Homeostasis of Extracellular ATP in Human Erythrocytes

Abstract

We explored the intra-and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferinluciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ␤-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin ؉ papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ( Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (ϳ28 fmol ؋ (10 6 cells min) ؊1 ), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.All cell types appear to possess mechanisms that enable a controlled, nonlytic release of ATP, that is, a release not involving cell membrane rupture, and which occurs in response to osmotic, mechanical, and/or neurohormonal stimuli (1). Specifically human erythrocytes release ATP following exposure to ␤-adrenergic stimulation, mechanical deformation, reduced oxygen tension, or acidosis (2). All of these represent physiological conditions to which erythrocytes are exposed in the vasculature, e.g. when passing through constricted vessels or in the contracting striated muscle (3). Once in the extracellular medium, extracellular ATP (ATPe) 5 can trigger different cellular responses by interacting with P receptors on the cell surface while at the same time its concentration is controlled by the activities of one or more ectonucleotidases (4, 5). Several reports published over recent years have shown that an increase in intracellular cyclic AMP (cAMP) concentration triggered ATP release from human erythrocytes (6, 7). Receptor-mediated ATP release in human erythrocytes involves activation of heterotrimeric G proteins G s or G i/o (3, 8, 9). Regarding the G s pathway, activation of ␤-adrenergic receptors by various agonists was reported to stimulate adenylyl cyclase, with concomitant increases in cAMP levels and protein kinase A activity (6, 10). Moreover, direct activation of adenylyl cyclase by forskolin resulted in both ATP release and cAMP increases in human and rabbit erythrocytes (6).Despite the accumulated knowledge regarding the intracellular signaling events mediating ATP release, comparatively little is known regarding the processes governing the kinetics of ATPe accumulation at the surface in animal cells (11,12), with rates of intracellular ATP release and extracellular ATP hydrolysis being the main actors. The human non-nucleated erythrocyte is an excellent model in this respect, because it lacks intracellular compartments and...

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

“…The same concentration (50 M) of scrambled 10 Panx also did not significantly alter CR1-mediated ATP release. Of note, when used in our system, both compounds caused RBC lysis (8%) at 200 M, the concentration previously shown to inhibit RBC ATP release (24). Of note, the greater ATP values in Fig.…”

Bacterial RTX Toxins Allow Acute ATP Release from Human Erythrocytes Directly through the Toxin Pore

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Liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export

“…[2][3][4][6][7][8]10,11,13 Even the earliest investigations, from Bergfeld and Forrester, 9 parsed authentic ATP release from that due to cell damage, and numerous publications document regulated ATP export from RBCs while controlling for hemolysis. If increases in [ATP] ec are solely from hemolysis, then [ATP] ec should be unaffected by antagonizing ATP-export signaling.…”

“…Lysis can mask regulated ATP release because the expulsion of intracellular nucleotidases leads to rapid ATP hydrolysis (as in Figure 2 3,11 ), RBC temperature shift (promotes vulnerability), and poly-L-lysine (higher concentrations perturb distribution of band 3, 3 a determinant of controlled ATP export 7,9 ). In the case of the ATP-release stimulus hypoxia, special caution and controls using normoxic gas flow are lacking in Sikora's study but necessary because the force of gas flow itself can lyse cells.…”

“…In contrast, adding 100% DMSO lysed RBCs, but 10% DMSO was innocuous. Consequently, they propose that hemolysis rather than cAMP could have caused ATP release in Montalbetti et al 11 This argument is unsustainable because in Montalbetti 11 : (1) identical volumes of 3V (as 10 times stock) and vehicle (phosphate-buffered saline [PBS]) were added to cells in 1% DMSO; (2) cAMP-elevating isoproterenol (in PBS) markedly potentiated forskolin-evoked ATP release; (3) dibutyryl-cAMP (aqueous solution) promoted ATP release; and (4) inhibitors of Px1 abolished ATP release in the absence of measurable hemolysis. Lastly, Sikora and colleagues ignored the observation that 3V promoted ATP release in RBCs from wild-type but not Px1 2/2 mice.…”