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In addition to playing a fundamental role in diverse processes, such as vision, growth and differentiation, vitamin A and its main biologically active derivative, retinoic acid (RA), are clearly involved in the regulation of testicular functions. The present study was undertaken to examine the direct effect of RA treatment on Leydig (TM-3) cells. TM-3 cells were cultured and treated with varying concentrations of RA for 24h. High doses of RA (1-20microM) induced a decrease in cell vitality and an increase in lipid peroxidation. RA treatment also induced a corresponding increase in apoptosis in the same cells in a dose-dependent manner. Apoptosis proceeded via the mitochondrial dependent pathway, as demonstrated by the release of cytochrome c, caspase-3 enzymatic activation and DNA fragmentation. Conversely, at physiological doses (0.1-500nM) RA did not increase lipid peroxidation or cell death and resulted in an increase of antioxidant enzyme activity.

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. The effects of RA are thought to be mediated by the nuclear retinoid receptors; however, not all the effects of RA can be explained by the nuclear receptor pathways. Indeed, retinoylation is another mechanism of action elicited by RA. In growing TM-3 Leydig cell cultures, the extent of retinoylation depends in a saturable manner on the initial concentration of 3H-RA, time and cell number. In addition, dose-response curves for RA-induced testosterone production and retinoylation are concomitant and exhibit a positive correlation. In the present study we demonstrate that RA is able to influence a retinoylation reaction on protein(s) probably involved on steroidogenesis.

All-trans-retinoic acid (atRA) is incorporated covalently into proteins of rat testes mitochondria. In this study, the effect of three diets with different fatty acid composition on the retinoylation of proteins of rat testes mitochondria has been investigated. Different groups of rats were fed on a basal diet supplemented with 15% of either coconut oil (CO), olive oil (OO) or fish oil (FO). We found that, when compared with CO, the binding of retinoic acid was decreased in FO- and OO-fed rats. Mitochondrial phospholipids composition was differently influenced by dietary treatments; minor changes were observed in fatty acid composition of phospholipids. Few differences were observed in the Arrhenius plots among the three groups of rats. Kinetic analysis revealed a decrease in the V (max) value in FO- and OO- as compared with CO-fed rats. No difference among the three groups were observed in the K ( M ) value. The retinoylation reaction was inhibited by 13-cis-RA and 9-cis-RA.

Inclusion compounds of eleven dihydropyridine drugs were formed and investigated for protection against photo-induced drug degradation. Formulations of cyclodextrins and liposomes were prepared and their photoprotective ability for the encapsulated drug was monitored. Drug photodegradation was spectrophotometrically followed during exposure of the formulations to light of a Xenon lamp. ICH guidelines for photostability testing were applied. A comparison with common pharmaceutical formulations revealed optimal protection for both formulations. The use of the liposome and cyclodextrin inclusion complexes resulted in a mean drug recovery of 77 and more then 90% respectively, after a light exposure until to 30 minutes with an intensity of 21 kJ x min(-1) m(-2). Lercanidipine and Manidipine only did not show a satisfactory increase of photostabilization in the studied supramolecular complexes, due to their low inclusion in both the systems.

The covalent incorporation of [(3)H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710+/-29 mean+/-SD) fmoles/8 x 10(4) cells at 37 degrees C. About 90% of [(3)H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65--75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC(50) values of 0.9 microM and 0.65 microM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 microM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 microM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [(3)H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [(3)H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells., ((Mol Cell Biochem 276: 55-60, 2005).)

The covalent incorporation of [(3)H]all-trans-retinoic acid into proteins has been studied in Leydig (TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570+/- 27 fmoles/8 x 10(4) cells at 37( composite function)C. About 95% of [(3)H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC(50) values of 0.6 and 1.2 microM respectively. Dibutyryl-cAMP and forskolin increased the retinoylation activity by 75 and 81% at 500 and 25 microM respectively. Also hCG increased the retinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydig cells the binding activity of [(3)H]RA was about the same that in the control, suggesting that the bond occurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (500 microM); on the contrary, there was an increase of the binding activity of about 60 and 50% respectively.

Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.