Rafael Peñafiel

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).

Gender-related differences in carnosine, anserine and lysine content of murine skeletal muscle

Summary.The aminoacyl-imidazole dipeptides carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-1-methyl-histidine) are present in relatively high concentrations in excitable tissues, such as muscle and nervous tissue. In the present study we describe the existence of a marked sexual dimorphism of carnosine and anserine in skeletal muscles of CD1 mice. In adult animals the concentrations of anserine were higher than those of carnosine in all skeletal muscles studied, and the content of aminoacyl-imidazole dipeptides was remarkably higher in males than in females. Postnatal ontogenic studies and hormonal manipulations indicated that carnosine synthesis was up-regulated by testosterone whereas anserine synthesis increased with age. Regional variations in the concentrations of the dipeptides were observed in both sexes, skeletal muscles from hind legs having higher amounts of carnosine and anserine than those present in fore legs or in the pectoral region. The concentration of L-lysine in skeletal muscles also showed regional variations and a sexual dimorphic pattern with females having higher levels than males in all muscles studied. The results suggest that these differences may be related with the anabolic action of androgens on skeletal muscle.

Antizyme Inhibitor 2 (AZIN2/ODCp) Stimulates Polyamine Uptake in Mammalian Cells

One of the processes that regulate intracellular levels of polyamines in mammalian cells is polyamine uptake. We have measured polyamine uptake in COS7 cells for putrescine, spermidine, and spermine, obtaining Km values of 4.5, 1.0, and 0.8 μm, respectively. Treatment of nonconfluent cells with cycloheximide stimulated polyamine uptake and prevented the inhibitory effect found in cells preloaded with polyamines, suggesting the existence of a feedback repression mechanism mediated by antizymes. Transient transfected cells with mutated antizyme forms of AZ1, AZ2, and AZ3, which do not require frameshifting, showed a total blockade of polyamine uptake. Transfection of COS7 cells with mouse or human AZIN2, a novel member of the antizyme inhibitor family, recently characterized by our group, markedly stimulated polyamine uptake and counteracted the action of any of the three antizymes in co-transfected cells. The stimulatory effect of AZIN2 on polyamine uptake was abrogated when the putative antizyme binding sequence, formed by residues 117–140 in AZIN2, was deleted. Real time reverse transcription-PCR analysis of antizyme inhibitor transcripts revealed that in brain and testes AZIN2 is more expressed than AZIN1, especially in the testes where the relative expression was about 25-fold higher. Collectively, our results clearly indicate that AZIN2 affects polyamine homeostasis not only by increasing ornithine decarboxylase activity but also by stimulating polyamine uptake, through negating the inhibitory effect of the antizymes. This finding may have physiological relevance, mostly in testes where AZ3 and AZIN2 are mainly expressed.

Antizyme inhibitor 2: molecular, cellular and physiological aspects

Polyamines are small organic polycations essential for cell proliferation and survival. Antizymes (AZs) are small proteins regulated by polyamines that inhibit polyamine biosynthesis and uptake in mammalian cells. In addition, antizyme functions are also regulated by antizyme inhibitors, homologue proteins of ornithine decarboxylase lacking enzymatic activity. There are two antizyme inhibitors (AZIN), known as AZIN1 and AZIN2, that bind to AZs and negate their effects on polyamine metabolism. Here, we review different molecular and cellular properties of the novel AZIN2 with particular emphasis on the role that this protein may have in brain and testis physiology. Whereas AZIN1 is ubiquitously found in mammalian tissues, AZIN2 expression appears to be restricted to brain and testis. In transfected cells, AZIN2 is mainly located in the endoplasmic reticulum–Golgi intermediate compartment and in the cis-Golgi network. AZIN2 is a labile protein that is degraded by the proteasome by a ubiquitin-dependent mechanism. Regarding its physiological role, spatial and temporal analyses of AZIN2 expression in the mouse testis suggest that this protein may have a role in spermiogenesis.

Equilibrium between active and inactive forms of rat liver ornithine decarboxylase mediated by L-ornithine and salts

Ornithine decarboxylase Polyamine Association equilibrium Rat liver Ionic strength

Diazepam potentiates the positive inotropic effect of isoprenaline in rat ventricle strips: role of cyclic AMP

The responses of the electrically driven right ventricle strip of the rat heart to isoprenaline and other cyclic AMP-related inotropic agents were recorded in the absence and in the presence of diazepam. Isoprenaline, in concentrations ranging from 10 nM to 1 microM, significantly increased, in a concentration-dependent manner, the contractile force in this preparation. Diazepam (10 microM) produced a leftward shift in the isoprenaline concentration-response curve and significantly reduced its EC50. Higher concentrations of diazepam (100 microM) produced no further shift, but reduced the maximum of the concentration-response curve of isoprenaline. Forskolin (0.5-10 microM), which directly stimulates adenyl cyclase, also produced a concentration-dependent increase in cardiac contractility. Diazepam (10 microM) displaced to the left the concentration-response curve for forskolin and reduced its EC50. The cyclic AMP analogous dibutyryl cyclic AMP (0.1-1 mM) produced concentration-dependent positive inotropic effects which were not significantly modified in the presence of diazepam (10 microM). Diazepam (10 microM) significantly enhanced the cyclic AMP production induced by isoprenaline (0.1 microM) and forskolin (10 microM) by about 136% and 35% respectively. These results indicate that diazepam potentiates the positive inotropic effect induced by beta-adrenoceptor agonists, probably by increasing cyclic AMP production induced by these agents.

Expression of antizyme inhibitor 2 in male haploid germinal cells suggests a role in spermiogenesis

Recently, we have found that the antizyme inhibitor 2, a novel member of the antizyme binding proteins related to polyamine metabolism, was expressed mainly in the adult testes, although its function in testicular physiology is completely unknown. Therefore, in the present work, the spatial and temporal expression of antizyme inhibitor 2, and other genes related to polyamine metabolism were studied in the mouse testis, in an attempt to understand the role of antizyme inhibitor 2 in testicular functions. For that purpose, the temporal expression of different genes, during the first wave of spermatogenesis in postnatal mice, was studied by real-time RT-PCR, and the spatial distribution of transcripts and protein in the adult testis was examined by both RNA in situ hybridization and immunocytochemistry. The results indicated that antizyme inhibitor 2 was specifically expressed in the haploid germinal cells, similarly to antizyme 3, the testis specific antizyme. Conversely, ornithine decarboxylase mRNA was mainly found in the outer part of the seminiferous tubules where spermatogonia and spermatocytes are located. Functional transfection assays and co-immunoprecipitation experiments corroborated that antizyme inhibitor 2 counteracts the negative action of antizyme 3 on polyamine biosynthesis and uptake. All these results indicate that the expression of antizyme inhibitor 2 is postnatally regulated and strongly suggest that antizyme inhibitor 2 may have a role in spermiogenesis.

Comparative study of tyrosinases from different sources: Relationship between halide inhibition and the enzyme active site

The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed.

The preovulatory rise of ovarian ornithine decarboxylase is required for progesterone secretion by the corpus luteum

Ovarian progesterone secretion during the diestrus stage of the estrous cycle is produced by luteal cells derived from granulosa and thecal cells after the differentiation process that follows ovulation. Our results show that blockade of the preovulatory rise of ovarian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by treatment with the specific inhibitor alpha-difluoromethylornithine (DFMO) leads to a significant decrease in the ovarian progesterone content and a dramatic fall in the plasma levels of this hormone during the following diestrus. The same inhibition was produced in spite of the fact that both luteinizing and follicle stimulating hormones were given concomitantly with DFMO. On the other hand, the acute rise in the plasma progesterone levels observed after administration of human chorionic gonadotropin to mice at different periods of the estrous cycle was not affected by DFMO administration. Our results indicate that although elevated levels of ODC are not required for acute ovarian steroidogenesis, the preovulatory peak of ovarian ODC activity observed in the evening of proestrus may be critical for the establishment of a constitutive steroidogenic pathway and progesterone secretion by the corpus luteum during the diestrus stage of the murine estrous cycle.

Postnatal Development of Ornithine Decarboxylase and Polyamines in the Mouse Kidney: Influence of Testosterone

The role of ornithine decarboxylase (ODC) and polyamines in kidney growth during the postnatal development of mice and the influence of testosterone were investigated. A marked sexual dimorphism in renal size was evident after the 3rd week of life, corresponding with the rise in circulating testosterone and the increase in renal ODC and urinary excretion of putrescine in male mice. Renal putrescine and spermidine gradually decreased during the first 3 weeks of life and did not correlate with ODC activity. Treatments with alpha-difluoromethylornithine, and ODC-specific inhibitor, and the antiandrogen flutamide during weeks 4 and 5 showed that both compounds decreased renal ODC activity but only flutamide impaired kidney growth, suggesting that renal growth in mice is regulated by androgens but is independent of the induction of ODC activity.