Evidence for location of the CFTR in human placental apical membrane vesicles

Abstract

Ion transport (36Cl uptake) and immunochemical studies were undertaken to detect the cystic fibrosis transmembrane conductance regulator (CFTR) in apical membrane vesicles prepared from human placenta. 36Cl uptake into membrane vesicles was studied in the absence and presence of inwardly directed potassium gradients and valinomycin (Ko = Ki and Ko > Ki, where Ko is potassium concentration outside and Ki is potassium concentration inside the vesicles). The sensitivities of 36Cl uptake to the inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), bumetanide, and diphenylamine-2-carboxylate were investigated. Each compound significantly inhibited uptake under both sets of conditions. Additional inhibition of 36Cl uptake was found when the compounds were added together, indicating that they were acting at least partly on different components of the 36Cl uptake. The DIDS- and bumetanide-insensitive component of transport was more selective for Cl than I. These findings suggested that this component may, at least in part, represent Cl transport via CFTR. Addition of adenosine 5'-O-(3-thiotriphosphate) (0.8 mM) led to a decrease in total 36Cl uptake but masked in the overall decrease was an increase in the DIDS- and bumetanide-insensitive component of 36Cl uptake. Western blot analysis of the apical membrane proteins with an antibody specific for a region of human CFTR detected a protein band of approximately 190 kDa. These ion transport and immunochemical studies provide evidence that CFTR is located in human placental apical membrane vesicles.

ATP-stimulated Ca2+-activated K+ efflux pathway and differentiation of human placental cytotrophoblast cells

“…Although the mechanism for ATP exit in human placenta has not been determined directly, the components for this are in place, and there is evidence for expression of ABC transporters in the syncytiotrophoblast of the human placenta (2,14). After ATP exits the cell, it is thought to act in a paracrine/autocrine manner as a regulator of cell function (17,40,49).…”

“…It is thought that ATP is released from cells following shear stress/cell activation, in response to hyposmotic challenge and/or via ATP-binding cassette (ABC) transporters (1,5,13,40,44). In the human placenta there is evidence for the ABC transporters cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) on the microvillous membrane of the syncytiotrophoblast, which might provide a pathway for cellular ATP exit (2,14). There are also data demonstrating increased intracellular Ca 2ϩ ([Ca 2ϩ ] i ) in isolated cytotrophoblast cells following exposure to extracellular ATP (26, 39).…”

Regulation of cell volume via microvillar ion channels

“…• Epithelial Na ϩ channel (Cantiello et al, 1991;Smith et al, 1991Smith et al, , 1997Menco et al, 1998), anion channels with Cl Ϫ , ATP, and osmolyte permeability such as MDR (Dutt et al, 1994;Hunter et al, 1993;Sussman-Turner and Renfro, 1995), CFTR (Faller et al, 1995), MRP/MOAT (Chu et al, 1999), monocarboxylate transporter MTC (Bergersen et al, 1999), and occasionally water channels (Deen et al, 1997;Funaki et al, 1998) were found on apical microvilli of polarized epithelial cells.…”

“…Under these conditions, autocrine ATP signaling is confined to the apical surface and is most likely the result of the CFTR and MRP type ATP channels of the apical pole (Elgavish and Elgavish, 1985), which have been detected on apical microvilli (Hunter et al, 1993;Faller et al, 1995;Sussman-Turner and Renfro, 1995). Stimulation of apical purinergic receptors by released ATP results in insertion of additional Na ϩ and water channels into the apical surface and subsequently activates these pathways at the basal surface, too.…”

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes

“…So far, the Cl -channels reported in the human placenta include: the "maxi" channel [8,36,37], CFTR [15], Ca 2+ -activated [25] and volume-activated [7,25] channels. The latter three conductive pathways could not account for the Cl -conductance observed in hSM oocytes, given their activation mechanisms, but the "maxi" Cl -channels could indeed be responsible for such conductance.…”