Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells.

Tight junctions (TJ) between adjacent epithelial cells play an important role in maintaining mammary function in the differentiated mammary gland. Mouse mammary cell lines (HC11 and Comma-1D) were used to investigate the effect of the lactogenic hormones prolactin (PRL) and glucocorticoids on the formation of mammary TJ. TJ formation was assessed by an increase in transepithelial electrical resistance and a decrease in paracellular flux of radiolabeled inulin. Both PRL and the synthetic glucocorticoid dexamethasone (DEX) stimulated TJ formation. The biggest effect on TJ formation was observed when both hormones were used in combination, but only when cells were pretreated with DEX. The effects of PRL and DEX are mediated, at least in part, via expression of the transmembrane TJ protein occludin. In summary, these data are the first to show an effect of PRL on mammary TJ formation and the expression of TJ proteins, and confirm the TJ-stimulating effects of glucocorticoids that have been reported previously.

Alteration of the sodium to potassium ratio in milk and the effect on milk secretion in goats.

Saanen goats were used to determine the effect of the alteration of the intramammary Na to K ratio on milk secretion. Udders were infused via the teat with an isosmotic solution that was high in Na or K to increase or decrease, respectively, the intramammary Na to K ratio. Control glands received an isosmotic sucrose solution. To ensure that the results were not confounded by a decrease in milk secretion as a result of enhanced permeability of mammary tight junctions, the latter was monitored throughout the experiments. An increase in the Na to K ratio caused a significant transient reduction in milk secretion. Therefore, an increase in Na and a decrease in K in milk, commonly observed as a result of the leakiness of tight junctions, may at least partially explain the reduction in milk secretion when the permeability of tight junctions was increased. These experiments further showed that the adverse effects on secretion were not due to a high intracellular concentration per se but were related to a change in the Na to K ratio because a reduction in the ratio also lowered milk secretion. These data support the evidence for activity of Na(+)-K(+)-ATPase in the basolateral secretory cell membranes and passive movement of these ions across the apical cell membranes.

Elevated plasma cortisol reduces permeability of mammary tight junctions in the lactating bovine mammary epithelium.

Induction of tight junction permeability in the mammary epithelium decreases milk secretion, and in cows tight junctions become leaky after 17 h of milk accumulation. In vitro studies demonstrate the importance of glucocorticoids for the formation and maintenance of tight junctions. In this study we examined whether cortisol can prevent mammary tight junction permeability in the lactating gland in vivo, and inhibit the associated milk loss, using our milk-accumulation model to challenge tight junction patency. Following a 4-day control period Jersey cows were subjected to a 24-h period in which they were milked twice at 0700 and 1500 h (TM;n=6), once at 0700 h (OM;n=7), or once and treated with ACTH (40 IU per 2 h, starting after 14 h of milk accumulation) to increase endogenous cortisol levels (OM+ACTH;n=7). Frequent blood samples for cortisol, lactose and glucose analyses were taken via indwelling jugular catheters. ACTH treatment resulted in a sustained elevation of systemic cortisol concentrations. Plasma lactose, an indicator of tight junction leakiness, was not changed in TM cows, but began to increase rapidly at 17 h of milk accumulation in OM cows. Treatment with ACTH prevented the increase in plasma lactose, although levels were slightly, but not significantly, higher than in TM cows, indicating that elevated plasma cortisol reduced mammary tight junction leakiness. Milk yield was reduced by 12% in both once-milked groups, despite cortisol preventing tight junction leakiness. However, the milk loss in the latter group may not be related to leaky tight junctions, but be due to a reduction in milk precursor uptake by the mammary gland. Consistent with this notion was a 34% increase in plasma glucose levels in OM+ACTH cows only.

No evidence for basolateral secretion of milk protein in the mammary gland of lactating goats.

Recent research suggests that a small percentage of milk proteins may be secreted basolaterally, which would have implications for our work on the permeability of tight junctions in the mammary epithelium. In our work, the presence of alpha-lactalbumin (LA) or lactose in plasma is used as an indicator of permeability. The aim of this study was to examine basolateral secretion by determining the presence of milk proteins in efferent mammary lymph. Five Saanen goats were fitted with mammary lymph catheters and were administered intramammary isosmotic bolus infusions of sucrose control solutions or ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to induce leaky tight junctions. Lymph samples were collected before and approximately 5 h after infusion. Lymph was analyzed by Western blotting for the presence of alpha-casein (CN), beta-CN, and alpha-LA No alpha-CN or beta-CN was detected in lymph, but alpha-LA was detected in all lymph samples. Moreover, the signal was much stronger in samples from goats that were treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and concentrations of alpha-LA in lymph were significantly increased with this treatment. These changes and the absence of casein in lymph suggest increased permeability of tight junctions rather than basolateral secretion. In summary, these data do not support basolateral secretion.

Time course of milk accumulation-induced opening of mammary tight junctions, and blood clearance of milk components.

Eight cows in early lactation were used to study the effect of milk accumulation on the state of mammary tight junctions and to examine alpha-lactalbumin as an indicator of tight junction permeability in vivo. During three successive periods, the cows were milked twice (4 days), once (6 days), and twice daily (4 days). Plasma lactose, alpha-lactalbumin, and milk sodium concentrations were used as indicators of tight junction permeability. Furthermore, four cows were used to study the clearance of lactose and alpha-lactalbumin from the blood. Milk yield during once-daily milking decreased by 15.4% (P < 0.001). All indicators of mammary tight junction patency increased (P < 0.05) transiently during once-daily milking and indicated that tight junctions opened after approximately 18 h. Plasma alpha-lactalbumin and lactose were highly correlated (r = 0.82, P < 0.001), indicating the suitability of plasma alpha-lactalbumin as an indicator of tight junction status in vivo. Clearance of alpha-lactalbumin and lactose from the blood was best described by a biexponential model. Elimination half-lives for lactose and alpha-lactalbumin were 44 and 40 min, respectively. This study showed that milk stasis during early established lactation induces tight junctions to switch to a leaky state after approximately 18 h and to revert to the closed state shortly after milking.