Uptake of Fluorescein via a pH-Dependent Monocarboxylate Transporter by Human Kidney 2 (HK-2) Cells.

Herein, we investigated whether a fluorescent probe for an organic anion transporter (OAT), fluorescein (FLS), could be accumulated by human kidney 2 (HK-2) cells derived from human kidney proximal tubular epithelia. HK-2 cells took up FLS in a pH-dependent and concentration-dependent manner. FLS accumulation by HK-2 cells was inhibited by monocarboxylic acids, ibuprofen, rosuvastatin, and indoleacetic acid but not by typical substrates for OATs. A typical protonophore, carbonyl cyanide p-trichloromethoxyphenylhydrazone completely abolished FLS accumulation by HK-2 cells. The FLS efflux process from the preloaded HK-2 cells exhibited substantial trans-stimulation by the excess amount of extracellular FLS transport inhibitable monocarboxylate compounds such as 2,4-dichloro phenoxyacetic acid, fluvastatin, ibuprofen, indoleacetic acid, salicylic acid and rosuvastatin, indicating that the FLS transporter can recognize and accumulate them into the cells in a pH-dependent manner. The involvement of the FLS transporter in the reabsorption of monocarboxylic compounds was indicated by demonstrating that the pH-dependent FLS uptake is inhibited by various monocarboxylates in rabbit renal brush border membrane vesicles. pH-dependent FLS uptake was trans-stimulated by the inhibitable monocarboxylates. Collectively, the present data indicate that the pH-dependent transporters expressed in HK-2 cells are involved in the reabsorption of monocarboxylates from the urinary fluid into the tubular epithelia.

Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket.

Here, we have elucidated the substrate recognition mechanism by a prokaryotic H+/oligopeptide cotransporter, YdgR, using isothermal titration calorimetry. Under acidic conditions (pH 6.0), the binding of a dipeptide, Val-Ala, to YdgR elicited endothermic enthalpy, which compensated for the increase in entropy due to dipeptide binding. A series of dipeptides were used in the binding titration. The dipeptides represent Val-X and X-Val, where X is Ala, Ser, Val, Tyr, or Phe. Most dipeptides revealed endothermic enthalpy, which was completely compensated by the increase in entropy due to dipeptide binding. The change in enthalpy due to binding correlated well with the change in entropy, whereas the Gibbs free energy involved in the binding of the dipeptide to YdgR remained unchanged irrespective of dipeptide sequences, implying that the binding reaction was driven by entropy, that is, the release of bound water molecules in the binding pocket. It is also important to clarify that, based on the prediction of water molecules in the ligand-binding pocket of YdgR, the release of three bound water molecules in the putative substrate binding pocket occurred through binding to YdgR. In the comparison of Val-X and X-Val dipeptides, the N-terminal region of the binding pocket might contain more bound water molecules than the C-terminal region. In light of these findings, we suggest that bound water molecules might play an important role in substrate recognition and binding by YdgR.

Protonation State of a Histidine Residue in Human Oligopeptide Transporter 1 (hPEPT1) Regulates hPEPT1-Mediated Efflux Activity.

To clarify the role of an amino acid residue in the pH-dependent efflux process in Chinese hamster ovary (CHO) cells expressing the human oligopeptide transporter hPEPT1 (CHO/hPEPT1), we determined the effect of extracellular pH on the hPEPT1-mediated efflux process. The efflux of glycylsarcosine (Gly-Sar), a typical substrate for hPEPT1, was determined using an infinite dilution method after cells were preloaded with [3H]-Gly-Sar. The efflux of [3H]-Gly-Sar was stimulated by 5 mM unlabeled hPEPT1 substrates in the medium. This trans-stimulation phenomenon showed that hPEPT1 mediated the efflux of [3H]-Gly-Sar from CHO/hPEPT1 and that hPEPT1 is a bi-directional transporter. We then determined the effect of extracellular pH (varying from 8.0 to 3.5) on the efflux activity. The efflux activity by hPEPT1 decreased with the decrease in extracellular pH. The Henderson-Hasselbälch-type equation, which fitted well to the pH-profile of efflux activity, indicated that a single amino acid residue with a pKa value of approximately 5.7 regulates the efflux activity. The pH-profile of the efflux activity remained almost unchanged irrespective of the proton gradient across the plasma membrane. In addition, the chemical modification of the histidine residue with diethylpyrocarbonate completely abolished the efflux activity from cells, which could be prevented by the presence of 10 mM Gly-Sar. These data indicate that the efflux process of hPEPT1 is also regulated in a pH-dependent manner by the protonation state of a histidine residue located at or near the substrate recognition site facing the extracellular space.

Transport Characteristics of 5-Aminosalicylic Acid Derivatives Conjugated with Amino Acids via Human H+-Coupled Oligopeptide Transporter PEPT1.

5-Aminosalicylic acid (5-ASA) is used as first line therapy for symptom remission and maintenance of inflammatory bowel disease (IBD). Because 5-ASA is well absorbed from the small intestine when orally administered, several 5-ASA formulations for selective delivery to the colon have been developed and used in clinical practice. However, its delivery efficiency to local inflamed colonic sites remains low. Intestinal H+-coupled oligopeptide transporter 1 (PEPT1) expression in the colon is low, whereas its expression is induced in the colon under chronic inflammation conditions, such as IBD. Therefore, we considered that PEPT1 would be a target transporter to improve 5-ASA delivery efficiency to local colonic lesions. We evaluated the transport characteristics of dipeptide-like 5-ASA derivatives, which were coupling glycine (Gly), lysine, glutamic acid (Glu), valine (Val) and tyrosine to amino or carboxyl group of 5-ASA, in Caco-2 cells. [3H]Glycylsarcosine (Gly-Sar) uptake into Caco-2 cells was inhibited by all 5-ASA derivatives. In addition, 5-ASA derivatives (Gly-ASA, Glu-ASA and Val-ASA), which were coupled by glycine, glutamic acid and valine to amino group of 5-ASA, were taken up in a pH- and concentration-dependent manner and their uptake was inhibited by excess Gly-Sar. Two-electrode voltage-clamp experiment using human PEPT1 expressing Xenopus oocytes showed that Gly-ASA, Glu-ASA and Val-ASA induced marked currents at pH 6.0. Taken together, these results showed that these 5-ASA derivatives are transportable substrates for PEPT1.

Extrapolation of In Vivo Hepatic Clearance from In Vitro Uptake Clearance by Suspended Human Hepatocytes for Anionic Drugs with High Binding to Human Albumin: Improvement of In Vitro-to-In Vivo Extrapolation by Considering the "Albumin-Mediated" Hepatic Uptake Mechanism on the Basis of the "Facilitated-Dissociation Model".

We investigated whether human serum albumin (HSA) in suspended human hepatocytes would affect the uptake clearance of anionic drugs with high binding to HSA and improve the extrapolation of in vivo hepatic clearance from in vitro uptake clearance by the hepatocytes via the "albumin-mediated" hepatic uptake mechanism. The uptake clearances for total forms (PS inf) and for unbound forms (PS u,inf) of 11 anionic drugs [all of which were organic anion-transporting polypeptide (OATP) substrates] were determined with suspended human hepatocytes in varying concentrations of HSA. The fraction of unbound drugs (f u) was determined using an equilibrium dialysis at the various HSA concentrations. The PS inf values decreased with increasing concentrations of HSA, whereas the unbound uptake clearances (PS u,inf(+) = PS inf/ f u) in the presence of HSA increased substantially, thus demonstrating the "albumin-mediated" hepatic uptake mechanism. The relationships between PS inf and HSA concentration were well described by the previously proposed facilitated-dissociation model, in which the drug-albumin complex interacts with the cell surface, enhancing the dissociation of the complex and providing unbound drug for hepatic uptake. Furthermore, the PS u,inf (+) values in in vivo conditions (at 5% HSA) were predicted from those obtained in isolated hepatocytes on the basis of the facilitated-dissociation model, revealing compatibility with the overall hepatic intrinsic clearance in vivo. We conclude that the "facilitated-dissociation" model is useful for describing the "albumin-mediated" hepatic uptake phenomenon of OATP drugs and to predict hepatic uptake clearance in vivo.

Transport Mechanisms for the Nutritional Supplement ß-Hydroxy-ß-Methylbutyrate (HMB) in Mammalian Cells.

PURPOSE: ß-Hydroxy-ß-methylbutyrate (HMB), a nutritional supplement, elicits anabolic activity in muscle. Here we investigated the mechanism of HMB uptake in muscle cells. METHODS: Murine muscle cells (C2C12) and human mammary epithelial cells (MCF7) were used for uptake. As HMB is a monocarboxylate, focus was on monocarboxylate transporters, monitoring interaction of HMB with H+-coupled lactate uptake, and influence of H+ directly on HMB uptake. Involvement of MCT1-4 was studied using selective inhibitors and gene silencing. Involvement of human Na+/monocarboxylate transporter SMCT1 was also assessed using Xenopus oocytes. RESULTS: H+-coupled lactate uptake was inhibited by HMB in both mammalian cells. HMB uptake was H+-coupled and inhibited by lactate. C2C12 cells expressed MCT1 and MCT4; MCF7 cells expressed MCT1-4; undifferentiated C2C12 cells expressed SMCT1. SMCT1 mediated Na+-coupled HMB transport. Inhibitors of MCT1/4, siRNA-mediated gene silencing, and expression pattern showed that MCT1-4 were responsible only for a small portion of HMB uptake in these cells. CONCLUSION: HMB uptake in C2C12 and MCF7 cells is primarily H+-coupled and inhibited by lactate, but MCT1-4 are only partly responsible for HMB uptake. SMCT1 also transports HMB, but in a Na+-coupled manner. Other, yet unidentified, transporters mediate the major portion of HMB uptake in C2C12 and MCF7 cells.

The Phenomenon of Albumin-Mediated Hepatic Uptake of Organic Anion Transport Polypeptide Substrates: Prediction of the In Vivo Uptake Clearance from the In Vitro Uptake by Isolated Hepatocytes Using a Facilitated-Dissociation Model.

The effects of bovine serum albumin and human serum albumin on the unbound hepatic uptake clearance (PSu,inf) of the organic anion-transporting polypeptide substrates 1-anilino-8-naphthalene sulfonate (ANS) and pitavastatin (PTV) were determined using primary cultured rat hepatocytes and isolated human hepatocytes, respectively. The PSu,inf value of hepatocytes was estimated by dividing the initial uptake rate of these anions by their unbound concentrations. The PSu,inf values for ANS and PTV were enhanced in the presence of albumin, thereby demonstrating the phenomenon of "albumin-mediated" hepatic uptake. We previously constructed a "facilitated-dissociation" model, in which the interaction of the ligand-albumin complex with the cell surface enhanced the dissociation of that complex to provide unbound ligand for uptake to the hepatocytes [J Pharmacokinet Biopharm 16:165-181 (1988)]. That model was able to describe accurately the relationship between the enhancement of the PSu,inf values and the albumin concentration. By considering the enhancement of hepatic uptake clearance by albumin using this facilitated-dissociation model, we could predict accurately the PSu,inf in vivo from that obtained in isolated hepatocytes. In the light of these findings, we suggest that the facilitated-dissociation model is applicable to describing the phenomenon of albumin-mediated hepatic uptake via organic anion transporters and to evaluating hepatic uptake clearance in vivo.

Carbidopa is an activator of aryl hydrocarbon receptor with potential for cancer therapy.

Carbidopa is used with l-DOPA (l-3,4-dihydroxyphenylalanine) to treat Parkinson's disease (PD). PD patients exhibit lower incidence of most cancers including pancreatic cancer, but with the notable exception of melanoma. The decreased cancer incidence is not due to l-DOPA; however, the relevance of Carbidopa to this phenomenon has not been investigated. Here, we tested the hypothesis that Carbidopa, independent of l-DOPA, might elicit an anticancer effect. Carbidopa inhibited pancreatic cancer cell proliferation both in vitro and in vivo Based on structural similarity with phenylhydrazine, an inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1), we predicted that Carbidopa might also inhibit IDO1, thus providing a molecular basis for its anticancer effect. The inhibitory effect was confirmed using human recombinant IDO1. To demonstrate the inhibition in intact cells, AhR (aryl hydrocarbon receptor) activity was monitored as readout for IDO1-mediated generation of the endogenous AhR agonist kynurenine in pancreatic and liver cancer cells. Surprisingly, Carbidopa did not inhibit but instead potentiated AhR signaling, evident from increased CYP1A1 (cytochrome P450 family 1 subfamily A member 1), CYP1A2, and CYP1B1 expression. In pancreatic and liver cancer cells, Carbidopa promoted AhR nuclear localization. AhR antagonists blocked Carbidopa-dependent activation of AhR signaling. The inhibitory effect on pancreatic cancer cells in vitro and in vivo and the activation of AhR occurred at therapeutic concentrations of Carbidopa. Chromatin immunoprecipitation assay further confirmed that Carbidopa promoted AhR binding to its target gene CYP1A1 leading to its induction. We conclude that Carbidopa is an AhR agonist and suppresses pancreatic cancer. Hence, Carbidopa could potentially be re-purposed to treat pancreatic cancer and possibly other cancers as well.

X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

Simple Evaluation Method for CYP3A4 Induction from Human Hepatocytes: The Relative Factor Approach with an Induction Detection Limit Concentration Based on the Emax Model.

We investigated the robustness and utility of the relative factor (RF) approach based on the maximum induction effect (Emax) model, using the mRNA induction data of 10 typical CYP3A4 inducers in cryopreserved human hepatocytes. The RF value is designated as the ratio of the induction detection limit concentration (IDLC) for a standard inducer, such as rifampicin (RIF) or phenobarbital (PB), to that for the compound (e.g., RFRIF is IDLCRIF/IDLCcpd; RFPB is IDLCPB/IDLCcpd). An important feature of the RF approach is that the profiles of the induction response curves on the logarithmic scale remain unchanged irrespective of inducers but are shifted parallel depending on the EC50 values. A key step in the RF approach is to convert the induction response curve by finding the IDLC of a standard inducer. The relative induction score was estimated not only from Emax and EC50 values but also from those calculated by the RF approach. These values showed good correlation, with a correlation coefficient of more than 0.974, which revealed the RF approach to be a robust analysis irrespective of its simplicity. Furthermore, the relationship between RFRIF or RFPB multiplied by the steady-state unbound plasma concentration and the in vivo induction ratio plotted using 10 typical inducers gives adequate thresholds for CYP3A4 drug-drug interaction risk assessment. In light of these findings, the simple RF approach using the IDLC value could be a useful method to adequately assess the risk of CYP3A4 induction in humans during drug discovery and development without evaluation of Emax and EC50.

Chemical Modulation of the Human Oligopeptide Transporter 1, hPepT1.

In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target.

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum.

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

Converting a light-driven proton pump into a light-gated proton channel.

There are two types of membrane-embedded ion transport machineries in nature. The ion pumps generate electrochemical potential by energy-coupled active ion transportation, while the ion channels produce action potential by stimulus-dependent passive ion transportation. About 80% of the amino acid residues of the light-driven proton pump archaerhodopsin-3 (AR3) and the light-gated cation channel channelrhodopsin (ChR) differ although they share the close similarity in architecture. Therefore, the question arises: How can these proteins function differently? The absorption maxima of ion pumps are red-shifted about 30-100 nm compared with ChRs, implying a structural difference in the retinal binding cavity. To modify the cavity, a blue-shifted AR3 named AR3-T was produced by replacing three residues located around the retinal (i.e., M128A, G132V, and A225T). AR3-T showed an inward H(+) flux across the membrane, raising the possibility that it works as an inward H(+) pump or an H(+) channel. Electrophysiological experiments showed that the reverse membrane potential was nearly zero, indicating light-gated ion channeling activity of AR3-T. Spectroscopic characterization of AR3-T revealed similar photochemical properties to some of ChRs, including an all-trans retinal configuration, a strong hydrogen bond between the protonated retinal Schiff base and its counterion, and a slow photocycle. From these results, we concluded that the functional determinant in the H(+) transporters is localized at the center of the membrane-spanning domain, but not in the cytoplasmic and extracellular domains.

Electrophysiological characterization of human Na⁺/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes.

The Na(+)/taurocholate cotransporting polypeptide (NTCP) plays a major role in Na(+)-dependent bile acid uptake into hepatocytes. The purpose of the present study was to establish the heterologous expression of human NTCP (hNTCP) in Xenopus laevis oocytes and to elucidate whether the transport of bile acid via hNTCP is electrogenic using electrophysiological techniques. First, we evaluated the uptake of taurocholate (TCA) by hNTCP heterologously expressed in Xenopus oocytes utilizing [(3)H]-labeled TCA. The uptake of 1.2 µM TCA by cRNA-injected oocytes increased more than 100-fold compared to H2O-injected oocytes, indicating that hNTCP is robustly expressed in the oocytes. hNTCP-mediated transport of TCA is saturable with a Michaelis constant of 10.5 ± 2.9 µM. The Na(+)-activation kinetics describing the relationship between the concentration of Na(+) and the magnitude of the TCA uptake rate by hNTCP were sigmoidal with a Hill coefficient of 2.3 ± 0.4, indicating the involvement of more than one Na(+) in the transport process. Ntcp in primary cultured hepatocytes from rats exhibited similar Na(+)-activation kinetics of TCA uptake rate with a Hill coefficient of 1.9 ± 0.1, suggesting that hNTCP could be expressed properly in the oocytes and exhibit the electrogenic property of Na(+)-coupled TCA transport. The transport of TCA via hNTCP was subsequently determined in the oocytes by the inward currents induced via TCA uptake under voltage (-50 mV). Two hundred micromolar TCA induced significant inward currents that were entirely abolished by the substitution of Na(+) with N-methyl-d-glucamine (NMDG) in the perfusate, indicating that the TCA-induced currents were obligatorily dependent on the presence of Na(+). The TCA-induced currents were saturable, and the substrate concentration needed for half-maximal induction of the current was consistent with the Michaelis constant. Transportable substrates, such as rosuvastatin and fluvastatin, also induced currents. These results in the hNTCP heterologously expressed in Xenopus oocytes directly demonstrated that hNTCP is an electrogenic Na(+)-dependent transporter.

Pharmacological evidence showing significant roles for potassium channels and CYP epoxygenase metabolites in the relaxant effects of docosahexaenoic acid on the rat aorta contracted with U46619.

Docosahexaenoic acid (DHA) shows more pronounced relaxation when blood vessel is contracted with prostanoid receptor agonists than other stimulants. The present study was carried out to obtain information on the mechanisms underlying prostanoid receptor-selective relaxant action of DHA, particularly focusing on the possible roles for K(+) channels and its CYP epoxygenase (EOX) metabolites. In endothelium-denuded rat thoracic aorta, DHA (10(-5) M) almost completely relaxed U46619 (a thromboxane A2 (TP) receptor agonist)-contracted muscle without substantially affecting noradrenaline (NA)-induced contraction. DHA-induced relaxation was not affected by a large conductance, calcium- and voltage-activated K(+) (BK) channels inhibitor iberiotoxin (IbTX, 10(-7) M) but was almost abolished by high-KCl (8×10(-2) M) or 10(-2) M tetraethylammonium (TEA) which non-selectively inhibits K(+) channel activity. DHA also prominently relaxed U46619-contracted aorta even in the presence of CYP inhibitors (SKF525A or miconazole, each at 10(-5) M). However, in the presence of these CYP inhibitors, the relaxant action of DHA was not affected by 10(-2) M TEA. In supporting a significant role for CYP EOX metabolites in the blood vessel relaxation to DHA, 16,17-epoxy docosapentaenoic acid (16,17-EpDPE), but not 19,20-EpDPE, showed a potent relaxation in U46619-contracted aorta, and this action was significantly attenuated by 10(-2) M TEA. The present findings suggest that the relaxant action of DHA shown in the rat aorta contracted through the stimulation with TP receptor is generated by DHA itself and its CYP EOX metabolites. The relaxant effect of DHA metabolites seems to be partly triggered by the activation of K(+) channels though the role for BK channel is insignificant.

Pharmacological characteristics of the inhibitory effects of docosahexaenoic acid on vascular contractions studied in rat mesenteric artery.

BACKGROUND/AIMS: Effects of docosahexaenoic acid (DHA) on blood vessel contractions to various constrictors were investigated in rat mesenteric artery and compared with those of eicosapentaenoic acid (EPA) and linoleic acid (LA). METHODS: Tension changes in mesenteric ring segments were isometrically recorded. RESULTS: On sustained contractions induced by a thromboxane A2 mimetic (U46619), DHA exerted a strong inhibitory effect. This inhibitory effect of DHA on U46619 appeared both in endothelium-intact and endothelium-denuded preparations. Although the inhibitory effect of DHA on prostaglandin F2α (PGF2α)-induced contractions was also significant, contractions to phenylephrine (PE) and high-KCI were not affected by DHA. As well as DHA, EPA strongly diminished U46619- and PGF2α-induced contractions without showing a substantial inhibition of PE- and high-KCl-induced contractions. By contrast, LA did not show any significant inhibitory effects on any contractions. The DHA-induced inhibitory actions exerted on U46619 and PGF2α also emerged if ring preparations were pretreated with this ω-3 polyunsaturated fatty acid (PUFA). CONCLUSION: DHA and EPA are found to more pronouncedly inhibit prostanoid receptor-mediated contractions than other constrictor responses of the mesenteric artery via endothelium-independent mechanisms. These inhibitory effects of ω-3 PUFAs on prostanoid receptor-mediated contractions may partly underlie the mechanisms by which these ω-3 PUFAs elicit protective actions against circulatory disorders.

Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state.

Salinarum halorhodopsin (HsHR), a light-driven chloride ion pump of haloarchaeon Halobacterium salinarum, was heterologously expressed in Escherichia coli. The expressed HsHR had no color in the E. coli membrane, but turned purple after solubilization in the presence of all-trans retinal. This colored HsHR was purified by Ni-chelate chromatography in a yield of 3-4 mg per liter culture. The purified HsHR showed a distinct chloride pumping activity by incorporation into the liposomes, and showed even in the detergent-solubilized state, its typical behaviors in both the unphotolyzed and photolyzed states. Upon solubilization, HsHR expressed in the E. coli membrane attains the proper folding and a trimeric assembly comparable to those in the native membranes.

Consideration of albumin-mediated hepatic uptake for highly protein-bound anionic drugs: Bridging the gap of hepatic uptake clearance between in vitro and in vivo.

The accuracy in predicting in vivo hepatic clearance of drugs from in vitro data (often termed as in vitro-to-in vivo extrapolation, IVIVE) has improved in part by applying the extended-clearance concept that considers the interplay between hepatic metabolism and uptake/efflux processes. However, the IVIVE-based prediction performs poorly in predicting the hepatic uptake clearance of highly albumin-bound anionic drugs. Their hepatic uptake clearances tend to be much higher than expected based on the free-drug theory. Such observation can be attributable to a phenomenon called albumin-mediated hepatic uptake, for which various models have been thus far proposed. Our group has been applying a facilitated-dissociation model, which assumes the enhanced dissociation of the drug-albumin complex upon interaction with the cell surface. By considering the albumin-mediated hepatic uptake (using the facilitated-dissociation model or alternative kinetic models), a number of investigations demonstrated the improvement in the prediction accuracy for the hepatic clearance of highly protein-bound anionic drugs that are substrates for hepatic uptake transporters. This review summarizes the reported kinetic analyses of the albumin-mediated hepatic uptake of highly albumin-bound drugs concerning the IVIVE and the clinical and physiological relevance.

Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum.

Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.