EXTRACELLULAR PH DETERMINES THE RATE OF CA2-DARBY CANINE KIDNEY-FOCUSCELLS( ENTRY INTO MADIN)

We investigated the relationship between intracellular Ca2+ and pH hom eostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell lin e exhibiting spontaneous oscillations of intracellular Ca2+ concentrat ion (Ca-i(2+)). Ca-i(2+) and intracellular pH (pH(i)) were measured wi th the fluorescent dyes Fura-2 and BCECF by means of video imaging tec hniques. Ca2+ influx from the extracellular space into the cell was de termined with the Mn2+ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontane ous Ca-i(2+) oscillations were observed in virtually all cells investi gated. Successive alkalinization and acidification of the cytoplasm in duced by an ammonia ion prepulse had no apparent effect on Ca-i(2+) os cillations. On the contrary, changes of extracellular pH value strongl y affected Ca-i(2+) oscillations. Extracellular alkalinization to pH 7 .6 completely suppressed oscillations, whereas extracellular acidifica tion to pH 6.8 decreased their frequency by 40%. Under the same condit ions, the respective pH(i) changes were less than 0.1 pH units. Howeve r, experiments with the Mn2+ quenching technique revealed that extrace llular alkalinization significantly reduced Ca2+ entry from the extrac ellular space. Large increases of Ca-i(2+) triggered by the blocker of the cytoplasmic Ca2+-ATPase, thapsigargin, had no effect on pH(i). We conclude: intracellular Ca2+ homeostasis in MDCK-F cells is pH depend ent. pH controls Ca2+ homeostasis mainly by effects on the level of Ca 2+ entry across the plasma membrane. On the contrary, the intracellula r pH value seems to be insensitive to rapid changes of Ca-i(2+).