Na+-independent proton secretion in MDCK-C11 cells.
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.
Fernandez
R
R
Depto de Fisiologia, Setor de Ciências Biológicas, Universidade Federal do Parana, Brazil.
Oliveira-Souza
M
M
Malnic
G
G
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007668:Kidney / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides; D008856:Microscopy, Fluorescence; D054328:Proton Pump Inhibitors; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D012964:Sodium / Q000008:administration & dosage / Q000494:pharmacology*; D012996:Solutions
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D007093:Imidazoles; D018942:Macrolides; D054328:Proton Pump Inhibitors; D012996:Solutions; C035235:Sch 28080; D000643:Ammonium Chloride; C036978:concanamycin A; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D006180:Proton-Translocating ATPases