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United States Department of Agriculture

Agricultural Research Service

Title: Ca2+ Atpase Protein Expression in Mammary Tissue: Alternate Splicing and Secretion of Plasma Membrane Ca2+ Atpase 2bw

Authors
item Reinhardt, Timothy
item Filoteo, Adelaida - MAYO FOUNDATION, MN
item Penniston, John - MAYO FOUNDATION, MN
item Horst, Ronald

Submitted to: American Journal of Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 15, 2000
Publication Date: N/A

Interpretive Summary: Most metabolic and infectious diseases in dairy cows are problems whose genesis starts with the stresses of the initiation of lactation. Calcium flows rapidly into the mammary gland, prior to the start of lactation, into as yet unidentified storage sites. The rapidity of this calcium flux contributes to milk fever and the complications that result from milk fever and subclinical hypocalcemia. Fine control of mammary milk cell calcium also is key to the health, hormone responsiveness and the quality of milk produced by the secretory cell. The well-being of the cow and her profitability could be greatly enhanced by understanding those factors that regulate the mammary glands transition to milk production and the concomitant excretion of calcium. This paper represents the second in a series of studies examining factors controlling calcium flow and storage in the mammary at and around calving. We studied the synthesis of 4 calcium pumps in the milk-producing mammary gland. All pumps increased when lactation started, but one pump increased prior to the start of milk production. Two pumps stand out as candidates for controlling most of the calcium movement in the mammary gland and, thus, the hypocalcemia (milk fever) that causes a disease complex in the transition cow. Their scientific names are plasma membrane calcium ATPase 2b and the secretory pathway calcium ATPase. Further studies on their function and roles are underway in cows. Initially, these data benefit the scientific community of lactation biologists. Ultimately, the dairy farmer will be the beneficiary of this work.

Technical Abstract: Plasma membrane Ca2+ ATPases (PMCA) and the putative Golgi secretory pathway Ca2+ ATPase (SPCA) protein expression were examined in developing and lactating mammary tissue. Small amounts of PMCA protein were found, in the mammary tissue prior to parturition. As lactation started, PMCA protein increased dramatically and this increased expression paralleled milk production. Isoform-specific antibodies showed that this increased mammary PMCA was primarily PMCA2b, but ca. 4000 daltons larger than expected. RT-PCR showed that the primary mammary PMCA2b transcript was alternatively spliced to include an additional 135 bp resulting in the insertion of 45 amino acids. This splice form is designated 2bw and accounts for the 4000 dalton increase in PMCA2b's size. We found that PMCA2bw is secreted into milk, associated with the milk fat globule membrane. This result demonstrated that significant amounts of PMCA2bw are located on the apical membrane of the secretory cell. Relatively smaller amounts of PMCA1b and 4b protein were found in lactating mammary tissue. PMCA4b was the major PMCA expressed in developing mammary tissue and declined as lactation started. PMCA1b increased moderately during lactation. While major increases in mammary PMCA2b expression occurred only after lactation started, SPCA protein increased 1 wk prior to parturition and increased further as lactation proceeded. PMCA2b's abundance, cell location, high affinity for Ca2+, and high constitutive activity suggests that PMCA2b is important for macro-Ca2+ homeostasis in lactating tissue. SPCA's pattern of expression and abundance suggest that SPCA is a candidate for the Golgi Ca2+ ATPase shown to be important in maintaining Golgi Ca2+ concentration required for casein synthesis.

Last Modified: 12/29/2014
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