Overexpression of carboxylesterase contributes to the attenuation of cyanotoxin microcystin-LR toxicity.

Microcystin-LR is a hepatotoxin produced by several cyanobacteria. Its toxicity is mainly due to a inhibition of protein phosphatase, PP1 and PP2A. Previously, we used a cell line stably expressing uptake transporter for microcystin-LR, OATP1B3 (HEK293-OATP1B3 cells). In this study, to determine whether overexpression of carboxylesterase (CES), which degrades ester-group and amide-group, attenuates the cytotoxicity of microcystin-LR, we generated the HEK293-OATP1B3/CES2 double-transfected cells. HEK293-OATP1B3/CES2 cells showed high hydrolysis activity of p-nitrophenyl acetate (PNPA), which is an authentic substrate for esterase. CES activity in HEK293-OATP1B3/CES2 cells was approximately 3-fold higher than that in the HEK293-OATP1B3 cells. HEK293-OATP1B3/CES2 cells (IC50: 25.4±7.7nM) showed approximately 2.1-fold resistance to microcystin-LR than HEK293-OATP1B3 cells (IC50: 12.0±1.5nM). Moreover, the CES inhibition assay and microcystin-agarose pull down assay showed the possibility of the interaction between CES2 and microcystin-LR. Our results indicated that the overexpression of CES2 attenuates the cytotoxicity of microcystin-LR via interaction with microcystin-LR.

Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning.

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.

Distinct patterns of aging effects on the expression and activity of carboxylesterases in rat liver and intestine.

The age-associated alteration in expression levels of carboxylesterases (CESs) can affect both intestinal and hepatic first-pass metabolism after oral administration of xenobiotic esters such as prodrugs. In this study, the age-related expression of CES isozymes and hydrolase activities were simultaneously investigated in liver, jejunum, and ileum from 8-, 46-, and 90-week-old rats. Rat liver expresses three major CES1 isozymes, Hydrolase A, Hydrolase B, and Hydrolase C, as well as one minor CES1 (Egasyn) and three minor CES2 isozymes (RL4, AY034877, and D50580). The mRNA and protein levels of major hepatic CES1 isozymes were decreased in an age-dependent manner, while those of minor CESs were maintained in all age groups. The hepatic hydrolase activity for temocapril was decreased in an age-dependent manner, accompanied by downregulation of Hydrolase B/C mRNA, while age-independent hydrolysis of propranolol derivatives was observed in rat liver, due to the contribution of Egasyn. Rat small intestine expresses one major CES2 (RL4) and four minor CESs (Hydrolase B, Hydrolase C, Egasyn, and AY034877). Interestingly, the expression of RL4 was age-dependently increased in both jejunum and ileum, while minor isozymes showed a constant expression across a wide age range. The up-regulation of RL4 expression with aging led to an increase of intestinal hydrolase activities for temocapril and propranolol derivatives. Consequently, age-dependent changes in the expression of CES isozymes affect the hydrolysis of xenobiotics in both rat liver and small intestine.

Involvement of Carboxylesterase in Hydrolysis of Propranolol Prodrug during Permeation across Rat Skin.

The use of a prodrug, a conjugate of an active drug with a lipophilic substituent, is a good way of increasing the cutaneous absorption of a drug. However, the activity of dermal hydrolases has rarely been investigated in humans, or experimental animals. In the present study, we focused on the identification of rat dermal esterases and the hydrolysis of a prodrug during permeation across rat skin. We found that carboxylesterase (CES), especially the rat CES1 isozyme, Hydrolase A, is expressed in rat skin and that the hydrolysis of p-nitrophenyl acyl derivatives and caproyl-propranolol (PL) was 20-fold lower in the 9000g supernatant fraction of skin homogenate than in liver microsomes. A permeation study of caproyl-PL was performed in rat full-thickness and stripped skin using a flow-through diffusion cell. Caproyl-PL was easily partitioned into the stratum corneum and retained, not only in the stratum corneum, but also in viable epidermis and dermis. Caproyl-PL could barely be detected in the receptor fluid after application to either full-thickness or stripped skin. PL, derived from caproyl-PL, was, however, detected in receptor fluid after extensive hydrolysis of caproyl-PL in viable skin. Permeation of PL was markedly decreased under CES inhibition, indicating that the net flux of caproyl-PL is dependent on its conversion rate to PL.

Comparison of two expression systems using COS7 cells and yeast cells for expression of heart/muscle-type carnitine palmitoyltransferase 1.

Carnitine palmitoyltransferase 1 (CPT1), catalyzing the transfer of the acyl group from acyl-CoA to carnitine to form acylcarnitine, is located at the outer mitochondrial membrane. Because it is easily inactivated by solubilization, expression systems using living cells are essential for its functional characterization. COS7 cells or yeast cells are often utilized for this purpose; however, the advantages/disadvantages of the use of these cells or the question as to how the CPT1 enzyme expressed by these cells differs are still uncertain. In this study, we characterized the heart/muscle-type isozyme of rat CPT1 (CPT1b) expressed by these two cellular expression systems. The mitochondrial fraction prepared from yeast cells expressing CPT1b showed 25% higher CPT1 activity than that obtained from COS7 cells. However, the expression level of CPT1b in the former was 3.8 times lower than that in the latter; and thus, under the present experimental conditions, the specific activity of CPT1b expressed in yeast cells was estimated to be approximately five times higher than that expressed in COS7 cells. Possible reasons for this difference are discussed.

[Structural and functional properties of the C-terminal region of mitochondrial ADP/ATP carrier].

Mitochondrial ADP/ATP carrier (AAC) is a protein catalyzing the transport of adenine nucleotides across inner mitochondrial membrane. In this review article, we first briefly introduce structural and functional properties of this protein. Next, we describe the results of our recent studies on the difference in the C-terminal region between yeast type 2 AAC isoform and bovine type 1 AAC isoform. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier are discussed.

Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier.

Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.

An oral adsorbent, AST-120 protects against the progression of oxidative stress by reducing the accumulation of indoxyl sulfate in the systemic circulation in renal failure.

PURPOSE: The effect of AST-120, an oral adsorbent, on oxidative stress in the systemic circulation in chronic renal failure (CRF) was examined and the potential role of indoxyl sulfate (IS), an uremic toxin adsorbed by AST-120, in inducing the formation of reactive oxygen species (ROS) in the vascular system was studied, in vitro and in vivo. MATERIALS AND METHODS: The level of oxidized albumin, a marker for oxidative stress in the systemic circulation was determined by HPLC, as previously reported. The mRNA levels of TGF-beta (1) and Oat1 were measured by quantitative RT-PCR. The IS induced ROS generation in cultured human umbilical vein endothelial cells (HUVECs) was estimated using a fluorescence microplate reader. RESULTS: An increase in the ratio of oxidized to unoxidized albumin was determined using 5/6 nephrectomized rats (CRF rats) compared to a control group. The ratio was significantly reduced in the group that received AST-120 of 4 weeks, suggesting that AST-120 inhibits oxidative stress in CRF. An anti-oxidative effect of AST-120 was also observed in CRF rats with a similar renal function. The ratio of oxidized albumin was correlated with serum IS levels in vivo. The same relationship was also observed in CRF rats with the continued administration of IS. In addition, IS dramatically increased the generation of ROS in both a dose- and time- dependent manner in HUVEC, suggesting that accumulated IS may play an important role in enhancing intravascular oxidative stress. CONCLUSION: We propose that AST-120 reduces IS concentrations in the blood that induces ROS production in endothelial cells, thereby inhibiting the subsequent occurrence of oxidative stress in the systemic circulation in renal failure.

Intestinal first-pass metabolism via carboxylesterase in rat jejunum and ileum.

To determine the activity of a major intestinal esterase in the first-pass hydrolysis of O-isovaleryl-propranolol (isovaleryl-PL), a model ester compound, rat intestinal jejunum and blood vessels were perfused simultaneously after inhibition of a carboxylesterase (CES) by bis-p-nitrophenyl phosphate (BNPP). BNPP specifically inhibits approximately 90% of CES activity without influencing aminopeptidase activity or the transport of L-leucyl-p-nitroanilide and p-nitroaniline, nonester compounds. When isovaleryl-PL was perfused into the jejunal lumen after BNPP treatment, its absorption clearance (7.60 +/- 0.74 microl/min) increased approximately 3-fold compared with control, whereas its degradation clearance (32.5 +/- 5.40 microl/min) decreased to 23% of control. Therefore, CES seems to be mainly responsible for the intestinal first-pass hydrolysis of isovaleryl-PL. This finding is consistent with the results from studies of in vitro BNPP inhibition in the mucosal S9 fraction. V(max) values for valeryl-PL, isovaleryl-PL, and p-nitrophenyl acetate in the jejunal S9 fraction were 1.7- to 2.5-fold higher than that in the ileal S9 fraction, which agreed with the jejunum/ileum ratio (approximately 1.5-fold) of mRNA expression levels for the CES2 isozymes, AB010635 and AY034877. These findings indicated that CESs expressed in the intestine markedly contribute to first-pass hydrolysis in both jejunum and ileum.

Roles of adjoining Asp and Cys residues of first matrix-facing loop in transport activity of yeast and bovine mitochondrial ADP/ATP carriers.

The mitochondrial ADP/ATP carrier (AAC) transports substrate by interconversion of its conformation between m- and c-states. The 1st loop facing the matrix (LM1) is extruded into the matrix in the m-state and is suggested to intrude into the mitochondrial membrane on conversion to the c-state conformation [Hashimoto, M., Majima, E., Goto, S., Shinohara, Y., and Terada, H. (1999) Biochemistry 38, 1050-1056]. To elucidate the mechanism of the translocation of LM1, we examined the effects of site-directed mutagenesis of two adjoining residues, Cys56 and Asp55 in the bovine type 1 AAC and Cys73 and Asp72 in the yeast type 2 AAC, on the substrate transport activity. We found that (i) replacement of the Cys by bulky and hydrophilic residues was unfavorable for efficient transport activity, (ii) the carboxyl groups of the Asp residues of the bovine and yeast AACs were essential and strictly position-specific, and (iii) hence, the mutation to Glu showed transport activity comparable to that of the native AACs. Based on these results, we discussed the functional role of LM1 in the transport activity of AAC.

Identification of esterases expressed in Caco-2 cells and effects of their hydrolyzing activity in predicting human intestinal absorption.

The absorption characteristics of temocapril were investigated using Caco-2 cells, and the esterases expressed in Caco-2 cells were identified. Temocapril was almost completely hydrolyzed to temocaprilat during transport across Caco-2 cells. Hydrolysis experiments of temocapril in Caco-2 cell 9000g supernatant (S9) and brush-border membrane vesicles showed that temocapril was mainly hydrolyzed within the cells after uptake, after which the temocaprilat formed was transported to both the apical and basolateral surfaces. In native polyacrylamide gel electrophoresis by detection of hydrolase activity for 1-naphthylbutyrate, Caco-2 cell S9 showed a band with high esterase activity and another band with extremely low activity. The proteins in the major and minor bands were identified as carboxylesterase-1 (hCE-1) and carboxylesterase-2 (hCE-2). The abundant expression of hCE-1 in Caco-2 cells was supported by reverse transcription-polymerase chain reaction. In the normal human small intestine, hCE-2 is abundantly present, although the human liver expresses much higher levels of hCE-1 and lower levels of hCE-2. The expression pattern of carboxylesterases in Caco-2 cells is completely different from that in human small intestine but very similar to that in human liver. Since the substrate specificity of hCE-1 differs from that of hCE-2, it is suggested that the prediction of human intestinal absorption using Caco-2 cell monolayers should be performed carefully in the case of ester- and amide-containing drugs such as prodrugs.

Kinetic and regulatory properties of HK I(+), a modified form of the type I isozyme of mammalian hexokinase in which interactions between the N- and C-terminal halves have been disrupted.

A modified form (HK I(+)) of rat Type I hexokinase (HK I) has been expressed. HK I(+) contains a centrally located polyalanine insert which, along with the known helical propensity of adjacent sequence, was expected to lead to alpha-helix formation, with resulting distension of the molecule and disruption of interactions between the N- and C-terminal halves. The properties of HK I(+) are consistent with this expectation and with previous proposals that (1) inhibition of HK I by Glc-6-P or its analogs and antagonism of this inhibition by P(i) result from competition of these ligands for a binding site in the N-terminal half of HK I, with resulting conformational changes propagated through interactions with the catalytic C-terminal half, and (2) binding of Glc-6-P to a site in the C-terminal half of HK I is obstructed by interactions between the halves, present in HK I but not HK I(+).