2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and gluconic-acid

Fragments of small interlobular bile ducts averaging 20 microns in diameter can be isolated from rat liver. These isolated bile duct units form luminal spaces that are impermeant to dextran-40 and expand in size when cultured in 10 microM forskolin for 24-48 hr. Secretion is Cl- and HCO3- dependent and is stimulated by forskolin > dibutyryl cAMP > secretion but not by dideoxyforskolin, as assessed by video imaging techniques. Secretin stimulates Cl-/HCO3- exchange activity, and intraluminal pH increases after forskolin administration. These studies establish that small polarized physiologically intact interlobular bile ducts can be isolated from rat liver. These isolated bile duct units should be useful preparations for assessing the transport properties of small bile duct segments, which are the primary site of injury in cholestatic liver disorders, known as "vanishing bile duct syndromes."

Topics: 1-Methyl-3-isobutylxanthine; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bicarbonates; Bile Ducts; Bucladesine; Chlorides; Cholestasis; Colforsin; Fluoresceins; Gluconates; Hydrogen-Ion Concentration; Kinetics; Liver; Male; Microscopy, Electron; Microscopy, Video; Rats; Rats, Sprague-Dawley; Secretin; Time Factors

1. Intracellular pH (pHi) was measured in single rat cerebellar Purkinje cells maintained in primary culture using microspectrofluorescence analysis of the intracellularly trapped pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF). 2. The ratio of the fluorescence signals measured at 530 nm in response to an alternating excitation at 450 and 490 nm was calibrated using the K(+)-H+ ionophore nigericin. This calibration gave a steady-state pHi of 7.06 +/- 0.02 (S.E.M., n = 17) when cells were perfused by a 5% CO2-25 mM-HCO3(-)-buffered solution at an external pH of 7.40 at 37 degrees C. 3. Replacement of external chloride with gluconate in the presence of bicarbonate induced a cytoplasmic alkalinization of about 0.3 pH unit. This alkalinization was independent of external sodium and was greatly reduced by 0.5 mM-DIDS, indicating the presence of a chloride-bicarbonate exchange. 4. In bicarbonate-free (HEPES-buffered) solution the steady-state pHi was 7.37 +/- 0.02 (n = 19), significantly higher than in bicarbonate-buffered solution. Recovery from an intracellular acid load brought about by the ammonium chloride pre-pulse technique was blocked by the removal of external sodium or the addition of 1.5 mM-amiloride, indicating the presence of a sodium-hydrogen exchange. 5. In bicarbonate-buffered solution pHi recovery after an acid load was also completely blocked by addition of 1.5 mM-amiloride indicating the absence of a bicarbonate-dependent acid extrusion mechanism. 6. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) induced an amiloride-sensitive alkalinization of about 0.3 pH unit in bicarbonate-buffered solution but had no effect in HEPES-buffered solution. This observation suggests that in cultured Purkinje cells the sodium-hydrogen exchanger could be activated through a protein kinase C pathway only when pHi is maintained at a low physiological value by the activity of the chloride-bicarbonate exchange.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Bicarbonates; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; HEPES; Hydrogen-Ion Concentration; Ion Exchange; Purkinje Cells; Rats; Sodium; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Amiloride; Animals; Bicarbonates; Carrier Proteins; Cell Survival; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; Hydrogen-Ion Concentration; Liver; Male; Monensin; Oxygen; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers