Sa. Weinman et Lm. Maglova, FREE CONCENTRATIONS OF INTRACELLULAR FLUORESCENT ANIONS DETERMINED BYCYTOPLASMIC DIALYSIS OF ISOLATED HEPATOCYTES, American journal of physiology: Gastrointestinal and liver physiology, 30(5), 1994, pp. 70000922-70000931
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.