Erich Pfaff

Development of Intrahepatic Cholestasis by α-Naphthylisothiocyanate in Rats

Development of Intrahepatic cholestasis induced by α-naphthylisothiocyanate was studied in rats. At various times after α-naphthylisothiocyanate application, livers were isolated from treated rats and perf used hemoglobin-free to assess cholestatic parameters. Unstimulated bile flow was found to only slightly decrease, up to 10 h after α-naphthylisothiocyanate administration. In contrast, secretion into bile of sulfobromophthalein and taurocholate declined markedly between 4 and 7 h as their concentrations in the perfusate increased, and stimulation of bile flow by taurocholate decreased. The permeability of the bile-to-perfusate barrier to [14C] sucrose and [32P]orthophosphate increased in parallel with the changes in sulfobromophthalein and taurocholate distributions. This correlation of changes in the distribution of cholephilic substances with biliary accessibility for extracellular markers suggests that, in α-naphthylisothiocyanate-induced cholestasis, increased leakage of tight junctions may contribute to regurgitation of bile constituents into the vascular system.

Red cell ouabain binding sites, Na+K+-ATPase, and intracellular Na+ as individual characteristics

Red cells were used as model cells to assess the relationship between the number of ouabain binding sites and the Na+K+-ATPase activity in humans. (3H) ouabain binding to intact red cells was found to be a reversible reaction and rectilinear Scatchard plots were obtained. Interindividual comparison revealed identical slopes, whereas the abscissa intercepts differed, indicating variations in the number of ouabain binding sites. The number of sites ranged from 330 to 890 (x = 453 ± 104 SD) per red cell in 73 individuals and was intraindividually stable during the course of 3–12 months. The number of sites was significantly lower in the middle-age females compared to younger ones. A linear relationship (r=0.96) was found between the total number of ouabain binding sites of intact red cells and the Na+K+-ATPase activity of red cell ghosts. The catalytic activity varied little among individuals; a turnover number of 5500 ± 250 (SD) molecules ATP hydrolyzed per site and per min was calculated. A highly significant correlation was established between [Na+]i of red cells in vivo and the number of ouabain binding sites.

Cholestatic steroid hormones inhibit taurocholate uptake into isolated rat hepatocytes

Abstract The effect of three cholestatic steroids (norethandrolone, 17-β-estradiol and progesterone) on hepatocellular uptake and secretion of taurocholate was studied in isolated rat liver cells. The steroids decreased the rate of taurocholate uptake. Norethandrolone inhibited uptake noncompetitively with a Ki of 18 μM, but had no effect on the activation energy of uptake. 17-β-estradiol and progesterone reduced taurocholate uptake by 50 per cent at concentrations between 40 μM and 50 μM. The secretion of taurocholate from taurocholate-loaded cells was slightly increased by all three steroids at concentrations below 100 μM. A 60 per cent inhibition of secretion was observed in the presence of 500μM norethandrolone. Interference of cholestatic steroids with hepatocellular bile acid uptake may be an important step in the pathogenesis of intrahepatic cholestasis.

Regulation of canalicular bile formation by α-adrenergic action and by external ATP in the isolated perfused rat liver

In isolated perfused rat liver, addition of adrenaline induced a complex response of bile flow including rapid, reversible stimulation (1/2-2 min), reversible inhibition (2-10 min), and prolonged stimulation. Both the reversible stimulation and the inhibition were mimicked by the alpha-sympathomimetic agonist phenylephrine but not by the beta-agonist isoproterenol. The reversible stimulation was a very early effect being terminated prior to all other alpha-adrenergic responses of liver. External ATP considerably lowered bile flow while inducing release of glucose and lactate, inhibition of respiration, and a reversible efflux of Ca2+. Variations of mannitol clearance parallel to those of bile flow indicate a canalicular origin of all changes.

Viability control and special properties of isolated rat hepatocytes

The need for quick viability tests is stressed. As these should achieve more than statistically categorizing dead or non-dead cells, several procedures are suggested that picture the energetic state of the cells. The almost classical criterion of this category, namely stimulation of respiration by succinate, must be questioned on the basis of the present results. It is shown, that restricted respiration by succinate is not due to limited permeability of the plasma membrane, but to competition by endogenous substrates for uptake into mitochondria. Distribution equilibria for succinate appear to be according to (ΔpH)2 with regard to cytoplasm. They are attained within 5–20 s or faster. Uptake is in part regulated by the surface charge density. Permeability changes caused by effectors of surface charge, such as amphiphilic ions, are examplified for succinate, chloride, phosphate, Na+, K+, and Ca2+. Such changes repeatedly also occur after pulses of BSP. They are counterregulated by the cell within a minute in a manner dependent on BSP concentration and the state of the cells. During the preincubation phase, that is the time of readaptation after transfer of cells from 0° C to higher temperature, a special labile state transiently occurs, where cyclic permeability changes for Ca2+, Na+, K+ can be caused by substrate addition, especially succinate, and/or ATP. The extent of these changes and their sequence again depend on the energetic state of the cells. In a probably narrow energetic window a sequence of cation movements reminding of that after depolarization of an excitable cell, is observed. Manipulation of the Na+/K+-ratio by variation of preincubation time and by ouabain shows that this is not simply the denominator for reversible calcium uptake. As the surface charge appears to reflect the energetic state, ANS fluorescence is applied to monitor the state of the plasma membrane, though difficulties arising from a slow ANS permeation are not yet solved.

Drug-induced intrahepatic cholestasis: characterization of different pathomechanisms

The pathogenesis of intrahepatic cholestasis in rats was studied using isolated perfused livers as an experimental model. Three basic mechanisms were differentiated: 1. Permeabilization of the bilio-sinusoidal barrier associated with electron microscopic alterations of the tight junctional complexes was found in livers of rats treated with α-naphthylisothiocyanate (ANIT, 250 mg/kg body weight). Consequences of these alterations were: reflux of bile constituents such as taurocholate and sulfobromophthalein and increased access to the biliary space of paracellular markers such as inulin and sucrose. The clear-cut mechanism of ANIT cholestasis was used to distinguish other mechanisms of intrahepatic cholestasis. 2. Inhibition of the basic process of fluid secretion was found to be the primary event in the development of cholestasis induced by estrogens. After 5 days of treating rats with ethinyl estradiol (5 mg/kg/day), bile flow was diminished in isolated livers while the permeability of the biliary tract to sucrose and inulin was not affected. Accordingly, the maximal concentration of taurocholate in bile was increased, indicating that its secretion was sustained. The same effect was observed after 1 week of treatment with the depot estrogen estradiol valerate (1 mg/kg/week). After 3 weeks of treatment, however, the taurocholate concentration in bile was lowered and the clearance of sucrose was increased. Bile flow remained at the same cholestatic level for 20 weeks. These results suggest that estrogens have the potency to increase tight junctional permeability only in a second step in the development of cholestasis, following the inhibition of bile flow. 3. An additional mode of secretory inhibition was induced by lowering the concentration of Ca2+ in the perfusate of isolated liver. Using ANIT-pretreated livers, i. e., livers with very low capacity to secrete foreign dyes, a high rate of efflux of sulfobromophthalein into the perfusate of preloaded livers suggests stimulation of the efflux of cholephilic solutes across the sinusoidal membrane of liver cells.The results demonstrate that the term intrahepatic cholestasis comprises a number of different sites of interference with the complex process of bile secretion.

Interaction of bromosulfophthalein with mitochondrial membranes--inhibition of respiration.

Abstract Bromosulfophthalein reversibly inhibits mitochondrial respiration. Most sensitive is state 3 respiration (Ki about 3 nmole/mg protein independent of substrate). At higher concentrations (20–100 nmole/mg protein) state 4 respiration and uncoupled respiration are also inhibited. This inhibition is substrate dependent. With succinate, inhibition appears to be noncompetitive at low concentrations and competitive above 1 mM succinate (half-maximal inhibition at 9–17 nmole/mg protein, dependent on succinate concentration). Substrate permeation seems to be not the only sensitive step in oxidation, as is deduced from similar results obtained on glycerol phosphate respiration. By use of artificial electron shunts and by difference spectroscopy, individual dehydrogenases have been made probable as the site of bromosulfophthalein action. It is suggested that bromosulfophthalein acts via the electro-static effects of the increased negative surface charge, making dehydrogenases less accessible for their substrates.

Interaction of bromosulfophthalein with mitochondrial membranes: Effect on ion movements

Abstract The increase in negative surface charge density resulting from binding of the anionic dye bromosulfophthalein to mitochondrial membranes affects the movement of a variety of ions, depending on the dye concentration. At low concentrations, a sort of membrane-stabilization with decreased permeabilities is observed, whereas at high concentrations, a detergent like action occurs. (1). Acidification on respiration of mitochondrial suspensions is increased by very small concentrations (= 3 nmol/mg protein) of bromosulfophthalein. It reflects an increased transmembranal pH gradient. The effect is mainly due to inhibition of phosphate translocation. (2), Mitochondrial swelling experiments under specific conditions showed that transport of inorganic phosphate is inhibited in either direction. (3). Concentrations of > 10 nmol bromosulfophthalein/mg protein result in a membrane-destabilization. This is reflected in a release of Ca 2+ ions and a subsequent increase in membrane permeability for succinate and inorganic phosphate. (4). All effects of bromosulfophthalein are freely reversible by the addition of albumin, to which the dye is bound with high affinity. (5). Similar effects can be observed with other compounds which are similar in structure, although none of those tried is effective in such small concentrations.

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na+-free, Na+ and K+-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP2+) and ATP. It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K+. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na+/K+ ratio, both effects were partially overcome by GSH. After treatment by DIP2+, Na+/K+ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K+ and accumulation of Na+. Here neither effect could be reversed by GSH; consequently, high cellular amounts of K+ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake. It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.

Influence of viability on bromosulfophthalein uptake by isolated hepatocytes.

SummaryThe initial rates of BSP uptake by isolated hepatocytes were compared in cells of good and poor viability. Cells with impaired viability were obtained by ageing or by accident also in fresh preparations. Viability was judged by trypan blue stainability, membrane potential and respiratory parameters indicative for energy state, substrate supply and plasma membrane permeability changes. It was found that concomitant with impaired viability there was a decline of uptake rates at low and an increase at high BSP concentrations with a crossover point at 10 μM as manifest in an increase of Km and V. Simultaneously, the affinity and size of the membrane bound fraction decreases. The results give kinetic support to the supposition that it is the decreased uptake from plasma to liver that is responsible for the prolonged plasma retention times in the liver function test of patients with impaired hepatobiliary function.