Isolation and purification of human placental plasma membranes from normal and pre-eclamptic pregnancies. a comparative study.
Human placental syncytiotrophoblast is the main barrier for materno-fetal exchange. Analysis of transplacental transport involves the study of ion channels in both the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Difficulties in having access to intact placenta with conventional electrophysiological methods favour alternative methodologies, such as isolation and reconstitution of membranes in artificial lipid systems. Pre-eclampsia is a major health problem of human pregnancy. The search for altered physiological processes in pre-eclamptic placentae requires the investigation of events at both the microvillous and basal surfaces. The aim of this study was to obtain reliable syncytiotrophoblast plasma membranes from human normal (N) and pre-eclamptic (PE) pregnancies. We describe a protocol which allows for the simultaneous isolation of MVM and BM. The purity of the membranes isolated was evaluated using enzymatic assays, binding studies, Western blotting and immunohistochemistry. Enrichment of alkaline phosphatase activity for MVM was 17 to 21-fold, with 13-16 per cent protein recovery, for both N and PE. Enrichment of adenylate cyclase activity for BM was 9-fold for N, and enrichment of dihydroalprenolol binding to beta-adrenergic receptors was 12-fold for N and 6-fold for PE, with 14 per cent protein recovery for both N and PE. Cross contamination was low and mitochondrial membrane contamination was negligible. We conclude that MVM and BM isolated from placentae of pre-eclamptic women are similar in enrichment and purity to those of healthy women, thus allowing their use in comparative electrophysiological studies.
Jimenez
V
V
Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile.
Henriquez
M
M
Llanos
P
P
Riquelme
G
G
D000199:Actins / Q000032:analysis; D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D045304:Cytochromes c / Q000032:analysis; D004082:Dihydroalprenolol / Q000378:metabolism; D005260:Female; D005615:Freezing; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D010920:Placenta / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D011225:Pre-Eclampsia / Q000201:enzymology / Q000378:metabolism*; D011247:Pregnancy; D011485:Protein Binding; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014327:Trophoblasts / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism
D000199:Actins; D008565:Membrane Proteins; D011943:Receptors, Adrenergic, beta; D004082:Dihydroalprenolol; D045304:Cytochromes c; D013385:Succinate Dehydrogenase; D000469:Alkaline Phosphatase; D000262:Adenylyl Cyclases