NA-BORDER MEMBRANES - ARE THERE SEVERAL MECHANISMS( TRANSPORT BY HUMAN PLACENTAL BRUSH)

Na+ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na+ uptake was assayed by th e rapid filtration technique in the presence and the absence of an uph ill pH gradient. Amiloride strongly decreased Na+ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na+ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na+ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na+ uptake c onsistently showed a single kinetics. Increasing the pH gradient decre ased Km values of the amiloride-sensitive Na+ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive N a+ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na+. Since ethylisop ropylamiloride, the specific Na/H exchanger inhibitor mimicked the eff ects of amiloride, decreasing by 98% the 10 mM Na+ uptake, whereas ben zamil, the Na+ channel blocker, had no effect, it was concluded that t he amiloride sensitive Na+ uptake was predominantly or exclusively due to a Na+-H+ exchanger activity. K+ in trans-position significantly de creased the amiloride sensitive uptake. In contrast, the presence of t he cation in cis-position had no effect. The amiloride resistant Na+ t ransport was neither influenced by pH, nor saturable. Incubation of th e placental tissue with 100 mu M or 1 mM dibutyryl cAMP, 0.1 or 1 mu M phorbol myristate acetate, 10(-7) M insulin, 10(-10) M angiotensin II , or 10(-8) M human parathyroid hormone (PTH) did not influence Na+ tr ansport by subsequently prepared brush border membranes. Finally, we f ailed to demonstrate any Na+-H+ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of cosubstrat es such as phosphate and aminoacids, the Na+-H+ exchange is probably t he unique mechanism of Na+ transport by the placental brush border mem brane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the is oform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. (C) 1996 Wiley-Liss, Inc.