Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney

Ornithine decarboxylase, the first enzyme of the polyamine biosynthetic pathway, is induced by androgens in the mouse kidney. We have isolated from a kidney cDNA clone bank two plasmids, pODC1440 and pODC934 , that contain different cDNA inserts corresponding to ornithine decarboxylase mRNA. Identification was based upon the ability of plasmid-specific mRNAs to encode a 53,000-dalton polypeptide that reacts with antibody to purified mouse kidney ornithine decarboxylase. Plasmid pODC1440 hybridizes predominantly to a mRNA that is 2.1 kilobases (kb) long and is induced about 20-fold in the kidneys of female mice treated with testosterone. Plasmid pODC934 hybridizes to mRNAs of lengths 2.2 and 2.6 kb, which are induced about 8-fold by testosterone. There are probably no more than 1-2 copies of the pODC1440 -specific sequence in the mouse genome, while there may be as many as 12 copies of the pODC934 -specific sequence. That the two plasmids correspond to different ornithine decarboxylase mRNAs is suggested by two observations. First, several mouse strains express normal levels of pODC934 -specific RNA and little or no pODC1440 -specific RNA; furthermore, pODC934 -specific RNA is expressed in several tissues while pODC1440 -specific RNA is kidney-specific. Thus, androgen-mediated stimulation of kidney ornithine decarboxylase activity levels involves alterations in the concentrations of at least two distinct mRNAs.     

Dietary induction of ornithine decarboxylase in male mouse kidney

In male mouse kidney, ornithine decarboxylase (ODC) is induced after feeding, and the induction depends on dietary protein content. 24 h after feeding with 50% casein-containing meal, ODC activity and amount of immunoreactive ODC protein increased more than 10-fold, ODC mRNA level increased 2-fold, and the ODC half-life extended 7-fold. The renal ODC induction after feeding is, therefore, due mainly to stabilization of ODC protein. Urinary excretion of putrescine increased in response to the ODC induction, but the renal polyamine contents scarcely changed. Consistently, the level of antizyme, a polyamine-inducible protein, determined as the ODC-antizyme complex level, scarcely changed after feeding, and the antizyme/ODC ratio in the kidney largely decreased, resulting in the stabilization of ODC protein. The present results suggest that the strong excretion system of the kidney for newly synthesized polyamines enables renal ODC escape from antizyme-mediated feedback regulation.     

Very fast purification of ornithine decarboxylase with high yield from mouse kidney and generation of a monoclonal antibody

Based on methods for ornithine-decarboxylase purification published previously we developed an improved procedure for purification of the enzyme from the kidneys of testosterone-treated NMRI mice. Advantages of the new procedure are, that inactivation of the enzyme during purification is largely reduced by fast methods for purification and by the use of proteinase inhibitors. That way we got pure ornithine decarboxylase within 60 h with a yield of about 70%. A part of the highly purified ornithine decarboxylase was used for the generation of monoclonal antibodies.     

Androgen and estrogen stimulation of ornithine decarboxylase activity in mouse kidney

In the present work, the activity of mouse renal ornithine decarboxylase (ODC) from CBA female mice was used as a biological marker to detect (anti)androgenic activity of different groups of endocrine disruptors and steroids. Daily injections of testosterone or dihydrotestosterone (DHT) into 60 day old female mice for 4 days increased renal ODC activity in a dose-dependent manner that reached up to 100-fold (testosterone) or 250-fold (DHT) above the baseline when the highest dose, 200 microg/mouse, was used. Administration of flutamide concurrently with testosterone (75 microg/mouse) caused a potent decrease of ODC induction in a dose-dependent manner, suppressing the enzyme activity at the doses of 0.1 and 0.5 mg/mouse by about 88 and 95%, respectively. In contrast, estradiol at the doses of 0.5 and 1 mg/mouse induced a significant stimulation of renal ODC activity in a dose-dependent manner when it was given alone or in combination with testosterone. Using a sensitive increase in ODC activity in response to androgens as an end point, we did not detect an antiandrogenic effect of several antiandrogens, such as cyproterone acetate, spironolactone, p,p'DDE and vinclozolin. Also, none of these antiandrogens were able to change the basal level of renal ODC activity, with the exception of cyproterone acetate that at a dose of 0.1 mg/mouse stimulated ODC activity. The data obtained suggest that mouse renal ODC from CBA females is not strictly androgen-specific and cannot be used for estimation of antiandrogenic effects of compounds having an affinity to different types of receptors.     

Multiple species of ornithine decarboxylase in mouse kidney. Effect of testosterone

Multiple species of ornithine decarboxylase were separated by chromatography of mouse kidney extract on DEAE-Sepharose CL-6B. The elution patterns of ornithine decarboxylase activity and immunoreactive enzyme protein in the kidneys of untreated and testosterone-treated male mice did not differ otherwise than in order of magnitude. The immunoblots of the chromatography fractions neither revealed any differences in enzyme subunit size between two experimental groups. These findings suggest that the stabilization of ornithine decarboxylase by androgens is not due to the molecular changes of enzyme protein.     

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.     

Steroid regulation of kidney histidine decarboxylase and ornithine decarboxylase levels in mouse kidney: effects of the mutation testicular feminization, Tfm

1. Testosterone represses kidney histidine decarboxylase levels in both normal male and female mice. Tfm/Y mutant mice lack an androgen receptor and are phenotypically female. It has been suggested that the testosterone induction of HDC levels in these animals is a result of aromatisation to oestrogens in the absence of the androgen receptor; the oestrogens then induce the enzyme. 2. It is shown that the induction of HDC in Tfm/Y mice is specific to testosterone and not other androgens and can be mimiced by low doses of beta-oestradiol in normal female mice. 3. Analysis of Tfm/+ mice indicates that the testosterone induction effect is a function of individual kidney cells.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

Entamoeba histolytica: purification and characterization of ornithine decarboxylase

Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.     

Effects of testosterone on renal ornithine decarboxylase and kidney function

Testosterone injection caused a 2,000% increase in renal ornithine decarboxylase activity in intact male mice. A single injection of testosterone produced the same effect as repeated injections. The response was dose-dependent and could be blocked by actinomycin, diaminopropane, and cadaverine. Cycloheximide and putrescine had no inhibitory effect. Renal ODC response to arginine vasopressin was altered after castration; however, urine specific gravity and serum osmolality were unaffected by changes in renal ornithine decarboxylase activity.     

Heterogenous expression of ornithine decarboxylase gene in the proximal tubule of the mouse kidney following testosterone treatment

The expression of the ornithine decarboxylase (ODC) gene in the mouse kidney following testosterone treatment was examined using in situ hybridization histochemistry. Testosterone (n=5) or vehicle (n=5) was subcutaneously injected (1 mg/animal) into male BALB/c mice (8 weeks in age) 14 h before sacrifice. Animals were sacrificed under ether anesthesia, their kidneys were removed and immediately frozen in liquid nitrogen. Frozen sections (10-m-thick) were cut on a cryostat. Sections were hybridized with ³⁵S-labeled sense or antisense RNA probe. The hybridization continued for 24 h at 50° C and emulsion autoradiography was subsequently performed. A marked increase in ODC mRNA was exclusively detected in the proximal tubule of the renal cortex in the testosterone-treated animals. The hybridization signal was greater in the outer portion of the proximal tubule than in the inner portion. No significant hybridization signal was detected either in the distal tubule, renal corpuscle or peritubular tissues. These results indicate that testosterone induces the expression of the ODC gene in the proximal tubule of the renal cortex, leading to the increase in ODC activity in the same region.