Factors affecting chloride conductance in apical membrane vesicles from human placenta

Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. 36Cl- uptake into these vesicles was studied in the presence of an outwardly directed Cl- gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 microM valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl- were examined in the presence and absence of 5 microM valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC50 values of 0.83 +/- 0.35 microM and 3.43 +/- 0.37 microM, respectively, in the absence of valinomycin. Although valinomycin reduced 36Cl- uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC50 of 5.6 +/- 2.1 microM. Under these conditions, NPA was without effect at concentrations below 100 microM. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced 36Cl- uptake significantly at 100 microM concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed. The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl- uptake with IC50 values of 37.6 +/- 14.9 microM and 4.59 +/- 0.51 microM, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl- gradient and this channel is sensitive to Cl- channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.