Free concentrations of intracellular fluorescent anions determined by cytoplasmic dialysis of isolated hepatocytes

Abstract

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 develop a method for measurement of the free cytosolic concentration of organic ions. This was accomplished by measuring initial rates of diffusion between patch-clamp pipettes and cell cytoplasm and determining the null-point concentration of this process. Carboxydimethylfluorescein (CF) was used as a model compound. It readily diffused between cytoplasm and pipette, and there was a linear relationship between concentration in the pipette and equilibrium cell fluorescence. When cells previously loaded with CF were patched, intracellular fluorescence rapidly changed in a positive or a negative direction, depending on the concentration of CF in the pipette. The null point, defined as the concentration at which cells neither gained nor lost fluorescence, described the same relationship between free concentration and total cell fluorescence as that determined by direct loading of the cells to equilibrium. In hepatocytes preloaded with a fluorescent bile acid derivative, cholylglycylamidofluorescein (CGamF), by exposure (0.05 microM) for 30 min, the null point occurred at a CGamF concentration in the pipette of 0.6 microM. This value is 12 times greater than that in the bath. In conclusion, a new method is described that can measure free cytosolic concentrations of fluorescent molecules. It should prove useful in determining the intracellular location and state of transported organic ions.

Bile Acid Uptake via the Human Apical Sodium-Bile Acid Cotransporter Is Electrogenic

“…Our previous studies have determined that diffusion of bile acids from cell to pipette is small and alters apparent uptake rates by only about 5% (23). The small magnitude of the effect is due to the high degree of intracellular binding of this hydrophobic substrate as well as the relatively small surface area of the pipette (26). Intracellular diffusion coefficients of bile acids are approximately 1000-fold lower than that predicted for simple aqueous diffusion (27).…”

Bile acid transporters

“…This vectorial trans-hepatocellular movement of bile acids is a remarkably concentrative transport process that is driven by a distinct set of primary (ATP-dependent), secondary (Na + gradient-dependent), and tertiary (OH Ϫ or HCO 3 Ϫ -dependent anion exchange) transport systems at the sinusoidal and canalicular plasma membranes ( 2,3 ). Hepatocellular uptake of bile acids occurs against a presumed unfavorable electrochemical gradient ( 18,19 ) via sodium-dependent and independent mechanisms. The uptake of conjugated (i.e., N-acyl amidated with taurine or glycine) bile acids, such as taurocholate, at the sinusoidal membrane is mediated predominantly (>75%) by secondary active Na + -dependent transport.…”

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

“…The pathway across hepatocytes is not yet fully understood and different mechanisms may step in depending on the bile salt load imposed to hepatocytes. At a physiologic exposition, the monomeric form of bile salts in the cytoplasm is likely < 1 μM [344]. Part of the bile salts are bound to cytoplasmic bile salt binding proteins, e.g.…”