Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity.
Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.
Kusinski
L C
LC
Maternal and Fetal Health Research Group, School of Clinical and Laboratory Sciences, University of Manchester, St. Mary's Hospital, Oxford Road, Manchester M13 9WL, United Kingdom.
Jones
C J P
CJ
Baker
P N
PN
Sibley
C P
CP
Glazier
J D
JD
D000469:Alkaline Phosphatase / Q000378:metabolism; D026924:Amino Acid Transport System A / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000737:chemistry* / Q000378:metabolism; D004622:Embryo, Mammalian; D005260:Female; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010920:Placenta / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D011247:Pregnancy; D047929:Term Birth / Q000378:metabolism / Q000502:physiology; D000099247:Taurine Transporters
D000469:Alkaline Phosphatase; D026924:Amino Acid Transport System A; D026905:Amino Acid Transport Systems; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D000099247:Taurine Transporters