Interaction of angiotensin II type 1 receptor blockers with P-gp substrates in Caco-2 cells and hMDR1-expressing membranes

AimsThe inhibitory effect of angiotensin II type 1 receptor blockers (ARBs) on P-glycoprotein (P-gp) was examined to evaluate their clinical drug–drug interaction (DDI) potential.Main methodsWe performed an inhibition study on the vectorial transport of digoxin, a typical substrate for P-gp, using a human colonic adenocarcinoma cell line, Caco-2 cells, and verapamil-stimulated ATPase activity using human multidrug resistance 1 (hMDR1)-expressing membrane.Key findingsThe vectorial transport of digoxin was inhibited by candesartan cilexetil, irbesartan and telmisartan with the IC₅₀ values of 14.7, 34.0 and 2.19 µM, respectively. Those values were 7.4–426-fold higher than their theoretical clinical gastrointestinal concentration [I] at doses in clinical DDI studies. Other ARBs failed to show interaction with P-gp.SignificanceIt was demonstrated that candesartan cilexetil, irbesartan and telmisartan had the potential to inhibit the transport of various drugs via P-gp. Telmisartan, which caused an increase in the serum digoxin concentration in humans, had a sufficiently high [I]/IC₅₀ value, suggesting that DDI between digoxin and telmisartan was caused by the inhibition of digoxin efflux via intestinal P-gp.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Responses of the pancreatic digestive enzyme secretion to amino acids, glucose and cholecystokinin in chicks

1. Pancreatic secretion of digestive enzymes, amylase, trypsinogen and chymotrypsinogen, was studied in response to the wing vein injection of digestive end products, various single amino acids and glucose, with or without cholecystokinin (CCK) in chicks. 2. Among amino acids administered, only phenylalanine significantly (P less than 0.05) increased trypsinogen and chymotrypsinogen secretions, while other amino acids did not. 3. Simultaneous injection of single amino acids with CCK increased digestive enzyme secretion to various extents depending on the kind of amino acids whereas the injection of glucose with CCK did not affect when compared with that of CCK alone. 4. By varying doses, a synergetic action of CCK plus amino acid on the secretion of pancreatic digestive enzymes was observed at 0.5 mM for valine and 5 mM for arginine.     

Crystal structure of a hypothetical protein,TTHA0829 from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8, composed of cystathionine-β-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-β-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four β-strands and two α-helices in a βαββαβ motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.     

Activation of glucuronidation through reduction of a disulfide bond in rat UDP-glucuronosyltransferase 1A6

UDP-glucuronosyltransferase- (UGT-) dependent glucuronidation is an important detoxification process for many endogenous and exogenous compounds in mammals. Treatment of rat hepatic microsomes with the reducing reagent dithiothreitol (DTT) resulted in a significant increase in p-nitrophenol (p-NP) glucuronidation in a time- and concentration-dependent manner. The DTT-dependent activation of glucuronidation was specific for planar phenols but not for bilirubin or testosterone without membrane perturbation of the microsomes. p-NP glucuronidation in Gunn rat hepatic microsomes lacking UGT1 isozymes was not affected by DTT, indicating that UGT1A6 in the microsomes is mainly involved in the activation. The DTT-dependent activation was inhibited by 1,6-bis(maleimido)hexane (BMH) but not by N-ethylmaleimide, indicating that cross-linking between cysteine residues in UGT1A6 is responsible for the activation. Immunoblot analysis of rat hepatic microsomes on nonreducing SDS-PAGE gels revealed that most of the UGT1A6 migrated as a monomer, suggesting that DTT could affect an intramolecular disulfide bond in the UGT1A6 that may be responsible for the activation. To identify which of the ten cysteines in UGT1A6 are involved in the disulfide bond, rat UGT1A6 wild type and a set of mutants, each with a cysteine to serine substitution, were constructed and expressed in COS cells. Treatment of COS microsomes with DTT had no effect on the activity of the wild type but BMH showed significant inhibition, suggesting that UGT1A6 expressed in COS cells may be in the reduced and activated state. Replacement of either Cys 121 or Cys 125 with serine showed insensitivity to the BMH-dependent inhibition. These results demonstrate that both Cys 121 and Cys 125 are responsible for the activation of the activity through the disulfide bond in rat UGT1A6.     

cDNA cloning and expression of rat tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor for the factor VIIa-tissue factor complex. We isolated cDNA for rat TFPI by screening a lambda gt10 rat liver cDNA library. We determined the 1,228 bp nucleotide sequence, comprising a 88 bp 5' non-coding region, a 906 bp open reading frame, and a 234 bp 3' non-coding region, which encodes a protein of 302 amino acid residues. On Northern blot analysis of rat TFPI mRNA, rat TFPI mRNA was detected as two forms with different molecular sizes, 4.0 and 1.4 kb, which were expressed abundantly in heart, lung, kidney, and aortic endothelial cells. The homology of the amino acid sequence of rat TFPI with those of human and rabbit TFPI was found to be 60.7 and 57.4%, respectively. The lengths of the three tandem Kunitz-type inhibitor domains were strictly conserved not only among TFPI from the three species, but also among other proteins containing Kunitz-type inhibitor domains. The homology of the Kunitz-type domains in TFPI among the three species was 57, 86, and 69% in the 1st, 2nd, and 3rd domains, respectively. There was no significant difference in hydropathy profiles of TFPI from man, rabbit, and rat.     

A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district,eastern Japan

To clarify the effects of forest fragmentation and a change in tree species composition following urbanization on endophytic fungal communities, we isolated fungal endophytes from the foliage of nine tree species in suburban (Kashiwa City, Chiba) and rural (Mt. Wagakuni, Ibaraki; Mt. Takao, Tokyo) forests and compared the fungal communities between sites and host tree species. Host specificity was evaluated using the index of host specificity (Si), and the number of isolated species, total isolation frequency, and the diversity index were calculated. From just one to several host-specific species were recognized in all host tree species at all sites. The total isolation frequency of all fungal species on Quercus myrsinaefolia, Quercus serrata, and Chamaecyparis obtusa and the total isolation frequency of host-specific species on Q. myrsinaefolia, Q. serrata, and Eurya japonica were significantly lower in Kashiwa than in the rural forests. The similarity indices (nonmetric multidimensional scaling (NMS) and C_MH) of endophytic communities among different tree species were higher in Kashiwa, as many tree species shared the same fungal species in the suburban forest. Endophytic fungi with a broad host range were grouped into four clusters suggesting their preference for conifer/broadleaves and evergreen/deciduous trees. Forest fragmentation and isolation by urbanization have been shown to cause the decline of host-specific fungal species and a decrease in β diversity of endophytic communities, i.e., endophytic communities associated with tree leaves in suburban forests were found to be depauperate.     

Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase.

With specific monoclonal antibodies, we found that human endometrial stromal cells and decidual cells express two function-related surface antigens. Indirect immunofluorescence staining revealed that both endometrial stromal cells and decidual cells during the first trimester of pregnancy expressed cluster of differentiation (CD) 13 antigen and CD10 antigen, which are identical to aminopeptidase N and neutral endopeptidase, respectively. By flow cytometric analysis, CD13 antigen was detected on 82-93% of the examined cells, and CD10 antigen was detected on 75-93% of the examined cells in endometrial stromal cell-enriched preparations. Furthermore, peptidase activity was detected in these cell preparations by an assay based on the hydrolysis of alanine-p-nitroanilide into p-nitroaniline and alanine.