Inhibition of Rho Kinase by Y27632 Increases Low Oxygen Tension‐Induced ATP Release from Human Red Blood Cells (RBCs)

The effect of oxygenation on the dissipative fluxes of K in trout red blood cells has been determined. Unidirectional influx under low oxygen tension (PO2 = 1 kPa) was 0.56 +/- 0.07 mmol.l-1 packed cells.h-1. Within a few minutes of equilibration with high oxygen tension (PO2 = 120 kPa), influx was increased 14-fold, and this was associated with a progressive loss of KCl and a cell shrinkage. K influx progressively declined over the following 3 h to levels close to those characteristic of cells at low oxygen tension. Replacement of medium Cl by NO3- or methane sulfonate inhibited the stimulation due to high oxygen as did furosemide and low extracellular pH. The oxygenation-stimulated influx was highly volume sensitive, being increased by up to 100% by osmotic swelling and decreased by osmotic shrinkage. By contrast, the small influx under low oxygen tension was unaffected by either Cl replacement or by shrinkage and increased only with extreme swelling. Thus high oxygen tension activated a Cl-dependent and furosemide-sensitive K flux. Once activated, the mechanism was rapidly deactivated on transfer back to low oxygen tension but slowly deactivated when maintained at high PO2. The oxygenation-stimulated flux mechanism promotes a rapid and more complete volume regulatory decrease than in cells at low oxygen tension.

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Comparison of buffer and red blood cell perfusion of guinea pig heart oxygenation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00383.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. H1819-H1825 ◽

Cited By ~ 32

Author(s):

Kenneth A. Schenkman ◽

Daniel A. Beard ◽

Wayne A. Ciesielski ◽

Eric O. Feigl

Keyword(s):

Oxygen Consumption ◽

Guinea Pig ◽

Oxygen Saturation ◽

Red Blood Cells ◽

Ventricular Function ◽

Oxygen Tension ◽

Blood Cells ◽

Low Oxygen ◽

Guinea Pig Heart ◽

Perfused Hearts

Myocardial mean myoglobin oxygen saturation was determined spectroscopically from isolated guinea pig hearts perfused with red blood cells during increasing hypoxia. These experiments were undertaken to compare intracellular myoglobin oxygen saturation in isolated hearts perfused with a modest concentration of red blood cells (5% hematocrit) with intracellular myoglobin saturation previously reported from traditional buffer-perfused hearts. Studies were performed at 37°C with hearts paced at 240 beats/min and a constant perfusion pressure of 80 cmH2O. It was found that during perfusion with a hematocrit of 5%, baseline mean myoglobin saturation was 93% compared with 72% during buffer perfusion. Mean myoglobin saturation, ventricular function, and oxygen consumption remained fairly constant for arterial perfusate oxygen tensions above 100 mmHg and then decreased precipitously below 100 mmHg. In contrast, mean myoglobin saturation, ventricular function, and oxygen consumption began to decrease even at high oxygen tension with buffer perfusion. The present results demonstrate that perfusion with 5% red blood cells in the perfusate increases the baseline mean myoglobin saturation and better preserves cardiac function at low oxygen tension relative to buffer perfusion. These results suggest that caution should be used in extrapolating intracellular oxygen dynamics from buffer-perfused to blood-perfused hearts.

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ATP Release from Red Blood Cells Is Regulated by a Negative Feedback Pathway where ADP Acts on P2Y13 Receptors.

Blood ◽

10.1182/blood.v104.11.1576.1576 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1576-1576

Author(s):

Martin L. Olsson ◽

Lingwei Wang ◽

Goran Olivecrona ◽

Matthias Gotberg ◽

Stefan Amisten ◽

...

Keyword(s):

Red Blood Cells ◽

Negative Feedback ◽

Blood Cells ◽

Atp Release ◽

Pig Model ◽

Coronary Vein ◽

Human Red Blood Cells ◽

Adp Receptor ◽

Tissue Circulation ◽

Feedback Pathway

Abstract Background: Red blood cells regulate tissue circulation and O2 delivery by releasing the vasodilator adenosine triphosphate (ATP) in response to hypoxia. When released extracellularly, ATP is rapidly degraded to adenosine diphosphate (ADP) in the circulation by ectonucleotidases. ATP and ADP activate subtypes of the large P2 receptor family (15 subtypes). Here we show that ADP acting on P2Y13 receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release. Methods: mRNA was quantified with real-time PCR. Western blot was used to detect P2 receptors with available antibodies. cAMP levels were determined with an enzyme immunoassay. ATP release was measured in incubated red blood cells using microdialysis and a luciferase assay. In a pig model, catheters were inserted through the carotid artery to place a catheter in the left coronary artery, and through the jugular vein to place a microdialysis probe in the coronary vein. 2-MeSADP was injected in the artery and ATP levels were measured in the coronary vein. Results: mRNA of the ADP receptor P2Y13 was highly expressed in human red blood cells and reticulocytes, whilst other ADP receptors were not (Fig.1). Figure Figure The stable ADP analogue 2-MeSADP decreased ATP release from red blood cells by inhibition of cAMP. The P2Y12 and P2Y13 receptor antagonist AR-C67085 (30 mM), but not the P2Y1 blocker MRS2179, inhibited the effects of 2-MeSADP. At doses where AR-C67085 only blocks P2Y12 (100 nM), it had no effect. AR-C67085 and the nucleotidase apyrase increased cAMP per se, indicating a constant cAMP inhibitory effect of endogenous extracellular ADP. 2-MeSADP reduced plasma ATP concentrations in an in vivo pig model. Furthermore, a missense polymorphism in the coding region of P2Y13 has been found that is in total disequilibrium with 5 polymorphisms in P2Y12 (the important ADP receptor in platelets) forming a haplotype that could contribute to vascular disease. Conclusion: Our results show that P2Y13 is selectively expressed in human red blood cells. The ATP degradation product ADP inhibited ATP release by acting on this receptor. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation. Because blood consists of approximately 40% red blood cells, containing a 1000-fold higher ATP concentration than plasma (mM vs. uM), even a minor release of ATP from the high intracellular concentrations could have major circulatory effects. A negative system may therefore be of great physiological importance to mitigate ATP release. In addition, this finding could be of interest for efforts to preserve intracellular ATP in red blood cells during storage.

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Deformation-induced ATP release from red blood cells requires CFTR activity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.5.h1726 ◽

1998 ◽

Vol 275 (5) ◽

pp. H1726-H1732 ◽

Cited By ~ 94

Author(s):

Randy S. Sprague ◽

Mary L. Ellsworth ◽

Alan H. Stephenson ◽

Mary E. Kleinhenz ◽

Andrew J. Lonigro

Keyword(s):

Cystic Fibrosis ◽

Red Blood Cells ◽

Blood Cells ◽

Atp Release ◽

Chronic Obstructive Lung Disease ◽

Mechanical Deformation ◽

Chronic Obstructive ◽

Healthy Humans ◽

Human Red Blood Cells ◽

Human Rbcs

Recently, it was reported that rabbit and human red blood cells (RBCs) release ATP in response to mechanical deformation. Here we investigate the hypothesis that the activity of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP binding cassette, is required for deformation-induced ATP release from RBCs. Incubation of rabbit RBCs with either of two inhibitors of CFTR activity, glibenclamide (10 μM) or niflumic acid (20 μM), resulted in inhibition of deformation-induced ATP release. To demonstrate the contribution of CFTR to deformation-induced ATP release from human RBCs, cells from healthy humans, patients with cystic fibrosis (CF), or patients with chronic obstructive lung disease (COPD) unrelated to CF were studied. RBCs of healthy humans and COPD patients released ATP in response to mechanical deformation. In contrast, deformation of RBCs from patients with CF did not result in ATP release. We conclude that deformation-induced ATP release from rabbit and human RBCs requires CFTR activity, suggesting a previously unrecognized role for CFTR in the regulation of vascular resistance.

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