R.C. Gorewit

Association and Coexpression of Fatty‐Acid‐Binding Protein and Glycoprotein CD36 in the Bovine Mammary Gland

The involvement of glycoprotein CD36 and fatty-acid-binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C-terminus (amino acid residues 121-131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti-FABP and anti-CD36, Northern-blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C-terminus of FABP with the sequence Val-Thr-Cys, identical to the sequence found in the CD36-binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val-Cys-Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period.

Effect of mastitis on plasminogen activator activity of milk somatic cells

This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay system, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p-nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0.05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 x 10(4) to > 10(6) cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (< 4 months in lactation) or late lactation (> 8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.

Half-life, clearance and production rate for oxytocin in cattle during lactation and mammary involution I

Abstract Basal concentration, production rate, metabolic clearance rate and T 1 2 for oxytocin were measured in Holstein cows at three lactational stages and during mammary involution. At each lactational stage, oxytocin was given intravenously at .5 IU/min or 1.0 IU/min for 60 min. Infusions were preceded by priming. During involution, the high dose was used. Mean basal concentrations of oxytocin ranged from 8.7 to 21.4 uU/ml. Mean basal values at early, middle, late lactation and involution were 17.57, 12.33, 15.15, 21.13 uU/ml, respectively and differed significantly. The mean “rapid” T 1 2 was 3.87 ± .1 min. Early, middle, late lactation and involution T 1 2 s were 4.2, 3.7, 4.0 and 3.5 min, respectively. The rapid T 1 2 was not affected by lactational stage. The mean “slow” T 1 2 was 25.53 ± 1 min. Early lactation and dry period means differed significantly. The overall mean oxytocin clearance rate was 8.41 ± .1 ml/kg · min. Clearance rate declined through lactation and into the dry period. Mean values of 9.3, 8.8, 7.8 and 7.1 ml/kg · min were obtained at early, middle, late lactation and involution, respectively. Clearance rates at late lactation and involution differed significantly from one another and from the early and middle stages. Mean entry rates for oxytocin at early, middle, late lactation and involution were 168.79, 106.03, 111.1 and 146.8 uU/kg · min, respectively. Measurements at early lactation and involution were greater than values for middle and late lactation. To summarize, basal oxytocin concentrations can be measured in cows that are lactating or undergoing mammary involution and changes in concentrations during lactation are related to hormone production (entry rate) and metabolic clearance rates.

Mammary-derived growth inhibitor in lactation and involution☆

Presence of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating and involuted cows was investigated. Eighteen lactating, non-pregnant high-producing Holstein cows were randomly assigned to 3 experimental groups of 6 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group were slaughtered at 2-3 d, and the others at 4-8 d following sudden cessation of milking. Cessation of milking occurred at approximately 300 d in lactation. Western blot analysis revealed the presence of MDGI in the cytosolic and microsomal fractions of mammary tissue homogenates. High levels of MDGI were detected in mammary tissue obtained from lactating non-pregnant cows. A dramatic reduction in MDGI was observed in early involution (2-3 or 4-8 d following cessation of milking). These data suggest that a relationship exists between MDGI levels and the physiological status of the gland. Lack of MDGI may play a role during the processes of mammary involution and development prior to parturition.

Oxytocin concentrations of cattle in response to milking stimuli through lactation and mammary involution II

Abstract Patterns of oxytocin release to milking stimuli over a lactation and during mammary involution, were examined in seven Holstein cows used in the previous study. Blood samples were taken before, during and after milking or udder massage. Oxytocin as measured by radioimmunoassay increased within O to 2 min after attachment of the milking unit. Oxytocin levels fluctuated during milking and declined after the initial increase. Oxytocin often dropped below basal levels after milking. Milking-induced oxytocin release decreased as lactation advanced. The maximal increment for oxytocin release was significantly different between early and late lactation. The time taken to reach peak hormone concentrations declined across lactation. Relative amounts of oxytocin released in response to milking stimuli were significantly more in early than during late lactation. Cows released oxytocin during mammary involution with relatively large, rapid increases to udder massage. A distinct peak was observed and return to basal concentrations was rapid. The mean increment of oxytocin concentration above basal was 51.6 ±10.1 uU/ml. Maximal oxytocin levels occurred 1.6 ±.2 min (0 to 2 min) after initial stimulation. The total amount of oxytocin released in response to stimulation was 1.2 ±.1 uU/ml. In summary, a continuous or multiple release of oxytocin occurs during milking. The sensitivity of the neuroendocrine reflex for oxytocin appears dynamic. Changes in maximal concentrations and total amounts of hormone released in response to milking during lactation, and the relationship between these variables and basal concentrations suggest a gradual loss of sensitivity from the early stages of lactation to mammary involution.

Physiological and pharmacological studies of oxytocin in virgin cattle III

Abstract Resting concentrations of oxytocin were measured in virgin cattle and it was determined if these animals would release oxytocin in response to milking associated stimuli. Half-life, clearance and entry rates for oxytocin were also quantified, to compare previous data from experiments carried out on lactating and nonlactating cows. Six Holstein virgin heifers received a two minute udder massage. Blood samples were taken before, at and after massage and analyzed for oxytocin by radioimmunoassay. Resting concentrations of oxytocin averaged 24.8 ±2.27 uU/ml (mean ± SE) and increased to 28 ±2.3 uU/ml at 2 min. The increase in oxytocin was clearly apparent after post-stimulation concentrations were corrected for basal (pre massage) levels. Serum oxytocin concentrations remained below baseline for 25 min post stimulation. No increase in oxytocin was seen prior to udder stimulation. Heifers were infused with 1.0 IU/min oxytocin to measure pharmacokinetic parameters. Oxytocin disappearance from sera was monitored by radioimmunoassay. A two compartment model for oxytocin elimination produced two half-life components. A rapid half-life component with a mean value of 3.07 ±.32 min and a slow half-life component with a mean of 24.3 ±2.2 min. The overall clearance rate for oxytocin was 9.4 ±.58 ml/kg min. The mean entry rate of oxytocin into the body was 231.96 ±25.96 uU/kg min. The sensitivity of the neuroendocrine reflex, eliciting oxytocin release, appears dependent upon the physiological state of the animal. Half-life and clearance measurements for oxytocin compare closely to those obtained in previous studies for cows in either early lactation, late lactation or undergoing mammary involution. It appears that the clearance and production of oxytocin are also related to the physiological state of the animal.