Erythrocytes’ Reactions to Osmotic, Ammonium, and Oxidative Stress Are Inhibited under Hypoxia

Hypoxic conditions, in addition to effects on the CO2 and O2 transport, may have an impact on various functions of red blood cells (RBCs) including ion and metabolic regulation, oxidative stress induction, and transport of nitrites, ammonia, urea, etc. We used laser diffraction, spectrometry, and flow cytometry for evaluation of responses of RBCs of lower vertebrates and human RBCs to hypoosmotic conditions, ammonia, and oxidative stress in normoxia and hypoxia. Under hypoosmotic conditions RBCs of lower vertebrates swell and immediately initiate the regulatory volume decrease (RVD) reaction. In our experiments, the RVD reaction in RBCs of Carassius carassius and Rana temporaria was inhibited by hypoxic conditions and restored by reoxygenation. Transport of ammonia/ammonium in human RBCs is facilitated via functional interaction of ammonium (RhAG) and anion (AE1) transporters. Ammonium transport in human RBCs was inhibited in hypoxic conditions, whereas the switch of hemoglobin from R- to T-state in hypoxia restored the rate of ammonia/ammonium transport. Oxidative stress inhibits esterase activity, triggers hemoglobin oxidation to ferri-forms, Fe(III), Fe(IV), phosphatidylserine externalization, AE1 clusterization, and microparticles formation. In the conditions of oxidative stress, hypoxia prevents cell death via apoptosis by inhibiting phosphatidylserine externalization, AE1 clusterization, and microparticle formation. The obtained results indicate that hemoglobin conformation plays a significant role in the process of volume regulation not only in higher but in lower vertebrates as well. In human RBCs transport of CO2 ($${\text{HCO}}_{3}^{ - }$$) and NH3 ($${\text{NH}}_{4}^{ + }$$) is dependent on the AE1 activity, which is regulated by hemoglobin conformational changes. Hypoxic conditions prevent apoptosis of RBCs by preserving them from oxidative stress. Thus, our data clearly show that hypoxic conditions have beneficial effects on the RBC long-term storage.