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Activation of the cAMP pathway by β-adrenergic stimulation and cGMP pathway by activation of guanylate cyclase substantially affects red blood cell (RBC) membrane properties in mammals. However, whether similar mechanisms are involved in RBC regulation of lower vertebrates, especially teleosts, is not elucidated yet. In this study, we evaluated the effects of adenylate cyclase activation by epinephrine and forskolin, guanylate cyclase activation by sodium nitroprusside, and the role of Na + /H + -exchanger in the changes of osmotic fragility and regulatory volume decrease (RVD) response in crucian carp RBCs. Western blot analysis of protein kinase A and protein kinase G substrate phosphorylation revealed that changes in osmotic fragility were regulated via the protein kinase A, but not protein kinase G signaling pathway. At the same time, the RVD response in crucian carp RBCs was not affected either by activation of adenylate or guanylate cyclase. Adenylate cyclase/protein kinase A activation significantly decreased RBC osmotic fragility, i.e., increased cell rigidity. Inhibition of Na + /H + -exchanger by amiloride had no effect on the epinephrine-mediated decrease of RBC osmotic fragility. NO donor SNP did not activate guanylate cyclase, however affected RBCs osmotic fragility by protein kinase G-independent mechanisms. Taken together, our data demonstrated that the cAMP/PKA signaling pathway and NO are involved in the regulation of crucian carp RBC osmotic fragility, but not in RVD response. The authors confirm that the study has no clinical trial., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Fish red blood cells (RBCs) exhibit an oxygen-dependent regulatory volume decrease (RVD) in hypoosmotic environment. In higher vertebrates, membrane-associated hemoglobin is involved in the regulation of osmotic ion movements across the cellular membrane. However, whether the hemoglobin conformational state plays a role in the regulation of osmotic responses in fish red blood cells is still not fully understood. We found that changes in hemoglobin conformation influence the pattern of the regulatory volume decrease in Carassius carassius red blood cells. In oxygenated cells (96.4 ± 3.7% oxygenated hemoglobin), the volume recovery was completed within 125 min. Deoxygenation of hemoglobin (96.5 ± 2.7% of deoxygenated hemoglobin) inhibited the volume decrease in hyposmotically swollen red blood cells. Reoxygenation restored regulatory volume decrease in cells within 5 min. Induced methemoglobinemia (48.4 ± 1.8% of methemoglobin and 41.3 ± 2.3% of deoxygenated hemoglobin) blocked the process of volume recovery and significantly decreased osmotic stability of red blood cells.