The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.     

Intravesicular pH and iron uptake by immature erythroid cells

The intravesicular pH of intact rabbit reticulocytes was measured by two methods; one based on the intracellular:extracellular distribution of DMO (5, 5, dimethyl + oxazolidin-2,4-dione), methylamine, and chloroquine and the other by quantitative fluorescence microscopy of cell-bound transferrin. The latter method was also applied to nucleated erythroid cells from the fetal rat liver. A pH value of approximately 5.4 was obtained with both methods and in both types of cells. Treatment of the cells with lysosomotrophic agents, metabolic inhibitors, and ionophores elevated the intravesicular pH and inhibited iron uptake from transferrin. When varying concentrations of NH4Cl were used, a close correlation was observed between the inhibition of iron uptake and elevation of the intravesicular pH. At pH 5.4 iron release from rabbit iron-bicarbonate transferrin in vitro was much more rapid than from iron-oxalate transferrin. The bicarbonate complex donates its iron to rabbit reticulocytes approximately twice as quickly as the oxalate complex. It is concluded that the acidic conditions within the vesicles provide the mechanism for iron release from the transferrin molecule after its endocytosis and that the low vesicular pH is dependent on cellular metabolism.     

Non-transferrin donors of iron for heme synthesis in immature erythroid cells

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.     

Calmodulin dependence of transferrin receptor recycling in rat reticulocytes.

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.     

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN₃, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl₂, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the sameThus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.     

Amines as inhibitors of iron transport in rabbit reticulocytes

The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I-transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin.     

Acid Intracellular Vesicles and the Cytolysis of Mammalian Target Cells by Entamoeba histolytica Trophozoites1

ABSTRACT. Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 ± 0.2 by spectrofluorimetry). Concentrations of NH₄Cl (1.0–10.0 mM) sufficient to increase vesicle pH to °5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of ³H-thymidine previously incorporated into CHO cell monolayers, and by the release of ¹¹¹indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 μM). In contrast, NH₄Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4°C and by the binding and ingestion of ³H-leucine-labeled bacteria. In the presence of NH₄Cl and and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 μg/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH₄Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 μg for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.     

Amine Transport in Riccia fluitans: Cytoplasmic and Vacuolar pH Recorded by a pH-Sensitive Microelectrode

The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH₄Cl) and methylamine (CH₃NH₃Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode.

On addition of 1 micromolar NH₄Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH₄Cl or 500 micromolar CH₃NH₃Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH₄⁺/CH₃NH₃⁺ uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH₄⁺; and second, alkalinization through protonation of NH₃ which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems.

     

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with ¹²⁵I and ⁵⁹Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of $\text{N-}\text{ethylmaleimide}$ which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37°C but nearly all that taken up 4°C was released when the cells were subsequently incubated with trypsin plus $\text{N-}\text{ethylmaleimide}$, despite the fact that about 80% of the ⁵⁹Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells.These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37°C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.     

An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells.

The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with ⁵⁹Fe and ¹²⁵I and concanavalin A (ConA) labelled with ¹³¹I. In concentrations of 50–200 μg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by α-methyl-d-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane.     

Transferrin recycling in reticulocytes: pH and iron are important determinants of ligand binding and processing

Iron uptake by rat reticulocytes is blocked by 20 mM NH₄Cl, while ¹²⁵I-diferric transferrin (Tf) uptake is relatively unaffected. At pH 5.0 both apo- and diferric Tf bind with high affinity; at pH 7.4 diferric Tf binds avidly, but apoTf binds very poorly. The dissociation rate (4°C) of diferric Tf is extraordinarily slow at pH 5.0 (extrapolated $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 32 hrs}$) and faster at pH 7.4 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 101 min}$). At pH 5.0 apoTf also dissociates slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 205 min}$), but at pH 7.4 apoTf exhibits a much faster dissociation rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 62 min}$). 20 mM NH₄Cl slows the release of Tf from cells at 37°C, but the rate of externalization of ligand is unaffected. Ligand dissociation at 37° involves both externalization of receptor-ligand complexes and receptor-ligand separation; the NH₄Cl effect may result from an increased fraction of externalized Tf in the differric form which may dissociate more slowly. Receptor-mediated movement of Tf through acid intracellular compartments provides a mechanism to remove iron from Tf and for apoTf to remain receptor-bound for externalization to the cell surface and subsequent dissociation.     

Specific inhibition by NH4Cl of autophagy-associated proteolysis in cultured fibroblasts

Rat embryo fibroblasts, prelabeled with [¹⁴C]leucine, showed an enhanced degradation of cell protein as well as increased peptide release when placed in a serum-deficient medium. NH₄Cl inhibited only the induced proteolysis, but had no effect on basal protein turnover. Electron microscopy studies showed that enhanced proteolysis was associated with an increase in autophagic vacuoles containing amorphous and membranous debris, and that NH₄Cl markedly increased the number of these intracellular vacuoles. Upon release from NH₄Cl inhibition, these cells showed a compensatory enhanced release of ¹⁴C into the medium and a decrease in the number of intracellular degradative vacuoles. We conclude that enhanced proteolysis reflects an activation of the autophagic-lysosomal system in these cells and that NH₄Cl inhibits the final hydrolysis and release steps in this mechanism.     

Preparation of membrane vesicles in lithium chloride from cells of Nitrosomonas europaea

The preparation of membrane vesicles in lithium chloride is described which oxidizes NH₄Cl via hydroxylamine to nitrite. The following properties of the vesicles were determined: the uptake of NH₄Cl and O₂ by electrode methods, the production of NH₂OH and NO₂ from NH₄Cl as well as ATPase activity. The stoichiometry of NH₄⁺:O₂:NO₂^? was found to be 1:1.2:0.7 in vesicles compared with 1:1.5:1.0 in either spheroplasts or washed cells. It is shown that the membrane vesicles also contain a Cu and energy-dependent NH₄^? translocase as in spheroplasts and cells.     

Effect of pH and iron content of transferrin on its binding to reticulocyte receptors

The effect of pH on the binding of apotransferrin and diferric transferrin to reticulocyte membrane receptors was investigated using rabbit transferrin and rabbit reticulocyte ghosts, intact cells and a detergent-solubilized extract of reticulocyte membranes. The studies were performed within the pH range 4.5–8.0. The binding of apotransferrin to ghosts and membrane extracts and its uptake by intact reticulocytes was high at pH levels below 6.5 but decreased to very low values as the pH was raised above 6.5. By contrast, diferric transferrin showed a high level of binding and uptake between pH 7.0 and 8.0 in addition to binding only slightly less than did apotransferrin at pH values below 6.5. It is proposed that the high affinity of apotransferrin for its receptor at lower pH values and low affinity at pH 7.0 or above allow transferrin to remain bound to the receptor when it is within acidic intracellular vesicles, even after loss of its iron, but also allow ready release from the cell membrane when it is exteriorized by exocytosis after iron uptake. The binding of transferrin to the receptor throughout the endocytosis-exocytosis cycle may protect it from proteolytic breakdown and aid in its recycling to the outer cell membrane     

Ammonia (C-Methylamine) Transport across the Bacteroid and Peribacteroid Membranes of Soybean Root Nodules

[¹⁴C]Methylamine (MA; an analog of ammonia) was used to investigate ammonia transport across the bacteroid and peribacteroid membranes (PBM) from soybean (Glycine max) root nodules. Free-living Bradyrhizobium japonicum USDA110 grown under nitrogen-limited conditions showed rapid MA uptake with saturation kinetics at neutral pH, indicative of a carrier. Exchange of accumulated MA for added ammonia occurred, showing that the carrier recognized both NH₄⁺ and CH₃NH₃⁺. MA uptake by isolated bacteroids, on the other hand, was very slow at low concentrations of MA and increased linearly with increasing MA concentration up to 1 millimolar. Ammonia did not inhibit MA by isolated bacteroids and did not cause efflux of accumulated MA. PBM-enclosed bacteroids (peribacteroid units [PBUs]) were qualitatively similar to free bacteroids with respect to MA transport. The rates of uptake and efflux of MA by PBUs were linearly dependent on the imposed concentration gradient and unaffected by NH₄Cl. MA uptake by PBUs increased exponentially with increasing pH, confirming that the rate increased linearly with increasing CH₃NH₂ concentration. The results are consistent with other evidence that transfer of ammonia from the nitrogen-fixing bacteroid to the host cytosol in soybean root nodules occurs solely by simple diffusion of NH₃ across both the bacteroid and peribacteroid membranes.     

增氮对青藏高原东缘高寒草甸土壤甲烷吸收的早期影响

研究大气氮沉降对青藏高原高寒草甸土壤CH4吸收的影响,对于揭示氮素调节土壤CH4吸收的机制和评价氮沉降增加背景下大气CH4收支平衡至关重要.通过构建多形态、低剂量的增氮控制试验,测定土壤CH4净交换通量和相关土壤理化性质,分析高寒草甸土壤CH4通量变化特征及其主要驱动因子.研究结果表明:自然状态下高寒草甸土壤是大气CH4汇,CH4平均吸收量为(35.40±1.92) μg· m-2· h-1.土壤CH4吸收主要受水分驱动,其次为土壤NH4+-N和NO3-N含量.NH4+-N抑制CH4吸收,NO3--N促进CH4吸收;不同剂量氮素输入对土壤CH4吸收影响也不尽相同,低氮处理促进土壤CH4吸收,而中氮和高氮处理抑制土壤CH4吸收.结果显示青藏高原高寒草甸土壤是重要的大气CH4汇,在未来大气氮沉降加倍的情景下CH4汇功能增强,但当氮沉降量增加两倍以上时CH4汇功能将会减弱.     

The effect of serum and experimental variables on the transferrin and reticulocyte interaction

The transfer of iron from transferrin to the developing erythrocyte is a research area of high interest and considerable controversy. We have found that the results of transferrin-reticulocyte incubation studies are quite sensitive to the experiemental procedures that are utilized. Reticulocytosis has been induced in rabbits by phelbotomy and phenylhydrazine inhections. While the latter gives a higher reticulocyte count, the cells appear to exhibit an altered transferrin-membrane interaction. Transferrin has been iodinated by published methods utilizing chloramine-T and molecular iodine. The iodotransferrin products exhibit the same iron donation ability, however, evidence was found that the chloramine-T treatment leads to a nonspecific binding of transferrin to the reticulocyte. The means of saturating transferrin with ⁵⁹Fe is also of prime importance. Fe(NH₄)₂ (SO₄)₂ and especially FeCl₃ were found to yield non-specifically bound iron when added to transferrin or serum. This artifact was reflected in an altered transferrin-reticulocyte interaction. Using what we believe to be optimal conditions, the effect of serum on the transferrin-reticulocyte system was re-examined. The results clearly indicated an enhancement of iron uptake by reticulocytes in the presence of serum, as well as an accelerated incorporation of iron by the cytoplasmic fraction.     

Iron metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH₄Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (⁵⁵Fe/⁵⁹Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.     

A moderate increase in the annual CH4 efflux by raised CO2 or NH4NO3 supply in a boreal oligotrophic mire

Natural wetlands release about 20% of global emissions of CH₄, an effective greenhouse gas contributing to the total radiative forcing. Thus, changes in the carbon cycle in wetlands could have significant impacts on climate. The effect of raised supply of CO₂ or NH₄NO₃ on the annual CH₄ efflux from the lawn of a boreal oligotrophic mire was investigated over two years. Ten study plots were enclosed with mini‐FACE rings, five vented with CO₂‐enriched air and the other five with ambient air. In addition, five plots were sprayed with NH₄NO₃ so that the cumulative addition of N was 3 g m^?2 y^?1; and five plots were controls. The CO₂ enrichment (target concentration 560 ppm_v) increased CH₄ efflux about 30–40%, but half of this increase seemed to be caused by the air‐blowing system. The increasing atmospheric concentration of CO₂ would promote CH₄ release in boreal mires, but the increase in CH₄ efflux would be clearly smaller than that reported in studies made in temperate or subtropical temperature conditions. Addition of N enhanced the annual release of CH₄ only slightly. At least over the short‐term, the increase in N deposition would have little effect on CH₄ effluxes. The increase in CH₄ release would probably increase radiative forcing and thus accelerate climate change. However, CH₄ effluxes are only a small part in the whole matter balance in mires and thus further studies are needed to define the net effects of raised supply of CO₂ or N for carbon accumulation, trace gas fluxes and radiative forcing.     

Simulated Nitrogen Deposition Reduces CH4 Uptake and Increases N2O Emission from a Subtropical Plantation Forest Soil in Southern China

To date, few studies are conducted to quantify the effects of reduced ammonium (NH₄ ⁺) and oxidized nitrate (NO₃ ⁻) on soil CH₄ uptake and N₂O emission in the subtropical forests. In this study, NH₄Cl and NaNO₃ fertilizers were applied at three rates: 0, 40 and 120 kg N ha⁻¹ yr⁻¹. Soil CH₄ and N₂O fluxes were determined twice a week using the static chamber technique and gas chromatography. Soil temperature and moisture were simultaneously measured. Soil dissolved N concentration in 0–20 cm depth was measured weekly to examine the regulation to soil CH₄ and N₂O fluxes. Our results showed that one year of N addition did not affect soil temperature, soil moisture, soil total dissolved N (TDN) and NH₄ ⁺-N concentrations, but high levels of applied NH₄Cl and NaNO₃ fertilizers significantly increased soil NO₃ ⁻-N concentration by 124% and 157%, respectively. Nitrogen addition tended to inhibit soil CH₄ uptake, but significantly promoted soil N₂O emission by 403% to 762%. Furthermore, NH₄ ⁺-N fertilizer application had a stronger inhibition to soil CH₄ uptake and a stronger promotion to soil N₂O emission than NO₃ ⁻-N application. Also, both soil CH₄ and N₂O fluxes were driven by soil temperature and moisture, but soil inorganic N availability was a key integrator of soil CH₄ uptake and N₂O emission. These results suggest that the subtropical plantation soil sensitively responses to atmospheric N deposition, and inorganic N rather than organic N is the regulator to soil CH₄ uptake and N₂O emission.