Measurement of intracellular calcium and pH in avian neural crest cells

1. Intracellular pH (pHi) and calcium (Cai2+) were studied in freely migrating neural crest cells and in closely packed non-migrating cells derived from avian neural tubes in vitro, using the fluorescent dyes 2,3-dicyanohydroquinone (DCH) and Indo-1 to measure pHi and Cai2+ respectively. 2. In freely migrating crest cells the pHi was approximately 0.2 pH units more alkaline and Cai2+ 90 nM lower than in closely packed cells. 3. Experiments to establish the cellular mechanisms regulating pHi in isolated neural crest cells demonstrate the presence of Na(+)-H+ exchange in 66% of the cells and Na(+)-HCO3(-)-dependent pHi-regulating mechanisms in all cells examined. 4. Interactions between pHi and Cai2+ were examined. pHi was altered using either NH4Cl pulses resulting in small changes in Cai2+ or using a weak acid and base (propionate and trimethylamine), which produced a fall and a rise in Cai2+ respectively. 5. Exposure to Ca2(+)-free media caused a lowering of Cai2+ and induced a transient acidification. 6. Application of BAPTA-AM (50 microM), a cell-permeant analogue of EGTA, resulted in a fall in Cai2+ and an intracellular acidification. 7. Co2+ and La3+ (2 mM) each induced a reversible fall in Cai2+ that was accompanied by intracellular acidification. These data suggest the presence of a transmembrane flux of Ca2+ in the resting cells. 8. It would appear that the mechanisms influencing Cai2+ and pHi are linked. This idea is discussed in terms of possible mechanisms and roles for Ca2+ and pH as modulators of neural crest cell behaviour.