In-plane stress development in mesoporous thin films.

Ordered mesoporous thin films of TiO2 and CexZr1-xO2 (x = 0, 0.5, 1) were prepared via an evaporation-induced self-assembly (EISA) process and subsequently investigated in terms of the developing intrinsic and residual in-plane stress. These mechanical properties were determined by the curvature method, which is based on the determination of the deflection of light due to concave or convex bending of the films on a substrate. The films were investigated with regard to the intrinsic stress during heat treatment up to 500 °C and to the residual stress at room temperature for several annealing temperatures. Following this strategy, the influence of the decomposition of a block copolymer template on the intrinsic stress as well as the pore collapsing on the residual stress was analyzed. Nanoporous TiO2 thin films were prepared using two different block copolymers (PIB50-b-PEO45 and Pluronic® F127). A comparison between the templated and non-templated TiO2 films showed the lowest intrinsic and residual stress for the ordered mesoporous material prepared with PIB50-b-PEO45 indicating that the distributed polymer and the corresponding mesopores act as relaxing agents for the system. This was verified by mesoporous CexZr1-xO2 (x = 0, 0.5, 1) thin films showing a comparable behavior in terms of the experienced intrinsic stress. This work reveals an increase in the residual in-plane stress during pore collapse, which lays the foundation for further understanding of the stress-related mechanical properties of mesoporous thin films.

Pattern of N gene-mediated systemic hypersensitive response and turnover of viral replicase protein in tobacco.

We examined the pattern of the N gene-mediated systemic hypersensitive response (HR), which was induced by tobacco mosaic virus upon temperature shift, and analyzed the distribution of the coat protein and the virus-encoded 126 kDa replicase protein (126 K protein) by immunoblot analysis. In the middle- and lower-positioned leaves, HR occurred in the advancing edge of the infected area, where we detected both the coat protein and the 126 K protein. In the areas between the main vein and the advancing edge of these leaves, we observed no HR and did not detect 126 K protein, though virus was present in these areas. In the upper-positioned mosaic leaves, patterns of the HR were different depending on the leaf age. In these mosaic leaves, the mechanism preventing the virus from invading dark green tissue seemed to be broken down in 8-14 days old leaves, and HR was observed only in the tissue just invaded by the virus, where we detected the 126 K protein. Those results suggested that the viral 126 K protein was present when the viral replication was taking place, and easily degraded when the amount of the virus was saturated in the cells.

Primary active transport of organic anions on bile canalicular membrane in humans.

Biliary excretion of several anionic compounds was examined by assessing their ATP-dependent uptake in bile canalicular membrane vesicles (CMV) prepared from six human liver samples. 2, 4-Dinitrophenyl-S-glutathione (DNP-SG), leukotriene C4 (LTC4), sulfobromophthalein glutathione (BSP-SG), E3040 glucuronide (E-glu), beta-estradiol 17-(beta-D-glucuronide) (E2-17G), grepafloxacin glucuronide (GPFXG), pravastatin, BQ-123, and methotrexate, which are known to be substrates for the rat canalicular multispecific organic anion transporter, and taurocholic acid (TCA), a substrate for the bile acid transporter, were used as substrates. ATP-dependent and saturable uptake of TCA, DNP-SG, LTC4, E-glu, E2-17G, and GPFXG was observed in all human CMV preparations examined, suggesting that these compounds are excreted in the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV preparations but was negligible in the others. The ATP-dependent uptake of all the compounds exhibited a large inter-CMV variation, and there was a significant correlation between the uptake of glutathione conjugates (DNP-SG, LTC4, and BSP-SG) and glucuronides (E-glu, E2-17G, and GPFXG). However, there was no significant correlation between TCA and the other organic anions, implying that the transporters for TCA and for organic anions are different also in humans. When the average value for the ATP-dependent uptake by each preparation of human CMVs was compared with that of rat CMVs, the uptake of glutathione conjugates and nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans was approximately 3- to 76-fold lower than that in rats, whereas the uptake of glucuronides was similar in the two species. Thus there is a species difference in the primary active transport of organic anions across the bile canalicular membrane that is less marked for glucuronides.

Biliary excretion mechanism of CPT-11 and its metabolites in humans: involvement of primary active transporters.

After administration of CTP-11, a camptothecin derivative exhibiting a wide spectrum of antitumor activity, dose-limiting gastrointestinal toxicity with great interpatient variability is observed. Because the biliary excretion is a major elimination pathway for CPT-11 and its metabolites [an active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), and its glucuronide, SN38-Glu], several hypotheses for the toxicity involve biliary excretion. Here, we investigated whether primary active transport is involved in the biliary excretion of anionic forms of CPT-11 and its metabolites in humans using bile canalicular membrane vesicles (cMVs). Uptake of the carboxylate form of CPT-11 and the carboxylate and lactone forms of SN38-Glu by cMVs prepared from five human liver samples was ATP dependent. The concentration dependence of the ATP-dependent uptake of the carboxylate form of CPT-11 and SN38-Glu suggests the involvement of at least two saturable transport components, both with lower affinity and higher capacity than in rats. The ATP-dependent uptake of the carboxylate form of SN-38 showed a single saturable component but was detectable only in one human cMV sample. Both carboxylate and lactone forms of SN38-Glu uptake also showed a large intersample variability, although the variability was less than that observed for the carboxylate form of SN-38. On the other hand, the carboxylate form of CPT-11 exhibited much less variability. The carboxylate forms of SN38-Glu and SN-38 almost completely inhibited the ATP-dependent uptake of leukotriene C4, a well-known substrate of canalicular multispecific organic anion transporter, whereas the inhibition by the carboxylate form of CPT-11 was not as marked. Thus, multiple primary active transport systems are responsible for the biliary excretion of CPT-11 and its metabolites, and the major transport system for CPT-11 differs from that for the other two compounds. A greater degree of inter-cMV variability in the uptake of SN-38 and SN38-Glu may imply that interindividual variability in biliary excretion of these metabolites might contribute to interpatient variability in the toxicity caused by CPT-11.

Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide.

UNLABELLED: Eisai hyperbilirubinemic rat (EHBR) is a mutant strain with a hereditary defect in canalicular multispecific organic anion transporter (cMOAT). We examined the uptake and mutual inhibition of S-(2,4-dinitrophenyl)-glutathione (DNP-SG), which is a typical substrate for cMOAT, and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide (E-glu) with canalicular membrane vesicles (CMV) prepared from Sprague-Dawley (SD) and EHBR rats to investigate the multiplicity of the organic anion transporter. The ATP-dependent uptake by CMV from SD rats had an apparent Km of 17.6 microM for DNP-SG and 5.7 microM for E-glu, whereas the corresponding uptake by CMV from EHBR had an apparent Km of 44.6 microM for E-glu. The effects of E-glu, 4-methylumbelliferone glucuronide (4 MUG), E3040 sulfate (E-sul) and 4-methylumbelliferone sulfate (4 MUS) on the uptake of [3H]DNP-SG were also examined. The uptake of [3H]DNP-SG was inhibited by glucuronides (E-glu and 4 MUG) in a concentration-dependent manner, although it was enhanced by the sulfate conjugates (E-sul and 4 MUS). This enhancement was shown to be caused by an increased DNP-SG affinity for the transporter. In CMV from SD rats, although ATP-dependent uptake of [3H]DNP-SG was almost completely inhibited by E-glu, that of [14C]E-glu was only reduced to about 30% of controls by DNP-SG. On the other hand, in CMV from EHBR, the ATP-dependent uptake of [14C]E-glu was not inhibited at all by DNP-SG. Kinetic analysis indicated that E-glu inhibited DNP-SG uptake competitively. IN CONCLUSION: 1) cMOAT recognizes both DNP-SG and E-glu, and another transporter present in SD rats is also involved in E-glu transport along with cMOAT; 2) the latter transporter is kinetically similar to the E-glu transporter present in EHBR; 3) E-sul enhances the uptake of DNP-SG by increasing the affinity of glucuronide for the transporter.

Hepatobiliary transport mechanism for the cyclopentapeptide endothelin antagonist BQ-123.

The hepatobiliary transport of an anionic cyclopentapeptide endothelin antagonist, BQ-123, was studied in rats. Biliary excretion of [3H]BQ-123 was extensive in vivo (approximately 75% of intravenous infusion rates). Liver-to-plasma and bile-to-liver concentration ratios at steady state were approximately 3 and 200, respectively, suggesting that hepatic uptake and biliary excretion are concentrative processes. The biliary excretion clearance exhibited a saturation at a hepatic concentration of > 100 nmol/g liver and was markedly reduced in Eisai hyperbilirubinemic rats, which have a hereditary defect of canalicular multispecific organic anion transporter. An ATP-dependent and saturable uptake of BQ-123 by isolated canalicular membrane vesicles was observed in vitro. Impaired transport of BQ-123 was also confirmed in canalicular membrane vesicles prepared from Eisai hyperbilirubinemic rats. These results demonstrate that the biliary excretion process is ATP-driven primary active transport. It is proposed that a canalicular multispecific organic anion transporter is mainly responsible for the biliary excretion of BQ-123.

Carrier-mediated hepatic uptake of the cationic cyclopeptide, octreotide, in rats. Comparison between in vivo and in vitro.

The plasma concentration and biliary excretion profiles of a cationic cyclic octapeptide, octreotide, were compared between control rats and rats given an intravenous infusion of a bile acid, taurocholate (TCA), and an organic anion, dibromosulfophthalein (DBSP). Both TCA and DBSP reduced the plasma elimination and biliary excretion of octreotide after its intravenous bolus administration. Two mechanisms accounting for this phenomenon were considered a priori: decreased hepatic uptake from blood to liver and decreased biliary excretion from liver to bile. The tissue uptake clearance (CLup) of octreotide in plasma and several tissues was determined, and extensive uptake of octreotide (0.20 ml/min/g liver) was observed only in liver. The kinetic analysis indicated that CLup in liver fell to 10% of controls after administration of both TCA and DBSP. To compare CLup between in vivo and in vitro, the initial velocity of octreotide uptake by isolated hepatocytes and primary cultured hepatocytes was measured. The estimated kinetic parameters KM and Vmax were approximately 100 microM and 200 pmol/min/10(6) cells in both systems, respectively. Hepatic uptake clearance estimated from the in vitro data was comparable with that observed in vivo. Biliary excretion of octreotide is reduced in Eisai hyperbilirubinemic rats (EHBRs), which have a heredity defect of multispecific organic anion transporter on the bile canalicular membrane, compared with that of Sprague-Dawley rats. The kinetic analysis demonstrated that the hepatic uptake was reduced in EHBRs. The uptake study using primary cultured hepatocytes suggested that a high level of unidentified endogenous substrate(s) in EHBR plasma may be responsible for the reduction of hepatic uptake of octreotide in EHBRs. In conclusion, we have demonstrated in vivo that carrier-mediated hepatic uptake of octreotide is inhibited by TCA and DBSP and that the CLup obtained in vivo is comparable with the CLup obtained in vitro in isolated hepatocytes and primary cultured hepatocytes.

Multispecific organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats.

Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11), is a potent anticancer drug that is increasingly used in chemotherapy. A frequent limiting side effect involves gastrointestinal toxicity (diarrhea), which is thought to be related to the biliary excretion of CPT-11 and its metabolites. Accordingly, the biliary excretion mechanisms for both the lactone and carboxylate forms of CPT-11 and its metabolites, SN-38 and its glucuronide (SN38-Glu), were investigated using Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR), with the latter being mutant rats with a genetic deficiency of the canalicular multispecific organic anion transporter. After i.v. administration of CPT-11, the biliary excretion clearance, defined as the biliary excretion rate normalized to the hepatic concentration, of both the lactone and carboxylate forms of SN38-Glu was much lower in EHBR. The biliary excretion clearance for the carboxylate form of both CPT-11 and SN-38 was also substantially smaller in EHBR and showed marked saturation with increasing dose only in SD rats. On the other hand, the biliary excretion clearance for the lactone forms of CPT-11 and SN-38 showed only a minimal difference in EHBR, compared with SD rats. These results suggest that, for the carboxylate form of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu, there exists a specific transport system at the bile canalicular membrane that is deficient in EHBR. To confirm this hypothesis, the uptake of these substrates by isolated hepatic canalicular membrane vesicles (CMV) was examined. ATP-dependence was clearly observed for the uptake of these four compounds by CMV prepared from SD rats but not by CMV from EHBR. In addition, the compounds inhibited the ATP-dependent uptake of S-(2,4-dinitrophenyl) glutathione by CMV from SD rats, in a concentration-dependent manner. These results suggest that the biliary excretion of the carboxylate forms of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu is mediated by the multispecific organic anion transporter, which is deficient in EHBR.

Temocaprilat, a novel angiotensin-converting enzyme inhibitor, is excreted in bile via an ATP-dependent active transporter (cMOAT) that is deficient in Eisai hyperbilirubinemic mutant rats (EHBR).

Temocapril x HCl (alpha-((2S,6R)-6-[(1S)-1-ethoxy-carbonyl-3-phenyl-propyl]amino-5-oxo-2- (2-thienyl)perhydro-1,4-thiazepin-4-yl)acetic acid hydrochloride) is a novel prodrug of an angiotensin-converting enzyme (ACE) inhibitor. Unlike many other ACE inhibitors, its pharmacologically active metabolite,temocaprilat, is excreted predominantly in bile. To investigate the mechanism for the biliary excretion of temocaprilat, we performed in vivo and in vitro experiments using mutant Eisai hyperbilirubinemic rats EHBR) whose canalicular multispecific organic anion transporter (cMOAT) is hereditarily defective. Biliary clearance of temocaprilat after i.v. administration of [14C]temocapril x HCl (1.0 mg/kg) in EHBR was significantly lower than that in Sprague-Dawley rats (5.00 ml/min/kg for Sprague-Dawley rats vs. 0.25 ml/min/kg for EHBR). The uptake of temocaprilat into canalicular membrane vesicles (CMVs) prepared from Sprague-Dawley rats was stimulated in the presence of ATP, whereas little stimulation was observed in CMVs from EHBR. The initial uptake rate of ATP-dependent transport of temocaprilat showed saturation kinetics; we obtained an apparent V(max) value of 1.14 nmol/min/mg protein and a K(m) value 92.5 microM. ATP-dependent transport of temocaprilat was competitively inhibited by 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT with an inhibition constant (K(i)) of 25.8 microM. The K(m) value for the uptake of 2,4-dinitrophenyl-S-glutathione into CMVs (K(m) = 29.6 microM) was consistent with this K(i) value. In addition, the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione was inhibited by temocaprilat in a concentration-dependent manner. Active forms of some ACE inhibitors (benazepril, cilazapril, delapril, enalapril and imidapril) did not affect the transport of temocaprilat into CMVs even at concentrations as high as 200 microM. These data suggest that temocaprilat is effectively excreted in bile via cMOAT that is deficient in EHBR and that many of other ACE inhibitors have low affinity for cMOAT.

Mechanism of the tissue distribution and biliary excretion of the cyclic peptide octreotide.

The hepatobiliary transport and tissue distribution of the cationic cyclooctapeptide octreotide were studies at steady state after its infusion, at various rates, in rats. After an increase in steady-state plasma concentration, marked decrease in the tissue to plasma concentration ratio was observed only in pancreas, the target organ of octreotide. A marked decrease in the biliary excretion clearance, defined with respect to the concentration in the liver, was also observed, suggesting that a transport carrier was involved in the biliary excretion. The plasma elimination and biliary excretion profiles of octreotide were determined in Eisai hyperbilirubinemic rats (EHBR), which have an hereditary defect of the active transport carrier for organic anions in bile canalicular membranes. Although biliary excretion of octreotide was significantly reduced in EHBR, compared with normal Sprague-Dawley rats, no difference was observed in biliary excretion clearance, defined with respect to the concentration in the liver, between Sprague-Dawley rats and EHBR. On the other hand, the liver to plasma concentration ratio in EHBR fell to half that in Sprague-Dawley rats. These results suggest that the decreased biliary excretion of octreotide in EHBR is due not to reduced biliary excretion ability but to reduced hepatic uptake of octreotide. We studied in vitro transport using bile canalicular membrane vesicles. A significant increase in the transport of octreotide by bile canalicular membrane vesicles was observed in the presence of ATP, and the estimated kinetic parameters K(m) and Vmax were 6.5 microM and 370 pmol/min/mg of protein, respectively. Similar ATP-dependent uptake was observed in bile canalicular membrane vesicles prepared from EHBR. We concluded that the biliary excretion of octreotide is by ATP-dependent primary active transport and that the carrier system for octreotide differs from the so-called "canalicular multispecific organic anion transporter," which is absent in EHBR.

[Preparation of plasma membrane and the analysis of transport kinetics and driving forces].

The principles of isolating plasma membrane (basolateral membrane and brush border membrane), and separating right side-out and inside-out membrane vesicles were described. We then focused on the liver plasma membrane. Liver plasma membrane vesicles have been used to investigate the mechanism for the hepatobiliary transport of ligands. We precisely describe our protocol to isolate the sinusoidal (SMV) and canalicular membrane vesicles (CMV) from rat liver using the percoll gradient. Methods to determine the purity and orientation of SMV and CMV are also mentioned. Furthermore, procedures for the transport studies with SMV and CMV are provided; we refer to the electrogenic sodium dependent transport of taurocholic acid and alanine into SMV as an example to characterize the secondary active transport system. ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione (DNP-SG) into CMV is also cited as an example to clarify the primary active transport mechanism. Kinetic analysis for the ATP-dependent uptake of [3H] DNP-SG into CMV is also described.

Synthesis and storage of prolactin in the pituitary gland of bullfrog tadpoles during metamorphosis.

Changes in prolactin (PRL) levels and PRL-producing activities in the pituitary gland of tadpoles during metamorphosis were studied. PRL concentration in the pituitary gland as measured by a homologous radioimmunoassay was relatively low during pre- and prometamorphosis and early climax (2.5-3.5 micrograms/100 micrograms protein). PRL levels increased thereafter and reached maximum (7.3 micrograms/100 micrograms protein) at late climax (stage 24). It was also demonstrated that the pituitary cells which immunologically reacted with the antiserum to bullfrog PRL increased in number as metamorphosis proceeded. Measurement of incorporation of [3H]leucine into pituitary PRL in vitro revealed that elevation of PRL synthesis took place in the tadpoles which had reached climax. Radioimmunoassay and bioassay of PRL in the pituitary glands of adult frogs and tadpoles indicated that there was no difference in immunological and biological properties between adults and larvae.