Effects of alpha-hemolysin on cell volume and intracellular sodium and potassium of human erythrocytes.

LAURI, NATALIA; CANE, LUCIA; MOURO-CHANTELOUP, ISABELLE; LEAL DENIS, MARÍA FLORENCIA; ALVAREZ, CORA LILIA; LEFEVRE, SOPHIE DENISE; OSTUNI, MARIANO ANIBAL; HERLAX, VANESA; ALLEVA, KARINA; SCHWARZBAUM, PABLO JULIO

Rosario

Congreso; Sociedad Argentina de Fisiologíaa. Reunión Anual 2019.; 2019

Sociedad Argentina de Fisiología

The hemolytic toxin α-hemolysin (HlyA) is a virulencefactor produced by several strains of Escherichia coli. It isinvolved in urinary tract infections, peritonitis, meningitisand septicemia. HlyA causes lysis of mammalian cells,including human erythrocytes (RBCs).In this work, we studied the extent to which changes incell volume kinetics and the concentration of intracellulardiffusible ions affect cell volume regulation of RBCsexposed to HlyA. RBCs from healthy donors and fromaquaporin 1 KOs (AQP1KO) individuals were used.Cells at hematocrit 10% were treated with 0.1 ng/µl HlyAor vehicle at 37 °C for 1, 5 and 10 min. Cell volume wasmeasured using the coulter counter principle, while cellvolume changes at short periods of time were assessedby light scattering using a stopped flow rapid mixingequipment. For stopped flow experiments exponentialcurves were fitted to data and best fit exponentialcoefficients (k) were used to compare treatments.Intracellular concentrations of sodium and potassiumwere evaluated by flame photometry of lysed RBCs.HlyA induced changes in volume and shape of RBCs,which shrunk during the first min followed by continuousswelling, reaching 10% over control values after 10 min.Under a similar treatment, volume changes correlatedwith a 5-8 fold increase of intracellular Na+ and 5-7 folddecrease of intracellular K+ concentrations.In wild type RBCs, cell volume kinetics was affected bytoxin treatment. Values of k were 8.5 ± 0.5 seg-1 foruntreated cells vs 6.5 ± 0.4, 4.1 ±0.4 and 3.4 ±0.3 seg-1for cells treated with the toxin for 1, 5 and 10 min,respectively. In the absence of toxin, k values were 5 foldlower for AQP1KO cells, as compared to wild type cells. Nevertheless, the relative decrease of k values upon HlyAexposure was similar for both types of cells.HlyA dependent decrease of volume, followed byswelling, correlated with acute and simultaneous Na+uptake and K+ efflux. Values of the kinetic constant kdecrease with time of exposure to the toxin, irrespectiveof the presence of aquaporin 1 in the cells.