Differences In Uptake Kinetics Research Articles

Extraction of radium and actinium with Pb resin and Rose Bengal

The extraction of radium and actinium with Eichrom Pb resin and Rose Bengal has been studied. Radium and actinium both show a strong affinity (k’ > 10,000) for the resin at intermediate pHs (~ 5–7) from solutions with Rose Bengal. The pH range and magnitude of uptake for both radium and actinium is significantly increased by the presence of Rose Bengal compared to basic solutions without this counter ion. Radium uptake is extremely fast from basic solutions with and without Rose Bengal; actinium uptake is slower, particularly in solutions without Rose Bengal. Column studies show that despite the similar behavior of actinium and radium in this system, separations of these two elements with Pb resin and Rose Bengal solutions are possible with reasonable yields due to the differences in uptake kinetics.

Do environmentally relevant concentrations of fluoxetine and citalopram impair stress-related behavior in zebrafish (Danio rerio) embryos?

Selective serotonin reuptake inhibitors (SSRIs) have been shown to interfere with various physiological functions of aquatic organisms, yet the neuroactive potential of low concentrations of SSRIs in the aquatic environment is unclear. The current study investigated the effects of fluoxetine and citalopram on the visual motor response (VMR) of 107 h old zebrafish (Danio rerio) embryos. Results document a reduction in stress-related swimming activity of zebrafish embryos at environmentally relevant concentration levels, with fluoxetine being more effective than citalopram. Further experiments were designed to elucidate (1) if the lower neuroactive potential of citalopram is due to differences in uptake kinetics, (2) if the metabolite of fluoxetine, norfluoxetine, contributes to the neuroactive potential of fluoxetine, (3) and how SSRIs and their metabolites interact in equimolar mixtures.At the stage of 120 h, zebrafish embryos accumulate citalopram at significantly lower rates (up to 127 times) than fluoxetine. Moreover, it was demonstrated that norfluoxetine reduces the embryonic VMR similarly to fluoxetine resulting in additive effects of these substances on stress-related behavior in zebrafish embryos. In contrast, the interaction of fluoxetine, norfluoxetine and citalopram varied with test concentrations of the equimolar mixtures. Findings provide evidence that environmentally relevant concentrations of fluoxetine reduce stress-related behavior of zebrafish embryos, while these effects may be enhanced by the interaction of multiple SSRIs and their metabolites in environmental exposure scenarios.

Cellular Binding Kinetics of Altertoxin II: A Fungal Metabolite with Highly Selective Activity Against Ewing Sarcoma

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are characterized by chromosomal translocations that fuse EWSR1 with FLI1 (~85% of ES) or another ETS transcription factor gene, resulting in expression of an oncogenic fusion protein (typically EWSR1‐FLI1). Despite significant advances over the past 40 years in therapy for pediatric hematological malignancies, less progress has been made with therapies for ES and other childhood solid tumors. Thus, there is a need to develop new and more efficacious therapies for ES that improve patient survival. We recently identified the fungal metabolite altertoxin II (ATXII) as having highly selective activity against six ES cell lines compared to a panel of pediatric and adult cancer cells. On average, ATXII is 94‐fold (range 16‐ to 400‐fold) more potent against ES cells compared to rhabdomyosarcoma (RMS) cells, indicating high selectivity for ES. Mechanism of action studies indicated that ATXII selectively induces DNA damage in ES cells. To characterize the cellular uptake of ATXII and identify potential molecular binding partners, a tritiated sample of the compound was produced and evaluated in cellular assays. We observed significantly different kinetics for 3H‐ATXII accumulation between sensitive EW8 and resistant Rh30 cells. In EW8 cells, maximum 3H‐ATXII accumulation was observed five minutes after treatment, with cellular levels slowly decreasing over the next hour. In contrast, cellular abundance of 3H‐ATXII steadily increased in Rh30 cells for one hour following treatment, with final levels approximately 1.6‐fold higher than those in EW8 cells. These data suggest that ATXII rapidly binds and saturates its binding partner(s) in EW8, but not Rh30 cells. Additionally, these data indicate that the increased sensitivity of EW8 cells to ATXII is not simply due to greater drug accumulation. Differences in uptake kinetics were also observed between EW8 and Rh30 cells when cellular uptake was measured at 4°C. While we measured 2‐fold less uptake in Rh30 cells at 4°C compared to 37°C, uptake increased nearly 5‐fold in EW8 cells at 4°C. To determine whether uptake of ATXII is ATP dependent, we evaluated the uptake of 3H‐ATXII following treatment with the uncoupling agent carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP). While pretreatment with FCCP reduced 3H‐ATXII accumulation in Rh30 cells, we observed an increase in 3H‐ATXII accumulation in EW8 cells. Together, these data suggest that EW8 cells actively export ATXII, while uptake is a passive process. Ongoing studies are aimed at identifying the molecular binding partners of ATXII, and identifying genes that mediate resistance in RMS cells by performing a genome‐wide CRISPR/Cas9 knockout screen. Overall, these studies have the potential to identify new molecular targets for the development of ES therapies.Support or Funding InformationNCI R01 GM107490, PO1 CA165995This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro dosimetry of agglomerates.

Agglomeration of nanoparticles in biological fluids is a pervasive phenomenon that leads to difficulty in the interpretation of results from in vitro exposure, primarily due to differing particokinetics of agglomerates to nanoparticles. Therefore, well-defined small agglomerates were designed that possessed different particokinetic profiles, and their cellular uptake was compared to a computational model of dosimetry. The approach used here paves the way for a better understanding of the impact of agglomeration on the nanoparticle-cell interaction.

Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.

Spatial variability of atmospheric semivolatile organic compounds in Chile

Abstract In an effort to characterize the spatial variability of pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) in the Chilean atmosphere, XAD-based passive air samplers (PAS) were deployed for 12 months (2006–2007) along three elevational gradients in Northern, Central and Southern Chile, for a total of 20 sampling sites, ranging in elevation from 10 to 4400 m and ranging over 26 degrees of latitude. Hexachlorobenzene (HCB) had largely uniform air concentrations with latitude and altitude, and is therefore used to derive sampler specific sampling rates, that account for differences in uptake kinetics due to environmental factors such as altitude and temperature. Levels of pesticides in the atmosphere of Chile are very low and only α- and γ-hexachlorocyclohexane, α-endosulfan and chlorothalonil were present in all of the samples. The concentrations of these four pesticides in air decreased from North to South, with much steeper gradients for chlorothalonil and endosulfan. Chlorothalonil levels were greatly elevated in air samplers deployed in urban locations. Endosulfan concentrations in air tend to increase with elevation. Since endosulfan is particularly susceptible to mountain cold-trapping, such gradients may reflect the re-volatilization of endosulfan that had been preferentially deposited at higher altitudes. Alternatively, increasing endosulfan concentrations with elevation could be the result of transport to higher altitude sites from the other side of Andes. Airshed analysis indeed indicates that air masses originating outside of Chile have a higher influence on the higher elevation sites, especially along the Northern transect. Concentrations of four-ring PAHs ranged from 0.3 to 1300 pg m −3 , with higher levels in air samplers deployed in urban areas and close to roads. PCB concentrations were low (0.7–27 pg m −3 ), with slightly higher levels of volatile congeners in two urban sampling sites in Concepcion and Arica. PCB-52 is the only congener present in all samples, with levels that are remarkably uniform across the country.

Distribution and Dynamics of 99mTc-Pertechnetate Uptake in the Thyroid and Other Organs Assessed by Single-Photon Emission Computed Tomography in Living Mice

(99m)Tc pertechnetate is a well-known anion, used for clinical imaging of thyroid function. This gamma emitter is transported by the sodium iodide symporter but is not incorporated into thyroglobulin. Scintigraphy using (99m)Tc pertechnetate or (123)iodide represents a powerful tool for the study of sodium iodide symporter activity in different organs of living animal models. However, in many studies that have been performed in mice, the thyroid could not be distinguished from the salivary glands. In this work, we have evaluated the use of a clinically dedicated single-photon emission computed tomography (SPECT) camera for thyroid imaging and assessed what improvements are necessary for the development of this technique. SPECT of the mouse neck region, with pinhole collimation and geometric calibration, was used for the individual measurement of (99m)Tc pertechnetate uptake in the thyroid and the salivary glands. Uptake in the stomach was studied by planar whole-body imaging. Uptake kinetics and biodistribution studies were performed by sequential imaging. This work has shown that thyroid imaging in living mice can be performed with a SPECT camera originally built for clinical use. Our experiments indicate that (99m)Tc pertechnetate uptake is faster in the thyroid than in the salivary glands and the stomach. The decrease in (99m)Tc pertechnetate uptake after injection of iodide or perchlorate as competitive inhibitors was also studied. The resulting rate decreases were faster in the thyroid than in the salivary glands or the stomach. We have shown that a clinically dedicated SPECT camera can be used for thyroid imaging. In our experiments, SPECT imaging allowed the analysis of (99m)Tc pertechnetate accumulation in individual organs and revealed differences in uptake kinetics.

A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes

Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations.

Serotonin transporter in rat platelets: Level of protein expression underlies inherited differences in uptake kinetics

By breeding selection for the extreme values of platelet serotonin level (PSL), two sublines of Wistar-derived rats, with constitutionally high or low PSL and platelet serotonin uptake (PSU), have been developed. Searching for the basis of these differences, we performed quantitative western blot analysis of serotonin transporter (5HTt) in platelet membranes isolated from both rat sublines. A polyclonal anti-5HTt antibody labeled a single, 5HTt-related 94 kDa protein band in platelet membranes, with significantly stronger intensity in membranes from rats that exhibited a high PSL. We conclude that the inherited differences in PSL and PSU in rats, following breeding selection, are determined by the level of 5HTt expression in platelet membranes.

Serotonin transporter on rat platelets: levels of mRNA underlie inherited differences in uptake kinetics

By the breeding selection for the extreme values of platelet serotonin transporter activity, two sublines of Wistar-derived rats, with constitutionally high or low platelet serotonin uptake (PSU), were previously developed. In order to study the genetic background of these inherited differences, comparative Northern blot analysis of the platelet serotonin transporter messenger RNA levels of the animals from the two sublines was performed. If the values of animals from the high-PSU subline are taken as 100%, animals from the low-PSU subline demonstrated lower values of both platelet serotonin uptake and transporter mRNA levels (amounting to 62 and 76% respectively). Correlation between platelet serotonin uptake and the respective levels of messenger RNA for the serotonin transporter ( r = 0.829, P < 0.01, N = 8) points to the same direction, indicating that the process of breeding selection for the extreme values of transporter kinetics has influenced transcription mechanisms of the serotonin transporter gene.

Macroscopic and Microscopic Properties of a Cloned Glutamate Transporter/Chloride Channel

The behavior of a Cl- channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of approximately 5A. Glutamate flux exhibited a much greater temperature dependence than Cl- flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl- conductance and a maximum channel Po << 1. The glutamate binding rate was similar to estimates for receptor binding. After glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of L-glutamate or D-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the transporter cycle.

Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line

Uptake and efflux of colchicine and vinblastine, whose effects are related to their high affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.

Identification of a Chloroquine Importer in Plasmodium falciparum: DIFFERENCES IN IMPORT KINETICS ARE GENETICALLY LINKED WITH THE CHLOROQUINE-RESISTANT PHENOTYPE

We demonstrate that uptake of the antimalarial drug chloroquine is temperature-dependent, saturable, and inhibitable in Plasmodium falciparum. These features are indicative of carrier-mediated transport and suggest that a P. falciparum-encoded protein facilitates chloroquine import. Although both chloroquine-resistant and susceptible parasite isolates exhibit facilitated chloroquine uptake, the kinetics differ. Chloroquine-resistant parasite isolates consistently have an import mechanism with a lower transport activity and a reduced affinity for chloroquine. These differences in uptake kinetics are linked with chloroquine resistance in a genetic cross. These data suggest that changes in chloroquine import kinetics constitute a minimal and necessary event in the generation of the resistant phenotype. Competitive inhibition of chloroquine uptake by amiloride derivatives further suggests that chloroquine import is mediated by a plasmodial Na+/H+ exchanger.

The partition and fate of soluble and digesta particulate associated oxfendazole and its metabolites in the gastrointestinal tract of sheep

The disposition of oxfendazole (OFZ) containing a trace of [ 14C]OFZ was examined in the gastrointestinal tract and bloodstream of sheep fitted with rumen and abomasal cannulae. Within 2 h of intraruminal (IR) administration, OFZ and its metabolites were almost completely associated with rumen particulate digesta. The proportion of metabolites in digesta fluid increased with their passage from the rumen into the abomasum. To determine the fate of 14C-labelled metabolites after distribution throughout rumen digesta, the rumen particulate and fluid digesta phases from a donor sheep were separated and each transferred to the rumen of an untreated recipient sheep. The 14C-labelled metabolites which derived from the donor rumen fluid quickly associated with recipient rumen particulate material. The metabolites were then progressively desorbed, as were metabolites which were transferred already associated with rumen particulate digesta. Desorption occurred faster in the abomasum than in the rumen. There was no difference in uptake kinetics between administration routes, indicating rapid equilibrium. Consequently the disposition of [ 14C] OFZ and its metabolites in the bloodstream was similar in each group. It is suggested that the progressive desorption of particulate associated metabolites is a principal determinant of the duration of OFZ availability.

Differences in uptake kinetics of ammonium and nitrate in legumes and cereals

Influx isotherms were obtained for nitrate and ammonium from three legumes, Cajanus cajan (L.) Millsp., Cicer arietinum L. and Arachis hypogaea L. and three cereals, Sorghum bicolor (L.) Moench., Pennisetum glaucum L. and Zea mays L. The transition in influx isotherms for both nitrogen sources was found to be within the concentration range (0.05–2.5 mM) tested. There were significant differences in Km and Vmax for ammonium between legumes and cereals. The difference in the kinetic properties for nitrate uptake between the two groups of plants only became apparent at the higher concentration tested. Legumes translocated absorbed nitrate and ammonium to shoots more rapidly than cereals. Results show that there are significant differences in uptake and translocation of ammonium and nitrate between legumes and cereals.

Mechanism of Action of Ginsenoside Rh2: Uptake and Metabolism of Ginsenoside Rh2 by Cultured B16 Melanoma Cells

The uptake and metabolism of ginsenoside Rh2 (Rh2) by B16 melanoma cells were studied. In a medium containing 2% fetal calf serum, the uptake of Rh2 reached a maximum of 3 nmol/106 cells at 3–6 h after Rh2 (12.5 μM) was added, but gradually decreased to 0.8 nmol/106 cells. In these cells, protopanaxadiol (PPD), which is an aglycon of Rh2, increased inversely with the decrease in Rh2 as a result of deglycosylation by the cells. When PPD (8 μM) was added to the medium, the uptake reached a plateau of 2.4 nmol/106 cells within 0.5 h. The association constant of Rh2 (1.74 ± 1.08 × 106 M−1) for bovine serum albumin (BSA) was significantly higher than that of PPD (9.90 ± 1.10 × 104 M−1). In a serum-free medium, both Rh2 and PPD were incorporated within 1.5 h. The uptake rate constant of Rh2 (1.20 ± 0.20 h−1) was not significantly different from that of PPD (1.02 ± 0.15 h−1), but the release rate constant of PPD (2.12 ± 0.38 h−1) was significantly lower than that of Rh2 (3.03 ± 0.57 h−1). These differences in affinity for BSA and the release rate constants were thought to be the cause of the difference in uptake kinetics between these drugs. The effects of Rh2 and PPD on the cells were identical, and there was no difference in the lag periods before the appearance of their effects, despite their differing rates of uptake. However, in the serum-free culture, PPD inhibited cell growth at a lower level of incorporated drug as compared with Rh2, indicating that the effect of PPD was stronger than that of Rh2. These results indicate that the effects of Rh2 on B16 melanoma cells are not derived from its aglycone PPD and that the effects of PPD are stronger than those of Rh2.

Differences in uptake kinetics of cis-3-aminocyclohexane carboxylic acid into neurons and astrocytes in primary cultures

The uptake of [ 3H]ACHC and [ 3H]GABA into cultured neurons and astrocytes was studied. [ 3H]ACHC uptake was less efficient than that of GABA in both cell types and K m values for ACHC uptake into neurons and astrocytes were 40.3 μM and 210.8 μM, respectively. The corresponding V max values were 0.321 and 0.405 nmol·min −1·mg −1 cell protein, respectively. Kinetic studies of the effects of GABA on ACHC uptake and vice versa showed that GABA is a linear competitive inhibitor of ACHC uptake in both cell types with a K i value of 15 μM. On the other hand, ACHC turned out to be a complex inhibitor of astrocytic GABA uptake being competitive at lower concentrations and non-competitive at higher concentrations. ACHC inhibited GABA uptake into neurons competitively with a K i of 69 μM. It is concluded that ACHC acts primarily on neuronal GABA uptake sites but its uptake is much more complicated than hitherto anticipated.

Dicarboxylic amino acid uptake in normal, Friedreich's ataxia, and dicarboxylic aminoaciduria fibroblasts.

Glutamic and aspartic acid uptake was measured in skin fibroblasts from patients with Friedreich's Ataxia, dicarboxylic aminoaciduria, and normal individuals. The results showed no difference in uptake kinetics of either dicarboxylic amino acids between Friedreich's Ataxia and normal cells, but reduced uptake velocities in dicarboxylic aminoaciduria fibroblasts. Friedreich's Ataxia fibroblasts were, however, less calcium-dependent and more magnesium and phosphate-dependent than controls in glucose-free incubation mixture. This difference might be related to some degree of glucose intolerance by Friedreich's Ataxia fibroblasts in culture.