Chloride currents in primary cultures of rabbit proximal and distal convoluted tubules

Abstract

Cl− conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl− conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl−currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl− currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl− currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl− current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl− pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.

Separation of two Cl^– Currents in Cultured Human and Murine Mesangial Cells: Biophysical and Pharmacological Characteristics of I_Cl.vol and I_Cl.Ca

“…Acetate (0.37 B 0.03; n = 3) and aspartate (0.18 B 0.01; n = 4) were less permeable than Cl -, as replacing extracellular Cl -with these anions shifted E rev to more positive values, indicating that these substitutes enter the channel pore less readily than Cl -. This permeability sequence of I Cl.vol in SV40-transformed murine mesangial cells is similar in order and magnitude to ClC-3 [23] and other native I Cl.vol [24,25], including that in renal epithelial cells [26][27][28]. The permeability sequence of I Cl.vol in human mesangial cells (n = 3) was very similar to that in murine cells: I -(1.50 B 0.11); Br -(1.15 B 0.02), and F -(0.53 B 0.03).…”

“…The nature of the Ca 2+ -independent Cl -current in mesangial cells has been less clear, but it is inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and the K + channel openers pinacidil and P1075 [31]. The volume sensitivity of the current in previous studies was not assessed; however, the NPPB-sensitive current is likely to be I Cl.vol , as NPPB inhibits I Cl.vol in renal epithelia [26,27]. Our data indicate the presence of a significant I Cl.vol , clearly separated from I Cl.Ca , in mesangial cells.…”

PTH stimulates a Cl−-dependent and EIPA-sensitive current in chick proximal tubule cells in culture

“…Regarding the possibility of CFTR localization to the PT, several studies have demonstrated CFTR mRNA in PT segments or in cultured cells (21,31,38), whereas expression at the protein level has been observed by some (6, 11), but not other, investigators (38). In the present study, attempts were made to detect CFTR protein in monolayer extracts using two different anti-human CFTR antibodies.…”

“…Glibenclamide, a sulfonylurea receptor inhibitor used to stimulate ␤-cell insulin release, has also been widely used as a blocker of CFTR Cl Ϫ channels (41,43). Similarly, the arylaminobenzoate NPPB is known as a potent inhibitor of a variety of Cl Ϫ channels (33,38,41). Both of these blockers, when added to the apical side, significantly reduced the PTH-induced I sc response (Fig.…”

Inhibition of ENaC by intracellular Cl−in an MDCK clone with high ENaC expression

“…2 and 3). A Ca 2ϩ -activated Cl Ϫ conductance has been described in mouse M-1 cortical collecting duct cells (41) and in primary cultures of rabbit proximal and distal tubule cells (52). We were unable to detect an RT-PCR product using various primer pairs based on the CaCC sequence from human or mouse; however, it is quite possible that the dog sequence is significantly different.…”