Heme oxygenase (HO) catalyzes the first and ratecontrolling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time-and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0·1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.
The present work aims to develop a growth medium to render a wild-type strain of Saccharomyces cerevisiae permeable to the antifungal drug Brefeldin A. In the current study, a synthetic medium containing 0.1% L-proline and supplemented with 3.0 × 10 −3 % SDS is employed. When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine. Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases. INTRODUCTIONBefore their delivery to the plasma membrane (PM), the different permeases involved in amino acid transport, like most of the membrane proteins, enter the membrane of the endoplasmic reticulum (ER). They then proceed through the protein secretory pathway of the ER, via the Golgi complex (GC) and exocytic vesicles, until they finally reach the PM [1].A very useful agent for investigating permease transport through the secretory pathway is the antifungal agent, Brefeldin A (BFA), which reversibly blocks the transport of proteins from the ER to the Golgi [2,3,4]. This drug can be used to create a temporary block in transport, allowing accumulation of permeases in the ER and depletion of these permeases downstream. In addition, when the BFA block is present, loss of permease molecules from the PM through endocytosis can be studied independent of their replacement via the secretory pathway. Moreover, release of the BFA block would permit the investigation of the dynamics of replacing the permeases in the depleted membrane. Because wild-type yeast has a very low apparent permeability to BFA, previous investigations have used strains bearing the erg6 mutation that blocks the final methylation reaction in ergosterol biosynthesis. The lack of ergosterol in the PM changes the permeability properties of the membrane and renders cells sensitive to several inhibitors, including BFA and the dye, crystal violet (CV) [2]. These changes appear to be at least partly due to decreases in activity of multidrug resistance pumps such as Pdr5p [5].There are several disadvantages of using the erg6 mutation to obtain BFA sensitivity. The mutation itself causes a marked increase in permeability to sodium and lithium ions [6]. Efficiency of genetic transformation is lowered dramatically, and sexual conjugation is also greatly reduced. Moreover, transport of tryptophan is lowered substantially [7].We have developed a simple method for obtaining BFA sensitivity without requiring the introduction of erg6. Because the method requires no genetic manipulation, it can be applied to wild-type cells and to strains already bearing various mutations related to secretion, to altered amino acid transport, and to modified permease turnover. The method depends upon the use of an SDS-supplemented synthetic growth medium in which the wild-type strain MMY2 presents increased sensitivity to BFA. At appropriate concentrations, BFA inhibits grow...
Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.
Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.
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