CONICET | Buscador de Institutos y Recursos Humanos

Escherichia coli α-hemolysin (HlyA) triggers sublytic and lytic processes on human erythrocytes (RBCs). We studied how HlyA affectsvolume regulation in RBCs.RBCs were treated with 0.1 ng/µl HlyA or vehicle at 37 °C for 1, 5 and10 min. Cell volume (Vt) was measured using the Coulter Counterprinciple. Rapid kinetics of Vt was assessed by light scattering usinga stopped flow rapid mixing equipment. Cells were preexposed 1, 5and 10 min to HlyA, and Vt changes were measured over 4 s after140 mosM osmotic gradient. Exponential curves were fitted to data.Best fit values of the exponential coefficient (k) were obtained, whichestimates osmotic water permeability (Pf). Intracellular contents ofNa+ and K+ were measured by flame photometry.HlyA-treated RBCs underwent a slight shrinkage, followed by continuous swelling up to 12% over control values after 10 min. Vtchanges were accompanied by parallel intracellular [Na+] increaseand [K+] decrease.Exposure to HlyA decreased k values from 7.6 ± 0.1 seg-1 to 3.4 ±0.2 seg-1 after 10 min. In hyposmotic media (190-280 mosM), Vtincreased over 10 min. Preexposure to these media showed thatk values decreased as Vt increased, so that a 54% reduction of kvalues was obtained when Vt was increased by 30%.In the presence of HlyA and suramin (blocker of purinergic receptors), Vt did not change, but k values were reduced by 30%.Comparing HlyA and hyposmotic media, the reduction of k valuesmay be partly assigned to cell swelling. However, results using suramin+HlyA, where changes in Vt and k were uncoupled, show thatanother factor is affecting k, and therefore Pf of RBCs.Paradoxically, HlyA causes RBC swelling, which ultimately leads tolysis, while simultaneously reduces water permeability.This work was supported by Grants PIP 112 201501 00459, CONICET; 200201701001 52BA, UBACyT; PICT 2016-1041, ANPCyT;ECOS sud-MINCyT A15S01 and Laboratory of Excellence GR-ExANR-11-LABX-0051.

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