2011, Vol. 6 No. 2, Article 86
Feed Back Inhibition of Lactation:
Joydip Mukherjee*, Mandira Chaudhury1 and Kalyan De2
*1Dairy Cattle Physiology Division,
*Corresponding Author; e-mail address: email@example.com
Frequency of milking or complete evacuation of udder regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. The active whey protein, which we term as FIL (Feedback Inhibitor of Lactation), decreases milk secretion temporarily when introduced into a mammary gland of lactating animals. FIL is synthesized by mammary epithelial cells, and secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. Local control of mammary secretion during weaning, and after peak lactation in dairy animals, may be due to the actions of this inhibitor, and can be manipulated by frequency of milk removal.
Milk Secretion, autocrine, mammary gland, epithelium, protein.
Frequency of milking or complete evacuation of udder regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. The secretory rate of mammary gland increases when milk is removed completely, even if the milk is replaced immediately by an inert solution to maintain the gland's distension . On the other hand, dilution of stored milk with an inert isotonic solution also increases the rate of milk secretion. An effect compatible with dilution of a chemical inhibitor. Explants of mammary tissue in organ culture which synthesize milk constituents when cultured in the presence of lactogenic hormones have been used to confirm the presence of a putative feedback inhibitor in milk. The tissue-explant bioassay showed initially that goat's milk whey proteins, but not caseins (the most abundant milk proteins), inhibited synthesis of both casein and lactose in a dose-dependent manner . Inhibition was rapid and readily reversible. Screening of goat's milk proteins in rabbit mammary explants cultures identified a single whey protein of Mr 7600 able to inhibit synthesis of milk constituents. This active whey protein was termed as FIL (Feedback Inhibitor of Lactation) and similar inhibitory proteins have since been found in cow of cows, macropod, marsupial and human being.
SYNTHESIS AND SECRETION
Feedback inhibitor of lactation or analogous proteins have been identified in milk from goats, cows , humans and wallabies .Their presence in milk is not in itself proof of mammary origin, so confirmation has been sought using the in vitro system of mammosphere culture. Mammary cells cultured on a reconstituted basement membrane (primary cultures of goat mammary epithelial cells on EHS matrix,) form multicellular spherical structures very reminiscent of intact alveoli and secreted vectorally into the central lumen. The contents of the lumen were obtained for analysis by temporarily opening cell:cell tight junctions with a chelating agent. FIL was detected immunologically using a monospecific antiserum raised against purified FIL in luminal contents at a considerably higher concentration than in the culture medium, showing it to be a secreted product of the epithelial cells. The synthesis of FIL within mammary secretory cells was also demonstrated using [35S ]methionine. Since milk present in the alveolar lumen is in contact only with secretory cells, it is apparent that FIL must exert its action on the same cells that produce it, indicating that the mechanism is truly autocrine.
PURIFICATION OF THE PROTEIN
Screening of milk fractions (goat milk) for a constituent fulfilling the criteria for the putative inhibitor was carried out using mammary explants in culture. Inhibitory activity in defatted milk appeared within the whey proteins rather than the caseins. In the whey, inhibition was associated with a fraction of nominal relative molecular mass (Mr) of 10,000-30,000, based on passage through or retention by ultra filtration membranes. The active constituent was subsequently purified by conventional FPLC anion exchange chromatography. One of eight major protein fractions was found consistently to inhibit both casein and lactose synthesis. Resolution of this fraction by chromate focusing revealed three components, one of which accounted in bioassay for all of the inhibitory activities , and represented a 40,000-fold purification of the inhibitory activity as compared to unfractionated whey. The inhibitor has a Mr by gel filtration of 7600. Again its mass is reduced by chemical deglycosylation, using trifluoromethanesulphonic acid, by -1000 indicating that the inhibitor is a glycoprotein. N-terminal analysis of the inhibitory protein produced the consensus amino acid sequence: Ala-Pro-Pro-Phe-Glu-Arg-Asn-Ser-Pro-Gly-Arg-Leu. It bears no homology either to other milk proteins or to any recorded sequence.
MODE OF ACTION OF FEEDBACK INHIBITOR OF LACTATION
Frequent drainage of milk from the udder cistern with avoidance of the milk ejection reflex does not lead to increased milk yield in the way that frequent milking does. This confirms that the site of action of FIL is within alveolar tissue. Since tight junctions prevent para cellular flux of small molecules and ions during established lactation, the precise site must be the apical surface of the secretory cell. Mammosphere culture supports this: to be effective, FIL must be placed within the lumen of the mammosphere, rather than in the culture medium. The nature of the FIL receptor remains to be determined, but preliminary data do support the notion of specific binding sites on the apical membrane. Within the cell, the action of FIL occurs at an early stage in the constitutive secretory pathway for proteins. Pulse-chase experiments have shown that FIL inhibits the trafficking of radiolabelled protein through the Golgi apparatus, whereas the later stages of the secretory process are unaffected with similar effects which are seen in other cells in response to the fungal drug, brefeldin A. Within 1 h of addition of FIL to the cultures, the morphology of the Golgi and endoplasmic reticulum were markedly altered and protein secretion was reduced by as much as 50%. Recovery was equally swift, cell ultrastructure and protein secretion recovering to normal within 1h of removal of FIL. In addition, accumulation of protein within the cell may result in intracellular casein degradation, which FIL is known to increase.
HOW IS FEEDBACK INHIBITION RELIEVED?
Some form of processing must occur to explain how the concentration of FIL decreases in residual milk soon after milking. Either FIL is secreted in an inactive form and is then activated within the alvolar lumen or else preformed, active FIL is secreted and then metabolized to inactive forms. There is evidence from anion-exchange chromatography of the existence of multiple forms of the molecule, including some that are biologically inactive.
VARIOUS INHIBITORY FUNCTIONS OF FIL
Local feedback inhibition of milk secretion results from the action of a secreted milk protein (FIL), which is effective in milk stored within secretory tissue but not in milk that has moved down into the storage sinuses. In species with little or no extra alveolar storage space (for example, humans and rabbits), there can be no doubt that FIL is at its active site within the alveolar lumen. As milk accumulates between milkings it also gets distributed between secretory tissue storage and cisternal storage. The fact that FIL is only active when in contact with secretory cells means that it will be most effective when the ratio of cisternal to secretory storage is low. This ratio is quite variable among individuals, and goats or cows that have a large cistern would be expected to produce more milk per gram of secretory tissue than those with a small cistern. Alterations in milking frequency affect milk yield but not milk composition, so it demonstrates that the inhibition occurred approximately equally in casein and lactose synthesis. Rapidity of onset and reversibility of effect are good methods for discriminating between a specific inhibition and a generalized toxic action; the effect of the FIL was both rapid and reversible in vitro and in vivo. FIL does not inhibit milk fat synthesis in vitro instead certain medium chain fatty acids (MCFAs) are responsible.
ARE CHANGES IN CELL DIFFERENTIATION A CONSEQUENCE OF FIL ACTION?
Addition of FIL to primary cell cultures from mid pregnant mouse mammary gland inhibited hormone-induced cell differentiation (Wilde et al., 1991), possibly through a mechanism related to its main effect on the protein secretory pathway. Some cellular proteins, including hormone receptors, are synthesized and trafficked within the cell in a manner analogous to the secreted milk proteins, and this trafficking appears to be responsive to FIL. Bennett et al., 1992 demonstrated that milk accumulation reduced cell-surface receptors for prolactin (and probably IGF-I) an effect that can be reproduced by intraductal FIL injection. Therefore, it is likely that the effects of FIL on cell differentiation are mediated by altered sensitivity of the secretory cell to circulating lactogenic hormones.
DOES FIL AFFECT THE NUMBER OF CELLS?
The same endocrine sensitivity mechanism may come into play here; both prolactin and IGF-I are recognized mammary mitogens in a variety of species and both appear to be involved in mammary cell survival through a complex interaction with the binding protein, IGFBP5 (Flint and Knight, 1997). In short, there appear to be multiple effects of FIL, which can all be related to a primary action at the level of intracellular protein trafficking. The immediate effect on milk secretion is a direct consequence, and the subsequent effect on cell activity and the eventual effect on the number of cells are indirect consequences mediated through altered hormone sensitivity.
RECENT DEMONSTRATION OF SEROTONIN AS A FEEDBACK INHIBITOR OF LACTATION
Serotonin (5-HT) is a hormone and neurotransmitter synthesized from L-tryptophan and has been peoposed to be a component of the autocrine - paracrine homeostatic feedback mechanism (feed back inhibitor of lactation) which opposes endocrine stimulation of mammary development and milk secretion (Wilde., 1995). The gene coding for tryptophan hydroxylase 1,the rate limiting enzyme for 5-HT synthesis ,is expressed in bovine mammary epithelial cells in vitro and is up-regulated by prolactin. It is known that 5-HT is a potent mammary vasoconstrictor and affects teat smooth muscle motility in the bovine. Antagonizing the mammary epithelial cells 5-HT receptors with METH has increased milk protein mRNA expression indicating that overall milk protein synthesis should increase. Additionally treating mammary epithelial cells with 5-HT reduced milk protein mRNA expression, indicating that over all milk protein synthesis would be decreased. Down regulation of milk protein gene expression by 5-HT in cultures of BMEC and upregulation of these genes using inhibitors of the serotonergic system support the concept that 5-HT is a feedback inhibitor of lactation in the bovine. This has been proved by whole udder treatment rather than half udder treatment.
PROPOSED MODE OF ACTION IN HUMANs
Recently the 5-HT2A receptor subtype was identified in the MCP-7 human breast cancer cell line (Sonier, 2006). Furthermore, Stull et al (2007) recently demonstrated that the 5-HT7 receptor was present on the basolateral surface of human and mouse mammary epithelial cells and was involved in tight junction regulation. Antagonist and agonist for this receptor altered transepithelial electrical resistance, suggesting that one potential feed back action of 5-HT involves regulation of these junctions, which are critical in maintaining transepithelial gradients. Furthermore they suggested that the route of 5-HT transfer from milk to the basolateral surface of the mammary epithelial cell was paracellular. Recently , Hernandez et al., 2008 have identified presence of the 5HT-1b, 2a, 2b, 4, and 7 receptors in bovine mammary tissue. It is possible that some or all of these have direct or indirect effects on milk synthesis and secretion. Thus , there are several potential sites of action for 5-HT on milk yield . The identity of the 5-HT receptor subtype(s) in mammary tissue is unknown. Therefore it is not possible to use a selective / specific 5-HT receptor subtype antagonist ,which would be effective on mammary tissue alone.
We have attempted to present the evidences for local control of the onset and maintenance of milk secretion by a small molecular weight glycoprotein (FIL) that has immediate and direct effects on protein and lactose secretion and long-term, probably indirect, effects on mammary development and involution. This local control co-exists very successfully with systemic endocrine and metabolic control.
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