Mechanism of Acyclovir Uptake in Rat Jejunum

The intestinal uptake mechanism of the purine analogue, acyclovir, was investigated in rat jejunum using in vitro and in situ methods. The pyrimidine, uracil, was used as a reference compound for carrier-mediated transport, while the purine analogue, caffeine, served as the reference compound for passive diffusion. With the in vitro intestinal ring method, acyclovir uptake was linear in the concentration range 0.01–5 mM. No significant competition for uptake was observed with uracil, 6-mercaptopurine, hypoxanthine, caffeine, or adenine. In addition, use of 2,4-dinitrophenol (DNP), ouabain, or K⁺ substituted buffer did not reduce the rate of acyclovir uptake. The in situ single-pass perfusion method yielded a wall permeability of 0.2, which did not vary consistently with increasing concentration. Coperfusion of acyclovir with DNP did not decrease the wall permeability. None of the data provided evidence of a carrier-mediated transport system, and it was concluded that the uptake mechanism of acyclovir in the rat jejunum is predominantly via passive diffusion.     

Placental and Foetal Metabolism of Acetaldehyde in Rat I. Contents of Ethanol and Acetaldehyde in Placenta and Foetus of the Pregnant Rat during Ethanol Oxidation

Abstract: Following the administration of ethanol to pregnant rats, the ethanol content of the maternal aortic blood was compared with that of the intact placenta and whole foetuses. The results support previous observations that ethanol is present in the placenta and foetus in about the same amount as in the maternal circulation. In contrast, only 25 % of the acetaldehyde content present in the maternal aortic blood could be found in the intact placenta and no acetaldehyde was found in the intact foetal tissue. These findings suggest that during maternal ethanol oxidation there is no accumulation of acetaldehyde in the foetal tissue.     

Placental and Foetal Metabolism of Acetaldehyde in Rat II. Studies on Metabolism of Acetaldehyde in the Isolated Placenta and Foetus

Abstract: The aldehyde-oxidizing capacity of placenta and foetal liver from the rat was studied using acetaldehyde as substrate. In the placenta the activity was approximately 8 % and in foetal liver 49 % of that found in the liver of adult animal. The placenta seems to prevent most of the acetaldehyde found in maternal blood during ethanol oxidation from entering the foetal circulation.     

Uptake Mechanism of Trientine by Rat Intestinal Brush-border Membrane Vesicles

The uptake characteristics of trientine by rat intestinal brush-border membrane vesicles were studied. The uptake characteristics of trientine were similar to those of the physiological polyamines with respect to the excessive accumulation in vesicles, the pH dependency, the temperature dependency and the ineffectiveness of K⁺ diffusion potential (inside negative). The initial uptake of trientine was saturable with a K_m value of 1.13 mM, which was larger than that of spermine and spermidine. Furthermore, the uptake rate of trientine was dose-dependently inhibited by spermine and spermidine. Spermine competitively inhibited the uptake of trientine with a K_i value of 18.6 μM, and it was close to the K_m value for spermine (30.4 μM). These data suggested that the uptake of trientine was similar to that of spermine and spermidine in rat small intestinal brush-border membrane vesicles, and these polyamines seem to inhibit the absorption of trientine from the gastrointestinal tract.     

Interaction of Nucleoside Analogues with Nucleoside Transporters in Rat Brain Endothelial Cells

A number of nucleoside analogues, consisting of antiviral compounds and agents designed as adenosine A₁ receptor agonists, were examined for nucleoside transporter affinity using an in vitro model of the blood–brain barrier (BBB), the rat brain endothelial cell line, RBE4. Structure–activity relationships (SAR) were also performed to identify the key structural requirements for transporter recognition and the suitability of these systems for carrier-mediated strategies to deliver therapeutics across the BBB. Adenosine receptor agonists did not show transport affinity for concentrative nucleoside carriers, but exhibited affinity for equilibrative systems (K_i=10.8–97.9 μM) within the range of K_ms for natural substrates. However, none of the antiviral compounds tested in this study showed affinity for either class of nucleoside transporter. SAR studies suggest that the hydroxyl group located at the 3′-position of the ribose moiety is an essential requirement for transporter recognition. This may explain the inability of nucleoside derived anti-viral compounds to use these systems despite the significant structural homology with naturally occurring nucleosides. Sites have also been identified which accommodate structural additions with retention of carrier affinity, suggesting that compounds which fail to penetrate the BBB could be attached to these sites for carrier-mediated delivery using a prodrug strategy.     

PEPT2-Mediated Uptake of Neuropeptides in Rat Choroid Plexus

Purpose. The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus. Methods. Whole-tissue rat choroid plexus uptake studies were performed on GlySar in the absence and presence of neuropeptides and on carnosine. Results. The neuropeptides NAAG, CysGly, GlyGln, kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg²]-kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 M. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km = 39.3 M; Vmax = 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or -alanine. Conclusions. These findings indicate that PEPT2 mediates the uptake of a diverse group of neuropeptides in choroid plexus, and suggests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid.     

Placental Transfer of Diethylhexyl Phthalate in the Guinea-pig Placenta Perfused in Situ

Abstract: Placental transport of diethylhexyl phthalate (DEHP) in guinea-pigs has been studied with a placental perfusion technique. This transfer was shown to be independent of perfusion flow rate within 1.5–2.3 ml/min. when heparinized blood was used as perfusion media. When dextran solution (Macrodex^®) or dextran solution with the addition of Intralipid^® was used as perfusion medium there was no transport of DEHP from the maternal to the foetal compartment. The placental transport of DEHP was shown to be dependent on the albumin concentration in the perfusion media, up to an albumin concentration of 54 mg/ml. With γ-globulin solution as perfusion medium there was no placental transfer of DEHP.     

2型糖尿病大鼠模型的建立与评价

目的：建立具有胰岛素抵抗特征、发病过程近似于人类2型糖尿病的动物模型。方法：雄性Wistar大鼠喂以富含不饱和脂肪酸的特殊高脂肪膳食5周，诱发出胰岛素抵抗．继之以小剂量链脲佐菌素（STZ，25mg／kg，腹腔注射）导致胰岛素代偿性分泌障碍，诱发高血糖症。应用正常血糖-高血浆胰岛素钳夹技术评价大鼠的胰岛素敏感性。结果：与常规饲料喂养大鼠相比，高脂膳食组的葡萄糖输注率显著降低，空腹血浆胰岛素显著升高。STZ注射后1周高脂膳食组大鼠血糖中度升高，血胰岛素下降，但仍高于普通饲料喂养组，且高脂血症和胰岛素抵抗继续存在。高脂饲料喂养组及高脂饲料＋小剂量STZ注射组大鼠的葡萄糖输注率显著低于正常对照组。结论：通过高脂膳食结合小剂量STZ注射成功复制出了实验性2型糖尿病动物模型，它是研究2型糖尿病发病机制、药物研究和胰岛素抵抗相关疾病的理想动物模型．     

Involvement of Rat Organic Cation Transporter 2 in the Renal Uptake of Jatrorrhizine

Jatrorrhizine is a protoberberine alkaloid derived from Coptis chinensis concentrated extremely in rat kidney. In the present study, the involvement of rat organic cation transporter 2 (rOCT2) in the renal uptake of jatrorrhizine in rat was investigated through in vitro and in vivo experiments. Saturable and nonsaturable uptakes of jatrorrhizine were observed in rat kidney slices and rOCT2–Madin–Darby canine kidney (MDCK) cells. Michaelis–Menten constants of 677.8 and 21.0 μM, maximum uptake rate of 123 (pmol/min)/mg kidney and 13.7 (pmol/min)/mg protein, and nonsaturable uptake clearance of 0.054 (μL/min)/mg kidney and 0.032 (μL/min)/mg protein were observed in rat kidney slices and rOCT2–MDCK cells, respectively. As inhibitors of rOCT2, corticosterone, verapamil, and cimetidine can inhibit jatrorrhizine uptake in rat kidney slices and rOCT2–MDCK cells. Their median inhibitory concentration in rat kidney slices was 7, 78, and 538 μM, whereas that in rOCT2–MDCK cells was 1.07, 86.5, and 151.8 μM. Coadministration with 20 mg/kg corticosterone, a selective inhibitor of rOCT2, reduced the jatrorrhizine concentration in the cortex and medulla in the in vivo experiment. Thus, rOCT2 is mainly responsible for the renal uptake of jatrorrhizine in rat and in the regulation of jatrorrhizine concentration in the kidney.     

Uptake of 14C-Neostigmine in Rat Striated Muscles

The uptake of ¹⁴C-neostigmine in striated muscles was studied after intravenous injection into rats and in the isolated rat diaphragm. 120 minutes after the intravenous injection of ¹⁴C-neostigmine (100 μg/kg) the ¹⁴C-concentration in the quadriceps, sternomastoid and intercostal muscles was 1/3-1/2 of the concentration in the plasma, whereas the ¹⁴C-concentration in the diaphragm was about double the concentration in the plasma. After continuous intravenous infusion of ¹⁴C-neostigmine the distribution pattern was the same as after a single injection. Paper chromatography showed that the radioactivity in the diaphragm was due partly to unchanged neostigmine, partly to a metabolite. In the isolated diaphragm unchanced ¹⁴C-neostigmine accumulates against an apparant concentration gradient. The uptake is partly saturable and energy-dependent. It is suggested that intracellular accumulation of neostigmine and/or its metabolites may be causally related to the respiratory failure following overdosage of neostigmine.     

Mechanism of silica-induced ROS generation in Rat2 fibroblast cells

Reactive oxygen species (ROS) play an important role in cell signaling pathway. Previously, we found that silica induced immediate ROS generation and sequential cellular responses such as kinase activation in Rat2 cells as well as an increase of intracellular calcium concentration in A549 cells. However, the detailed mechanism underlying the immediate ROS generation induced by silica in fibroblast cells remains to be elucidated. Therefore, in the present study, we investigated the mechanism of ROS generation by silica within Rat2 fibroblast cells by examining the effects of a diverse group of inhibitors for the enzymes related with signal transduction events. Inhibitors for protein tyrosine kinase (PTK), phospholipase C (PLC), protein kinase C (PKC) and calmodulin (CaM) kinase II effectively suppressed ROS generation in silica-stimulated Rat2 cells, whereas those for protein kinase A and phospholipase A(2) did not. Diphenyleneiodonium chloride (DPI), an inhibitor for NADPH oxidase was also found to be effective in inhibiting silica-induced ROS generation. These results suggest that PTK, PLC, PKC, CaM kinase II, and NADPH oxidase are all involved in signal transduction pathways for ROS generation in silica-stimulated Rat2 cells.     

Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.     

Characterization of 5-Hydroxytryptamine Uptake in Sliced Rat Lung

Abstract: Slices of rat lung were incubated with tritiated 5-hydroxytryptamine and the tissue uptake of tritium was studied after separating the free and bound radioactivity by filtration on glass fiber filters. At 37° a rapid uptake occured during the first 10 min. After that time the uptake was less marked but it was still present after 60 min. The uptake was moderately potentiated by the MAO inhibitor iproniazid (3 μM) after 30–60 min. incubation. The 5-hydroxytryptamine uptake was inhibited by some uptake inhibitors, their order of potency beeing: clomipramine > imipramine ≥ nortriptyline ≥ cocaine ≥ desipramine > maprotiline. At 0° the uptake of 5-hydroxytryptamine was negligible. Non-linear regression analysis of uptake data indicated that 5-hydroxytryptamine was taken up by two different mechanisms. One of the uptake processes was saturated by high concentrations of 5-hydroxytryptamine and showed an apparent K_m of 8×10^?7 M. The other uptake was linearly related to the 5-hydroxytryptamine concentration and could not be saturated even by concentration up to 5×10^?5 M of the amine.     

Physicochemical Modification of Lidocaine Uptake in Rat Lung Tissue

Abstract The uptake of lidocaine, methyllidocaine, bupivacaine, etidocaine was studied in rat lung slices at different pH-values. The accumulation of the quaternary analogue, methyllidocaine, was not changed in the pH interval 7.0-8.0. The uptake of the three other substances was about 3-4 times lower at pH 7.0 than at pH 8.0. The rank order of distribution at a fixed catiodbase ratio was bupivacaine>etidocaine>lidocaine. Interactions between lidocaine and other substances were studied in lung slices and in isolated perfused lungs. Bupivacaine and nortriptyline counteracted the accumulation of ¹⁴C-lidocaine in lung slices in a dose-dependent manner. Nortriptyline was more effective than bupivacaine. In isolated perfused lung, bolus injections of nortriptyline and lidocaine rapidly displaced ¹⁴C-lidocaine from the tissue. In this study we suggest that the base form of local anaesthetics accumulate in the lung tissue, while the cationic form binds to accessible binding sites in the cell membranes.     

Quantification of ENT1 and ENT2 Proteins at the Placental Barrier and Contribution of These Transporters to Ribavirin Uptake

The aims of this study are to quantify the protein levels of nucleoside transporters in placental microvillous membranes (MVMs) and to clarify the contributions of these transporters to ribavirin uptake at the placental barrier. Placental MVMs of human and rat expressed equilibrative nucleoside transporter (ENT) 1 protein, whereas the expression of ENT2 protein was obscure. Maternal-to-fetal transfer of [³H]ribavirin in rats was much higher than that of [¹⁴C]sucrose. The uptake of [³H]ribavirin by rat placental trophoblast TR-TBT 18 d-1 cells, which functionally express both ENT1 and ENT2 proteins, was saturable, and was significantly inhibited by 0.1 μM nitrobenzylthioinosine, which selectively abolishes ENT1-mediated uptake. Dipyridamole at 10 μM is capable of inhibiting ENT2 as well as ENT1, but a degree of inhibition by 10 μM dipyridamole on [³H]ribavirin uptake was not much different from that by 0.1 μM nitrobenzylthioinosine (ENT1-specific inhibitor). Therefore, ENT2 may contribute little to [³H]ribavirin uptake by these cells. Rat ENT1 cRNA-injected oocytes showed increased [³H]ribavirin uptake compared with water-injected oocytes, while rat ENT2 cRNA-injected oocytes did not. In conclusion, ENT1 protein expressed in placental MVMs appears to play a predominant role in the uptake of ribavirin.     

Renoprotective Effect of Total Glucosides of Paeony (TGP) and Its Mechanism in Experimental Diabetes

Total glucosides of paeony (TGP), extracted from the root of Paeonia lactiflora pall, has been shown to have ant-inflammatory and antioxidative actions. The aims of this study were to elucidate the renoprotective effect of TGP and its mechanism in experimental diabetes. Streptozotocin-induced diabetic rats were treated with TGP for 8 weeks. Treatment with TGP at 50, 100, and 200 mg/kg significantly lowered 24-h urinary albumin excretion rate in diabetic rats. TGP treatment in all doses markedly attenuated glomerular volume, and treatment with TGP at 100 and 200 mg/kg markedly reduced indices for tubulointerstitial injury in diabetic rats. Western blot analysis showed that the expressions of 1α (IV) collagen, intercellular adhesion molecule (ICAM)-1, interleukin (IL)-1, tumor necrosis factor (TNF)-α, NF-κB p65, and 3-nitrotyrosine (3-NT) protein were increased in the kidneys of diabetic rats; the increases in these proteins were all dose-dependently and significantly inhibited by TGP treatment. The expression of nephrin protein was significantly reduced in the kidneys from diabetic rats and markedly increased by TGP treatment. The expression of transforming growth factor (TGF)-β1 protein in the kidney was also significantly increased in diabetic rats, which was significantly inhibited by treatment with TGP at all doses. Our data suggest that TGP treatment ameliorates early renal injury via the inhibition of expression of ICAM-1, IL-1, TNF-α, and 3-NT in the kidneys of diabetic rats.     

Substrate Specificity and Mechanism of the Intestinal Clonidine Uptake by Caco-2 Cells

Purpose This study was performed to characterize the substrate specificity and mechanism of the intestinal clonidine transport. Methods Uptake of [³H]clonidine into Caco-2 cells was investigated. Interaction with drugs was studied in competition assays. Results Uptake of [³H]clonidine was linear for up to 2 min, Na⁺-independent, and insensitive to changes in membrane potential, but strongly H⁺-dependent. The uptake rate of clonidine was saturable with kinetic parameters of 0.5 ± 0.1 mM (K_t) and 16.6 ± 1.8 nmol/2 min per mg of protein (V_max) at an outside pH of 7.5. Many drugs such as clonidine, guanabenz, methamphetamine, imipramine, clomipramine, nortriptyline, quinine, xylazine, ephedrine, and diphenhydramine strongly inhibited the [³H]clonidine uptake with K_i values between 0.15 and 1 mM. Conclusions Clonidine is transported by a carrier-mediated process. Substrate specificity and mechanism are very similar to the transport described in blood–brain barrier endothelial cells. The transport characteristics do not correspond to carriers for organic cations of the SLC22 family or the choline transporters CHT1 and CLT1. The system might be identical to the H⁺/tertiary amine antiporter. It interacts with a large number of both hydrophilic and lipophilic cationic drugs, and also, interestingly, with opiates.     

Cellular Uptake of Liposomal Daunorubicin and the Induction of Apoptosis

Liposomal daunorubicin (L-DnR) has clinical activity against Kaposi's sarcoma, leukemias and lymphomas, but the uptake and cytotoxic mechanisms of its actions remain unclear. Uptake of LDnR and daunorubicin (DnR) by tumor cells and J774 (murine monocyte/macrophage) cells were compared and shown to occur both at 4 C and after formalin-fixation, suggesting that endocytosis is not the sole mechanism of liposome uptake even by phagocytic cells. L-DnR cytotoxicity in vitro was equivalent to that of DnR in standard, 48-hr assays, but substantial cytotoxicity (50% growth inhibition) also occurred following brief 1exposures to DnR and LDnR (5 and 30 min, respectively). Similar degrees of cytotoxicity were induced by DnR and L-DnR immobilized by adherence to tissue culture wells. As these data suggested that the induction of cytotoxicity was a rapid, early, and possibly cell-membrane-mediated event, we examined cell lysates for evidence of apoptosis. DNA fragmentation in J774 cells was demonstrable within 1-4 hr exposure to DnR and L-DnR by an ELISA method and by DNA laddering. Studies with human leukemia and lymphoma cell lines confirmed DnR- and L-DnR-induced apoptosis in HL60, JVM-2, and WSU-NHL cells. In studies of apoptosis-induction by the related anthracyclines doxorubicin (DoX) and liposomal doxorubicin (LPEG-DoX, polyethylene glycol-coated liposome), it was found that doxorubicin activity closely resembled that of L-DnR but LPEG-DoX showed no or little effect. These data show, for the first time, the induction of apoptosis by a conventional cytotoxic agent encapsulated in a liposome and suggest that the uptake and intracellular pharmacology may be similar to that of the free drug.