Effects of unilateral orchidectomy on rat epididymal delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase

The effects of unilateral orchidectomy on the adult rat epidiymal testosterone metabolizing enzymes, delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, are investigated. Five weeks following unilateral orchidectomy, it is found that the activity of 3 alpha-hydroxysteroid dehydrogenase per organ is not altered, whereas delta 4-5 alpha-reductase activity decreased by more than 80% on the side of the orchidectomy. Neither accessory sex tissue weights, ventral prostate and seminal vesicles, nor the concentration of circulating testosterone, luteinizing hormone, follicle-stimulating hormone, or prolactin is altered by unilateral orchidectomy. These data indicate that (1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity can be maintained by circulating androgens and that (2) the major factor regulating delta 4-5 alpha-reductase activity is not a substance secreted by the testes into the peripheral circulation. It is suggested that a substance directly secreted into the epididymis by the testis regulates epididymal delta 4-5 alpha-reductase activity.     

The sexually differentiated delta 4-3-ketosteroid 5 beta-reductase of rat liver. Purification, characterization, and quantitation

delta 4-3-Ketosteroid 5 beta-reductase was purified from male rat liver cytosol. The purification scheme consisted of column chromatographies on hydroxylapatite and DEAE-Sepharose, chromatofocusing, and Sephadex G-75 gel filtration followed by sodium dodecyl sulfate-gel electrophoresis. The column chromatography steps gave a 100-fold purification and resulted in a 90% pure preparation as judged by sodium dodecyl sulfate-gel electrophoresis. Kinetic properties with 4-androstene-3,17-dione as substrate were established for the enzyme, and its activity regarding three other delta 4-3-ketosteroids, testosterone, progesterone, and cortisol, was investigated. The relative rates of reduction of these steroids were 1.0, 0.8, 0.7, and 0.62, respectively. The electrophoretically purified 5 beta-reductase, with an Mr of 38,000, was used for immunization of rabbits. The antiserum was shown to be monospecific as judged from immunoblotting of electrophoretically separated rat liver cytosolic proteins. Immunological reactive protein and enzymatic 5 beta-reductase activity co-purified in the chromatographic steps. The sex difference in enzyme activity, 0.26 versus 0.10 nmol of product/mg of proteins/min for males and females, respectively, was shown to be due to a difference in concentration of enzyme protein. The 5 beta-reductase was calculated to constitute 1% of the total cytosolic proteins in male livers, whereas the corresponding figure for female livers was 0.3%.     

Protein kinase C (PKC) delta regulates PKCalpha activity in a Syndecan-4-dependent manner

The phosphorylation state of Ser(183) in the cytoplasmic tail of syndecan-4 determines the binding affinity of the cytoplasmic tail to phosphatidylinositol 4,5-bisphosphate (PIP(2)), the capacity of the tail to multimerize, and its ability to activate protein kinase C (PKC) alpha. We sought to identify the kinase responsible for this phosphorylation and to determine its downstream effects on PKCalpha activity and on endothelial cell function. Among several PKC isoenzymes tested, only PKCalpha and -delta were able to specifically phosphorylate Ser(183) in vitro. However, studies in cultured endothelial cells showed that the phosphorylation level of syndecan-4 was significantly reduced in endothelial cells expressing a dominant negative (DN) PKCdelta but not a DN PKCalpha mutant. Syndecan-4/PIP(2)-dependent PKCalpha activity was significantly increased in PKCdelta DN cells, while PKCdelta overexpression was accompanied by decreased PKCalpha activity. PKCdelta-overexpressing cells exhibited a significantly lower proliferation rate and an impaired tube formation in response to FGF2, which were mirrored by similar observations in PKCalpha DN endothelial cells. These findings suggest that PKCdelta is the kinase responsible for syndecan-4 phosphorylation, which, in turn, attenuates the cellular response to FGF2 by reducing PKCalpha activity. The reduced PKCalpha activity then leads to impaired endothelial cell function. We conclude that PKCdelta regulates PKCalpha activity in a syndecan-4-dependent manner.