Short communication: Arginase inhibition reduces the synthesis of casein in bovine mammary epithelial cells

Abstract

The main purpose of this work was to determine the effect of arginase inhibition via N-hydroxy-nor-l-arginine (nor-NOHA) on casein synthesis in bovine mammary epithelial cells (BMEC). Passage 2 BMEC isolated from dairy cows were seeded to 6-well plates and randomly divided into 4 treatments: (1) control [Dulbecco's modified Eagle medium:Nutrient Mixture F-12 medium (DMEM/F12)]; (2) nor-NOHA (DMEM/F12 + 1 mmol/L nor-NOHA); (3) nor-NOHA + arginine (DMEM/F12 + 1 mmol/L nor-NOHA + 3.2 mmol/L Arg); and (4) nor-NOHA + ornithine (DMEM/F12+ 1 mmol/L nor-NOHA + 1 mmol/L Orn). Then, we determined the activity of enzymes related to Arg metabolism and casein synthesis in BMEC and the proliferation of cells. The addition of nor-NOHA reduced the activity of arginase and ornithine decarboxylase but had no effect on the activity of nitric oxide synthase, and these responses were the same at the gene expression level. In addition, supplementation of nor-NOHA in BMEC reduced cellular proliferation and casein synthesis. Addition of Arg to nor-NOHA resulted in cellular proliferation and casein synthesis similar to that of nor-NOHA alone. In contrast, addition of Orn to the medium with nor-NOHA increased the synthesis of casein and cellular proliferation compared with Nor-NOHA. In conclusion, suppression of the Arg-arginase-Orn pathway reduced casein synthesis and cellular proliferation, which indicated that this pathway is an important regulator of the synthesis of casein in BMEC.

Inhibition of arginase via jugular infusion of Nω-hydroxy-nor-l-arginine inhibits casein synthesis in lactating dairy cows

“…In the present study, the concentration of α-casein and β-casein in milk, and gene expression of CSN1S1, CSN1S2, and CSN2 in mammary gland also decreased when arginase activity was reduced by nor-NOHA. These results were similar to previous in vitro findings (Wang et al, 2017).…”

“…N ω -Hydroxy-nor-l-arginine is a natural and powerful inhibitor of arginase and has no effects on the activity of NOS (Custot et al, 1997). Similar to a previous in vitro study (Wang et al, 2017), the activity of arginase in coccygeal artery blood and the expression of ARG1 and ARG2 in mammary tissue decreased after infusing nor-NOHA for 7 d. Reid et al (2007) infused nor-NOHA in mice and reported lower activity of total arginase in blood. In addition to the effects on the activity of arginase in blood, nor-NOHA was also reported to had efficient negative effects on the activity of arginase in different tissues like mammary artery, heart, kidney, and aorta (Kropf et al, 2005;Shemyakin et al, 2012;Rodríguez-Gómez et al, 2015).…”

“…Decreasing activity of the Arg-Orn pathway with an arginase-specific inhibitor led to a decrease in the synthesis of casein and mammary cell proliferation. However, addition of Orn to the BMEC cultured with arginase inhibitor resulted in a recovery of casein synthesis and rate of cellular proliferation (Wang et al, 2017). These in vitro results provided strong indication that the Arg-Orn pathway is a key regulator of the synthesis of casein.…”

“…Commonly used reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) were selected as internal control genes. Arginase 1 (ARG1), arginase 2 (ARG2), endothelial nitric oxide synthase (NOS3), inducible nitric oxide synthase (NOS2), ornithine decarboxylase 1 (ODC1), α S1 -casein (CSN1S1), α S2 -casein (CSN1S2), β-casein (CSN2), κ-casein (CSN3), and insulin-like growth factor 2 (IGF2) were chosen to allow for comparison with previous in vitro results (Wang et al, 2017). In addition, 6 additional genes associated with transport mechanisms also were selected: solute carrier family 7 member 1 (SLC7A1), solute carrier family 7 member 2 (SLC7A2), solute carrier family 7 member 5 (SLC7A5), solute carrier family 7 member 6 (SLC7A6), solute carrier family 7 member 7 (SLC7A7), and solute carrier family 7 member 8 (SLC7A8).…”

Influence of arginine on enzymes related to arginine metabolism in bovine mammary epithelial cells in vitro

“…Arg appears to meditate the casein synthesis through regulating cellular proliferation and transcriptional regulators. Further study on arginase inhibition found that the casein synthesis was regulated by Arg/Orn pathway, which implies the involvement of enzymes related to Arg metabolism in the casein synthesis of BMEC (Wang et al 2017). Previous studies have identified the interaction of related enzymes in Arg metabolic pathways.…”