Regulatory Mechanism Of Arginase On Casein Synthesis In Bovine Mammary Gland

Our group has found that Arginine (Arg) has some active effects on the synthesis of casein in bovine mammary epithelial cells. Besides, Arg also has some effects on the activity of enzymes related to Arg metabolism. On the basis of the results above, we tried to make sure wether Arg regulates casein synthesis through enzymes. So we treated epithelial cells with inhibitors of arginase, nitric oxide synthase and ornithine decarboxylase, and then detected the activity of different enzymes related to Arg metabolism and casein concentration as well. We also infused arginase inhibitor (nor-NOHA) through jugular vein in Holstein cows and detected the milk yield and quality, feed digestibilities, blood routine and blood biochemistry.In the first experiment, Nω-hydroxy-nor-Arginine (Cayman, nor-NOHA,10006861) was used as arginase inhibitor,2-(difluoromethyl)ornithine (Sigma, DFMO, D193) was selected as ODC inhibitor and the 2-(2-Aminoethyl) isothiourea dihydrobromide (Sigma, A5879) was used as NOS inhibitor. Bovine mammary epithelial cells were cultured with growth medium which added with different inhibitors of enzymes. The activity of different enzymes related to Arg metabolism, the concentrations of casein in cells, and the proliferation of cells was detected. We found that the proliferation and casein synthesis was inhibited (P<0.05) in groups treated with nor-NOHA and DFMO. These results indicated that Arg was likely to regulate the synthesis of casein through "Arg-Orn-polyamine" pathway.On the basis of the first experiment, we added nor-NOHA, nor-NOHA+Orn, nor-NOHA+Arg into growth mediums to treat mammary epithelial cells in the second experiment. The activity of different enzymes related to Arg metabolism and concentration of casein in cells were determined at 24 h, the proliferation of cells was detected at 24 h,48 h and 72 h. We found the synthesis of casein and proliferation of cells were inhibited (P<0.05) after treating with nor-NOHA, and this result was not affected by adding Arg into growth medium (P>0.05). But the concentration of casein and proliferation of cells increased in group treated with nor-NOHA+Orn (P<0.05). In conclusion, the synthesis of casein was inhibited in bovine mammary cells when the "Arg-Orn" pathway was inhibited.In the third experiment, nor-NOHA and nor-NOHA+Arg was infused through jugular vein in Helstein cows. The activity of enzymes related in Arg metabolism in blood, the milk quality and milk yield, the casein concentration in milk and gene expression of casein and Arg transport carrier feed digestibilities, blood routine and blood biochemistry were detected. The result showed that, the activity of total arginase and ODC decreased significantly (P<0.05), the concentration of casein, protein and fat in milk reduced (P<0.05), and the casein expression in bovine mammary also decreased (P<0.05) after infusing nor-NOHA, but the activity of NOS, the expression of Arg transport carriers and milk yield was not affected (P>0.05). When supplementary perfusion with Arg, the activity of total arginase and ODC, the concentration of casein, protein and fat in milk did not change (P>0.05) compared with "nor-NOHA" group, but the activity of NOS, the expression of some Arg transport carriers and milk production increased (P<0.05). Considering all the results above, the casein synthesis was regulated by Arg through "Arg-Orn" pathway.The fourth experiment was aimed to determine the effects of nor-NOHA and nor-NOHA+Arg infusion on apparent digestibility of dietary nutrients, nitrogen cycle and blood index. The experimental design were the same as the 3rd experiment. We found that, the infusion of nor-NOHA had no effects on the digestibility of nutrients (P>0.05), increased the concentration of nitrogen in urine but decreased that in milk (P<0.05). However, when infused nor-NOHA+Arg, the digestibility of crude fat in diets increased (P<0.05), and the concentration of nitrogen in urine and milk return to normal level (P<0.05). Additionally, the blood routine index and the concentration of immune globulin were not affected by different treatments (P<0.05). The results indicated that, the different infusions had no effects on the health of dairy cows, while the "Arg-Orn" pathway was inhibited, the utilization ratio of nitrogen decreased.