• Archives of Pharmacal Research
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• 제26권4호
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• pp.338-343
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• 2003

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a $K_m of 29.1\pm3.2 \mu M and V_{max} of 2.9\pm0.1$ mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was $2.2\pm0.1 \mu$ L/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at $4^{\circ}C$, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The $V_{max} and K_m$ values were 0.54 mmol/min/mg protein, and 10.0 $\mu$ M, respectively. Based on the comparison of the ratios of $V_{max} to K_m (V_{max}/K_m)$ corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.

• Journal of Pharmaceutical Investigation
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• 제31권4호
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• pp.209-216
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• 2001

The purpose of the present study was to investigate the hepatic uptake and biliary excretion of l-anilino-8-naphthalene sulfonate (ANS) in vivo. The plasma concentration and liver concentration of ANS were determined after its i.v. bolus administration at a dose of $30\;{\mu}mol/kg$ in rats. The hepatic uptake clearance $(CL_{uptake})$ of ANS was 0.1 ml/min/g liver. On the basis of the unbound concentration of ANS, the permeability-surface area product $(PS_{influx})$ was calculated to be l0.4 ml/min/g liver, being comparable of in vitro data. On the other hand, we determined the plasma concentration, liver concentration and biliary excretion rate of ANS at steady-state after its i. v. infusion $(0.2-1.6\;{\mu}mol/min/kg)$ in rats. The excretion clearance $(CL_{excretion})$ of ANS showed Michaelis-Menten kinetics with increasing the infusion rate. The permeability-surface area product $(PS_{excretion})$ based on the unbound concentration in the liver was calculated to be 0.0165 ml/min/g liver, which is negligible compared with the intrinsic clearance $(CL_{int}=3.3\;ml/min/g\;liver)$ by rat liver microsomes. The sequestration process of ANS, therefore, was considered to be mainly due to the metabolic process in the liver $(PS_{seq}{\risingdotseq}CL_{int})$. Furthermore, $PS_{efflux}$ value calculated from $PS_{influx}$ and $PS_{seq}$ was 4.4 ml/min/g liver, which was comparable of in vitro data. In conclusion, in vivo parameters such as $PS_{influx}$, $PS_{efflux}$ and $PS_{seq}$ in the present study showed good in vivo-in vitro relationship. Thus, the kinetic analysis method proposed in the present study would be useful to analyze the hepatic transport of drugs in vivo.

• Archives of Pharmacal Research
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• 제21권6호
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• pp.677-682
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• 1998

The present study was designed to examine the metabolism of 1-anilino-8-naphthalene sulfonate (ANS), an anionic compound which is transported into liver via "multispecific organ ic anion transporter", with rat hepatic microsomes. TLC analysis indicated that the fluorescent metabolites were not produced to a measurable extent, which made it possible to assess the ANS metabolism by measuring the fluorescence disappearance. The metabolism of ANS was remarkably inhibited by the presence of SKF-525A as well as by the substitution of 02 by CO gas. ANS metabolism by microsomes also required NADPH as a cofactor. These results indicated that the microsomal monooxygenase system might be mainly responsible for the ANS metabolism. The maximum velocity ($V_{max}$) and Michaelis constant ($K_m$) were calculated to be $4.3{\pm}0.2$ nmol/min/mg protein and $42.1{\pm}2.0\;{\mu}M$, respectively. Assuming that 1g of liver contains 32mg of microsomal protein, the $V_{max}$ value was extrapolated to that per g of liver ($V_{max}^I$). The intrinsic metabolic clearance ($CL_{int}$) under linear conditions calculated from this in vitro metabolic study was 3.3ml/min/g liver, being comparable with that (3.0ml/min/g liver) calculated by analyzing the in vivo plasma disappearance curve in a previous study. Furthermore, the effects of other organic anions on the metabolism of ANS were examined. Bromophenolblue (BPB) and rose bengal (RB) competitively inhibited the metabolism of ANS, while BSP inhibited it only slightly. The inhibition constant ($K_i$) of BPB ($6\;{\mu}M$) was much smaller than that of RB ($200\;{\mu}M$). In conclusion, the microsomal monooxygenase system plays a major role in the metabolism of ANS, and other unmetabolizable organic anions (BPB and RB) compete for this metabolism.

• Journal of Pharmaceutical Investigation
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• 제21권1호
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• pp.43-50
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• 1991

The hepatic uptake of an anionic fluorescence probe, l-anilino-8-naphthalene sulfonate (ANS) was characterized using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was fitted well to the Michaelis-Menten equation with a saturable component. The $V_{max}$ and $K_m$ values were $2.9{\pm}0.1\;nmol/min/mg$ protein and $29.1{\pm}3.2\;{\mu}M$, respectively. The uptake clearance $(CL_{up})$ based on the ratio of $V_{max}$ to $K_m$ was 11.7 ml/min/g liver, revealing the good coincidence with that assessed from the analysis of the plasma disappearance curve in previous report. Furthermore, the effect of serum protein on the hepatic uptake of ANS into isolated hepatocytes was investigated. The permeability clearances $(PS_{inf})$ of ANS uptake were much higher than those predicted based on the unbound fractions in the presence of serum. These suggested that the hepatic uptake of extensively serum protein-bound ANS is mediated not only by the unbound form of ligand but also by the serum protein-mediated uptake mechanism.

• Journal of Pharmaceutical Investigation
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• 제22권4호
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• pp.289-300
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• 1992

The counter-transport phenomena in the hepatic transport of 1-anilino-8-naphthalene sulfonate (ANS) were kinetically investigated by analyzing the plasma disappearance-time profiles and the transport into the isolated hepatocytes. In vivo "counter transport phenomena" were simulated based on the perfusion model which incorporated the carrier-mediated transport and the saturable intracellular binding. The condition that the mobility of carrier-ligand complex is greater than that of free carrier is not essential for the occurrence of counter-transport phenomenon. To examine the inhibitory effects on the initial uptake of a ligand by the liver, it is necessary to judge whether the true counter-transport mechanism (trans-stimulation) is working or not. The initial plasma disappearance curves of ANS were then kinetically analyzed based on a two-compartment model, in which the ligand is eliminated only from the peripheral compartment (liver compartment). No effects on the initial plasma disappearance rates of ANS were observed after preloading of bromophenol blue (BPB) or rose bengal (RB) in the liver. Inhibitory effect of BPB or RB on the initial uptake (or efflux) rates of ANS by the isolated hepatocytes were not observed, suggesting that the true counter transport mechanism is not working. In conclusion, checking the preloading effects of transstimulation on the initial uptake of a ligand by the liver could be a useful criterion for carrier cycling and common use of the same carrier between two ligands. However, one cannot exclude those possibilities even if the preloading effects cannot be observed.

• 한국미생물·생명공학회지
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• 제11권3호
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• pp.155-161
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• 1983

Bacteriophage f2에 대한 자외광선의 살균효과와 외투막 단백질의 구조에 미치는 영향을 Ray-onet photoreactor PPR-208을 사용하여 300nm의 광선으로 연구하였다. 처음 20분간의 조사에서는 약 4 log의 phage가 감소되고 그후 완만한 살균효과를 보이다가 90분 이상의 조사에서는 생존 바이러스가 발견되지 않았다. Tryptophan residue의 fluorescenve quenching, 자외선으로 조신한 phage에 부착시킨 ANS (8-anilino-1-napht-halene sulfonate)의 fluorescence emission의 감소, tryptophan에서 ANS로의 energy transfer 의 감소 등 spectroscopic technique에 의한 결과와 자외선 조사에 의하여 단백질 외투만이 파손되는 전자현미경 관찰의 결과에 의하여 자외광선은 phage f2의 외투막 단백질의 구조에 변화를 일으킨다는 것이 밝혀졌다.

• BMB Reports
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• 제39권5호
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• pp.636-641
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• 2006

Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. In the present study, an apo form of the enzyme was prepared by removal of zinc and a comparative study was performed on some characteristic features of the apo and native forms by far- and near-UV circular dichroism (CD), intrinsic fluorescent spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, fluorescence quenching by acrylamide, and Tm measurement. Results indicate that protein flexibility is enhanced and the hydrophobic sites become more exposed upon conversion to the apo form. Accordingly, the apo structure showed a greater affinity for interaction with hydrophobic adsorbents as compared with the native structure. As observed for the native enzyme, heat denaturation of the apo form promoted interaction with alkyl residues present on the adsorbents and, by cooling followed by addition of zinc, catalytically-active immobilized preparations were obtained.

• 약학회지
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• 제49권4호
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• pp.275-283
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• 2005

The purpose of the present study was to kinetically investigate the carrier-mediated uptake in the hepatic transport of organic anions, and to simulate the 'in vivo counter-transport' phenomena, using kinetic model which was developed in this study. The condition that the mobility of carrier-ligand complex is greater than that of free carrier is not essential for the occurrence of 'counter-transport' phenomenon. To examine the inhibitory effects on the initial uptake of organic anions by the liver, it is necessary to judge whether the true counter-transport mechanism (trans-stimulation) is working or not. Effects of bromophenol blue (BPB) or bromosulfophthalein (BSP) on the plasma disappearance curves of a 1-anilino-8-naphthalene sulfonate (ANS) were then kinetically analyzed based on a flow model, in which the ligand is eliminated only from the peripheral compartment (liver compartment). Moreover, 'in vivo counter-transport' phenomena were simulated based on the perfusion model which incorporated the carrier-mediated transport and the saturable intracellular binding. The 'in vivo counter-transport' phenomena in the hepatic transport of a organic anions were well demonstrated by incorporating the carrier-mediated process. However, the 'in vivo counter-transport' phenomena may be also explained by the enhancement of back diffusion due to the displacement of intracellular binding. In conclusion, one should be more cautious in interpreting data obtained from so-called 'in vivo counter-transport' experiments.