FREE CONCENTRATIONS OF INTRACELLULAR FLUORESCENT ANIONS DETERMINED BYCYTOPLASMIC DIALYSIS OF ISOLATED HEPATOCYTES

Intracellular organic ions exist in free solution bound to cytoplasmic proteins, partitioned within intracellular membranes, and enclosed in intracellular vesicles and organelles. The aim of this study was to d evelop a method for measurement of the free cytosolic concentration of organic ions. This was accomplished by measuring initial rates of dif fusion between patch-clamp pipettes and cell cytoplasm and determining the null-point concentration of this process. Carboxydimethylfluoresc ein (CF) was used as a model compound. It readily diffused between cyt oplasm and pipette, and there was a Linear relationship between concen tration in the pipette and equilibrium cell fluorescence. When cells p reviously loaded with CF were patched, intracellular fluorescence rapi dly changed in a positive or a negative direction, depending on the co ncentration of CF in the pipette. The null point, defined as the conce ntration at which cells neither gained nor lost fluorescence, describe d the same relationship between free concentration and total cell fluo rescence as that determined by direct loading of the cells to equilibr ium. In hepatocytes preloaded with a fluorescent bile acid derivative, cholylglycylamidofluorescein (CGamF), by exposure (0.05 mu M) for 30 min, the null point occurred at a CGamF concentration in the pipette o f 0.6 mu M. This value is 12 times greater than that in the bath. In c onclusion, a new method is described that can measure free cytosolic c oncentrations of fluorescent molecules. It should prove useful in dete rmining the intracellular location and state of transported organic io ns.