Effect of hydrocortisone on alkaline proteinase activity in rat target organs

Сергеев, П. В.; Ogurtsov, S. I.; Dukhanin, A. S.; Mitroshina, S. Yu.

doi:10.1007/bf00837656

Bull Exp Biol Med

1992

DOI: 10.1007/bf00837656

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Effect of hydrocortisone on alkaline proteinase activity in rat target organs

П. В. Сергеев¹,

S. I. Ogurtsov²,

A. S. Dukhanin³

et al.

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1998

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“…The mechanism of this effect is not clear. Glucocorticoids in doses causing skeletal muscle atrophy increase the activity of proteolytic enzymes with optimum effect at alkaline pH [7].Previously, we demonstrated that dexamethasone (DM), a synthetic glucocorticoid, increases thymic alkaline protease (AP) activity in animals [5,6]. Proteolytic activity of homogenate depended on pH of incubation medium.…”

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Effect of dexamethasone on activity of alkaline proteases and thymocytic pH

et al. 1998

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The effect of dexamethasone on activity of "alkaline proteases and intracellular p H of rat thymocytes is studied. Dexamethasone increases the activity of alkaline proteases in the thymus. Intracellular pH changes under the effect of the hormone in two phases both in vivo and in vitro. Possible mechanism of rapid acidification of the cytoplasm followed by its alkalization is discussed.Key Words: dexamethasone, thymocytes, intracellular pH; alkaline proteases; phenylmethyl- sulfonyl fluorideClhfical use of glucocorticoid hormones and their synthetic analogs involves many complications. One of the effects of steroids with glucocorticoid activity is lymphoid tissue catabolism and involution of the thymus associated with it. The mechanism of this effect is not clear. Glucocorticoids in doses causing skeletal muscle atrophy increase the activity of proteolytic enzymes with optimum effect at alkaline pH [7].Previously, we demonstrated that dexamethasone (DM), a synthetic glucocorticoid, increases thymic alkaline protease (AP) activity in animals [5,6]. Proteolytic activity of homogenate depended on pH of incubation medium. In this study we investigated the effect of DM on intracellular pH(pHi) of thymocytes and AP activity. MATERIALS AND METHODSThymus of male Wistar rats weighing 150-200 g was examined. After decapitation, the thymus was placed in the medium containing 0.14 M NAG1, 5 mM KCI, t mM Mg2SO~) 4 mM NaHCO v 0.5 mM KH2PO4, 0.4 mM Na2HPO 2 12xH20, 5 mM glucose, pH 7.2. Cells were obtained by careful disintegration of the thymus with Teflon brushes, and suspension was then filtered through a Kapron filter. Suspension of thymocytes (2x107 cell/ml)was stored as a thin (no more than 3 mm) layer at 37~ Viability of cells was assessed by Tie/pan Blue absorption [4]. The number of viable cells during incubation in vitro was 90-92%.For assessing protease activity, 1 ml cell suspension was centrifuged at 1000g for 5 rain. The precipitate was resuspended in 350 ~tl 0.05 M Tris-HCI buffer (pH 8.5) with 0.14 M NaCI. The cells were destroyed by homogenization. AP activity was estimated by azocasein (Sigma) hydrolysis 1101. 350 I.d homogenate was incubated for 30 min at 37"C, then 50 M fresh azocasein solution (2 mg/ml) in 0.05 M Tris-HCl buffer was added, and the mixture was incubated at 370C for 60 min. The reaction was stopped by adding 400 pJ cold 10% aqueous solution of trichloroacetic acid. The samples were kept on the cold (0-4"C) for 10 min and then centrifuged at 3000g for 15 min. Light absorbance of the supernatant was measured at 366 nm in a CPC-2CP photocolorimeter.Intracellular (pHi) was measured as described previously [1]. Cells were stained with fluorescein diacetate, which was "added to a fmal concentration of 10 -s M, and cells were incubated at 37~ for 5 min.

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Effect of dexamethasone on activity of alkaline proteases and thymocytic pH

et al. 1998

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Effect of hydrocortisone on alkaline proteinase activity in rat target organs

Cited by 1 publication

References 4 publications

Effect of dexamethasone on activity of alkaline proteases and thymocytic pH

Effect of dexamethasone on activity of alkaline proteases and thymocytic pH

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