Changes in intracellular pH and pH regulating mechanisms in somitic cells of the early chick embryo: a study using fluorescent pH-sensitive dye.
1. Measurements of intracellular pH (pHi) have been made using the fluorescent dye dicyano-hydroquinone (DCH) in somites isolated from 2-day-old chick embryos. Measurements of pHi, from freshly dissected somites, gave values of 7.18 +/- 0.02 (mean +/- S.D., n = 12) while in somites kept in media containing 10% fetal calf serum for 2-5 h pHi was 7.36 +/- 0.02 (n = 11). 2. In freshly dissected control tissue recovery from an acid load (NH4Cl pre-pulse), in the nominal absence of HCO3-, was 0.007 +/- 0.003 pH units/min (n = 11). In the presence of 5 mM-HCO3- the rate of recovery was increased to 0.132 +/- 0.003 pH units/min (n = 11). This HCO3(-)-dependent recovery was inhibited by pre-treatment with DIDS (5 X 10(-4) M) and stopped when Na+ was replaced in the bathing medium with K+ or N-methylglucamine. Amiloride (10(-3) M) had no effect. 3. Replacement of Cl- with gluconate had little effect on pHi in control somites suggesting that absence of Cl(-)-HCO3- exchange. 4. These observations are consistent with the presence of a coupled Na+-HCO3- entry and the absence of Na+-H+ exchange. 5. In serum-treated cells recovery from an acid load was 0.101 +/- 0.01 pH units/min (n = 11) in the nominal absence of HCO3-. Increasing the HCO3- concentration (5-10 mM) or pre-treatment with DIDS had no effect on the rate of acid extrusion. Recovery is Na+ dependent and inhibitable with amiloride, indicating the presence of Na+-H+ exchange. 6. These results suggest that during somitic development the mechanisms regulating pHi and recovery from acid loading are modified from a Na+-HCO3- influx to a Na+-H+ exchanger. This transition allows the cells to increase pHi by up to 0.2 pH units and may have a role in somitic cell development.
Gillespie
J I
JI
Department of Physiological Sciences, Medical School, University of Newcastle.
Greenwell
J R
JR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates; D002021:Buffers; D002642:Chick Embryo; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones; D008536:Meglumine; D008648:Mesoderm / Q000502:physiology*; D011188:Potassium; D012964:Sodium; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D001639:Bicarbonates; D002021:Buffers; D005456:Fluorescent Dyes; D006873:Hydroquinones; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C030753:2,3-dicyanohydroquinone; D008536:Meglumine; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium