In vitro effects of L-arginine on lysosomal cysteine proteases activity in isolated experiment and in the state of oxidative stress

Abstract

Aim. Assessment of direct influence of arginine on lysosomal cysteine proteases activity in vitro, in isolation as well as the stimulation of oxidative stress.

Methods. The study was conducted on the 72 female conventional mature Wistar rats 280-320 g divided into 6 series of 12 rats each. Lysosome slurries were isolated from the liver of intact animals with a subsequent in vitro incubation in a sucrose solution, in the presence of L-arginine, as well as in the presence of L-arginine accompanied by the stimulation of oxidative stress. Samples of control groups were exposed in vitro with the addition of isolate and oxidant, respectively. Each batch was reproduced three times, incubation was performed at 37 °C in a water bath for 1, 2 and 4 hours. The activity of cathepsins B, L and H was studied using spectrofluorimetric method in two fractions - intra- and extralysosomal. Acid phosphatase activity was used as the main marker of membrane labialization.

Results. One hour Incubation with 5 mM arginine in vitro led to inhibition of the cathepsin H activity and lysosomal membrane damage, however, further increase in incubation time led to its stabilization. In vitro exposure to 5 mM H2O2 caused an increase in activity of cathepsines B and L and the drop in the cathepsin H activity without obvious changes in the distribution of enzymes between extra and intralysosomal fractions. In a state of oxidative stress 2-hour in vitro incubation with 5 mM arginine reduced the permeability of lysosomal membranes for cathepsines B, H and L; while 4-hour incubation led to the destabilization of lysosomal membranes.

Conclusion. The direct effect of arginine at a concentration of 5 mM within the 1,2 and 4-hour time intervals leads to a distinct change as a lysosomal cysteine protease activity and stability of lysosomal membranes.

Keywords

cathepsins B / L-arginine / lysosomal membrane stability

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Fomina M.A., Kudlaeva A.M.