Intermediary metabolism and its relationship with ion transport in isolated guinea pig colonic epithelial cells

Abstract

We describe a method to isolate surface cells from guinea pig distal colon that obtains a good yield and high viability, as demonstrated by a 99% exclusion of trypan blue, only a 10% liberation of lactate dehydrogenase after 30-min incubation at 37 degrees C, and the inability of succinate to stimulate oxygen consumption before plasma membrane permeabilization. Oxygen consumption (QO2) measured after the sequential addition of the following drugs showed that oligomycin inhibited QO2 by 67%, carbonyl cyanide p-trifluoromethoxyphenylhydrazone increased QO2 by approximately 70% of the basal consumption, and rotenone inhibited QO2 by 90%. Cells at 37 degrees C for 30 min maintained intracellular Na+ and K+ concentrations of 25 and 120 mM, respectively. The ATP consumed by the Na(+)-K+ pump was derived from oxidative phosphorylation (79%) and from glycolysis (21%). Initial rates for Na+ and K+ transported by the pump were 105 +/- 10 and 65 +/- 5 nmol.mg protein-1.min-1, respectively. The rates of Na+ and K+ transported per ATP consumed were estimated to be 2.5 Na+/ATP and 1.6 K+/ATP.