Local control of milk secretion

Milk secretion is controlled locally, within the mammary, gland, by milk removal. This control is believed to be mediated through feedback inhibition by milk constituents. The aim of this study was to investigate the effects of milk-borne peptides, implicated in the local control ofmilk secretion, on protein secretion in bovine mammosphere culture. These peptides, tenned A, Band C, had previously been shown in mammary acini cultures to acutely inhibit the secretion of milk proteins. To this end, a bovine mammosphere culture system was developed whereby bovine mammary epithelial cells cultured on EHS matrix were stimulated to express milk protein genes and secrete the major milk proteins casein, (3-lactoglobulin and lactoferrin. Additionally, the recent claim that the phosphopeptide (3casein 1-28 (Silanikove et ai, 2000) was capable of controlling milk secretion was investigated in'mouse mammary acini and bovine mammosphere culture. The secretory perfonnance of the bovine mammosphere system was validated by western blot measurement ofcasein, (3-lactoglobulin and lactoferrin in conditioned culture medium, . and mammosphere fonnation was investigated by SEM and cryosectioning of cultured cell aggregates. Experiments investigating the effects ofthe peptides tested all three peptides in combination (triple peptide treatment), the peptide pair AC, peptide B and (3-casein 1-28. Peptide effects were assessed by measuring 3H-Ieucine incorporation into TCA precipitable protein following 2 and 4 days oftreatment. Additionally, the effect 'ofthe triple peptide on (3-casein and lactoferrin mRNA abundance, and on the secretion of the major bovine milk proteins casein, (3-lactoglobulin and lactoferrin, was measured by real time RT-PCR and western blotting respectively. (3-casein and lactoferrin mRNA abundance was significantly reduced in bovine mammosphere culture following triple peptide treatment for two days. At concentrations of 1 pM and 10 pM of all three test peptides, triple peptide treatment caused 35 % and 47 % reductions, respectively, in (3-casein mRNA levels, and 21 % and 35 % reductions respectively in lactoferrin mRNA levels. This was not reflected in changes in milk protein secretion, with no significant effect observed on total protein secretion or individual milk protein secretion after two days of treatment. This may be a consequence of the insensitivity ofthe blotting technique used, resulting in trends in the data for the three milk proteins being rendered insignificant due to large variations between replicates. On the other hand, triple peptide treatment for two days significantly increased mRNA levels of the purported housekeeping gene GAPDH, which rose by 75 % when treated with 10 JlM of the triple peptide. These effects may have been associat~d with aggregation of the three peptide~in culture: incubation of peptides A, Band C together at 37°C for 25 h resulted in the formation of apeptide multimer, which was detected by gel exclusion fast protein liquid chromatography. Although no definitive role for these peptides in the local control of milk secretion was established, the results suggest that the peptides may control milk secretion by reducing milk protein gene expression. Furthermore, GAPDH has recently been implicated in the control of ER - Golgi transport in the early secretory pathway in NRK cells. This role offers a potential explanation for the increase in GAPDH mRNA induced by peptide treatment, and suggests that these peptides may effect milk secretion by blocking GAPDH mediated retrograde vesicular transport. The data obtained in this study has extended information on factors which appear able to control mammary function locally, and together with our increasing understanding of the control of secretory pathways, has provided scope for further investigation of the mechanism by which milk-borne factors control milk secretion.EThOS - Electronic Theses Online ServiceGBUnited Kingdo