Mammary Tight Junctions Research Articles

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.

Tight junction regulation in the mammary gland.

Tight junctions form a narrow, continuous seal that surrounds each endothelial and epithelial cell at the apical border, and act to regulate the movement of material through the paracellular pathway. In the mammary gland, the tight junctions of the alveolar epithelial cells are impermeable during lactation, and thus allow milk to be stored between nursing periods without leakage of milk components from the lumen. Nonetheless mammary epithelial tight junctions are dynamic and can be regulated by a number of stimuli. Tight junctions of the mammary gland from the pregnant animal are leaky, undergoing closure around parturition to become the impermeable tight junctions of the lactating animal. Milk stasis, high doses of oxytocin, and mastitis have been shown to increase tight junction permeability. In general changes in tight junction permeability in the mammary gland appear to be the results of a state change and not assembly and disassembly of tight junctions. Both local factors, such as intramammary pressure and TGF-beta, and systemic factors, such as prolactin, progesterone, and glucocorticoids, appear to play a role in the regulation of mammary tight junctions. Finally, the tight junction state appears to be closely linked to milk secretion. An increase in tight junction permeability is accompanied by decrease in the milk secretion rate, and conversely, a decrease in tight junction permeability is accompanied by an increase in the milk secretion rate.

Effect of cortisol on mammary tight junction (TJ) permeability in lactating dairy cows

Intact mammary epithelial TJ are important for milk synthesis. Recent in vitro studies indicate that glucocorticoids are important for maintenance of mammary TJ. In the present study, we examined the effects of cortisol on mammary TJ integrity in vivo. In two experiments, endogenous levels of cortisol in plasma were manipulated by challenging cows with (0.05 mg, 40 IU; i.v.) adrenocorticotropic hormone (ACTH 1–24 ) or by inducing transport-related stress. In experiment 1, 20 cows were subjected to an ACTH challenge 4, 2, or 1 h before milking, or no challenge (control). In experiment 2, 2 h before milking, 15 cows were challenged with ACTH, transported for 2 h, or used as controls. Blood samples were collected at −5, 60, and 120 min following the start of treatments. Decreased plasma lactose and increased milk K content were used as indicators of TJ permeability. Both ACTH and transport caused a 6-fold increase (P < 0.01) in plasma cortisol at 60 min. Time of ACTH challenge (exp. 1) did not affect plasma lactose, allowing the data to be pooled. ACTH decreased plasma lactose in experiment 1 (−5 vs 60 vs 120 min: 28.0 a vs 26.9 a vs 24.9 b ± 0.8 M, a,b P < 0.05) and experiment 2 (26.1 a vs 24.6 a vs 23.6 b ± 0.8 M, a,b P < 0.01), whereas K content in milk increased (exp. 1, control vs 4 vs 2 vs 1 h: 31.8 a vs 32.1 a vs 32.0 a vs 33.5 b ±0.2 mmol l , a,b P < 0.01; exp. 2, control vs ACTH: 41.1 vs 42.1 ±0.4 mmol l , P < 0.01). These changes are consistent with decreased TJ permeability. Moreover, these changes occurred rapidly (≤ 1 h). Despite elevated cortisol levels with transport, no changes in plasma lactose were observed, but K in milk increased (control vs transport: 40.9 vs 42.1 ± 0.4 mmol l , P < 0.05). This variable response with stress may be due to increased secretion of epinephrine, overiding the beneficial effects of cortisol on TJ. In conclusion, our data, obtained in vivo, support the findings of recent in vitro studies that glucocorticoids play a role in maintaining the integrity of TJ in the epithelium of the mammary gland.

17. Elevated sodium content in milk affects milk synthesis, but not mammary tight junction (TJ) integrity in goats

17. Elevated sodium content in milk affects milk synthesis, but not mammary tight junction (TJ) integrity in goats

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.

EGTA-induced disruption of epithelial cell tight junctions in the lactating caprine mammary gland.

The suitability of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to induce disruption of mammary tight junctions (TJ) and its effect on milk secretion were investigated in six goats. EGTA was administered via the teat of one gland as an isosmotic (300 mosmol/l) K-EGTA solution (68 mM EGTA), whereas the control gland received an isosmotic sucrose solution. Lactose, Na, K, and Cl in milk, blood lactose, and the presence of Evans blue (EB) in mammary lymph were used as indicators of TJ disruption. EGTA caused transient (approximately 60 h) changes (P < 0.05) in the concentration of lactose, K, Na, and Cl in milk, consistent with loss of TJ integrity. This was confirmed by a rapid (< 1 h) increase (P < 0.05) in blood lactose levels. Moreover, EB appeared in lymph < 1 h after EGTA+EB treatment. Milk secretion declined unilaterally by 15% (P < 0.05) after EGTA and did not return to baseline until approximately 60 h after EGTA. EGTA caused a unilateral, temporary (first 7 h) increase in mammary blood flow. This study shows that a rapid temporary disruption of mammary TJ can be successfully induced in vivo and that such disruption compromises milk secretion.

Effect of Once Daily Milking and Concurrent Somatotropin on Mammary Tight Junction Permeability and Yield of Cows

Six pairs of monozygous Friesian twin cows during late lactation were used to assess the effect of once daily milking and bST treatment on yields and tight junction permeability of mammary epithelial cells. During the first 7 d, all cows were milked twice daily; on d 8 through d 21, all cows were milked once daily, but one cow of each twin pair was treated daily with 20mg of bST on d 13 through 21; and, finally, during d 22 through 28, all cows were again milked twice daily. Once daily milking, a common management practice in New Zealand, resulted in a small (7%) but significant decrease in milk yield. Treatment with bST increased milk yield by 19%, thereby exceeding the milk yield loss from once daily milking. The integrity of mammary tight junctions was assessed indirectly by measuring concentrations of plasma lactose and milk BSA. Once daily milking temporarily disrupted tight junction integrity, based on a 4- to 5-fold increase in plasma lactose and a 42 to 55% increase in the concentrations of milk BSA. In the present study, bST did not affect the permeability of mammary tight junctions.

Mammary Epithelial Cell Tight Junction Integrity and Mammary Blood Flow During an Extended Milking Interval in Goats

The timing and relation of changes in mammary epithelial cell tight junction integrity and mammary blood flow during a 36-h milking interval were studied in six lactating Saanen goats. An increase in lactose concentration in plasma, a decrease in transepithelial potential difference, and changes in ionic milk composition were used to indicate tight junction patency. After 36h of milk accumulation, mammary tight junctions had become disrupted. Further analyses indicated that this disruption began after 21h of milk accumulation and that mammary blood flow also started to decline after 21h. The time when both events occurred was not significantly different from the time when milk secretion began to decline (19h). Moreover, positive but nonsignificant correlations existed between these events. Mammary tight junctions became disrupted when milk secretion declined, suggesting that impairment of mammary tight junction integrity is associated with decreased milk secretion during an extended milking interval. The decline in mammary blood flow may be the result of a negative feedback response to a reduced demand for metabolites, which is due to a reduced rate of milk secretion.

Milk Synthesis and Secretion Rates in Cows with Milk Composition Changed by Oxytocin1,2

Supraphysiological doses of oxytocin were used to investigate the association among milk composition, secretion rates, and cell electrolyte concentrations. Groups of 8 Holstein cows were injected i.v. with 1, 10, 100, or 1000 IU oxytocin after a normal milking. Milk yield over the following 12-h period declined 5.5, 8.1, 17.0, and 43.7% respectively. Milk Na increased in proportion to oxytocin dose. Yield and concentration of lactose and protein declined with increasing oxytocin doses, but milk fat yield remained unchanged. To determine whether the decreased milk lactose secretion was caused by a decline in synthesis or leakage into blood plasma and subsequent renal clearance, milk and urine were collected for 24-h periods before and after 100 IU oxytocin. Urinary lactose accounted for 2.7±3.19 and 12.9±8.9% of the total collected on the days before and after oxytocin. This increase (46.7 g/d) was only one-fifth of the 25% decline in total lactose recovery, suggesting that reduced mammary lactose synthesis is primarily responsible for lower milk yield. Measurement of Na and K in biopsied mammary tissue suggests that a high Na:K ratio in milk will increase that ratio in secretory cells, which may be part of the mechanism for lower milk yield under circumstances that increase permeability of mammary tight junctions.