Na + -independent proton secretion in MDCK-C11 cells

Abstract

In this work we studied the proton secretion mechanisms in recently cloned MDCK-C11 cells. We measured intracellular pH (pHi) in monolayers grown on permeable filters, using the pH-sensitive probe BCECF and an inverted epifluorescence microscope. The cells have a basal pHi of 7.20+/-0.01 (n=136) and after an acid-releasing NH4Cl pulse pHi recovered at a rate (dpHi/dt) of 0.167+/-0.006 pH units/ per minute (n=20). This rate decreased significantly when Na+ was removed from both cell surfaces, and was further reduced when they were both perfused with a solution containing no Na+ and K+. pHi recovery fell again in the presence of concanamycin (at a concentration of 4.6x10(-8) M; a specific inhibitor of the vacuolar H+-ATPase). When Na+ was removed from the apical or the basolateral side, pHi recovery (in pH units per minute) was significantly reduced to 0.099+/-0.008 (n=11) and 0.086+/-0.01 (n=10), respectively. The Na+-independent mechanism of pHi recovery was significantly inhibited by the presence of 5 x 10(-5) M Schering 28080 (an inhibitor of the H+-K+-ATPase) at the apical side (0.065+/-0.01 versus 0.099+/-0.008 pH units per minute, P<0.05), but not at the basolateral side (0.072+/-0.01 versus 0.086+/-0.01 pH units per minute). On the other hand, concanamycin inhibited the Na+-independent pHi recovery when applied apically (0.0304+/-0.005 pH units per minute, n=7) and basolaterally (0.025+/-0.004 pH units per minute, n=7). From these results we conclude that monolayers of MDCK-C11 cells have a Na+/H+ exchanger and a concanamycin-sensitive H+-ATPase on their apical and basolateral membranes; and a K+-dependent, Schering 28080-sensitive H+-K+-ATPase on their apical side.

The effect of angiotensin II on intracellular pH is mediated by AT1 receptor translocation

“…5A). However, a slight pH i recovery was observed, probably due to Na ϩ -independent proton extrusion mechanisms (8,9,20). Our results indicate that in nontransfected and transfected MDCK cells (Figs.…”

“…Cells were incubated for 5 h at 37°C with 5% CO 2 and then cultured for 24 h in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 2 mmol/l L-glutamine. The individual cell lines that stably expressed the recombinant hAT 1/pEGFP-N1 were selected with geneticin (G418 1 mg/ml) and used for all experiments (passages [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”

ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H&lt;sup&gt;+&lt;/sup&gt;-ATPase subcellular vesicle trafficking

“…Because two-thirds of these cells exhibited a peanut lectin binding capacity [23], they resemble intercalated cells in the renal collecting duct and are presumably the cells in which the H + -ATPase under investigation is localized. Supporting this proposal, the two Na + -independent proton secretion mechanisms found in these cells, the H + /K + -ATPase and the vacuolar H + -ATPase [28,29], are similar to the mechanisms found in the intercalated cells of the mammalian collecting duct. The experiments were performed in an Na + -free solution (to inhibit the Na + /H + exchanger) and in the presence of Schering 28080 (to inhibit the H + /K + -ATPase), experimental conditions under which H + -ATPase provides the only mechanism for pHi recovery in these cells [13], which is inhibited by concanamycin (a specific inhibitor of the vacuolar H + -ATPase) [29].…”

“…Supporting this proposal, the two Na + -independent proton secretion mechanisms found in these cells, the H + /K + -ATPase and the vacuolar H + -ATPase [28,29], are similar to the mechanisms found in the intercalated cells of the mammalian collecting duct. The experiments were performed in an Na + -free solution (to inhibit the Na + /H + exchanger) and in the presence of Schering 28080 (to inhibit the H + /K + -ATPase), experimental conditions under which H + -ATPase provides the only mechanism for pHi recovery in these cells [13], which is inhibited by concanamycin (a specific inhibitor of the vacuolar H + -ATPase) [29]. Under the preliminary experimental conditions in the absence of acid loading, our data indicate that the acridine orange was taken up rapidly by the cell, concentrated in the cytoplasmic vesicles and is not lost during the initial 12 min.…”

Cl- and regulation of pH by MDCK-C11 cells

“…Cell pH (pHi) was measured using the fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) (12,13). Cells grown to confluence on permeable filters were loaded with the dye by exposure for 20 min to 12 µM BCECF-AM in the control solution (solution 1, Table 1).…”