Biomedical subjects

P Llanos

Publications and source records attributed to P Llanos.

3 recordsLinked to original sources

PubMed2004

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.

Actins↗

PubMedDate not supplied

Large chloride channel from pre-eclamptic human placenta.

Chloride transport involving conductive pathways participates in numerous epithelial functions, such as membrane voltage maintenance, solute transport and cell volume regulation. Evidence points to involvement of transepithelial chloride transport in such functions in placental syncytiotrophoblast. A molecular candidate for physiologic conductive chloride transport in apical syncytiotrophoblast membrane is a Maxi-chloride channel with distinct biophysical properties: conductance over 200 pS, multiple substates, voltage dependent open probability, and permeation to anionic amino acids. Pre-eclampsia, a high incidence pathology of pregnancy, exerts great impact on fetal morbi-mortality. This relies, among others, on intrauterine growth restriction (IUGR), thought to be mediated by diminished blood flow to the placenta, with growing knowledge regarding contribution of other factors. The Maxi-chloride channel's properties suggest it could be altered in this pathology. We have characterized the apical chloride channels from pre-eclamptic placentae, reconstituted in giant liposomes suitable for patch-clamp electrophysiological studies. In n=33 experiments from n=6 pre-eclamptic placentae we observed a chloride-permeable channel with similar biophysical properties to the channel from normal tissue (n=29 experiments from n=15 placentae). However, the main conductance state showed diminished magnitude (<150 pS), and the open probability versus voltage relationship exhibited a flattened curve instead of the bell-shaped curve of normal placentae. These results are the first evidence of a functionally altered ionic channel from placental syncytiotrophoblast in pre-eclampsia. Considering the abundance of chloride-conducting channel activity in human apical membrane and their relevance in epithelial function in general, these alterations could greatly disturb numerous placental functions that rely on syncytiotrophoblast integrity.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMedDate not supplied

A low conductance, non-selective cation channel from human placenta.

Non-selective cation channels have been identified in the plasma membranes of many different cells. Previous research using fluorescent techniques has demonstrated the presence of cation conductances in membranes from human trophoblast. The purpose of this work was to explore, by electrophysiological methods, a non-selective cation channel in apical membranes from human placenta. Human placental apical membranes were purified by differential centrifugation and reconstituted in giant liposomes. These giant liposomes were then used for electrophysiological studies and were probed for the presence of cation channels by the patch-clamp method. The channel identified had a linear current-potential relationship with a conductance of around 16 pS in symmetrical Na(+) solution. Under asymmetrical conditions the reversal potential was close to the reversal potential for Na(+). The channel was equally permeable to sodium and potassium and the permeability sequence was NH+4>Cs(+) approximately Rb(+)>Na(+) approximately K(+)>Li(+). The channel also showed permeability to calcium and barium. The channel was insensitive to calcium but was blocked by millimolar concentration of Mg(2+). We have demonstrated the presence of a low conductance, non-selective cation channel in placental apical membranes. These channels share some properties with non-selective cation channels previously described in other different cells. The precise role of these channels in placental physiology has yet to be determined.

Adult↗