You are what you eat: nutrient and water relations between mistletoes and hosts.

Mistletoes play important roles in biogeochemical cycles. Although many studies have compared nutrient concentrations between mistletoes and their hosts, no general patterns have been found and the nutrient uptake mechanisms in mistletoes have not been fully resolved. To address the water and nutrient relations in mistletoes compared with their hosts, we measured 11 nutrient elements, two isotope ratios and two leaf morphological traits for 11 mistletoe and 104 host species from four sites across a large environmental gradient in southwest China. Mistletoes had significantly higher phosphorus, potassium, and boron concentrations, nitrogen isotope ratio, and lower carbon isotope ratio (δ13 C) indicative of lower water-use efficiency than hosts, but other elements were similar to those in hosts. Sites explained most of the variation in the multidimensional trait space. With increasing host nitrogen concentration, both mistletoe δ13 C and the difference between mistletoe and host δ13 C increased, providing evidence to support the 'nitrogen parasitism hypothesis'. Host nutrient concentrations were the best predictors for that of the mistletoe nutrient elements in most cases. Our results highlight the important roles of environmental conditions and host nutrient status in determining mistletoe nutrient pools, which together explain their trophic interactions with hosts in subtropical and tropical ecosystems.

Ion-Pairing with Spermine Targets Theophylline To the Lungs via the Polyamine Transport System.

Certain xenobiotics, such as paraquat, are sequestered into the lungs from the systemic circulation by the polyamine transporter system (PTS). The aim of this study was to investigate whether ion-pairing a drug (theophylline) with a PTS substrate (spermine) provides a means of using this active transport mechanism to target drug delivery to the lungs. Fourier transform infrared spectroscopy showed that two of the amine groups of spermine interact with C-N7 and C6═O of theophylline, leaving two free amines to interact with the PTS. In A549 cells, which possess a functional PTS (spermidine Km and Vmax, 0.6 ± 0.3 µM and 1.8 ± 0.3 pmol·min-1 per 105 cells, respectively), uptake of the theophylline-spermine ion-pair was increased 1.8-fold compared to free theophylline at 37 °C, but not at 4 °C. In an isolated perfused rat lung model (IPL) a 3.6-fold increase in lung theophylline concentration was observed after vascular administration of the ion-pair compared to free theophylline. Theophylline was cleared from the IPL with similar kinetics irrespective of whether it was delivered as the free drug or an ion-pair, although lung levels remained elevated after washout following delivery as an ion-pair. In vitro simulation of the theophylline-spermine break down demonstrated that a drop in pH from 9.6 to 7.4, such as that undergone by the ion-pair in biological matrices, induces rapid and almost complete dissociation of the ion-paired species. However, infusion of the ion-pair formulations via the vasculature provides almost immediate delivery to the pulmonary capillary bed permitting PTS-mediated active sequestering of ion-paired theophylline into the lungs.

Patterns and regulation of silicon accumulation in Synechococcus spp.

Six clones of the marine cyanobacterium Synechococcus, representing four major clades, were all found to contain significant amounts of silicon in culture. Growth rate was unaffected by silicic acid, Si(OH)4 , concentration between 1 and 120 µM suggesting that Synechococcus lacks an obligate need for silicon (Si). Strains contained two major pools of Si: an aqueous soluble and an aqueous insoluble pool. Soluble pool sizes correspond to estimated intracellular dissolved Si concentrations of 2-24 mM, which would be thermodynamically unstable implying the binding of intracellular soluble Si to organic ligands. The Si content of all clones was inversely related to growth rate and increased with higher [Si(OH)4 ] in the growth medium. Accumulation rates showed a unique bilinear response to increasing [Si(OH)4 ] from 1 to 500 µM with the rate of Si acquisition increasing abruptly between 80 and 100 µM Si(OH)4 . Although these linear responses imply some form of diffusion-mediated transport, Si uptake rates at low Si (~1 µM Si) were inhibited by orthophosphate, suggesting a role of phosphate transporters in Si acquisition. Theoretical calculations imply that observed Si acquisition rates are too rapid to be supported by lipid-solubility diffusion of Si through the plasmalemma; however, facilitated diffusion involving membrane protein channels may suffice. The data are used to construct a working model of the mechanisms governing the Si content and rate of Si acquisition in Synechococcus.

Accumulation of cytokinins in roots and their export to the shoots of durum wheat plants treated with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP).

Cytokinin flow from roots to shoots can serve as a long-distance signal important for root-to-shoot communication. In the past, changes in cytokinin flow from roots to shoots have been mainly attributed to changes in the rate of synthesis or breakdown in the roots. The present research tested the possibility that active uptake of cytokinin by root cells may also influence its export to shoots. To this end, we collapsed the proton gradient across root membranes using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to inhibit secondary active uptake of exogenous and endogenous cytokinins. We report the impact of CCCP on cytokinin concentrations and delivery in xylem sap and on accumulation in shoots of 7-day-old wheat plants in the presence and absence of exogenous cytokinin applied as zeatin. Zeatin treatment increased the total accumulation of cytokinin in roots and shoots but the effect was smaller for the shoots. Immunohistochemical localization of cytokinins using zeatin-specific antibodies showed an increase in immunostaining of the cells adjacent to xylem in the roots of zeatin-treated plants. Inhibition of secondary active cytokinin uptake by CCCP application decreased cytokinin accumulation in root cells but increased both flow from the roots and accumulation in the shoots. The possible importance of secondary active uptake of cytokinins by root cells for the control of their export to the shoot is discussed.

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells.

BACKGROUND: Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood-brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). METHODS: We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. RESULTS: We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min-1·mg protein-1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. CONCLUSIONS: We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.

Investigating how amine structure influences drug-amine ion-pair formation and uptake via the polyamine transporter in A549 lung cells.

Transiently associating amines with therapeutic agents through the formation of ion-pairs has been established both in vitro and in vivo as an effective means to systemically direct drug delivery to the lung via the polyamine transport system (PTS). However, there remains a need to better understand the structural traits required for effective PTS uptake of drug ion-pairs. This study aimed to use a structurally related series of amine counterions to investigate how they influenced the stability of theophylline ion-pairs and their active uptake in A549 cells. Using ethylamine (mono-amine), ethylenediamine (di-amine), spermidine (tri-amine) and spermine (tetra-amine) as counterions the ion-pair affinity was shown to increase as the number of protonated amine groups in the counterion structure increased. The mono and diamines generated a single hydrogen bond and the weakest ion-pair affinities (pKFTIR: 1.32 ± 0.04 and 1.43 ± 0.02) whereas the polyamines produced two hydrogen bonds and thus the strongest ion-pair affinities (pKFTIR: 1.93 ± 0.05 and 1.96 ± 0.04). In A549 cells depleted of endogenous polyamines using α-difluoromethylornithine (DFMO), the spermine-theophylline uptake was significantly increased (p < 0.05) compared to non-amine depleted cells and this evidenced the active PTS sequestering of the ion-pair. The mono-amine and di-amine failed to enhance theophylline uptake in these A549 cells, but the tri-amine and tetra-amine both almost doubled the theophylline uptake into the cells when compared to the uptake of free drug. As the data indicated that polyamines with at least 3 amines were required to form ion-pairs that could enhance A549 cell uptake, it suggested that at least two amines were required to physically stabilise the ion-pair and one to interact with the PTS.

High hepatic exposure of furanocoumarins in Radix Angelica dahuricae is associated with transporter mediated active uptake.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Angelica dahuricae (RAD), the roots of Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav, is a well-known traditional Chinese medicine (TCM) and has been used for centuries to treat headaches, toothaches, nose congestion, abscesses, furunculoses, and acne. This herb is also one of frequently reported TCMs showing the herb-drug interaction potential. Furanocoumarins are main bioactive components of RAD. AIM OF THE STUDY: This study is designed to characterize the tissue distribution profiles of furanocoumarins after oral administration of RAD extract in rats and to explore the mechanism underlying the high hepatic exposure of the major furanocoumarins. MATERIALS AND METHODS: The tissue distribution of nine furanocoumarins was determined in rats after an oral dose of 0.46g/kg RAD extract using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Unbound fractions (ƒu) of major furanocoumarins, including imperatorin (IM), isoimperatorin (IIM), bergapten (BER) and oxypeucedanin hydrate (OXYH), were measured in rat plasma and selected tissue homogenates (liver, kidney, lung and brain) with Rapid Equilibrium Dialysis (RED) method. The temperature dependent hepatic uptake of IM, IIM, BER and OXYH were evaluated in suspended rat primary hepatocytes at 4°C or 37°C by the oil-spin method. The uptake kinetics was conducted in the cells over a wide concentration range. The furanocoumarins were co-incubated with a panel of transporter inhibitors to investigate the involvement of uptake transporters in the hepatic uptake. The transcellular transport characteristics of IM, IIM, BER and OXYH were further assessed using Caco-2 cell monolayer model. RESULTS: IM, IIM, BER and OXYH were found to be the major bioactive furanocoumarins in rat plasma and tissues, representing more than 90% exposure for all the detected furanocoumarins. The most concentrative organ of major furanocoumarins was the liver, with liver-to-plasma exposure ratio (Kp,AUC) of 5.1, 6.5 and 4.7 for IM, IIM and BER, and 2.3 for OXYH, respectively. IM, IIM and BER also showed higher concentrations in the kidney with Kp above 2.2. The higher protein binding of the furanocoumarins partially contributed to their higher tissue exposure. In suspended rat primary hepatocyte, the hepatic uptake of IM, IIM, BER and OXYH was temperature-dependent, with considerably higher uptake at 37°C than at 4°C. Uptake kinetics indicated that the hepatic uptake of IM, IIM, BER and OXYH involved both active transport and passive diffusion processes. For IM, IIM and BER, the contribution of the active transport was greater than the passive process, with the CLactive/CLuptake > 72%. Ritonavir (RTN) and cyclosporine A (CsA), the known inhibitors of organic anion transporting polypeptide (Oatp) significantly inhibited the hepatic uptake of IM and BER, while the inhibitor of the organic anion transporters (Oat) probenecid (PBC) remarkably reduced IIM uptake. In the Caco-2 cell model, the furanocoumarins were highly permeable in the apical to basolateral direction without notable active efflux. CONCLUSION: The furanocoumarins rapidly and widely distributed into various tissues after oral dose of the RAD extract. IM, IIM, BER and OXYH were the major components detected in both plasma and tissues. Liver was the most distributed tissue of the total and free furanocoumarins. Non-specific protein binding contributed partially to the higher tissue exposures of these bioactive components. The Oatp and Oat mediated active uptake played the primal role in the high hepatic exposure of the furanocoumarins.

Inorganic mercury (Hg2+) accumulation in autotrophic and mixotrophic planktonic protists: Implications for Hg trophodynamics in ultraoligotrophic Andean Patagonian lakes.

Microbial assemblages are typical of deep ultraoligotrophic Andean Patagonian lakes and comprise picoplankton and protists (phytoflagellates and mixotrophic ciliates), having a central role in the C cycle, primary production and in the incorporation of dissolved inorganic mercury (Hg2+) into lake food webs. In this study we evaluated the mechanisms of Hg2+ incorporation in hetero- and autotrophic bacteria, in the autotrophic dinoflagellate (Gymnodinium paradoxum) and in two mixotrophic ciliates (Stentor araucanus and Ophrydium naumanni) dominating the planktonic microbial assemblage. The radioisotope 197Hg was used to trace the Hg2+ incorporation in microbiota. Hg uptake was analyzed as a function of cell abundance (BCF: bioconcentration factor), cell surface (SCF: surface concentration factor) and cell volume (VCF: volume concentration factor). Overall, the results obtained showed that these organisms incorporate substantial amounts of dissolved Hg2+ passively (adsorption) and actively (bacteria consumption or attachment), displaying different Hg internalization and therefore, varying potential for Hg transfer. Surface area and quality, and surface:volume ratio (S:V) control the passive uptake in all the organisms. Active incorporation depends on bacteria consumption in the mixotrophic ciliates, or on bacteria association to surface in the autotrophic dinoflagellate. Hg bioaccumulated by pelagic protists can be transferred to higher trophic levels through plankton and fish feeding, regenerated to the dissolved phase by excretion, and/or transferred to the sediments by particle sinking. In ultraoligotrophic Andean Patagonian lakes, picoplankton and planktonic protists are key components of lake food webs, linking the pelagic and benthic Hg pathways, and thereby playing a central role in Hg trophodynamics.

γ-Hydroxybutyric Acid (GHB) Pharmacokinetics and Pharmacodynamics: Semi-Mechanistic and Physiologically Relevant PK/PD Model.

An overdose of γ-hydroxybutyric acid (GHB), a drug of abuse, results in fatality caused by severe respiratory depression. In this study, a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed to characterize monocarboxylate transporter 1 (MCT1)-mediated transport of GHB, as well as effects of GHB on respiration frequency, for IV doses of 200, 600, and 1500 mg/kg in rats. The proposed PK/PD model for GHB consists of nonlinear metabolism of GHB in the liver, MCT1-mediated renal reabsorption with physiologically relevant concurrent fluid reabsorption, MCT1-mediated uptake into the brain, and direct effects of binding of GHB to GABAB receptors on the PD parameter, respiration frequency. Michaelis-Menten affinity constants for metabolism, renal reabsorption, and uptake into and efflux from the brain were fixed to the observed in vitro values. The IC 50 value for the effect of GHB on respiration frequency was fixed to a reported value for binding of GHB to GABAB receptors. All physiological parameters were fixed to the reported values for a 300-g rat. The model successfully captured the GHB PK/PD data and was further validated using the data for a 600-mg/kg dose of GHB after IV bolus administration. Unbound GHB brain ECF/blood partition coefficient (Kp u,u ) values obtained from the model agreed well with values calculated using experimental ECF concentrations obtained with brain microdialysis, demonstrating the physiological relevance of this model. Sensitivity analysis indicated that the PK/PD model was stable. In conclusion, we developed a semi-mechanistic and physiologically relevant PK/PD model of GHB using in vitro drug-transporter kinetics and in vivo PK/PD data in rats.

Root uptake and translocation of nickel in wheat as affected by histidine.

The role of histidine (His) on root uptake, xylem loading and root to shoot transport of nickel (Ni) was investigated in a winter (Triticum aestivum cv. Back Cross) and a durum wheat (Triticum durum cv. Durum) cultivar. Seedlings were grown in a modified Johnson nutrient solution and exposed to 10 µM of Ni and 100 µM of histidine (His) as no His, Ni (10) + His (100) and Ni(His) in a 1:1 mole ratio (1:1) complex. In our study, the presence of vanadate (a metabolic inhibitor) resulted in a significant decrease of root Ni uptake, indicating that a part of Ni uptake by the plant root is energy-dependent. Addition of His significantly increased the Ni content in shoots and roots of both wheat cultivars. The data suggest that the Ni(His) is most likely to be taken up as a complex or receptors at the membrane are able to enhance Ni uptake from Ni(His) complex. This result was indirectly supported by using EDTA as a strong chelating reagent to reduce the uptake of Ni(His) complexes. By using this ligand, the xylem loading of Ni and His was disproportionately reduced. Cycloheximide (a translation inhibitor) strongly decreased the release of His and Ni from the root into the xylem of wheat, suggesting the significance of a symplastic pathway for Ni loading into the xylem.