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The mystery of canalicular GSH transport: Lessons from EHBR and diabetic rats
310A
813
AASLD
SEX DIFFERENCES IN MEMBRANE BINDING AND HEPATIC TRANSPORT OF INDOCYANINE GREEN (ICG) IN RAT LIVER.
ABSTRACTS
814
MATURATIONAL INCREASE IN CANALICULAR GSH EFFLUX IS DUE TO A RISING Vmax OF THE LOW-AFFINITY TRANSPORTER. M. Ookhtens and A. Mitmr. Department of Medicine, USC, Los Angeles, CA. Previously, using isolated peffused livers, we showed that biliary GSH effiux increases with maturation (AJP 261:G648-G656, 1991). However, in the intact organ, hepatic GSH could not be increased to more than twice normal (<12 umol/g~mM). Thus, we could not defineate whether maturational changes were due to changes in K m or Vmax (or both) of transport. In these studies, maturational changes in membrane potential and paracellular permeability were ruled out as possible mechanisms. Subsequent work with canalicular liver plasma membrane vesicles (cLPM) has shown the major component of this transport to have lowaffinity (Km~17 mM), in addition to a relatively minor high-affinity component (Km~0.2 raM). Therefore, we used the cLPM model to study whether any maturational changes occur in kinetic parameters of canalicular GSH transport that may account as the mechanism(s) for changes observed earlier. Exnerimental: cLPMs were prepared by the method of Meier et al. with minor modifications. Livers from immature (I), 28-45 days old (175-200g) and mature (M), 120-160 days old (425-480g) male Harlan Sprague-Dawley rats were used. Marker enzyme assays showed uniform enrichments+relative purities and ATPdependent taurocholate uptake had similar profiles (overshoot+plateau) between the cLPM of I and M rats. Kinetics of GSH transport in cLPM were determined from initial rates (1O see) of uptake of [35S]GSH in the presence of varying GSH from 0.01 to 50 raM, Binding/transport at 4oc was measured by both methods utilized by previous investigators, i.e. GSH associated instantaneously with vesicles, or at 10 sec, the time used for measurement of 'raw' initial rates of uptake at 25oc. Kinetic data representing the 'net' uptake of GSH (after subtraction of instantaneous or 10 sec binding/transport at 4oc) were analyzed with the SAAM program. Results: There were no significant differences between the Km'S of the 'nef data sets obtained by either method of estimating the 4oc binding/transport; only Vmax'S were sealed accordingly. Kinetic data for each age group had a high-affinity and a low-affinity component. While the high-affinity components were characterized by Michaelis-Menten kineticssthe low-affinity components were sigmoidal (Hill coefficients ~2). The kinetic parameters estimated by the SAAM program, using the 'net' data after subtraction of 4oc uptake at 10 sec, were: Age Group Kml X/maxl Km2 Vmax2 Immature (I) 0.21±0.087 0.58±0.15 17.4±1.9 8.0-+4).54 [ Mature (M) [ 0.10±0.029 I 0'43+-0'063 [ 14.6±i.4 [ 15"1+1"0 I ~ : Our previously-reported maturational rise in biliary efflux of GSH is due to a rising Vmax of the low-affinity component of canalicular transport.
816
THE MYSTERY OF CANALICULAR GSH TRANSPORT: LESSONS FROM EHBR AND DIABETIC RATS. $. C. Lu. H. Wu. S: Horie. H. Takikawa. J. Yi and N. Kaolowitz. Division of GI and Liver Diseases, USC School of Medicine, LA, CA, Eisai Co,, Ltd., and Teikyo University, Japan. Both EHBR and chronic diabetic rats exhibit a profound selective decrease (90-99%) in biliary GSH secretion. We reported the Km and Vmax for GSH uptake into canalicular membrane (cLPM) vesicles in EHBR rats was not different from controls (J. Biol. Chem. 267:1667-1673, 1992). To further evaluate the mechanism for the defect we examined GSH transport in cLPM vesicles from 4 week diabetic rats and the abundance of liver mRNA and polypeptide of rat canaUcular GSH transporter (RcGshT)in the two models, cLPM vesicles prepared from 4 week streptozotocin-induced diabetic and agematched control rats showed similar enrichment of marker enzymes and similar kinetic parameters (Km and Vmax) for GSH uptake. Thus, diabetic rats resemble EHBR in showing decreased biliary GSH secretion in vivo bUt no change in vesicle studies in vitro. Moreover, both Northern and Western blot analysis of diabetic and EHBR liver showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared to controls. Treatment with phenobarbital, which increases steady state RcGshT mRNA by 5-6 fold and biliary GSH secretion in controls, also increased steady state RcGshT mRNA level in EHBR rats (by 1-2 fold) but did not increase biliary GSH secretion. In conclusion, the defect of biliary GSH secretion in both diabetic and EHBR rats is lost upon isolation of cLPM vesicles and is not reflected in a change in the RcGshT mRNA or polypeptide levels. Therefore, in both models the failure to secrete GSH into bile must be due to an endogenous inhibitor(s) or posttranslational modifications of RcGshT which are lost or reversed during vesicle isolation or impaired transfer o f RcGshT from pericanalicular vesicles to the canalicular membrane.
P. Ott. Med Dept. A, Rigshospitalet. 2100 El, Denmark. We studied sex differences in hepatic cyt0sol/membrane binding and transport of ICG in the perfused rat liver model. ICG is known to be excreted unmetabolized into bile. ICG was measured by HPLC. Cytosol/membrane bindino: 5 d and 5 g livers were homogenized, incubated with 5 h'M ICG at 37°C for 1 hour, and then ultracentrifuged (100.000g for 90 min). The membrane bound fraction of ICG (extracted from the pellet) was 0.66±0.06 in 9 and 0.45 + 0.05 in d' livers (p = 0.004). Hepatic uotake: The livers (6 9, 6 5) were perfueed once through with 0.20 hct human erythrocytes in Krebs-Henseleit Buffer (pH 7.4) with 2% bovine albumin. In each liver 5 concentrations of ICG were used: 1.2, 8.7, 29.1, 58.1, and 116.2pM. For 9/d livers we estimated by use of non-linear regression Vmax (nmol/min/g liver): 31.3 ± 2.1/28.9 ± 1.8 (NS), Km ~M): 17.0 ± 1.7/23.7 + 1.9 (p = 0.03), and Vmax/Km (ml/min/g)1.87±0.13/1.24± O.07(p =0,002). Transheoatic transoort: (6 9, 7 d). A 1 minute bolus of O.026,umol ICG was given through a side channel to the portal canula. Bile was cellected at 2 minute intervals for 60 minutes. After correction for delays caused by the intrahepatic bile ducts (0.25 p-L/g liver, e value taken from the literature) and by the biliary catheter (measured) the transhepatocellular transport time was calculated as the time from start of the ICG bolus to the time of the peak excretion rate. This was 8.3±0~5 min in 9 livers versus 10.9±0.5 min in 6 livers (p = 0.005). Bileflow rates (pL/min/g) were similar: 2.15 + O. 16 (9) and 2.25 ± O.10 (6), while maximum excretion rates (pmol/min/g liver) differed: 28.4±4.7 (9) and 13.0+3.7 (6). Thus, female livers had higher membrane binding, lower Km, and shorter transhepatic transport time than males. The differences in Km rather than Vmax and in membrane binding suggest that one or more transport steps may be qualitatively different.
815
HIGH AFFINITY TRANSPORTER OF REDUCED GLUTATHIONE PLAYS A MAIN ROLE IN GLUTATHIONE TRANSPORT IN RAT BASOLATERAL LIVER PLASMA MEMBRANE VESICLES (bLPM). K. Tankahara. 3"_ Ohmura. K_ Okano. Dept. of GI and Liver Diseases, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan. Two saturable transporters, a low and a high Km in MichaelisMenten constant (,, 0.3 mM and 3 ,~ 7 raM) of reduced glutathione (GSH), are thought to be responsible for GSH transport at the basolateral pole of hepatocytes. In a kinetic study, however, an insidenegative membrane potential induced by an accumulation of GSH in bLPM may inhibit further influx of GSH into the vesicles especially at higher GSH concentrations (>10 raM), which could generate false parameters. To address this issue, we voltage-clamped the vesicles to eliminate the effect of the membrane potential and re-evaluated the kinetic data by a computer program. We also studied the effect of BSP-GSH on the transporters to determine which of them was really affected by the specific inhibitor of GSH transport in bLPM. ~ bLPM were prepared according to Meier et al. Intactness of bLPM was confmaaed by Na÷-dependent overshoot in alanine uptake. Kinetic studies were performed by measuring theuptake of [35S]GSH into bLPM at pH 7.4, 25 °C, and 10 s at various GSH concentrations (0.05 - 50 raM) under voltage-clamped conditions induced by 100 mM K*-valinomycin equilibration. Data were analyzed by equations representing one or two carriermediated eornponent(s) either with or without a diffusion component. The only equation that fitted the data and provided significant parameters was "J = Jmax x [S]/([Kt + [S]) + P[S]", where Jmax is apparent maximal carrier-mediated influx, Kt is the apparent GSH concentration resulting in half-maximal uptake, [S] is the extetnal GSH concentration, and P is the rate constant of diffusional permeability. The equation revealed the parameters only corresponding to the high affinity GSH transporter; Kt = 0.12 ± 0.02 mM, Jmax = 0.32 ± 0.01 nmol*mg~ • l 0 s "~ and P = 0.08 ± 0.001 nmol*mg'M0 s "~ *ram4 (mean ± SEM of triplicate determinants in 3 vesicle preps.). 1 mM and 10 mM BSP-GSH inhibited 0.2 mM GSH uptake by 40 % and 33 % of the control values respectively, whereas those concentrations of BSP-GSH did not inhibit 10 raM GSH uptake. ~ Only the high affinity GSH transporter may be responsible for GSH transport in bLPM.
HEPATOLOGY O c t o b e r 1995
813
AASLD
SEX DIFFERENCES IN MEMBRANE BINDING AND HEPATIC TRANSPORT OF INDOCYANINE GREEN (ICG) IN RAT LIVER.
ABSTRACTS
814
MATURATIONAL INCREASE IN CANALICULAR GSH EFFLUX IS DUE TO A RISING Vmax OF THE LOW-AFFINITY TRANSPORTER. M. Ookhtens and A. Mitmr. Department of Medicine, USC, Los Angeles, CA. Previously, using isolated peffused livers, we showed that biliary GSH effiux increases with maturation (AJP 261:G648-G656, 1991). However, in the intact organ, hepatic GSH could not be increased to more than twice normal (<12 umol/g~mM). Thus, we could not defineate whether maturational changes were due to changes in K m or Vmax (or both) of transport. In these studies, maturational changes in membrane potential and paracellular permeability were ruled out as possible mechanisms. Subsequent work with canalicular liver plasma membrane vesicles (cLPM) has shown the major component of this transport to have lowaffinity (Km~17 mM), in addition to a relatively minor high-affinity component (Km~0.2 raM). Therefore, we used the cLPM model to study whether any maturational changes occur in kinetic parameters of canalicular GSH transport that may account as the mechanism(s) for changes observed earlier. Exnerimental: cLPMs were prepared by the method of Meier et al. with minor modifications. Livers from immature (I), 28-45 days old (175-200g) and mature (M), 120-160 days old (425-480g) male Harlan Sprague-Dawley rats were used. Marker enzyme assays showed uniform enrichments+relative purities and ATPdependent taurocholate uptake had similar profiles (overshoot+plateau) between the cLPM of I and M rats. Kinetics of GSH transport in cLPM were determined from initial rates (1O see) of uptake of [35S]GSH in the presence of varying GSH from 0.01 to 50 raM, Binding/transport at 4oc was measured by both methods utilized by previous investigators, i.e. GSH associated instantaneously with vesicles, or at 10 sec, the time used for measurement of 'raw' initial rates of uptake at 25oc. Kinetic data representing the 'net' uptake of GSH (after subtraction of instantaneous or 10 sec binding/transport at 4oc) were analyzed with the SAAM program. Results: There were no significant differences between the Km'S of the 'nef data sets obtained by either method of estimating the 4oc binding/transport; only Vmax'S were sealed accordingly. Kinetic data for each age group had a high-affinity and a low-affinity component. While the high-affinity components were characterized by Michaelis-Menten kineticssthe low-affinity components were sigmoidal (Hill coefficients ~2). The kinetic parameters estimated by the SAAM program, using the 'net' data after subtraction of 4oc uptake at 10 sec, were: Age Group Kml X/maxl Km2 Vmax2 Immature (I) 0.21±0.087 0.58±0.15 17.4±1.9 8.0-+4).54 [ Mature (M) [ 0.10±0.029 I 0'43+-0'063 [ 14.6±i.4 [ 15"1+1"0 I ~ : Our previously-reported maturational rise in biliary efflux of GSH is due to a rising Vmax of the low-affinity component of canalicular transport.
816
THE MYSTERY OF CANALICULAR GSH TRANSPORT: LESSONS FROM EHBR AND DIABETIC RATS. $. C. Lu. H. Wu. S: Horie. H. Takikawa. J. Yi and N. Kaolowitz. Division of GI and Liver Diseases, USC School of Medicine, LA, CA, Eisai Co,, Ltd., and Teikyo University, Japan. Both EHBR and chronic diabetic rats exhibit a profound selective decrease (90-99%) in biliary GSH secretion. We reported the Km and Vmax for GSH uptake into canalicular membrane (cLPM) vesicles in EHBR rats was not different from controls (J. Biol. Chem. 267:1667-1673, 1992). To further evaluate the mechanism for the defect we examined GSH transport in cLPM vesicles from 4 week diabetic rats and the abundance of liver mRNA and polypeptide of rat canaUcular GSH transporter (RcGshT)in the two models, cLPM vesicles prepared from 4 week streptozotocin-induced diabetic and agematched control rats showed similar enrichment of marker enzymes and similar kinetic parameters (Km and Vmax) for GSH uptake. Thus, diabetic rats resemble EHBR in showing decreased biliary GSH secretion in vivo bUt no change in vesicle studies in vitro. Moreover, both Northern and Western blot analysis of diabetic and EHBR liver showed nearly identical RcGshT mRNA and polypeptide levels, respectively, as compared to controls. Treatment with phenobarbital, which increases steady state RcGshT mRNA by 5-6 fold and biliary GSH secretion in controls, also increased steady state RcGshT mRNA level in EHBR rats (by 1-2 fold) but did not increase biliary GSH secretion. In conclusion, the defect of biliary GSH secretion in both diabetic and EHBR rats is lost upon isolation of cLPM vesicles and is not reflected in a change in the RcGshT mRNA or polypeptide levels. Therefore, in both models the failure to secrete GSH into bile must be due to an endogenous inhibitor(s) or posttranslational modifications of RcGshT which are lost or reversed during vesicle isolation or impaired transfer o f RcGshT from pericanalicular vesicles to the canalicular membrane.
P. Ott. Med Dept. A, Rigshospitalet. 2100 El, Denmark. We studied sex differences in hepatic cyt0sol/membrane binding and transport of ICG in the perfused rat liver model. ICG is known to be excreted unmetabolized into bile. ICG was measured by HPLC. Cytosol/membrane bindino: 5 d and 5 g livers were homogenized, incubated with 5 h'M ICG at 37°C for 1 hour, and then ultracentrifuged (100.000g for 90 min). The membrane bound fraction of ICG (extracted from the pellet) was 0.66±0.06 in 9 and 0.45 + 0.05 in d' livers (p = 0.004). Hepatic uotake: The livers (6 9, 6 5) were perfueed once through with 0.20 hct human erythrocytes in Krebs-Henseleit Buffer (pH 7.4) with 2% bovine albumin. In each liver 5 concentrations of ICG were used: 1.2, 8.7, 29.1, 58.1, and 116.2pM. For 9/d livers we estimated by use of non-linear regression Vmax (nmol/min/g liver): 31.3 ± 2.1/28.9 ± 1.8 (NS), Km ~M): 17.0 ± 1.7/23.7 + 1.9 (p = 0.03), and Vmax/Km (ml/min/g)1.87±0.13/1.24± O.07(p =0,002). Transheoatic transoort: (6 9, 7 d). A 1 minute bolus of O.026,umol ICG was given through a side channel to the portal canula. Bile was cellected at 2 minute intervals for 60 minutes. After correction for delays caused by the intrahepatic bile ducts (0.25 p-L/g liver, e value taken from the literature) and by the biliary catheter (measured) the transhepatocellular transport time was calculated as the time from start of the ICG bolus to the time of the peak excretion rate. This was 8.3±0~5 min in 9 livers versus 10.9±0.5 min in 6 livers (p = 0.005). Bileflow rates (pL/min/g) were similar: 2.15 + O. 16 (9) and 2.25 ± O.10 (6), while maximum excretion rates (pmol/min/g liver) differed: 28.4±4.7 (9) and 13.0+3.7 (6). Thus, female livers had higher membrane binding, lower Km, and shorter transhepatic transport time than males. The differences in Km rather than Vmax and in membrane binding suggest that one or more transport steps may be qualitatively different.
815
HIGH AFFINITY TRANSPORTER OF REDUCED GLUTATHIONE PLAYS A MAIN ROLE IN GLUTATHIONE TRANSPORT IN RAT BASOLATERAL LIVER PLASMA MEMBRANE VESICLES (bLPM). K. Tankahara. 3"_ Ohmura. K_ Okano. Dept. of GI and Liver Diseases, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan. Two saturable transporters, a low and a high Km in MichaelisMenten constant (,, 0.3 mM and 3 ,~ 7 raM) of reduced glutathione (GSH), are thought to be responsible for GSH transport at the basolateral pole of hepatocytes. In a kinetic study, however, an insidenegative membrane potential induced by an accumulation of GSH in bLPM may inhibit further influx of GSH into the vesicles especially at higher GSH concentrations (>10 raM), which could generate false parameters. To address this issue, we voltage-clamped the vesicles to eliminate the effect of the membrane potential and re-evaluated the kinetic data by a computer program. We also studied the effect of BSP-GSH on the transporters to determine which of them was really affected by the specific inhibitor of GSH transport in bLPM. ~ bLPM were prepared according to Meier et al. Intactness of bLPM was confmaaed by Na÷-dependent overshoot in alanine uptake. Kinetic studies were performed by measuring theuptake of [35S]GSH into bLPM at pH 7.4, 25 °C, and 10 s at various GSH concentrations (0.05 - 50 raM) under voltage-clamped conditions induced by 100 mM K*-valinomycin equilibration. Data were analyzed by equations representing one or two carriermediated eornponent(s) either with or without a diffusion component. The only equation that fitted the data and provided significant parameters was "J = Jmax x [S]/([Kt + [S]) + P[S]", where Jmax is apparent maximal carrier-mediated influx, Kt is the apparent GSH concentration resulting in half-maximal uptake, [S] is the extetnal GSH concentration, and P is the rate constant of diffusional permeability. The equation revealed the parameters only corresponding to the high affinity GSH transporter; Kt = 0.12 ± 0.02 mM, Jmax = 0.32 ± 0.01 nmol*mg~ • l 0 s "~ and P = 0.08 ± 0.001 nmol*mg'M0 s "~ *ram4 (mean ± SEM of triplicate determinants in 3 vesicle preps.). 1 mM and 10 mM BSP-GSH inhibited 0.2 mM GSH uptake by 40 % and 33 % of the control values respectively, whereas those concentrations of BSP-GSH did not inhibit 10 raM GSH uptake. ~ Only the high affinity GSH transporter may be responsible for GSH transport in bLPM.
HEPATOLOGY O c t o b e r 1995