Michael Schwenk

Uridine uptake by isolated intestinal epithelial cells of guinea pig.

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.

Transport systems of isolated hepatocytes

Hepatocellular transport of some substances with predominantly biliary elimination was studied by use of isolated rat liver cells. Active uptake of the hydrophilic compounds ouabain and cholate proceeded over 10–20 min and the final intracellular concentrations exceeded the extracellular concentrations more than 60-fold. Uptake of both substances was saturable and compatible with carrier-mediation. Uptake of the lipophilic compounds digitoxin and naphthol was not active, terminated within 1 min and compatible with simple diffusion.The velocity of uptake depended on 3 major parameters: substrate features, cellular factors, and experimental conditions. Substrate features included: lipophilicity or hydrophilicity of the compound. Cellular factors included: presence and activity of transport systems. Experimental conditions included: substrate concentration, amount of cells, composition of medium.Intracellular conjugation of cholate and naphthol produced the same polar metabolites which are formed in vivo. These metabolites were then in part released from the isolated hepatocytes into the medium. The transport systems for release are more difficult to characterize than those for uptake, and therefore little is known about their nature, despite their importance in detoxication.If isolated hepatocytes are used in toxicology to assess metabolism or biological effects of foreign compounds, transport across the liver cell membrane is potentially a rate limiting step.

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.

Uptake, accumulation and release of ouabain by isolated rat hepatocytes.

SummaryWe investigated uptake of ouabain into isolated rat hepatocytes and release of ouabain from preloaded hepatocytes, thus assessing separately the two membrane transport steps, involved in biliary elimination of the drug. The following results were obtained:Uptake of ouabain was saturable (Km=263±61μM, V=3.3±1.0 nmol/mg prot.×min), energy-dependent, sensitive to dinitrofluorobenzene and temperature-dependent (E=80−96 kJ/mol). It had no pH-optimum in the physiological pH-range and was independent of the extracellular cation composition.Uptake of ouabain was competitively inhibited by the cardiac glycoside digitoxin (Ki=0.3 μM).Uptake was not inhibited in the presence of the glycosidic sugar l-rhamnose, but it was competitively inhibited by the steroid taurocholate (Ki=6.3 μM).Ouabain was accumulated within hepatocytes 170-fold. The predominant fraction of intracellular ouabain beeing unbound.Release of ouabain from preloaded cells was energyindependent, independent of the Na+-concentration and not susceptible to inhibition by dinitrofluorobenzene or taurocholate.It is concluded, first that hepatocellular uptake of ouabain is mediated by a carrier for steroids and second that the pathway of release is distinct from that of uptake. We assume, that the high bile/plasma concentration-gradient of ouabain in vivo is generated during active uptake into the cell and not during release into bile.

Uptake of estrone sulfate by isolated rat liver cells

Abstract Hepatocellular uptake of the endogenous sulfate ester estrone sulfate was characterized by use of isolated liver cells: (1) Uptake was saturable with an apparent K M of 0.8 μM and a V max of 0.3 nmol/mg protein × min. (2) Uptake was inhibited by the sulfhydryl (SH-) poisons mersalyl and dinitrofluorobenzene. (3) Taurocholate and estrone sulfate competed for uptake. (4) Activation energy was 67 KJ/mol; pH optimum was within physiological limits. (5) Inhibition of mitochondrial respiration lowered the uptake rate by 93%. (6) Ouabain and extracellular K + decreased the uptake rate. These data indicate that estrone sulfate uptake into rat hepatocytes is energy dependent and carrier mediated. Mutual competition for uptake of estrone sulfate and taurocholate suggests that both steroids are taken up by a single carrier.

Drug transport in intestine, liver and kidney

Drug transport in intestine, liver and kidney is similar, because in each case transport occurs across a barrier of epithelial cells. However, the physiological conditions differ in each organ: intestinal drug absorption is largely influenced by physicochemical conditions in the intestinal lumen; actual transport across the epithelial barrier occurs mainly by diffusion; carrier-mediated transport plays a subordinate role. In contrast, hepatic uptake is mediated by specific carriers, which transport a wide variety of drugs into the liver cell and then release them either into bile, or back into the portal blood. It is unclear how many carrier systems are involved, how they are organized in the liver cell membrane, and to what extent their substrate specificities overlap. Renal secretion and reabsorption of drugs is mediated by highly active carrier systems for cations and anions. Their cooperative action results in either active reabsorption or active secretion of drugs.

Sulfation in isolated enterocytes of guinea pig: dependence on inorganic sulfate

Isolated intestinal epithelial cells of the guinea-pig were used to study uptake and metabolism of inorganic sulfate in the jejunum (proximal cells) and ileum (distal cells). Proximal enterocytes accumulated sulfate 1.5-fold and distal enterocytes 3.1-fold. Accumulation was almost linearly related to substrate concentrations up to 5 mM. In proximal cells, despite their lower intracellular sulfate levels, sulfate incorporation into acid-precipitable material and sulfate conjugation of 1-naphthol were faster than in distal cells. Formation of 1-naphthyl sulfate increased with extracellular sulfate concentrations up to 1 and 3 mM sulfate in distal and proximal enterocytes, respectively. The data suggest that the extent of intestinal sulfation of phenolic compounds may be enhanced by oral administration of sulfate.

First pass biotransformation of ethinylestradiol in rat small intestine in situ

SummaryThe intestinal absorption and biotransformation of ethinylestradiol and of ethinylestradiol glucuronide in rats were studied using a jejunal loop preparation in situ. Radioactivity associated with ethinylestradiol amost completely appeared in the venous outflow within 80 min. Forty two percent was present as unchanged compond, 56% in the glucuronide fraction and 2% as sulfate ester. Absorption of ethinylestradiol glucuronide was about 20 times slower than absorption of the nonconjugated molecule. The data indicate, that intestinal absorption of ethinylestradiol involves several superimposed kinetics and that glucuronidation in the gut may result in a decreased bioavailability of the compound.

The kinetics of hepatocellular transport and metabolism of estrogens (comparison between estrone sulfate, estrone and ethinylestradiol).

Abstract The turnover of estrone, estrone sulfate and ethinylestradiol∗ was studied in the whole rat and in isolated rat liver cells with regard to the sequential steps involved in hepatic uptake, metabolism and secretion of estrogens. After i.v. injection of estrone or estrone sulfate (5 nmol/250 g rat), the maximal biliary excretion of both compounds is observed within 20 min, and the fractions of biliary glucuronides (about 79%) and sulfates (about 19%) derived from the two estrogens, are almost identical. Isolated liver cells have preserved their ability to hydroxylate and conjugate estrogens and to release the newly formed conjugates. The major difference between the hepatocellular turnover of estrone and estrone sulfate is uptake, which proceeds much slower with the conjugated form. Uptake of estrone sulfate is followed by deconjugation. This can explain the similar conjugate patterns of metabolites derived from estrone and estrone sulfate in vivo. The free estrogens are conjugated within the isolated hepatocytes. The conjugates are released from the cells mainly as glucuronides (about 35%) and sulfates (about 13% in the case of estrone). The conjugation of ethinylestradiol is faster than conjugation of estrone and can be depressed by inhibitors of microsomal hydroxylation.

Taurolithocholate inhibits taurocholate uptake by isolated hepatocytes at low concentrations.

SummaryThe cholestatic bile acid taurolithocholate inhibits taurocholate uptake by isolated liver cells non-competitively. Inhibition is instantaneous and inversely related to the cell number in the incubate. The Kiamounts to 7 μM in the presence of 2 mg cellular protein per ml. Secretion of taurocholate by isolated liver cells is not affected by taurolithocholate up to a concentration of 50 μM. This indicates a difference between the carrier for taurocholate uptake and the carrier for taurocholate secretion. Inhibition of bile acid uptake by liver cells may be involved in the pathogenesis of lithocholate-induced cholestasis.