Zinc uptake by isolated rat hepatocytes

Isolated rat hepatocytes were used to investigate the uptake of zinc at early exposure times. Hepatocytes were incubated with ⁶⁵Zn (1–500 μM) and samples were withdrawn at times ranging from 25 s to 60 min. A biphasic pattern of uptake was observed with a rapid first phase of uptake followed by a slower second phase. The relationship between velocity of uptake and substrate concentration for the first phase was nonlinear, while that of the second phase was linear. The presence of 10 μM cadmium produced a decrease in the velocity of uptake of only the first phase. This suggests that the first phase is at least partly carrier mediated, while there is no indication of involvement of a carrier in the second phase. KCN (1 mM) and carbonyl cyanide m-chlorophenylhydrazone (2 μM), did not cause any change in the uptake of ⁶⁵Zn (1 μM), which suggests that there is no active component in the uptake of zinc.     

Iron uptake and metabolism by hepatocytes

The hepatocytes form part of the iron storage system of the body. In serving this function they exchange iron bidirectionally with the plasma iron transport protein transferrin (Tf). Iron uptake involves binding of the iron-Tf complex to cell membrane receptors and endocytosis into low-density vesicles, where the iron is released from its carrier protein before the Tf is returned undegraded to the extracellular medium. Two components of the iron uptake process can be distinguished, one saturable at low concentrations of diferric Tf and the other not saturable by increasing the Tf concentration. Both result in net uptake of iron by the cells and both appear to depend on specific binding to the cell membrane and endocytosis. Hepatocytes also obtain some iron from haptoglobin-hemoglobin, heme-hemopexin, and ferritin (Fn), in each case by interaction with membrane receptors and endocytosis. Within the cell iron from all sources enters one or more transit pools, where it is available for exchange with the iron storage protein Fn, and for release from the cell to plasma Tf or to iron chelators administered therapeutically or experimentally. Chelator-mediated iron release occurs to the plasma and/or to the bile, depending on the nature of the chelator and the source of the iron.     

Transferrin protein and iron uptake by cultured hepatocytes

The binding and uptake of ⁵⁹Fe-loaded ³H-labelled rat transferrin by cultured rat hepatocytes was investigated. At 4°C, there is no evidence for a specific binding of transferrin which could be related to the association of neo-synthesized transferrin with plasma membrane receptors. At 37°C, iron uptake is much more important than transferrin uptake; it proceeds linearly over the time of incubation, is largely proportional to the extracellular transferrin concentration, and is compatible with uptake by fluid phase endocytosis. The difference observed between iron and transferrin uptake implies the existence of a mechanism allowing the reutilization of transferrin after iron delivery.     

Leukotriene uptake by hepatocytes and hepatoma cells.

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial-rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low-Km system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS-30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S-decyl glutathione, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 microM each), but not by taurocholate, the amphiphilic cations verapamil and N-propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene-binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene-binding proteins with apparent molecular masses in the ranges of 48-58 kDa and 38-40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.     

Zinc uptake by cultured human lymphoblasts

Biological Trace Element Research - At physiological plasma concentrations (12–20 μM), zinc uptake by cultured human B lymphoblasts is biphasic with an early, rapid, saturable phase,...     

Free fatty-acid uptake by isolated rat hepatocytes

In isolated rat hepatocytes, the rate of palmitic acid binding and uptake is directly related to the concentration of free fatty acid (FFA) in the medium. After their entry into the cell, FFA are immediately incorporated into cellular phospholipids and triglycerides and no accumulation of free fatty acids can be demonstrated inside the cell. The rate of free fatty-acid uptake remains unchanged after incubation in a 2 mM KCN containing medium, indicating that in the range of fatty-acid concentrations used in this study, this phenomenon does not require energy.     

Zinc hyperaccumulation and uptake by Potentilla griffithii Hook

The ability of Potentilla griffithii Hook var. velutina Cardot to hypaeraccumulate zinc (Zn) was identified through field survey and hydroponic experiments. Our results showed that P. griffithii could be classified as a new Zn hyperaccumulator. Zn concentrations in the shoots of P. griffithii averaged 6250 mg kg(-1) (3870-8530 mg kg(-1)) growing in Zn-rich soils. The highest Zn concentration was observed in the leaves of P. griffithii at 22,990 mg kg(-1). The fact that P. griffithii was able to grow in a mining soil with a Zn concentration of 193,000 mg kg(-1) without showing a major sign of phytotoxicity demonstrated its high tolerance to Zn. When growing in hydroponic systems, P. griffithii accumulated a maximum 26700 mg kg(-1) zinc concentration in the shoots, indicating the ability of this species to effectively take up and translocate Zn. Translocation factors (the ratio of Zn concentration in shoot to root) of 1.1 to 1.6 were obtained. Compared to the control, dry biomass of P. griffithii in 160 mg L(-1) Zn treatment increased 66.6% (P < 0.05). The time-course experiment showed that the maximum Zn concentration at 100 mg L(-1) Zn treatment was found at 16 d, much later than that of the 10 mg L(-1) Zn treatment, which might be an attribution of a accumulating mechanism or detoxification of a plant. The report of a new Zn hyperaccumulator provides a new plant species for the phytoremediation of contaminated soil and for the research on mechanisms of Zn hyperaccumulation in plants.     

Mechanism of sucrose uptake by isolated rat hepatocytes

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C-sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration-dependent from 5 microM to 100 mM and follows first-order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid-phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.     

Receptor- and non-receptor-mediated uptake and degradation of insulin by hepatocytes

Native insulin inhibits the binding and degradation of ¹²⁵I-labelled insulin in parallel. Half-maximal inhibition of degradation occurs with 10nm-insulin, a hormone concentration sufficient to saturate the insulin receptor. The proportion of bound hormone that is degraded increases as the insulin concentration is increased, suggesting that low-affinity uptake is functionally related to degradation. Since only a small fraction (approx. 10%) of the overall degradation occurs at the plasma membrane, or in the extracellular medium, translocation of bound hormone into the cell is the predominant mechanism mediating the degradation of insulin. In the presence of 0.6nm-insulin, a concentration at which most cell-associated hormone is receptor-bound, chloroquine increases the amount of ¹²⁵I-labelled insulin retained by hepatocytes. However, chloroquine increases the retention of degradation products of insulin in incubations containing sufficient hormone (6nm) to saturate the receptor and permit occupancy of low-affinity sites. Glucagon does not compete for the interaction of ¹²⁵I-labelled insulin (1nm) with the insulin receptor. In contrast, 20μm-glucagon inhibits 75% of the uptake of insulin (0.1μm) by low-affinity sites. A fraction of the cell-bound radioactivity is not intact insulin throughout a 90min association reaction at 37°C. During dissociation, fragments of ¹²⁵I-labelled insulin are released to the medium more rapidly than is intact hormone. The production and transient retention of degradation products of the hormone complicates the characterization of the insulin receptor by equilibrium or kinetic methods of assay. It is proposed that insulin degradation occurs by receptor- and non-receptor-mediated pathways. The latter may be related to the action of glutathione–insulin transhydrogenase, with which both insulin and glucagon interact.     

Zinc uptake by corn as affected by vesicular-arbuscular mycorrhizae

Pot-grown mycorrhizal and non-mycorrhizal sweet corn were grown in a low Zn soil. All treatments received a complete nutrient solution with or without Zn. Treatments were harvested sequentially to detemine temporal mycorrhizal effects on: (a) tissue and water soluble Zn and (b) differential uptake of P and Zn. Plants grown with supplemental Zn had greater growth and Zn tissue concentration than those not receiving Zn. With no supplemental Zn, mycorrhizal treatments had greater growth and Zn concentration than non-mycorrhizal treatments. There was no indication of nutrient interaction between Zn and P. Over the range of tissue Zn found, there appeared to be no advantage to water soluble Zn analysis over total Zn in assessing plant Zn status.     

Cellobiose uptake and metabolism by Ruminococcus flavefaciens

The cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 utilizes cellobiose but not glucose as a substrate for growth. Cellobiose uptake by R. flavefaciens FD-1 was measured under anaerobic conditions (N2), using [G-3H]cellobiose. The rate of cellobiose uptake for early- or late-log-phase cellobiose-grown cells was 9 nmol/min per mg of whole-cell protein. Cellobiose uptake was inhibited by electron transport inhibitors, iron-reactive compounds, proton ionophores, sulfhydryl inhibitors, N,N-dicyclohexylcarbodiimide, and NaF, as well as lasalocid and monensin. The results support the existence of an active transport system for cellobiose. Transport of [U-14C]glucose was not detected with this system. Phosphorylation of cellobiose was not by a phosphoenolpyruvate-dependent system. Cellobiose phosphorylase activity was detected by both a coupled spectrophotometric assay and a discontinuous assay. The enzyme was produced constitutively in cellobiose-grown cells at a specific activity of 329 nmol/min per mg of cell-free extract protein.     

Preparation of EGF labeled liposomes and their uptake by hepatocytes

The preparation of epidermal growth factor (EGF) bearing liposomes (EGF-liposomes) and their uptake by rat hepatocytes were described. EGF was bound to the liposomal surface by the disulfide bridge linkage using a heterobifunctional cross-linking reagent, N-hydroxysuccinimidyl-3-(2-pyridyl-dithio) propionate. EGF-liposomes were taken up in a significant amount by rat hepatocytes in an EGF receptor mediated manner, and their uptake was dependent on the amount of labeled EGF coupled to the liposomal surface. These results raise the possibility that the EGF-liposomes may be an effective drug carrier to the cells having EGF-receptors like some cancer cells.     

Pathways in the binding and uptake of ferritin by hepatocytes

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.     

Ornithine uptake by isolated hepatocytes and distribution within the cell

Uptake of ornithine by isolated hepatocytes and its distribution within the cell was investigated. Ornithine uptake was energy independent and exhibited a saturable and a nonsaturable component. The Km value of the saturable component was 1.3 mM. At an external ornithine concentration of 0.5 mM the rate of ornithine uptake was 127 +/- 19 nmol/g. Lysine inhibited ornithine uptake, indicating the existence of an ornithine transport system. It was concluded that ornithine transport can limit urea synthesis in the state of transition from a low ammonia to a high ammonia supply.     

Zinc and insulin metabolism

A review of experimental studies of the effect of zinc nutrition on insulin metabolism is presented. In addition to a short introduction to the synthesis, secretion, and action of insulin, the effects of zinc deficiency—specifically on glucose tolerance, insulin secretion, insulin synthesis and storage, and on total insulin-like activity—are dealt with. The concentrations of zinc and chromium in serum, pancreas, and liver are compared to those of zinc-deficient animals and pair-fed controls. In contrast to pair-fed controls, zinc-deficient rats had unaltered proinsulin contents after glucose stimulation, but they showed a diminished glucose tolerance, lowered serum insulin content, and an elevated total insulin-like activity. The serum zinc concentration of the deficient animals was greatly reduced and did not change during glucose stimulation, whereas it rose in the case of the pair-fed controls. The serum chromium concentration increased in both groups in response to glucose stimulation. In the pancreas of the deficient animals, the zinc concentration was reduced 60% and it increased during the glucose tolerance test. In the liver there were no significant differences. The chromium concentrations were elevated in both the pancreas and liver of the zinc-deficient rats by 60 and 100%, respectively, and were not influenced by glucose injection. These studies show clearly that nutritional zinc deficiency influences insulin metabolism and action.     

Zinc uptake by human placental microvillous membrane vesicles

Zinc uptake by syncytiotrophoblast microvillous membrane vesicles (SMMV) from human placentas was characterized and the effects of maternal serum zinc levels at term and of gestational age on kinetic parameters were evaluated. Zinc uptake at pH 7.2 was rapid for the first 2 min, followed by a slower increase, approaching equilibrium after 30 min. Uptake was saturable at a zinc concentration of 30 micromol/L, higher than the upper range of the physiological serum zinc level. Kinetic analysis of uptake at 1 min in SMMV from term placenta showed similar Km values (mean: 6.9+/-0.6 micromol/L) for different levels of maternal serum zinc. However, Vmax was higher (p < 0.05) in SMMV from mothers with serum zinc lower than 7.6 micromol/L compared to those with higher serum zinc levels (35.8+/-1.6 and 26.6+/-1.6 nmol 65Zn/mg protein/min, respectively). Km values were similar in term (>37 wk of gestation) and preterm (20-25 wk of gestation) placentas, whereas Vmax was higher (p < 0.05) in the preterm (34.3+/-1.6 nmol Zn/mg protein/min) compared to term placentas from mothers with serum zinc levels above 7.6 micromol/L. These results suggest that whereas afffinity for zinc was not altered with gestational age or maternal serum zinc levels, zinc-uptake capacity in human placenta is influenced both by gestational age and by low levels of maternal serum zinc in order to ensure an adequate maternal-fetal zinc transfer.