Renal reabsorptive capacity for alpha 2u-globulin in the adult male rat.

Adult male rats were maintained on 0, 5, 10, 15, and 20% casein diets to produce a series of animals having serum alpha 2u-globulin levels varying linearly from a normal of 31 micrograms/ml to a minimum of 13 micrograms/ml. In this way, it was possible to titrate endogenously the renal reabsorption and urinary excretion of this low molecular weight protein. The average maximal reabsorption rate (Tm) was established to be 9.7 micrograms/min and was reached at a renal filtered load (F alpha 2u) of 13.6 micrograms/min. These data were expressed in terms of a Tm:F alpha 2u ratio of 0.71. Below this value, the reabsorption declined from 70% to 50% of the F alpha 2u. Above 0.71, where F alpha 2u is less than the Tm, the reabsorption increased to 80-90%. It was observed that the fractional renal uptake of the alpha 2u-globulin varied linearly with the filtered load.     

Pretranslational regulation of alpha 2u-globulin in rat liver by growth hormone

Hypophysectomy is known to cause complete suppression of the hepatic synthesis alpha 2u-globulin. The effect of hypophysectomy on the synthesis of alpha 2u-globulin can be reversed by multiple hormone treatment. The role of pituitary growth hormone in the multihormonal regulation of alpha 2u-globulin in rat liver was examined in the hypophysectomized male rats with and without growth hormone supplementation. Daily treatment of hypophysectomized rats with 5 alpha-dihydrotestosterone, corticosterone, thyroxine, and growth hormone for 8 days caused about 80% recovery in the hepatic content of alpha 2u-globulin and its corresponding mRNA as determined by radioimmunoassay, in vitro translation, and liquid hybridization with a cloned cDNA probe. However, omission of growth hormone from the treatment regimen failed to raise hepatic alpha 2u-globulin and its mRNA to more than 5% of the normal control. The possible effect of growth hormone on the translation of the mRNA for alpha 2u-globulin was examined with cultured hepatocytes derived from growth hormone-deficient rats. Culture of these cells in the presence of growth hormone for 24 h did not turn on the synthesis of alpha 2u-globulin. These results indicate that growth hormone regulates the synthesis of alpha 2u-globulin by acting at a step antecedent to mRNA translation.     

Hormonal induction of alpha2u-globulin synthesis in isolated rat hepatocytes.

Hepatocytes freshly prepared with collagenase synthesize alpha 2u-globulin and other hepatic proteins in vitro at approximately the same rate throughout 30 h of incubation. The newly synthesized proteins are efficiently secreted into the medium throughout this period. That the secretion of proteins by hepatocytes is not due to cell leakage is shown by the fact that 30 micrometer colchicine prevents the appearance of labeled alpha2u-globulin and other proteins in the medium. Hepatocytes isolated from animals in different endocrine states synthesize alpha2u-globulin in vitro at rates consistent with the hormonal effects upon its in vivo biosynthesis. In vitro addition of androgens to hepatocytes isolated from castrated male rats evokes an elevation of general protein synthesis in these hepatocytes. Glucocorticoids, added in vitro, specifically induce and elevated rate of alpha2u-globulin synthesis.     

Synthesis and processing of the dimorphic forms of rat alpha 2u-globulin

alpha 2u-Globulin the androgen-dependent male rat urinary protein, can be resolved into two distinct molecular forms by SDS-polyacrylamide slab gel electrophoresis. These two forms designated as alpha 2u-A (M, 18,800) and alpha 2u (Mr 18,100) are found both in urine and in the liver cells. Translation of rat liver mRNA in the rabbit reticulocyte lysate produced two preprotein forms of alpha 2u-globulin, designated as alpha 2uA' (Mr 20,300) and alpha 2uB' (Mr 19,600). Cell-free translation of rat liver mRNA in the presence of dog pancreas microsomal membrane or in Xenopus oocytes produced the two processed forms of alpha 2u-globulin (alpha 2uA and alpha 2uB). Quantitation of alpha 2uA and alpha 2uB within the in vitro translation products of the hepatic mRNA from albino rats of Yale, Sprague-Dawley and Fischer strains showed genetic differences in the proportion of translatable mRNA for alpha 2uA and alpha 2uB. The ratio of alpha 2uA: alpha 2uB in the translation products of liver mRNA from Yale rats was found to be 1:2.5 while in the case of both Sprague-Dawley and Fischer rats, the ratio was 1:4. A small portion of the alpha 2uA and alpha 2uB synthesized in the cultured hepatocytes, in the Xenopus oocytes or in the membrane-supplemented cell-free system appeared as two additional forms, designated as alpha 2uA" (Mr 21,200) and alpha 2uB" (Mr 20,600). Unlike alpha 2uA and alpha 2uB both alpha 2uA" and alpha 2uB" were found to bind to Con A-Sepharose, suggesting their glycoprotein nature.     

Transcortin and alpha 2u-globulin messenger RNA activities during turpentine-induced inflammation in the rat

Previously we have shown that the serum concentration of transcortin and alpha 2u-globulin markedly decreases during turpentine-induced inflammation. In the present study transcortin and alpha 2u-globulin mRNA from healthy rats and from animals with inflammation was translated in a rabbit reticulocyte lysate system. Female rats had higher levels of translatable transcortin mRNA than male animals and the level of mRNA for transcortin and alpha 2u-globulin decreased rapidly during inflammation. These results indicate that the sex difference in the serum level of transcortin and the changes in serum transcortin and alpha 2u-globulin during inflammation are mainly determined by differences in the mRNAs in the liver.     

Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line

A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.     

Renal reabsorption of low molecular weight proteins in adult male rats: alpha 2u-globulin

Urinary proteins are reabsorbed by the renal tubule cells by two processes, the first for high molecular weight (HMW) and the second for low molecular weight proteins (LMW). The purpose of this report is to establish that alpha 2u-globulin, the sex-dependent, major urinary protein of the adult male rat, is reabsorbed in the kidneys by the general mechanism for LMW proteins. Parameters such as clearance rates were determined to show that alpha 2u is reabsorbed by a process comparable to that for lysozyme. The aminoglycoside, gentamicin, was observed to inhibit the reabsorption of alpha 2u in a dose-dependent fashion. It increased the alpha 2u excretion rate from 4.2 to 13.5 micrograms/min; the clearance was increased from a normal of 0.33 to 0.91 ml/min. The excretion rate for alpha 2u was also increased by the injection of lysozyme from a normal of 7.4 to 18.1 micrograms/min. The effect of lysozyme was dose-dependent and reversible. Although gentamicin and lysozyme each increased the excretion of alpha 2u, they had no effect on albumin. Both were equally effective as inhibitors of alpha 2u reabsorption and were 80% as effective as sodium maleate. It is suggested that alpha 2u is reabsorbed by a mechanism which is shared with other LMW proteins. Furthermore, this process is independent of the one which serves to translocate HMW proteins such as albumin.     

Studies on the mode of oestrogenic inhibition of hepatic synthesis of alpha2u-globulin and its corresponding messenger ribonucleic acid in rat liver.

1. The possible mechanism of the oestrogenic inhibition of the androgen-dependent synthesis of alpha2u-globulin in rat liver was explored by a correlative study of the amounts of alpha2u-globulin, its corresponding mRNA and circulating testosterone in oestrogen-treated male rats. 2. Daily treatments of mature male rats with oestradiol-17beta (10 microgram/100g body wt.) decreased and ultimately stopped the hepatic synthesis of alpha2u-globulin as determined by both hepatic and urinary concentrations of the protein. The oestrogen-mediated decrease in the hepatic synthesis of alpha2u-globulin was correlated with a decrease in the mRNA for this protein. 3. Withdrawal of oestrogen resulted in the recovery of alpha2u-globulin synthesis and an increase in mRNA for alpha2u-globulin. 4. At higher doses of oestradiol-17beta (50 microgram/100g body wt.), synthesis of alpha2u-globulin was totally suppressed. In addition, this treatment resulted in an extended period of androgen-insensitivity during which treatment with androgens induced synthesis of neither alpha2u-globulin nor its corresponding mtrna. 5. it is concluded that the oestrogenic inhibition of alpha2u-globulin synthesis is mediated by an oestrogen-dependent decrease in the hepatic content of translatable mRNA for alpha2u-globulin.     

Electrophoretic variants of alpha 2u-globulin in the livers of adult male rats: a possible polymorphism

Two-dimensional polyacrylamide gel electrophoresis has been used to examine the microsomal fractions from the livers of 32 adult male Alpk/AP (Wistar-derived) rats for the presence of alpha 2u-globulin variants of differing isoelectric point. Three major such isoelectric variants are described. Different combinations of these three forms were found in the population examined, with one-half of the animals expressing all three variants in approximately equal proportions and with one variant being present in all the animals examined. An understanding of the relevance of such different alpha 2u-globulin profiles to the individual animal must now await the assignment of a biological role for alpha 2u-globulin.     

Glucocorticoid induction of hepatic alpha2u-globulin synthesis and messenger RNA level in castrated male rats in vivo.

alpha2u-Globulin is a male rat liver protein of Mr = 20,000 which is synthesized in the liver of adult male rats, secreted into the serum, and excreted in the urine. Its function is unknown. The hepatic synthesis of this protein is under complex hormonal control. We had previously shown that castration of male rats diminishes hepatic alpha2u-globulin synthesis and the level of its mRNA, and that administration of androgen to these castrated animals results in the reinduction of the synthesis of this protein and the level of its mRNA. We now report that alpha2u-globulin synthesis and the level of its mRNA can be fully reinduced in castrated males by administration of glucocorticoid alone. This induction is much more rapid than the androgenic induction and is inhibited by the glucocorticoid antagonist progesterone. Administration of glucocorticoid to intact male animals does not induce alpha2u-globulin synthesis above normal levels; however, if alpha2u-globulin synthesis has been depressed in intact male rats by pretreatment with estrogen or cyproterone acetate, the level of this protein can be reinduced by administration of glucocorticoids. The implications for the control of alpha2u-globulin gene expression are discussed.     

Localization of alpha 2u-globulin within protein droplets of male rat kidney: immunohistochemistry using perfusion-fixed, GMA-embedded tissue sections

We investigated the light microscopic subcellular localization and quantitation of alpha 2u-globulin (alpha 2uG) in male rat kidney after 2,2,4-trimethylpentane exposure using a monoclonal antibody to alpha 2uG. Slices of perfusion-fixed kidney were cold-processed in glycolmethacrylate and the antigen localized after an avidin-biotin-horseradish peroxidase procedure with Hanker-Yates reagent as the chromagen. Light microscopic examination revealed resolution comparable to low-magnification electron microscopy, with excellent morphological detail of tissue architecture, including subcellular localization of alpha 2uG within lysosomes of P2 segment cells of the proximal tubule epithelium. The anatomical relationship between alpha 2uG and TMP-induced protein droplets observed after staining with Lee's methylene blue-basic fuchsin was studied using serial sections. Image analysis of selected P2 segments in treated and control rats revealed a high correlation between subcellular localization of alpha 2uG and protein droplet deposition in the cytoplasm of P2 segment cells of the proximal tubule epithelium. Quantitative morphometry of alpha 2uG-stained proximal tubule epithelium 72 hr after treatment with 50 mg/kg 2,2,4-trimethylpentane p.o. demonstrated a 1.5- to 2-fold increase in staining area of tubules from treated rats compared with controls. Similar increases of Lee's methylene blue-basic fuchsin-stained protein droplets were also observed, but quantitative morphometry of the protein droplets was technically more difficult owing to a lower staining contrast between droplets and the surrounding cytoplasm. This immunohistochemical procedure provides a valuable technique for further studies on the pathological role of alpha 2uG in protein droplet nephropathy of male rats induced by many environmental chemicals, and demonstrates the value of cold glycolmethacrylate processing to improve morphological detail.