced form (GSH). In fact, 90% of the total glutathione in the RBCs of healthy, non-diabetic individuals is the reduced form, GSH, while in the RBCs of diabetic individuals, this value is closer to 80%. Thus, in RBCs that may be more oxidatively stressed (e.g., those of type II diabetics), the GSH ∶ GSSG ratio is only affected by about 10%.^45,46

Fig. 3 The dynamic redox status of GSH is shown as a function of time after a 0.1% hematocrit of RBCs were subjected to 20 µM diamide. As expected, the GSH levels decrease immediately after addition of the oxidant insult. However, the GSH levels return to near-initial levels after about 30 min. This demonstrates that the GSH levels are dynamic; however, because the lowest GSH level occurs at the same time point as the lowest recorded value of RBC-derived ATP in Fig. 2, it also suggests that ATP release from the RBCs is also related to the antioxidant status of the RBC. The crosses indicate GSH values that are statistically equivalent to the initial value at t = 0 min (p > 0.02).

The data in Fig. 4a provide further evidence that the pentose phosphate pathway may be a major determinant of RBC-derived ATP. Here, G6PD in the RBCs was inhibited by incubating a 7% hematocrit with 1 mM dihydroxyepiandrosterone (DHEA) for 30 min. DHEA has been shown previously to be an inhibitor of G6PD.^47,48 As shown in Fig. 4a, RBCs that were subjected to diamide alone were able to recover from the oxidant insult (diamide addition) as evident by the recovery of the ATP release for those measurements taken after 20 min. The ability to recover from the diamide insult is similar to the data found in Fig. 2, although the data in Fig. 4a were obtained with different rabbits on different days from those used in the experiments to generate the data in Fig. 2. The RBCs that had been incubated in the DHEA were not able to recover in the same amount of time that it took for RBCs incubated in diamide alone to recover. In fact, as shown, the ATP release never recovered, even after a period of 40 min. Fig. 4a also shows ATP release from RBCs that were incubated with DHEA and subjected to diamide. As shown, these RBCs had ATP release trends that were statistically equivalent to those RBCs subjected to the G6PD inhibitor alone. These data suggest that inhibition of the G6PD enzyme, a major component of the pentose phosphate pathway, may be a determinant in the ability of RBCs to release ATP upon deformation.

Fig. 4 The ability of RBCs to release ATP in the absence and presence of a G6PD inhibitor is shown in (a). The gray bars show the average ATP release (n = 6 rabbits) at various time intervals measured from RBCs that were subjected to 20 µM diamide alone. The black bars show the ATP release from aliquots of the RBCs after the addition of diamide; however, these RBCs were first incubated with 100 µM DHEA for 30 min in order to inhibit the G6PD activity. The bars with lines show ATP release from RBCs that were incubated with DHEA alone. Note the inability of the ATP release to recover for those cells whose G6PD activity was inhibited. The asterisks represent those values significantly different from the initial (t = 0 min, p < 0.001) while the crosses indicate ATP values that are statistically equivalent to the initial value at t = 0 min (p > 0.02). The effect of DHEA on G6PD activity is shown in (b). Diamide alone did not affect G6PD activity, nor did it have any further effect on G6PD activity in the presence of DHEA.

The data in Fig. 4a indicate that inhibition of G6PD results in a decreased ability of RBCs to release ATP upon mechanical deformation. However, the activity of the G6PD was never directly measured during the experiments that were performed in generating the data shown in Fig. 4a. Experiments were performed in a non-flow system to determine the effects of DHEA and diamide on G6PD activity. Fig. 4b shows the results of measuring G6PD activity in rabbit RBCs under various conditions. Diamide alone had no significant effect on G6PD activity, while DHEA (100 µM) reduced the apparent activity to a value that is ∼57% of the activity in the absence of the inhibitor. A combination of diamide and DHEA was not significantly different from the G6PD inhibitor alone. The results in Fig. 4 demonstrate that, when subjected to an oxidant insult, RBCs are able to recover and continue releasing ATP at levels similar to those prior to the addition of the oxidant insult. However, when G6PD is inhibited, the cell's ability to release ATP is diminished even in the absence of any external oxidant insult.

Based on the results from Fig. 2–4 that suggest that oxidant stress may affect the ability of RBCs to release ATP, the ATP release from the mechanically-deformed RBCs of type II diabetics (n = 7) were compared to the release from non-diabetic controls (n = 7). The results in Fig. 5 demonstrate that the overall RBC-derived ATP from the healthy controls (190 nM ± 10 nM) is approximately 100% greater than that of the diabetic patients (91 ± 10 nM). The data in Fig. 2–4 suggest that the reduced release of ATP from the RBCs of diabetic patients (shown in Fig. 5) may be due, at least in part, to the diabetic RBCs inability to fight off oxidant stresses. This inability to fight off oxidant insults may contribute to the less deformable RBCs of diabetics that are reported in the literature. It is well documented that the oxidant defense system of diabetic RBCs is not as strong as the RBCs from non-diabetics. However, prior to this work, there had never been an investigation that explains the possible physiological consequences of this weakened oxidant defense system.

Fig. 5 Determination of ATP release from the RBCs of diabetic (n = 7) and non-diabetic (n = 7) patients is shown. The RBCs were pushed through microbore tubing that had an inside diameter of 50 µm. The error bars represent standard errors about the mean. The asterisks represent those values significantly different from the controls (p < 0.001).

The results involving G6PD from rabbit RBCs in Fig. 4 are interesting because it has been demonstrated in cell types other than RBCs that G6PD activity is lower under hyperglycemic conditions.^24,35,36 Moreover, deficiencies in this enzyme, along with sickle cell trait, are associated with resistance to malaria.⁴⁹ Thus, the lower activity of the G6PD enzyme may contribute to the high number of diabetic cases seen among people of African descent.^50,51

In order to identify a potential consequence of diminished RBC-derived ATP release in diseased states, or upon oxidant insult, we set out to monitor NO production in endothelial cells following exposure to increasing concentrations of ATP. The images shown in Fig. 6 demonstrate the increase in fluorescence in diaminodifluoro-fluorescein diacetate (DAF-FM DA) loaded bovine pulmonary artery endothelial cells (bPAECs) before ATP addition (Fig. 6a) and in the same cells 30 min after the addition of 100 µM ATP (Fig. 6b). The data in Fig. 6 indicate the change in average fluorescence intensity 30 min after addition of the buffer, 10 µM ATP or 100 µM ATP to the bPAECs. The change in fluorescence intensity measured for the ATP standards are both significantly different from the buffer alone (p < 0.001). Thus, diminished ATP concentrations coincide with decreased endothelial cell NO production indicating a potential link between RBC-derived ATP and vascular complications such as hypertension and thrombosis.

Fig. 6 Effect of ATP as a stimulus of endothelium-derived NO production in bovine pulmonary artery endothelial cells (bPAECs). The fluorescence shown in (a) is a result of the bPAECs loaded with a fluorescence probe specific for NO (DAF-FM DA). The increase in fluorescence intensity shown in (b) is a result of the addition of 100 µM ATP to the same cells shown in (a). The graph shows summarized data from the addition of either buffer containing no ATP, or buffer containing 10 µM or 100 µM ATP. The results are reported as the average change in fluorescence intensity 30 min after addition of the buffer or ATP standards to the bPAECs. The change in fluorescence intensity measured for the ATP standards are both significantly different from the buffer alone (p < 0.001).

In a sense, the RBC, through its ability to release deformation-induced ATP, becomes a determinant of vascular resistance in the circulation. Importantly, it has been shown by Sprague et al. that in the isolated rabbit lung, a minimum of 300 nM ATP was a required component of the perfusate to induce any significant decrease in vascular resistance.³⁷ Infusion of the lung with 10 nM and 100 nM ATP did not result in any significant changes in vascular resistance. Such results suggest that nanomolar changes in the available extracellular ATP result in significant physiological events in vivo. The results reported here show that diabetic RBCs release less than 50% of the ATP released by the RBCs of non-diabetic patients (a concentration value of >100 nM). The inability of the RBCs obtained from the diabetic patients to release ATP at the same levels as non-diabetic controls may be one possible reason for the higher rates of cardiovascular complications seen in diabetic patients. Moreover, because NO is known to reduce platelet activation and adhesion to the walls of the endothelium, the RBC (via its ability to release NO-stimulating ATP) also becomes a possible determinant in mediating the involvement of platelets in various vascular complications including thrombosis and stroke.^52–54

Experimental

Collection of RBCs

All procedures involving the collection of blood samples from animals or humans were approved by the Wayne State University Animal Investigation Committee or Institutional Review Board, respectively. For studies involving rabbit RBCs, male New Zealand White rabbits (2.0–2.5 kg) were anesthetized with ketamine (8 mL kg⁻¹, i.m.) and xylazine (1 mg kg⁻¹, i.m.) followed by pentobarbital sodium (15 mg kg⁻¹ i.v.). A cannula was placed in the trachea and the animals were ventilated with room air at 20 breaths min⁻¹ and a tidal volume of 20 mL kg⁻¹. A catheter was placed into a carotid artery for administration of heparin and for phlebotomy. After heparin (500 units, i.v.), animals were exsanguinated. Human blood was obtained by venipuncture without the use of a tourniquet (antecubital fossa) and collected into a heparinized syringe. All type II diabetic patients involved in this study had glycosylated hemoglobin values (HA1c) of 7.0–9.5. Blood was centrifuged at 500 × g at 4 °C for 10 min. The plasma and buffy coat were discarded. RBCs were then resuspended and washed three times in a physiological salt solution [PSS; in mM, 4.7 KCl, 2.0 CaCl₂, 140.5 NaCl, 12 MgSO₄, 21.0 tris(hydroxymethyl)aminomethane, 11.1 dextrose with 5% bovine serum albumin (final pH 7.4)]. Cells were prepared on the day of use within 8 h of removal from animal or human subjects.

Measurement of ATP release

All reagents were from Sigma Chemical (St. Louis, MO) unless otherwise noted. A 100 µM stock solution of ATP was prepared by adding 0.0583 g of ATP to 100 mL of distilled and deionized water (DDW). ATP standards ranging from 0–1.5 µM were then prepared in a physiological saline solution (PSS). PSS was made by combining 25 mL of TRIS buffer (prepared by mixing 50.9 g of TRIS in 1 L of DDW) and 25 mL of Ringer's (164.2 g NaCl, 7.0 g KCl, 5.9 g CaCl₂·2H₂O, and 2.83 g MgSO₄ in 1 L of DDW). After the addition of 0.50 g of dextrose and 2.50 g of albumin bovine fraction V (fatty acid free) to the TRIS–Ringer's mixture, the entire solution was diluted to 500 mL with DDW and the pH was adjusted to 7.35–7.45. The PSS was then filtered three times using a 0.45 µm filter (Corning, Fisher Scientific) before use.

To prepare the luciferin–luciferase mixture used to measure the ATP by chemiluminescence, 5 mL of DDW were added to a vial containing luciferase and luciferin (FLE-50, Sigma). In order to enhance the sensitivity of the assay, 2 mg of luciferin were added to the vial. For ATP measurements involving RBCs, all RBC samples were diluted to a 7% hematocrit. In order to determine ATP release, the luciferin–luciferase mixture was placed in a 500 µL syringe (Hamilton, Fisher Scientific). Either the ATP standards or the RBCs were placed in another 500 µL syringe next to the luciferin–luciferase mixture. The syringes were loaded on to a syringe pump (Harvard Apparatus, Boston, MA) and were connected to 50 cm of microbore tubing with an internal diameter of 50 µm and an outer diameter of 362 µm (Polymicro Technologies, Phoenix, AZ). The RBCs or ATP standards were then pumped through the microbore tubing at a rate of 6.7 µL min⁻¹. The two separate microbore tubes containing either the luciferin–luciferase mixture or the RBCs/ATP standards were combined at a T-junction (Upchurch Scientific, Oak Harbor, WA). The now combined contents flowed through a 10 cm section of 75 µm tubing where the resultant chemiluminescence was detected by a photomultiplier tube that was housed in a light excluding box to decrease as much background light as possible. The tubing over the photomultiplier tube must have its polyimide coating removed in order to detect the chemiluminescence. The measured chemiluminescence is proportional to the amount of ATP present.

For studies involving diamide, a 2 mM stock solution of diamide was prepared by adding 0.0344 g of diamide to 100 mL of deionized water. An aliquot of 1 mL of diamide stock solution was added to 9 mL of wash buffer to create a buffered 200 µM solution of diamide. Next, an appropriate volume of RBCs were placed in a 5.0 mL volumetric flask containing 0.5 mL of the 200 µM diamide creating a 7% RBC solution in 20 µM diamide. This mixture was then placed in a 500 µL syringe and ATP release was measured at 5 minute intervals. The syringe was rotated periodically to ensure a homogeneous mixing of red blood cells.

Measurement of glutathione

Glutathione redox status was monitored using the GSH probe monochlorobimane (MCB, Molecular Probes, Eugene, OR). MCB has been shown to preferentially label GSH.^55–57 To determine GSH in the RBCs, 100 µL of 200 µM diamide were added to 100 µL of RBCs (1% hematocrit). After allowing the diamide to incubate with the RBCs for times ranging from 0 to 40 min, 100 µL of 2.5 mM MCB were added to form a fluorescent product with GSH. The MCB was allowed to react with GSH for 10 min before measuring the fluorescence emission at 525 nm (excitation at 370 nm). All fluorescence measurements of GSH with MCB were performed with a scanning spectrofluorometer (Shimadzu R5301). Due to the MCB incubation time, the earliest reading of GSH status after the addition of diamide is 10 min. However, we have included in the data reported here a GSH determination in the absence of diamide.

Determination of G6PD activity

The activity of G6PD was determined by monitoring the generation of NADPH using the Vybrant Cytotoxicity Assay Kit (Invitrogen Corp., Carlsbad, USA). In this assay NADPH reduces resazurin to produce the fluorescent product resorufin. Here the resazurin reaction mixture was added to a solution of RBCs (0.04% HCT) and incubated at 37 °C. Following a 30 min incubation, the fluorescence intensity of resorufin was measured (ex. 563 nm, em. 587 nm). To verify the change in fluorescence was the result of G6PD activity, RBCs were incubated with 100 µM DHEA, a known inhibitor of G6PD, for 30 min prior to addition to the reaction mixture. Finally to identify the involvement of G6PD in ATP release from RBCs upon deformation, RBCs were incubated with 2 µM diamide (± 1 mM DHEA) for 20 min and monitored for G6PD activity as described above.

Measurement of ATP-induced nitric oxide production

bPAECs were cultured in 35 mm tissue culture dishes which had been treated with 1 mL of 100 µg mL⁻¹ fibronectin (Sigma) for 1 h at room temperature. Once confluent, the culture media was aspirated and the cells were washed with HBSS to remove any residual media. The bPAECs were incubated in HBSS containing 5 µM diaminodifluoro-fluorescein diacetate (DAF-FM DA, Molecular Probes, Eugene, OR) for 30 min. Excess DAF-FM DA was removed by washing the cells twice with HBSS. A fluorescent image of the DAF-FM DA loaded bPAECs was then acquired using an Olympus IX71 microscope (Olympus America, Melville, NY) with an electrothermally-cooled CCD (Orca, Hamamatsu) fitted with a FITC filter cube to establish the intracellular basal level of fluorescence. Solutions of varying ATP concentrations (1 µM, 10 µM and 100 µM) prepared in HBSS were added to the tissue culture dish and fluorescent images were taken every 5 min for the next 30 min.

Conclusion

Endothelium-derived nitric oxide (NO) mediates vascular caliber and hypertension, and exhibits platelet inhibitory effects. Each of these physiological events or conditions is linked to a known complication associated with diabetes. When traversing the microcirculation system, erythrocytes are deformed and release sub-micromolar amounts of ATP, a stimulus of NO production in endothelial cells. The erythrocytes from people with type II diabetes are less deformable than those from healthy, non-diabetic individuals. In addition, it is well-established that the erythrocytes from people with type II diabetes are more susceptible to oxidant insult. Until now, there has been no link between these physical and biochemical characteristics of the erythrocytes from type II diabetics and the ability of these erythrocytes to release NO-stimulating ATP. Here, we establish that a reduced erythrocyte oxidant defense system results in less ATP release from these cells when traversing a biomimetic microcirculation system. Herein, we show that the erythrocytes from individuals with type II diabetes release 50% less ATP than the erythrocytes from healthy, non-diabetics. We demonstrate that there is a direct relationship between the ability of extracellular ATP to stimulate NO production in endothelial cells. Taken collectively, the results shown here represent a revolutionary new role for erythrocytes as a determinant in diabetic complications.

Acknowledgements

The authors would like to thank Michael Owczarek and Brandon King for their assistance in this work. Human blood samples obtained by Dr Michael Kleerekoper and Barbara Lloyd, RD, are greatly appreciated. This work was funded by the National Institutes of Health (NHLBI R01-073942) and Wayne State University.

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