Сhanges in subcellular distribution of lysosomal cysteine proteinases activity in parenchymatous organs of rats under the action of nitric oxide synthesis modulators

M A Fomina,A A Terent'Ev

doi:10.17709/2409-2231-2018-5-3-3

Abstract

Aim. To study the eﬀect of non-selective inhibitor of NO-synthase N-nitro-L-arginine methyl ester (L-NAME) and substrate of nitric oxide synthesis L-arginine on the activity of cathepsins B, L, H and its subcellular distribution in liver, kidney and lung tissues.Materials and methods. The object of study – male rats Wistar line, the material was the cytoplasmic and lysosomal fraction of homogenates of liver, kidney, lung tissues. A non-selective inhibitor of inducible NO-synthase N-nitro-L-arginine methyl ester (L-NAME) was applied at a dose of 25 mg/kg, the substrate of NO synthesis L-arginine – at a dose of 500 mg/kg. Activity of cathepsins B, L, H was defined separately in the cytoplasmic and lysosomal fractions by spectrofluorometry quantitative determination of the specific substrate cleavage product 7-amido-4-methylcoumarin.Results. Suppression of nitric oxide synthesis by non-selective inhibitor of NO-synthase L-NAME (25 mg/kg, 7 days) in the kidney tissue leads to a decrease in the activity of cathepsins В, L, H in lysosomal fraction with a parallel increase in non-lysosomal activity of cathepsin L, in the liver tissue leads to an increase in lysosomal activity of cathepsin H and a decrease in non-lysosomal activity of cathepsin L. The substrate of nitric oxide synthesis L-arginine (500 mg/kg, 10 days) only causes increased activity of cathepsin L in non-lysosomal fraction of liver tissue, leads to increased lysosomal activity of cathepsin H in kidney tissue, the lung tissue shows a significant increase in the activity of the all studied cathepsins in non-lysosomal fraction, accompanied by an increase in lysosomal activity of cathepsins B and H. The revealed changes are associated with the signs of changes in the ratio of pro-enzyme and active forms of cathepsins.Conclusion. The eﬀects of non-selective inhibitor and substrate of nitric oxide synthesis on the total activity of cathepsins B, L and H in parenchymatous organs and its subcellular distribution are tissue-specific and multidirectional in some cases and are accompanied by signs of changes in the ratio of pro-enzyme and enzymatically active forms mainly due to an increase of pro-enzyme forms.

Highlights

Suppression of nitric oxide synthesis by non-selec ve inhibitor of NO-synthase L-NAME (25 mg/kg, 7 days) in the kidney ssue leads to a decrease in the ac vity of cathepsins В, L, H in lysosomal frac on with a parallel increase in non-lysosomal ac vity of cathepsin L, in the liver ssue leads to an increase in lysosomal ac vity of cathepsin H and a decrease in non-lysosomal ac vity of cathepsin L
The substrate of nitric oxide synthesis L-arginine (500 mg/kg, 10 days) only causes increased ac vity of cathepsin L in non-lysosomal frac on of liver ssue, leads to increased lysosomal ac vity of cathepsin H in kidney ssue, the lung ssue shows a significant increase in the ac vity of the all studied cathepsins in non-lysosomal frac on, accompanied by an increase in lysosomal ac vity of cathepsins B and H
Cystein proteinases and their inhibitors in extracellular fluids: marker for diagnosis and prognosis in cancer

Summary

МАТЕРИАЛЫ И МЕТОДЫ

Исследование выполнено на 32 конвенциональных половозрелых крысах-самцах линии Wistar. Для моделирования дефицита синтеза оксида азота (Эксперимент 1, n = 8) осуществляли внутрибрюшинное введение неселективного ингибитора NO-синтазы N-нитро-L-аргининметилового эфира (L-NAME, «Sigma», США) в дозе 25 мг/кг [10] в виде водного раствора 1 раз в сутки в утренние часы ежедневно в течение 7 дней. Моделирование изменения уровня синтеза оксида азота субстратом NO-синтазы (Эксперимент 2, n = 8) осуществляли путем внутрижелудочного введения раствора L-аргинина («Sigma», США) на 0,9% растворе NaCl в дозе 500 мг/кг [11] 1 раз в сутки до утреннего кормления ежедневно в течение 10 дней. Полученный после центрифугирования при 20 000 g супернатант представлял собой неседиментируемую (цитоплазматическую) фракцию гомогената ткани и использовался в этом качестве для измерения изучаемых показателей. Поскольку отмечалось отсутствие согласия большинства данных с нормальным распределением, в качестве характеристик использовали медиану (Ме), верхний и нижний квартили (Q1и Q3 соответственно), результаты представляли в формате Ме [Q1; Q3], для оценки статистической значимости различий независимых выборок использовали ранговый критерий Манна–Уитни (U-тест)

РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ

Катепсин H