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E Petzinger

Publications and source records attributed to E Petzinger.

At least 37 records · Page 2Linked to original sources
PubMed1995

Hepatobiliary transport of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors conjugated with bile acids.

To obtain prodrugs with affinity to liver parenchymal cells, the hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors HR 780 and lovastatin (syn. mevinolin) were conjugated with the bile acids cholic acid, taurocholic acid, and glycocholic acid. Hepatic uptake and biliary excretion of the coupled drugs were investigated and compared with the noncoupled drugs. Studies were performed with livers of normal Wistar rats, and TR-/GT- Wistar rats with deficient drug excretion. The experiments showed that the parent drug HR 780 was slowly excreted into bile. In contrast, the excretion of the bile acid-conjugated HR 780 derivatives S 3554 (conjugated with cholate), S 3898 (conjugated with glycocholate), and S 4193 (conjugated with taurocholate) was rapid and very efficient in both groups of rat strains. The bile acid-conjugated HMG-CoA reductase inhibitors showed a 10 to 20 times higher affinity for the uptake systems of bile acids than the noncoupled parent drug compounds, and even higher affinities than the bile acids themselves. The cholate conjugate of HR 780 (compound S 3554) was shown to be a noncompetitive inhibitor of taurocholate uptake and a competitive inhibitor of sodium-independent cholate uptake (Ki = 1 mumol/L). Uptake of radiolabeled S 3554 into isolated rat hepatocytes was observed to be rapid, cell specific, saturable, energy dependent, and carrier mediated. However, the carrier for S 3554 uptake was found not to be the cloned Na(+)-dependent taurocholate cotransporting polypeptide Ntcp. Expression of this carrier cRNA in Xenopus laevis oocytes did not stimulate S 3554 uptake.

ATP-Binding Cassette Transporters↗
PubMed1994

Deficiency of bile acid transport and synthesis in oval cells from carcinogen-fed rats.

Freshly isolated oval cells, which we obtained from the livers of rats fed a choline-deficient/DL-ethionine-supplemented diet, did not transport bile acids. Compared with freshly isolated rat hepatocytes they took up only negligible amounts of [3H]taurocholate or [14C]cholate. The cells bound small amounts of radioactive bile acids. This portion of the total cell-associated radioactivity was enhanced on membrane permeabilization. In contrast to cultured liver parenchymal cells from untreated rats, no bile acid synthesis was detected in cultured oval cells. Cultured oval cells also lost the ability to conjugate exogenously added cholate (100 mumol/L) with taurine or glycine. However, when liver parenchymal cells were isolated from carcinogen-fed rats, bile acid uptake was diminished compared with that in hepatocytes from control animals. In particular, the maximum values of taurocholate and cholate uptake were decreased by 75% and 50%, respectively, whereas the Michaelis-Menten constant values were not altered. The study demonstrates that (a) oval cells lack typical liver parenchymal cell-specific properties such as bile acid uptake, bile acid synthesis and conjugation of bile acids with taurine/glycine and therefore do not contribute to bile acid dependent bile formation (b) proliferating in livers of rats fed a choline-deficient/DL-ethionine-supplemented diet are part of the bile duct epithelial cell compartment); and (c) bile acid uptake is reduced in liver parenchymal cells of rats fed a choline deficient/DL-ethionine-supplemented diet, and this effect is due to a decrease in transport capacity without a decrease in transport affinity.

Animals↗
PubMed1994

Immortalization of rat hepatocytes by fusion with hepatoma cells. I. Cloning of a hepatocytoma cell line with bile canaliculi.

Hepatocytoma (HPCT) hybrid cells were obtained by fusion of cultured rat hepatocytes with Fao Reuber hepatoma cells H35 by polyethylene glycol treatment. Surviving cells were cloned in HAT (hypoxanthine-aminopterine-thymidine)/ouabain medium and propagated in cell lines over 80 passages. Morphological criteria were chosen to allow differentiation of the clones into two types of cells: 1) Type I cells which formed irregular cell layers, lacked contact inhibition and resembled the parental Fao hepatoma cells and 2) type II cells, which proliferated in monolayer cultures, exhibited contact inhibition during growth in culture plates and formed bile canaliculi thereby resembling cultured hepatocytes by phenotype. Bile canaliculi were absent in type I clones and Fao cells. One particular type II clone 1E3 was studied in detail. These cells formed bile canaliculi sealed by tight junctions and were comparably polarized as cultured hepatocytes. They expressed canalicular antigen B10, canalicular aminopeptidase N, and even secreted the fluorescent bile acid derivative NBD-cholate into the canalicular lumen. This type of HPCT cells lacked malignancy by tests in vivo and in vitro, and contained 110 +/- 5 chromosomes. The cells were considered to represent an immortalized hepatocyte-like cell line.

Animals↗
PubMed1994

Immortalization of rat hepatocytes by fusion with hepatoma cells. II. Studies on the transport and synthesis of bile acids in hepatocytoma (HPCT) cells.

Hepatocytoma (HPCT) cells were screened for the uptake of bile acids, the organic anion bumetanide and phalloidin. In addition, the rate of bile acid synthesis and the ability to conjugate exogenously added free bile acids was investigated. The unconjugated bile acid cholate is transported via carrier-mediated uptake into HPCT cells of the clone 1E3. This transport is expressed less by HPCT clones 1F9 and 2E9. Cholate uptake is 40% sodium-dependent, temperature-sensitive, and reveals a km-value of 47 +/- 9 pmol/l and a Vmax-value of 94 +/- 29 pmol x min-1 x mg-1. However, all of the HPCT clones lack carrier-mediated uptake of glycocholate and taurocholate. The clones which possess cholate transport are sensitive to phalloidin and take up bumetanide. In higher cell passages cholate transport and the sensitivity to phalloidin are reduced. This process is not prevented by a selection procedure with HAT-medium, nor is it stimulated by nicotinamide, dimethyl sulfoxide, or sodium butyrate. The cells of the 1E3, 1F9 and 2E9 clones, but not Fao hepatoma cells synthesize bile acids endogenously even after 60 passages, and conjugate exogenously added cholate with taurine and glycine. These results indicate that liver-specific properties of freshly isolated hepatocytes which are lacking in the parental hepatoma cells are maintained in HPCT cells.

Animals↗
PubMed1993

A membrane-bound form of protein disulfide isomerase (PDI) and the hepatic uptake of organic anions.

Protein disulfide isomerase (PDI) was considered to be involved in the hepatic uptake of certain organic anions because the protein is photoaffinity labeled by photolabile derivatives of the bile acid taurocholate. Several lines of evidences including photoaffinity labeling experiments indicated a close relationship between the uptake of bile acids and the organic anion bumetanide. The possible involvement of PDI in hepatic transport processes of these organic anions was tested with polyclonal antibodies raised against a PDI-beta-galactosidase fusion protein. Western blot analysis and immunofluorescence of intact hepatocytes showed that protein disulfide isomerase is located in sinusoidal rat liver plasma membranes. This protein is immunologically identical with microsomal PDI prepared from bovine liver. The plasma membrane form of PDI is, however, not labeled by photoactivated bumetanide as revealed by two-dimensional gel electrophoresis. These results indicate that, although a membrane-bound form of the PDI is present in the sinusoidal plasma membrane of rat hepatocytes, this protein is not involved in the hepatocellular uptake of the organic anion bumetanide.

Animals↗
PubMed1993

Two different mRNAs from rat liver code for the transport of bumetanide and taurocholate in Xenopus laevis oocytes.

The aim of this study was to elucidate whether bumetanide, which is a competitive inhibitor of carrier mediated bile acid uptake in liver cells, is transported by bile acid carriers. The expression of hepatocellular transport proteins for bile acid uptake and the uptake of the loop diuretic bumetanide was therefore studied in Xenopus laevis oocytes by injection of rat liver poly(A)(+)-RNA. Three hours after injection, a 70% increase in [3H]taurocholate uptake versus noninjected oocytes was accompanied by an increase in only 24% in the uptake of [3H]bumetanide. Size fractionation of the poly(A)(+)-RNA yielded 33 mRNA fractions of which fraction 21 accounted for an 800% increase of taurocholate transport with only a slight increase in bumetanide uptake. Bumetanide transport was coded by mRNA-fraction 18, which stimulated uptake by 160-200% with a concomitant small increase in taurocholate uptake. Uptake of cholate was induced by both mRNA fractions with almost 2.5 fold greater expression by the bumetanide fraction. Oocyte transport of taurocholate (expressed by fraction 21) and bumetanide transport (expressed by fraction 18) were characterized in terms of Na+ dependency, inhibition by 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (DIDS) and mutual competition. The results indicate that the bumetanide transporter mRNA is clearly different from the mRNA for the taurocholate transport protein. The mRNA fraction 18 was used for the construction of a cDNA library.

Animals↗
PubMed1993

Two distinct types of SH-groups are necessary for bumetanide and bile acid uptake into isolated rat hepatocytes.

Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 microM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [3H]bumetanide and [3H]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic abd interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 microM, DTONB 141 microM) and for cholate uptake (DTSNB 250 microM, DTONB 115 microM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Characterization of bumetanide transport in isolated skate hepatocytes.

The uptake of [3H]bumetanide was studied in isolated skate hepatocytes in an albumin-free elasmobranch Ringer solution and compared with the uptake of bile acids in the presence of other cholephilic organic anions. [3H]bumetanide uptake was energy dependent, temperature sensitive, and exhibited saturation kinetics. In contrast to taurocholate and cholate, which are transported only by Na(+)-independent mechanisms, removal of Na+ reduced the maximal uptake rate (Vmax) for bumetanide from 404 +/- 80 to 230 +/- 47 pmol.mg-1 x min-1 without a change in the apparent Michaelis constant (Km). The apparent Km for the Na(+)-dependent portion of bumetanide uptake was 58 +/- 24 microM, and Vmax was 151 +/- 38 pmol.min-1 x mg-1. Taurocholate (100 and 200 microM) inhibited Na(+)-independent bumetanide transport competitively but was a noncompetitive inhibitor for Na(+)-dependent bumetanide uptake. Furosemide (100 microM) and two bumetanide analogues, PF-3034 (500 microM) and PF-2203 (500 microM), preferentially inhibited the Na(+)-dependent bumetanide uptake system, whereas cholate (100 microM) and probenecid (100 microM) preferentially inhibited Na(+)-independent bumetanide transport. The sulfhydryl (SH) reagents N-ethylmaleimide, 2,2'-dithio-bis(5-nitropyridine), and p-chloromercuribenzenesulfonic acid (PCMBS) inhibited both bile acid and bumetanide uptake. Dithiothreitol (500 microM) completely reversed the PCMBS-induced inhibition of bumetanide uptake. These results indicate that bumetanide is transported into hepatocytes of the small skate, Raja erinacea, by both Na(+)-dependent and Na(+)-independent mechanisms; the latter is shared by bile acids and probably sulfobromophthalein and other organic anions. Their uptake requires free SH groups.

Animals↗
PubMed1993

Interaction of bumetanide derivatives with hepatocellular bile acid uptake.

The loop diuretic bumetanide is an organic monocarboxylic organic anion assumed to be transported into hepatocytes by a transport system for bile acids. The structural requirements of 22 bumetanide analogues were analyzed for an interaction with bile acid uptake into isolated rat hepatocytes. Whereas bumetanide inhibited the hepatocellular uptake of [14C]cholate to the same degree as its own uptake, derivatization altered affinity and specificity and yielded compounds that selectively inhibited either cholate or taurocholate uptake or uptake of both. No correlation was found between the diuretic potency of bumetanide derivatives, reflecting the affinity to the Na(+)-K(+)-Cl- cotransporter, and their affinity to hepatic bile salt transport. Computer-aided model building combined with the calculation of potential energy maps showed a strictly amphipathic charge separation in bumetanide analogues as in bile acids. Ranking bumetanide compounds by their mean inhibitory concentration values, inhibition constants, and their type of competition, we conclude that at least three binding domains in the proteins are essential for recognition by bile acid transporters, namely two hydrophobic and an anionic side, and that for the anionic binding region a carbonyl atom in the ligands as an electron donor group is sufficient for ligand interaction.

Animals↗
PubMed1993

Secretion of plasma proteins and its insulin-dependent regulation in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of primary rat hepatocytes with Fao hepatoma cells. Synthesis and secretion of the liver-specific plasma proteins albumin and transferrin were preserved in these proliferating hybrids, called hepatocytoma cells. In the hybrid clone 1E3, secretion of albumin was 75% and that of transferrin was 45% of the secretion in normal hepatocytes. The level of albumin mRNA was fivefold higher in hepatocytoma cells than in normal hepatocytes. Essentially no albumin and transferrin secretion or albumin mRNA was found in the parental Fao hepatoma cells. Insulin decreased the albumin mRNA level in hybrid cells, while it increased the level in normal hepatocytes. This indicates an inverse effect of the hormone due to this regulation in immortalized hepatocytoma cells compared to normal hepatocytes.

Animals↗
PubMed1992

Separation and purification by two-dimensional gel electrophoresis of a 52-54 kDa bumetanide binding protein from rat liver plasma membranes.

By affinity labeling with photolabile [3H]bumetanide, a 52-54 kDa bumetanide binding protein was identified in the sinusoidal plasma membrane fraction from rat liver. The protein is assumed to represent the carrier for hepatic uptake of loop diuretics. By two-dimensional (2D) gel electrophoresis we have purified this protein from hepatocytes, sinusoidal plasma membranes and subfractions of associated and integral plasma membrane proteins. Amongst more than 20 protein spots, a single integral plasma membrane protein was detected. The apparent pI of this molecule is 6.7. Specific labeling of this protein was not found in the fraction of associated plasma membrane proteins. To detect possible binding of radioactive bumetanide to microsomal cytochrome P450s, photolabeling experiments with integral plasma membrane proteins were performed under nitrogen/carbon monoxide atmosphere and in the presence of piperonyl butoxide. Labeling of the 52-54 kDa protein was not affected by these inhibitors of P450 enzymes. Taken together, these results indicate that the bumetanide binding protein is very likely to be a non-microsomal integral plasma membrane protein.

Affinity Labels↗
PubMed1992

Evidence for a saturable, energy-dependent and carrier-mediated uptake of oral antidiabetics into rat hepatocytes.

The hepatic uptake of two sulfonylureas, glisoxepide and glibenclamide, was investigated in isolated rat hepatocytes. Two transport processes were defined: passive physical diffusion and saturable carrier transport. For diffusion at pH 7.4 the permeability coefficients were 3.3 x 10(-6) cm/s for glisoxepide and 10.6 x 10(-6) cm/s for glibenclamide. Saturable uptake differed among the sulfonylureas. Glibenclamide uptake was neither energy- nor sodium-dependent and temperature dependence was linear. The apparent activation energy for saturable glibenclamide uptake was 15.2 kJ/mol and Q10 values for uptake between 7 and 37 degrees C were 1.17 +/- 0.12. Saturable glibenclamide uptake exhibited a Km = 3.1 microM and a Vmax = 416 pmol/mg cell protein per min. Thus glibenclamide uptake was defined kinetically as a facilitated diffusion process. Glisoxepide uptake revealed two Km values: Km1 = 2-3 microM and Vmax1 = 200 pmol/mg protein per min, and Km2 = 110 microM and Vmax2 = 1600 pmol/mg protein per min. Uptake at low and high substrate concentration was energy-dependent, sodium-dependent and was inhibited by ouabain. Temperature dependence increased markedly beyond 22 degrees C and the apparent activation energy was 59.7 kJ/mol at low Km1 glisoxepide concentrations and 60.3 kJ/mol at high Km2 concentrations. Whereas glisoxepide was slowly taken up into AS-30D hepatoma cells, glibenclamide was not. The hepatic uptake of glibenclamide was not inhibited by glisoxepide but glibenclamide inhibited glisoxepide uptake. The inhibition by glibenclamide was noncompetitive. Isolated hepatocytes accumulated the sulfonylureas markedly and metabolized both. The metabolized radioligands were slowly released into the incubation buffer. The results indicate that the hepatic uptake of the two sulfonylureas is by carrier-mediated transport. The uptake processes are, however, strikingly different, indicating heterogeneity of sulfonylurea transporters.

Administration, Oral↗
PubMed1992

Interaction of sulfonylureas with the transport of bile acids into hepatocytes.

The sulfonylurea compounds glisoxepide and glibenclamide inhibit the uptake of bile acids into isolated rat hepatocytes. The Ki values for the inhibition of cholate uptake was 9 microM with glibenclamide and 200 microM with glisoxepide. The inhibition of cholate uptake by both sulfonylureas was noncompetitive. Uptake of the conjugated bile acid taurocholate was inhibited by glibenclamide, Ki = 75 microM. Again the inhibition was noncompetitive. Glisoxepide inhibited taurocholate uptake only in the absence of sodium ions. Under sodium-free conditions glisoxepide also strongly inhibited cholate uptake. The inhibition was competitive, Ki = 42 microM. Both bile acids interfered with the hepatocellular uptake of [3H]glisoxepide, with IC50 values of 375 and 467 microM for cholate and taurocholate, respectively. The uptake of [3H]glibenclamide was inhibited by cholate, IC50 = 328 microM, but not by taurocholate. Glisoxepide uptake was further inhibited by blockers of the hepatocellular monocarboxylate transporter, by the loop diuretic bumetanide, by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and by sulfate. Glibenclamide uptake was weakly inhibited by DIDS and by anthracene-9-carboxylic acid (A-9-C) but not by bumetanide and sulfate. Neither bromosulfophthalein nor the fatty acid oleate inhibited glisoxepide or glibenclamide uptake. These results are consistent with the transport of glisoxepide via the transport system for the unconjugated bile acid cholate. Glibenclamide uptake is mediated by a still unknown hepatocellular transport system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Xenobiotic-metabolizing enzyme activities in hybrid cell lines established by fusion of primary rat liver parenchymal cells with hepatoma cells.

1. The activities of xenobiotic-metabolizing enzymes were determined in hybrid cell lines (hepatocytoma, HPCT) which have been established by fusion of liver parenchymal cells from adult rat (PC) with cells from a Reuber hepatoma cell line (FAO). 2. Cytochrome P450 was not measurable spectrophotometrically in FAO and HPCT. P450-dependent conversion of testosterone was below the detection limit in FAO and only marginally present in HPCT. 3. Microsomal and cytosolic epoxide hydrolase, glutathione S-transferase and phenol sulphotranserase were low or even below detection limit in FAO. These enzyme activities were significantly higher in HPCT and correspond to about 1-10% the activities measured in PC. 4. 1-Naphthol UPD-glucuronosyl transferase activity was about 20% in FAO and about 100% in HPCT compared to PC. 5. Metabolic conversion of benzo[a]pyrene was low in FAO, high in PC, and intermediate in HPCT. The presented data, however, do not allow the conclusion whether this intermediate rate is catalyzed by similar P450 isoenzymes as in PC. 6. Due to the easily measurable phase II-metabolizing enzyme activities HPCT may, however, be useful for in vitro enzyme induction or repression studies.

Animals↗
PubMed1992

Hormone-sensitive carbohydrate metabolism in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of hepatocytes from adult rats and Fao hepatoma cells in the presence of polyethylene glycol. These hybrids were called hepatocytoma cells. The preservation of liver-specific enzyme activities and metabolic functions was studied in the hybrid clone 1E3. 1) The proliferating hepatocytoma cells formed monolayers presenting morphological similarity to primary cultures of hepatocytes. 2) In contrast to Fao hepatoma cells, activities of all gluconeogenic key enzymes were preserved at normal or reduced levels. 3) Lactate-dependent glucose formation was maintained at a state reduced to 36% of the gluconeogenesis in hepatocytes; no glucose formation was detected in Fao hepatoma cells. 4) The activity of the liver-specific glucokinase was reduced in hepatocytoma cells, but it was still present in contrast to Fao cells. The liver-specific isoenzyme pyruvate kinase type L was replaced by the isoenzyme type M2. 5) Gluconeogenic and glycolytic enzyme activities were regulated in hepatocytoma cells by glucagon (0.1 microM) and by insulin (0.1 microM). 6) The genome of hepatocytoma cells and its expression were stable for at least one year, when spontaneously dedifferentiating cells were removed by recloning in hypoxanthine-aminopterine-thymidine (HAT) medium.

Animals↗
PubMed1992

[The loop diuretic bumetanide as a tool in physiology and pharmacology].

Loop diuretics are derivatives of 4-sulfamoylbenzoic acid, which derived originally from sulfonamides. Their diuretic effect is due to the inhibition of the Na-K-Cl-cotransport system in the distal part of Henle's loop. The compounds react with different affinity with the chloride binding site of the transporter. Bumetanide is among the most potent blockers with an IC50 of 0.2 microM. The compound was developed by P. W. FEIT, 1971; the saluretic response was described first by H. H. FREY 1972. Bumetanide has outgrown to become a tool for physiologists and pharmacologists in renal transport research. The compound has essentially contributed to the elucidation of the mechanisms of volume regulation of cells. Bumetanide is taken up into tubule cells and hepatocytes by active transport. The uptake in tubule cells ist mediated by an organic anion transporter, which is involved in the renal secretion of drugs. In the liver bumetanide is transporter by the bile acid carrier. The carrier is a multispecific drug transporter, too. It is not yet known, whether both drug transport systems contain identical membrane proteins. For this purpose bumetanide is currently used to investigate by photoaffinity labeling and functional expression cloning molecular principles of the drug elimination in kidney and liver.

Animals↗
PubMed1991

Photoaffinity labeling of plasma membrane proteins involved in the transport of loop diuretics into hepatocytes.

To identify proteins involved in the hepatocellular uptake of loop diuretics, [3H]bumetanide was photoactivated by light flash in the presence of either intact isolated rat hepatocytes, rat liver basolateral plasma membranes or integral membrane proteins extracted from the basolateral plasma membranes. Proteins of 52-54, 48, 33, 27, 25 and 23 kDa in sodium dodecyl sulfate (SDS) gel electrophoresis were radiolabeled on intact hepatocytes. On liver basolateral plasma membranes a 50-52 kDa protein was the most intensely labeled protein. After separation into integral and associated membrane proteins by extraction with Triton X-114, radioactive labeling was only found in integral membrane proteins with a molecular weight of 50-52 kDa. Photoactivated bumetanide irreversibly inhibited the hepatocellular uptake of cholate, taurocholate but not of serine. Binding proteins for photoactivated bumetanide were absent on AS 30-D ascites hepatoma cells. Labeling of all proteins was sodium dependent in intact hepatocytes but was sodium independent in plasma membranes. Labeling was prevented by non-labeled bumetanide and by the loop diuretics piretanide and furosemide. Labeling protection was further achieved with organic anions such as bromosulfophthalein, rifampicin, probenecid and by the bile acids taurocholate, deoxycholate and dehydrocholate. The radiolabeled proteins did not belong to the bumetanide-sensitive NaCl/KCl co-transport system which apparently does not occur in intact isolated rat hepatocytes.

Affinity Labels↗