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Ca(2+)-ATPase protein expression in mammary tissue
T A Reinhardt1, A G Filoteo, J T Penniston
1Periparturient Diseases of Cattle Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010, USA. treinhar@nadc.ars.usda.gov
Abstract:
Protein expression of plasma membrane Ca(2+)-ATPases (PMCAs) and the putative Golgi secretory pathway Ca(2+)-ATPase (SPCA) was examined in rat mammary tissue. As lactation started, PMCA protein expression increased dramatically, and this increased expression paralleled milk production. Mammary PMCA was primarily PMCA2b but was approximately 4,000 daltons larger than expected. RT-PCR showed that the primary mammary PMCA2b transcript was alternatively spliced, at splice site A, to include an additional 135 bp, resulting in the insertion of 45 amino acids. This splice form is designated 2bw. PMCA2bw is secreted into milk, associated with the milk fat globule membrane. Therefore, PMCA2bw is located on the apical membrane of the secretory cell. Smaller amounts of PMCA1b and 4b protein were found in mammary tissue. PMCA4b was the major PMCA expressed in developing tissue, and its level declined as lactation started. PMCA1b expression increased moderately during lactation. SPCA protein expression increased 1 wk before parturition and increased further as lactation proceeded. The abundance and cell location of PMCA2b suggest that it is important for macro-Ca(2+) homeostasis in lactating tissue. The pattern of expression and abundance of SPCA suggest that it is a candidate for the Golgi Ca(2+)-ATPase.
Insights
During lactation, plasma membrane Ca(2+)-ATPase (PMCA) protein expression significantly increases in rat mammary tissue, with a novel splice variant, PMCA2bw, secreted into milk. This suggests a key role for PMCA2bw in calcium regulation during lactation.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Calcium ATPases, including plasma membrane Ca(2+)-ATPases (PMCAs) and secretory pathway Ca(2+)-ATPases (SPCAs), are crucial for cellular calcium homeostasis.
- Understanding the specific isoforms and their roles in specialized tissues like the mammary gland during lactation is essential for comprehending calcium transport mechanisms.
Purpose of the Study:
- To investigate the protein expression and localization of PMCAs and SPCAs in rat mammary tissue during lactation.
- To identify novel splice variants of PMCAs and determine their functional significance in milk production.
Main Methods:
- Western blotting was used to examine protein expression levels of PMCAs and SPCAs.
- RT-PCR was employed to analyze alternative splicing of PMCA transcripts.
- Immunohistochemistry or similar techniques were likely used to determine protein localization within mammary tissue.
Main Results:
- PMCA protein expression, predominantly PMCA2b, dramatically increased with lactation onset, correlating with milk production.
- A novel splice variant, PMCA2bw, was identified, characterized by an additional 45 amino acids, and found to be secreted into milk, associated with the milk fat globule membrane.
- SPCA protein expression increased pre-parturition and continued to rise during lactation, suggesting its involvement in Golgi calcium transport.
Conclusions:
- The increased abundance and apical localization of PMCA2bw indicate its significant role in macro-calcium homeostasis in lactating mammary tissue.
- The expression patterns of SPCA suggest it is a likely candidate for the Golgi Ca(2+)-ATPase in this tissue.