Hemolysis is a primary ATP-release mechanism in human erythrocytes

Abstract

The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

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

“…Although we began with the hypothesis that regulated ATP release mechanisms do operate in RBCs, after extensively verifying each experimental approach, we came to the conclusion expressed in the title: ie, that "Hemolysis is a primary ATP release mechanism in human erythrocytes." 1 In the course of the study, while attempting to reproduce previously published findings, we identified a number of methodological pitfalls (the major ones are discussed in the supplemental Methods available on Blood Web site), which likely precluded a proper evaluation of ATP release and also the contribution of hemolysis in the earlier studies 2-10 cited by Kirby et al We agree that "liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export." 11 Our point is that without the controls described in our paper, a confounding effect of hemolysis cannot be excluded.…”

Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors

“…Hence, the mechanism of ETX-induced hemolysis is different from those of HlyA- and leukotoxin A-induced hemolysis. It was also reported that hemolysis is a primary ATP-release mechanism in human erythrocytes stimulated by several stimuli, including shear stress and hypoxia [37].

10.1080/21505594.2018.1528842-F0008Figure 8.Inferred model for ETX-induced hemolysis.

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Reducing Resistance to Acid Hemolysis by Iron-Contained Drug Increases the Level of Hemoglobin in the Erythrocytes of Aging Animals

“…Однією з причин зниження стійкості до кислотного гемолізу є наявність у еритроцитів старих тварин оксидативно/нітрозативного стресу, тобто підвищеної генерації супероксиду (O 2 -) і над-лишкового оксиду азоту (NO) [2]. Відомо, що при гемолізі еритроцитів звільняється АТФ [3], який активує ендотеліальну NO-синтазу (еNOS) [4], але руйнування пламатичної мембрани еритроцитів (гемоліз) і звільнен-ня гемоглобіну має за наслідок утворення газового трансмітера оксиду вуглецю (СО) через руйнування гему гемоксидазою [5], так і сприяє підвищенню синтезу гемоглобіну [6]. В еритроцитах інтенсивно синтезується не лише NO при відновленні нітрит-аніона (NO 2 -) дезоксигемоглобіном [7], але і H 2 S ферментом меркаптопіруватсульфотранс-феразою (MPST) [8].…”