The effect of hormones on glucocorticoid binding capacity of rat liver cytosol.

The relationship between the glucocorticoid binding capacity of rat liver cytosol and the activity of tyrosine aminotransferase has been studied in adrenalectomized male rats. Bilateral adrenalectomy of male rats caused an increase within 3 days in the level of specific dexamethasone binding of liver cytosol accompanied by a rapid decrease in tyrosine aminotransferase activity. Known inducers of tyrosine aminotransferase were administered in vivo to test their effect on dexamethasone binding capacity, in order to determine whether the induction was by an indirect mechanism involving an increase in glucocorticoid binding capacity. Insulin, adrenalin, glucagon, dibutyryl cyclic AMP and oestradiol caused a significant increase in the activity of the enzyme, with no change in the specific dexamethasone binding. Tetracosactrin, a synthetic analogue of ACTH, had no effect on either parameter. It was concluded that the induction of tyrosine aminotransferase by the compounds tested was not mediated by an increase in glucocorticoid receptor activity.     

Subunit structure and autophosphorylation mechanism of casein kinase-TS (type-2) from rat liver cytosol

Type-2 casein kinase-TS (Ck-TS) purified to homogeneity from rat liver cytosol exhibits a molecular mass of 130000 daltons in non-denaturating media and a subunit composition consistent with an alpha 2 beta 2 heterotetramer. The quaternary structure of Ck-TS is not compromised by limited proteolysis with trypsin which converts the 38-kDa alpha subunit into 36-kDa (alpha') and 34-kDa (alpha") derivatives, inducing a parallel decrease of enzymatic activity. Since the 25-kDa beta subunit is unaffected under comparable conditions, the catalytic activity seemingly resides in the alpha subunits. The beta subunit, on the other hand, undergoes a very rapid phosphorylation upon incubation of Ck-TS with ATP/Mg2+: 0.8-1.5 mol P/mol Ck-TS are incorporated within 30 s. Such a fast autophosphorylation is neither prevented nor slowed down by the addition of a large excess of phosphorylatable substrates and takes place through an intra-molecular rather than inter-molecular process. This conclusion is supported by the following data. (a) The autophosphorylation rate is linearly proportional to the concentration of Ck-TS. (b) Thermally inactivated Ck-TS is not phosphorylated by catalytic amounts of active enzyme. (c) Basic polypeptides like protamine and polylysine stimulate the activity of Ck-TS toward phosphorylatable substrates while preventing the autophosphorylation reaction. Since the effectors that inhibit autophosphorylation also induce a remarkable decrease of the Km values for the protein substrates, the possibility is discussed that autophosphorylation might represent a regulatory device by which Ck-TS could be converted into a partially inactivated form exhibiting reduced affinity toward its endogenous targets.     

Effect of sex hormones on lipid peroxidation in the necrotic liver of rat

Influence of sex-hormones on hepato-toxicosis induced by 2 halogenalkanes viz. carbon tetrachloride and trichloroethylene has been studied in rats. Rate of peroxidative decomposition thus measured by determining malonaldehyde has shown the protective effects of testosterone on CCl4 toxicity but an additive effect on injury caused by trichloroethylene, whereas progesterone failed to offer protection against their toxicity. Modification in histopathological lesions thus caused by these hormones have also been studied and discussed.     

Calcium uptake and release by rat liver mitochondria in the presence of rat liver cytosol or the components of cytosol

Summary A study has been made of factors present in rat liver cytosol that might regulate the calcium content of mitochondria. A cytosol preparation containing all the components of molecular weight greater than 10,000 prevented uptake and caused early release of accumulated calcium. These effects were due to free long-chain fatty acids and their coenzyme A derivatives present in the cytosol, and these inhibitory effects were controlled by inclusion of Mg²⁺, carnitine, and adenosine triphosphate at physiological levels in the incubation medium. Palmitoyl carnitine was a good substrate for calcium uptake and did not cause release of calcium from mitochondria. A specific fatty acid-binding protein was found in cytosol which may be the intracellular transport protein for fatty acids.     

Acetaldehyde-dependent oxidation of glutathione catalyzed by rat liver cytosol

We have identified a novel reaction in which acetaldehyde promotes rat hepatic cytosolic catalysis of O2 consumption coupled with glutathione oxidation without apparent release of activated forms of O2. Acetaldehyde is not consumed in the reaction. The reaction (O2 consumption or oxidized glutathione production) is saturable with respect to varying glutathione (K'm congruent to 20-45 microM) but not at high acetaldehyde concentrations. However, activity in the range of acetaldehyde found in liver from alcohol metabolism (10-100 microM) appeared to be saturable (K'm congruent to 25-50 microM). Since neither acetaldehyde-dependent glutathione loss nor O2 consumption is detectable in guinea pig hepatic cytosol or hepatic cytosol from selenium-deficient rats, we propose that acetaldehyde interacts with glutathione peroxidase, converting the enzyme into a glutathione oxidase.     

The esterification of dolichol by rat liver microsomes.

The incubation of 1-[3H)dolichols with cell-free preparations from various rat tissues resulted in the formation of a labeled material which possessed the characteristics of synthetic dolichol palmitate. Rat liver microsomes were found to be a good source of the acyltransferase activity, and the properties of the reaction were investigated using microsomal preparations. The reaction did not require ATP, CoA, or Mg2+ and was stimulated by the addition of phosphatidylcholine. The esterification of dolichol appears to be similar to the esterification of retinol. The fact that the esterification of dolichol is not depressed even in the presence of a several-fold excess of retinol is evidence that the two reactions are catalyzed by different enzymes.     

Modulators of rat liver cytosol casein kinases 1 and 2

Rat liver cytosol casein kinases 1 and 2 are similarly activated by spermine and inhibited by caffeine. On the contrary they are differently affected by heparin and basic proteins. Low concentrations of heparin inhibited selectively the phosphorylation of casein by casein kinase 2 whereas protamine and histones inhibited specifically casein kinase 1. On the contrary the basic proteins stimulated slightly the activity of casein kinase 2 and released its inhibition by heparin. Increasing the concentration of casein substrate reversed both the inhibition of casein kinase 1 by protamine as well as that of casein kinase 2 by heparin. The data suggest the existence of modulators having either similar or opposite effects on each type of casein kinase.     

Transthyretin is up-regulated by sex hormones in mice liver

The chaperone function of alpha-crystallin is significantly affected in diabetes. Increased formation of advanced glycation end products (AGEs) is the likely cause. This study was aimed to investigate the effect of AGE crosslinks on the chaperone activity of alpha-crystallin and to show the effect of an AGEs crosslink breaker, phenacyl-4,5-dimethylthiazolium bromide (DMPTB). Recombinant alphaA-crystallin was prepared by expressing it in Escherichia coli and purified by size exclusion chromatography. Glycation of alphaA-crystallin was performed with 1-100 mM glucose-6-phosphate (G6P) as the glycating agent for a period of 1-15 days. To break AGE crosslinks, pre-glycated alphaA-crystallin was treated with 0.1-20 mM DMPTB for 3 days. Excess G6P and DMPTB were removed by gel filtration before performing additional experiments. AGEs and crosslinked proteins were estimated by measuring non-tryptophan fluorescence and by SDS-PAGE. Chaperone activity was determined with alcohol dehydrogenase as the target protein. With increasing duration of glycation and G6P concentration, chaperone activity of alpha-crystallin decreased. When pre-glycated alphaA-crystallin was treated with 5-20 mM DMPTB, a DMPTB concentration-dependent recovery of chaperone activity was seen. Lower concentrations, 0.1, 0.5, and 1.0 mM, of DMPTB also showed significant recovery of the chaperone activity. SDS-PAGE analysis after DMPTB treatment showed 40% decrease in crosslinked proteins and fluorescence scan indicated 30% decrease in AGEs. DMPTB is expected to regain alpha-crystallin chaperone activity and provide structural stability to other eye lens proteins that are in aggregation mode which emphasizes the clinical importance of the present finding.     

性激素对大鼠诱发肝癌中胎盘型谷胱甘肽S—转移酶（GST—P）表达的影响

Using Solt-Farber method for the induction of rat hepatocarcinoma, the changes of the activity of glutathione S-transferase (GST) and the content of placenta form GST (GST-P) were studied during hepatocarcinogenesis, then the effects of sex hormones on the hepatic expression of GST-P were observed using immunohistochemical method. The results showed that both GST activity and GST-P content began to increase at the 3rd week, and reached the highest level at the 5th week (Table 1). Therefore, the 5th week was selected for the study of GST-P expression in the livers of rats treated with different protocol (Fig. 1). It was found that GST-P was highly expressed in the livers of sham-castrated male rats after chemically induced hepatocarcinoma (PLATE I, Fig. 1A, Table 2). When estradiol was administrated to these rats, both the number and area of GST-P positive(+) foci decreased significantly (PLATE I, Fig. 1B, Table 2). While testosterone was administrated instead of estradiol, the decrease of the area but slight increase of the number of GST-P positive foci were found (PLATE I, Fig. 1C, Table 2). After orchiectomy, the areas of GST-P (+) foci in carcinogen treated liver of male rats were smaller than those in rats with sham-orchiectomy and same carcinogen treatment (PLATE I, Fig. 2A, B, Table 3). When the orchiectomized male rats were administrated with estradiol, the areas of GST-P (+) foci decreased further (PLATE I, Fig. 2C, Table 3). In contrast, after ovariectomy of the female rats, the areas of GST-P (+) foci in carcinogen treated livers were slightly increased as compared with those in the rats with sham-ovariectomy and same carcinogen treatment (PLATE I, Fig. 2D, E, Table 3). While the ovariectomized female rats were administrated with testosterone, the areas of GST-P (+) foci increased further (PLATE I, Fig. 2F, Table 3). Regardless of whether castrations were done or not, GST-P expression in livers of male rats induced hepatocarcinoma was higher than in livers of female rats (PLATE I, Fig. 2A, B, D, E, Table 3). These results indicated that estrogen may inhibit but androgen may promote the GST-P expression in the rat liver during hepatocarcinogenesis. This may be related to the higher incidence of liver carcinoma in male than in female.     

Influence of adrenalectomy and sex on the binding of 3-methylcholanthrene to cytosol macromolecules of rat liver

The retention of 3-methylcholanthrene (3-MC) in rat hepatic cytosol was significantly enhanced by adrenalectomy. In contrast, there was no significant difference in 3-MC retention in females as compared with males. 3-MC present in the cytosol fraction was bound to macromolecules and could be separated into three fractions by ion-exchange column chromatography.     

The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.     

Metabolism of 2-hydroxy(4-14C)estradiol by rat liver microsomes

The metabolism of ¹⁴C-labeled estradiol and 2-hydroxyestradiol by rat liver microsomes has been compared under a variety of experimental conditions. An active liver microsomal system as well as NADPH was required to obtain high percentage yields of water-soluble metabolites from both steroids. Spermine stimulated the formation of polar products in both cases but the sex-difference in metabolism observed with estradiol was less marked with 2-hydroxyestradiol as substrate.     

Formation of acids shorter than palmitic by rat liver cytosol

Particle-free liver supernatant of rats maintained on stock diet synthesizes fatty acids of average chain lengths 9–11 carbon atoms when the protein concentration is high while palmitic acid is the main product when protein concentration is low. This ability to synthesize acids shorter than palmitic is lost on purification of fatty acid synthetase or by starvation of the rats followed by the ingestion of a high sucrose diet. The results are consistent with the presence of a factor in the cytosol, similar to that in lactating mammary glands, which shortens the chain length of the products of fatty acid synthetase.