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Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ~1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ~2-fold elevation of the content of membrane-bound protein with Mr of ~60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions. [ABSTRACT FROM AUTHOR]

In smooth muscles, inhibition of Na+,K+,2Cl- cotransport (NKCC) by bumetanide decreased intracellular Cl- content ([Cl-]i) and suppressed the contractions triggered by diverse stimuli. This study examines whether or not bicarbonate, a regulator of several Cl- transporters, affects the impact of NKCC in excitation-contraction coupling. Addition of 25 mM NaHCO3 attenuated the inhibitory action of bumetanide on mesenteric artery contractions evoked by 30 mM KCl and phenylephrine (PE) by 5 and 3-fold, respectively. In cultured vascular smooth muscle cells, NaHCO3 almost completely abolished inhibitory actions of bumetanide on transient depolarization and [Ca2+]i elevation triggered by PE. In bicarbonate-free medium, bumetanide decreased [Cl-]i by ∼15%; this effect was almost totally abrogated by NaHCO3. The addition of NaHCO3 resulted in 2-fold inhibition of NKCC activity and 3-fold attenuation of [Cl-]i. These data strongly suggest that extracellular HCO3- diminishes the NKCC-sensitive component of excitation-contraction coupling via suppression of this carrier. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]