Difference In Net Uptake Research Articles

Abstract 5491: Marked augmentation of the polyglutamate derivatives as the basis for the enhanced activity of pralatrexate relative to methotrexate.

Abstract Pralatrexate (PDX) is a new-generation dihydrofolate reductase (DHFR) inhibitor with high affinity for the reduced folate carrier (RFC) and for folypolyglutamyl synthetase (FPGS), the enzyme that mediates the addition of glutamate moieties at the γ-carboxyl of folates and antifolates (Sirotnak et al., Cancer Chemother Pharmacol. 42:313, 1998). The polyglutamate derivatives are better accumulated and retained into the cells. Recently, PDX received FDA approval for the treatment of relapsed or refractory peripheral T-cell lymphoma. This report represents the first analysis of PDX cellular pharmacology using the tritiated derivative, comparing its properties with methotrexate (MTX) in HeLa cells. The influx Kt was determined at ∼0.5 μM in comparison to a MTX influx Ki of ∼3 μM. Consistent with RFC-targeting, [³H]PDX was a lesser substrate for the proton-coupled folate transporter (PCFT) than MTX. While the net uptake of [³H]MTX reached steady-state conditions within 30 min, net uptake of [³H]PDX continued to increase, exceeding the [³H]MTX level by a factor of 15, by 6 hrs. This difference in net uptake could be attributed to a marked increase in PDX polyglutamates. At 24 hrs, the PDX mono-, di- and triglutamate levels in the cells were 14%, 12%, and 70% of the total antifolate, respectively. When polyglutamation was suppressed, the subsequent intracellular [³H]PDX level was markedly decreased now exceeded the [³H]MTX level by a factor of only 3. The importance of PDX polyglutamation was reflected in the marked difference in growth inhibition relative to MTX when exposure to the drugs was transient. When cells were exposed continuously to PDX or MTX for six days the IC50 for the former was ∼ 1/25th the latter (7 nM vs 180 nM, respectively). However, when the exposure was limited to 6 hrs followed by growth in drug-free medium, the pharmacological advantage of PDX over MTX was markedly increased with an IC50 of 70 nM and 30 μM,respectively, now ∼ 1/450th that of MTX. Hence, in the first direct measurement of the transport and polyglutamation of PDX, the data confirm a much higher affinity for RFC relative to MTX along with a markedly greater rate of polyglutamation in cells. It is this difference in the extent of accumulation of these active polyglutamate derivatives that is the basis for the marked difference in the antitumor activities between these antifolates. The apparent low affinity of PDX for PCFT suggests that changes at the nitrogen-10 position are one determinant of the affinity of 4-amino antifolates for this transporter. Further, since PCFT is the mechanism by which folates and antifolates are absorbed across the proximal small intestine, the data suggests that the low affinity of PDX for this carrier would result in a low rate of re-absorption of this antifolate during its enterohepatic circulation, with a consequent higher level of fecal excretion, than for MTX. Citation Format: Michele Visentin, Ersin Selcuk Unal, Rongbao Zhao, I David Goldman. Marked augmentation of the polyglutamate derivatives as the basis for the enhanced activity of pralatrexate relative to methotrexate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2013-5491

Uptake, transport, accumulation and retranslocation of potassium in grapevine rootstocks (Vitis)

The concentration and content of potassium (K) within grapevine can be regulated by selective use of rootstocks. However, the mechanisms by which rootstocks differ in the accumulation of K in the shoot of grapevine are not well understood. Therefore, the current study addresses these issues. Ungrafted rootstocks 140 Ruggeri (140 R) and 101-14 Millardet de Grasset (101-14) were exposed to K tracer rubidium (Rb) in a glasshouse, for 4 hours through roots to study uptake and transport of Rb from roots to shoot, and for 24 hours through leaf to study downward transport of Rb, in each case assessed up to 48 hours following exposure to Rb. Transpiration rates and root traits were also measured. Results showed similar total Rb uptake and similar downward transport of Rb between the two rootstocks but lower accumulation of Rb in the shoot and lower shoot/roots Rb concentration (as a result of higher retention of Rb in roots), in 140 R than that in 101-14. Transpiration rates and total root length (but not total root surface area) remained similar between two rootstocks. The study shows that short term differences in the accumulation of K in roots and shoot between rootstocks are due to differences in the transport of K from roots to the shoot, rather than by differences in net uptake or retranslocation of K. In addition, such differences were not affected by transpiration rates. The results highlight that accumulation of K in shoot of grapevine rootstocks is regulated mainly by roots.

Control of Net Uptake of Nutrients by Regulation of Influx in Barley Plants Recovering from Nutrient Deficiency

Rates of influx and net uptake of nitrate, phosphate and sulphate were measured in intact barley plants, and concurrent efflux was obtained by difference. Net uptake of these anions varied widely depending on the nutrient status of the plants, and the differences in net uptake could be accounted for almost entirely by changes in influx. Efflux played only a minor role in regulating net uptake of nitrate, phosphate or sulphate during recovery from N-, P-, or S-deficiency.Nitrate influx and short-term ammonium absorption by N-deficient barley plants were closely correlated, and varied in parallel with rates of net uptake of nitrate or ammonium by similar plants. Again, it would seem that net uptake of ammonium is controlled predominantly by changes in the rate of influx.

Autoregulation of Auxin Transport in Corn Coleoptile Segments

Polar transport of auxin decreased and its net uptake increased in maize (corn) coleoptile segments preincubated in buffer following excision from the intact seedling. Reduced rate of cellular efflux of auxin accounted for these changes in preincubated segments where the endogenous auxin level was depleted. The difference in net uptake of auxin between freshly excised and preincubated tissues was completely abolished by the phytotropin N-1-naphthylphthalamic acid. Studies on in vitro binding of NPA to membrane vesicles isolated from the tissue indicated that the number of binding sites on the plasma membrane was likewise reduced following excision of the tissue. Presence of exogenous auxin in the preincubation medium prevented the decay of both auxin transport and the putative auxin efflux carriers. These results suggest that polar auxin transport in corn coleoptiles is autoregulated by endogenous auxin level through its influence on the auxin efflux carrier.

Lysinuric protein intolerance mutation is not expressed in the plasma membrane of erythrocytes.

We measured mediated fluxes of L-lysine and L-ornithine across the plasma membrane of erythrocytes from control subjects and patients homozygous for the lysinuric protein intolerance (LPI) mutation. We found no differences in net uptake or efflux of cationic amino acids in control and LPI cells, contrary to our findings in cultured skin fibroblasts. We conclude that expression of the LPI (y+) transport system for cationic amino acids varies between tissues and that measurements of fluxes in erythrocytes cannot be used for diagnosis of LPI.

Uptake of free amino acids by bacteria-free larvae of the sand dollar Dendraster excentricus.

Larvae of Dendraster excentricus were produced by collecting gametes and carrying out fertilization under aseptic conditions. Since gametes are free of bacteria in the gonad, bacteria-free (axenic) suspensions of larvae result. Net rates of entry of 14 amino acids and the rate of production of ammonia were simultaneously determined by high-performance liquid chromatography. The net rates of uptake of neutral amino acids were an order of magnitude greater than rates for basic and acidic amino acids. Influx of 14C-labeled leucine, arginine, and glutamate accurately reflects the net entry rate of these substrates. Uptake of amino acids by axenic suspensions of larvae was compared with uptake by suspensions prepared without aseptic precautions. There was no significant difference in net uptake of the 14 amino acids or in the pattern of oxidation and assimilation of [14C]leucine during short-term experiments of 4-h duration or less.

Varietal Differences in Rubidium Uptake Efficiency of Barley Roots

AbstractUptake efficiency for Rb+ and SO2−4 was different in eight cultivars of barley, Hordeum vulgare L. Rb+ uptake was studied in more detail in low‐and high‐K+ plants of cv. Salve and cv. Hellas at different Rb+ concentrations in the experimental solutions.The K+/Rb+ selectivity of the roots was strongly dependent on the experimental conditions, but changed similarly in the two cultivars. After six days the fresh weight of the shoots was c. 15% higher in ‘Salve’ than in ‘Hellas’. Root development and the average concentration of K+ in the plants were the same. Influx of Rb+ in the roots was up to 75% faster in ‘Salve’ than in ‘Hellas’. Experiments with short‐term Rb+ influx (10 min) + wash (5 min) indicated that the differences in Rb+ uptake efficiency were due to variations in Rb+ influx at the plasmalemma of the root cells. Efflux of 86Rb+ from 86Rb+‐labelled roots was higher in ‘Salve’ than in ‘Hellas’ during the initial 30 min in inactive solution, indicating that passive permeation (leakage) of Rb+ and probably also of K+ at the plasmalemma of root cells of cv. Salve is faster than that of cv. Hellas. The parallel differences between influxes and effluxes explain the comparatively small difference in net uptake between the two varieties.

Triglyceride, free fatty acid and carbohydrate metabolism in hyperlipaemic (type IV) and normolipaemic subjects on carbohydrate- of fat-rich diets.

1. Five patients with hyperlipoproteinaemia type IV and five normolipaemic controls were maintained on diets containing either a fat or a carbohydrate content of approximately 60%. Leg exchanges of triglycerides, free fatty acid (NEFA), glucose and other metabolites were measured in the basal state. The metabolic response to an intravenous glucose load and the turnover of intravenously infused (14C)oleic acid were determined. Individual fatty acids were analysed in plasma NEFA and very-low-density lipoprotein triglyceride. 2. No differences were detected between patients and controls with regard to leg uptake of triglyceride or NEFA on either diet. On shifting from a fat-rich to a carbohydrate-rich diet, the control subjects showed reduced values for arterial NEFA and for NEFA turnover, accompanied by a lowered release of NEFA and glycerol from the leg. In patients, on the other hand, the arterial concentration and turnover of NEFA remained unchanged and the release of glycerol from the leg increased. Glucose administration gave a less-pronounced fall of arterial NEFA on either diet in the patients and the incorporation of (14C)oleic acid into triglycerides after glucose administration was more marked than in the controls. 3. No difference in net uptake of triglyceride by the leg was demonstrable between patients and controls. 4. it is concluded that hyperlipaemic patients show an altered metabolic response to a carbohydrate-rich diet, possibly contributing to the development of a hyperlipaemic state, and that acute glucose loading results in a greater incorporation of circulating NEFA into very-low-density lipoproteins in these patients.