Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells.
We investigated the relationship between intracellular Ca2+ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca2+ concentration (Ca2+i). Ca2+i and intracellular pH (pHi) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca2+ influx from the extracellular space into the cell was determined with the Mn2+ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca2+i oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca2+i oscillations. On the contrary, changes of extracellular pH value strongly affected Ca2+i oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pHi changes were less than 0.1 pH units. However, experiments with the Mn2+ quenching technique revealed that extracellular alkalinization significantly reduced Ca2+ entry from the extracellular space. Large increases of Ca2+i triggered by the blocker of the cytoplasmic Ca(2+)-ATPase, thapsigargin, had no effect on pHi. We conclude: intracellular Ca2+ homeostasis in MDCK-F cells is pH dependent. pH controls Ca2+ homeostasis mainly by effects on the level of Ca2+ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rap changes of Ca2+i.
Wojnowski
L
L
Department of Physiology, University of Würzburg, FRG.
Mason
W T
WT
Schwab
A
A
Oberleithner
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins; D016257:Fura-2; D006706:Homeostasis / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D005452:Fluoresceins; D013729:Terpenes; D019284:Thapsigargin; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2