The cellular heat stress (HS) response is one component of the acute systemic response to HS. Gene networks within and across cells and tissues respond to environmental heat loads above the thermoneutral zone with both intra- and extracellular signals that coordinate cellular and whole-animal metabolism. Activation of these systems appears to be initiated at skin surface temperatures exceeding 35 degrees C as animals begin to store heat and rapidly increase evaporative heat loss (EVHL) mechanisms. Gene expression changes include 1) activation of heat shock transcription factor 1 (HSF1); 2) increased expression of heat shock proteins (HSP) and decreased expression and synthesis of other proteins; 3) increased glucose and amino acid oxidation and reduced fatty acid metabolism; 4) endocrine system activation of the stress response; and 5) immune system activation via extracellular secretion of HSP. If the stress persists, these gene expression changes lead to an altered physiological state referred to as "acclimation," a process largely controlled by the endocrine system. In the acclimated state, metabolism is adjusted to minimize detrimental effects of increased thermal heat load. The role of secreted HSP in feedback regulation of the immune and endocrine system has not yet been investigated. The variation in EVHL among animals and the central role that HSF1 has in coordinating thermal tolerance suggest that there is opportunity to improve thermal tolerance via gene manipulation. Determining the basis for altered energy metabolism during thermal stress will lead to opportunities for improved animal performance via altered nutritional management.

Recent studies in dairy cows have demonstrated that serotonergic ligands affect milk yield and composition. Correspondingly, serotonin (5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution in mouse and human mammary cells. We determined the mRNA expression of bovine 5-HT receptor (HTR) subtypes in bovine mammary tissue (BMT) and used pharmacological agents to evaluate functional activities of 5-HT receptors. The mRNAs for five receptor isoforms (HTR1B, 2A, 2B, 4, and 7) were identified by conventional real-time (RT)-PCR, RT quantitative PCR, and in situ hybridization in BMT. In addition to luminal mammary epithelial cell expression, HTR4 was expressed in myoepithelium, and HTR1B, 2A, and 2B were expressed in small mammary blood vessels. Serotonin suppressed milk protein mRNA expression (α-lactalbumin and β-casein mRNA) in lactogen-treated primary bovine mammary epithelial cell (BMEC) cultures. To probe the functional activities of individual receptors, caspase-3 activity and expression of α-lactalbumin and β-casein were measured. Both SB22489 (1B antagonist) and ritanserin (2A antagonist) increased caspase-3 activity. Expression of α-lactalbumin and β-casein mRNA levels in BMEC were stimulated by low concentrations of SB224289, ritanserin, or pimozide. These results demonstrate that there are multiple 5-HT receptor isoforms in the bovine mammary gland, and point to profound differences between serotonergic systems of the bovine mammary gland and the human and mouse mammary glands. Whereas human and mouse mammary epithelial cells express predominately the protein for the 5-HT7 receptor, cow mammary epithelium expresses multiple receptors that have overlapping, but not identical, functional activities.

Serotonin (5-HT) is a homeostatic regulator of lactation. Selective 5-HT reuptake inhibitors (SSRI) are commonly prescribed pharmaceuticals that inhibit activity of the 5-HT reuptake transporter, increasing cellular exposure to 5-HT. Use of SSRIs has been shown to alter lactation performance in humans and 5-HT has been shown to reduce milk yield in cattle. However, it has not been determined how SSRI treatments affect the bovine mammary gland. We evaluated the effects of SSRI (fluoxetine (FLX)) administration on tight junctions (TJs) and milk protein gene expression in a lactogenic culture model, using primary bovine mammary epithelial cells (pBMEC). Additionally, we evaluated the effects of intramammary infusions of FLX and 5-hydroxytryptophan on milk production and TJ status in multiparous Holstein cows at dry-off. Treatment of pBMEC cultured on permeable membranes disrupted TJs, as measured by transepithelial resistance and immunostaining for zona occludens 1. Correspondingly, treatment of '3D', collagenembedded lactogenic cultures of pBMEC with FLX suppressed milk protein gene expression (a-lactalbumin and b-casein) in a concentration-dependent manner. Finally, intramammary treatment of Holstein cows with FLX resulted in an accelerated rate of milk decline. Additionally, TJ permeability increased in FLX-treated animals, as measured by plasma lactose and milk Na C and K C levels. Results of these experiments imply that SSRI administration accelerates the rate of mammary gland involution through disassembly of TJs and inhibition of milk protein gene expression in vitro and in vivo, leading to reduction of milk yield.

Betaine (BET), a natural, organic osmolyte, improves cellular efficiency by acting as a chaperone, refolding denatured proteins. To test if dietary BET reduced the effect of heat stress (HS) in lactating dairy cows, multiparous, lactating Holstein cows (n=24) were blocked by days in milk (101.4±8.6 d) and randomly assigned to 1 of 3 daily intakes of dietary BET: the control (CON) group received no BET, mid intake (MID) received 57mg of BET/kg of body weight, and high dose (HI) received 114mg of BET/kg of body weight. Cows were fed twice daily and BET was top-dressed at each feeding. Cows were milked 2 times/d and milk samples were taken daily for analysis. Milk components, yield, feed intake, and water intake records were taken daily. Rectal temperature and respiration rate were taken 3 times/d at 0600, 1400, and 1800h. Cows were housed in environmentally controlled rooms and were allowed acclimation for 7d at thermoneutral (TN) conditions with a mean temperature-humidity index of 56.6. Cows were then exposed to 7d of TN followed by 7d of HS represented by a temperature-humidity index of 71.5 for 14d. This was followed by a recovery period of 3d at TN. Dietary BET increased milk yield during the TN period. No differences were found between BET and CON in total milk production or milk composition during HS. The increase in water intake during HS was not as great for cows fed BET compared with controls. The cows on CON diets had higher p.m. respiration rate than both MID and HI BET during HS, but lower rectal temperature compared with BET. No difference was found in serum glucose during TN, but cows given HI had elevated glucose levels during HS compared with CON. No differences were found in serum insulin levels between CON and BET but an intake by environment interaction was present with insulin increasing in HI-treated lactating dairy cows during HS. The heat shock response [heat shock protein (HSP) 27 and HSP70] was upregulated in bovine mammary epithelial cells in vitro. Blood leukocyte HSP27 was downregulated at the HI dose under TN conditions and HSP70 was upregulated at the HI dose and this effect was increased by HS. No effect was seen with the MID dose with HSP27 or HSP70. The lack of effect of BET at MID may be associated with uptake across the gut. We conclude that BET increased milk production under TN conditions and was associated with reduced feed and water intake and slightly increased body temperatures during HS of cows fed BET. The effect of BET on milk production was lost during HS with HI BET, whereas serum glucose levels increased during HS.

Biochemical changes in living cells are detected using a fiber probe system composed of a single chalcogenide fiber acting as both the sensor and transmission line for infrared optical signals. The signal is collected via evanescent wave absorption along the tapered sensing zone of the fiber. We spectroscopically monitored the effects of the surfactant Triton X-lOO, which serves as a toxic agent simulant on a transformed human lung carcinoma type II epithelial cell line (A549). We observe spectral changes between 2800-3000 cm-1 in four absorptions bands, which are assigned to hydrocarbon vibrations of methylene and methyl groups in membrane lipids. Comparison of fiber and transmission spectra shows that the present technique allows one to locally probe the cell plasma membrane in the lipid spectral region. These optical responses are correlated with cellular metabolic activity measurements and LDH (lactate dehydrogenase) release assays that indicate a loss of cellular function and membrane integrity as would be expected in response to the membrane solubilizing Triton. The spectroscopic technique shows a significantly greater detection resolution in time and concentration.