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ABSTRACT: Sub-optimal nutrition during pregnancy has been shown to have long-term effects on the health of offspring in both humans and animals. The most common outcomes of such programming are hypertension, obesity, dyslipidaemia and insulin resistance. This spectrum of disorders, collectively known as metabolic syndrome, appears to be the consequence of nutritional insult during early development, irrespective of the nutritional stress experienced. For example, diets low in protein diet, high in fat, or deficient in iron are all associated with programming of cardiovascular and metabolic disorders when fed during rat pregnancy. In this paper, we hypothesise that the nutritional stresses act on genes or gene pathways common to all of the insults. We have termed these genes and/or gene pathways the "gatekeepers" and hence developed the "gatekeeper hypothesis". In this paper, we examine the background to the hypothesis and postulate some possible mechanisms or pathways that may constitute programming gatekeepers.
Medical Hypotheses 10/2011; 78(1):88-94. · 1.39 Impact Factor
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ABSTRACT: Iron and copper are both essential micronutrients and are required for a wide variety of enzymatic and other processes within the developing foetus. Transfer of both nutrients across the placenta is tightly regulated. In this review, we consider their mechanisms of transport, how the transfer is modulated in response to nutritional requirements and how the two metals interact. Iron uptake is via the transferrin receptor, followed by endocytosis, acidification of the vesicle, and release of the iron into the cytosol, and transfer across the basolateral membrane. Many of the genes involved have been identified, and, to varying extents, their mechanisms of regulation clarified, but there are still unanswered questions and conundrums. For example, although the ion channel DMT1 (now formally known as slc11a2) is essential for iron uptake in the gut, knockout mice, which have no slc11a2 protein, have apparently normal transfer across the placenta. There must, therefore, be an alternative mechanism, which remains unclear, although nonspecific calcium channels have been proposed as one possibility. For copper, uptake is a carrier-mediated process, and intracellular transfer is mediated by proteins known as chaperones. Efflux is through ATPases, but their localisation and how they are regulated is only now being elucidated. Regulation of copper proteins appears to be different from that of iron, with localisation of the protein, rather than changing levels, being responsible for altering rates of transfer. This may not be true for all the proteins and genes involved in the delivery of copper, and, again, there is much that remains to be clarified. Finally, we consider the interactions that occur between the two metals, reviewing the data that show how alterations in levels of one of the nutrients changes that of the other, and we examine the hypotheses explaining the interactions.
Journal of Neuroendocrinology 05/2008; 20(4):427-31. · 3.14 Impact Factor
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ABSTRACT: During pregnancy, the developing fetus is dependent on its mother for all nutritional requirements. It is not surprising, therefore, that variations in maternal nutrition can be reflected in alterations in fetal health and well-being. Interestingly, the changes can persist into adulthood and may result in increased risk of diseases such as diabetes, obesity and cardiovascular disease. The first observations of these phenomena resulted in the development of hypotheses collectively brought under the heading of "fetal" or, more recently, "developmental" programming. In this review, we will examine some of the animal models used to understand the mechanisms involved and attempt to determine whether there are common, "gatekeeper", pathways or genes, altered by the different nutritional stresses. We will concentrate primarily on nutrition related to post-natal development of hypertension and will restrict the review to studies in rodents, since that is where most of the mechanistic studies are being undertaken. Our conclusions are that, while there may well be some common gatekeeper pathways, there is also some diversity of mechanism which may contribute to the generation of the same or similar phenotypes.
Placenta 05/2006; 27 Suppl A:S56-60. · 3.69 Impact Factor
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ABSTRACT: At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.
Journal of Inorganic Biochemistry 06/2005; 99(5):1018-22. · 3.35 Impact Factor
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ABSTRACT: We have previously shown that maternal iron (Fe) deficiency not only reduces fetal size, but also increases blood pressure in the offspring when they are adults. In this paper we examine whether there are critical periods when supplementation reverses or fails to reverse the effect both on size and on expression of genes of Fe metabolism. We made dams Fe deficient, mated them and provided supplements of Fe in the diet from the beginning of gestation (0.5 days), from 7.5 days or from 14.5 days. Within 12 h of birth, dams and neonates were killed and tissues taken and examined. Fe deficiency throughout pregnancy reduces neonatal size. Supplementation from the beginning of the first, second or third week all reduced the effect. Maternal haematocrit was restored to normal levels only in animals given supplements for at least 2 weeks. In contrast, the neonates' Fe levels were normal in all supplemented groups. These results were mirrored in liver Fe levels and in transferrin receptor mRNA. Iron-responsive element (IRE)-regulated divalent metal transporter 1 (DMT1) increased in maternal and neonatal liver. Non-IRE-regulated DMT1 levels did not change in the maternal liver, but decreased in the neonatal liver. H and L ferritin mRNA levels also showed different patterns in the mother and her offspring. Finally, the neonatal size correlated with maternal Fe stores, and not with those of the fetus. The data demonstrate that Fe supplementation during pregnancy is most effective when given early, rather than later, in gestation.
The Journal of Physiology 12/2004; 561(Pt 1):195-203. · 4.72 Impact Factor
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ABSTRACT: During pregnancy, iron is transferred from the mother to the fetus across the placenta. The mechanism has been extensively studied. Altered iron metabolism changes transfer, but also has other consequences. In this review, we examine how the placenta adapts to altered iron supply, both in terms of changing cytokine expression and in relation to the proteins of iron transfer. Changing iron levels alters the levels of other metals, especially copper, and we review how this is related to changing function. There are also consequences to the placenta itself, to vascularisation and other aspects of the physiology. In turn, this has effects on the fetus and we review how growth and development are modified. Finally, we examine in more detail the efflux process, how it is regulated and, especially, the putative role of the placental Cu oxidase in the efflux process. As appropriate, we draw on data from humans, from animal models and from cell culture systems to illustrate the information.
BioMetals 04/2003; 16(1):161-7. · 2.82 Impact Factor
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ABSTRACT: During pregnancy, iron is transferred from the mother to the fetus across the placenta. The mechanism has been extensively studied. Altered iron metabolism changes transfer, but also has other consequences. In this review, we examine how the placenta adapts to altered iron supply, both in terms of changing cytokine expression and in relation to the proteins of iron transfer. Changing iron levels alters the levels of other metals, especially copper, and we review how this is related to changing function. There are also consequences to the placenta itself, to vascularisation and other aspects of the physiology. In turn, this has effects on the fetus and we review how growth and development are modified. Finally, we examine in more detail the efflux process, how it is regulated and, especially, the putative role of the placental Cu oxidase in the efflux process. As appropriate, we draw on data from humans, from animal models and from cell culture systems to illustrate the information.
BioMetals 02/2003; 16(1):161-167. · 2.82 Impact Factor
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ABSTRACT: Maternal iron deficiency during pregnancy induces anaemia in the developing fetus; however, the severity tends to be less than in the mother. The mechanism underlying this resistance has not been determined. We have measured placental expression of proteins involved in iron transfer in pregnant rats given diets with decreasing levels of iron and examined the effect of iron deficiency on iron transfer across BeWo cell layers, a model for placental iron transfer. Transferrin receptor expression was increased at both mRNA and protein levels. Similarly, expression of the iron-responsive element (IRE)-regulated form of the divalent metal transporter 1 (DMT1) was also increased. In contrast, the non-IRE regulated isoform showed no change in mRNA levels. Protein levels of DMT1 increased significantly. Iron efflux is thought to be mediated by the metal transporter protein, IREG1/ferroportin1/MTP1, and oxidation of Fe(II) to Fe(III) prior to incorporation into fetal transferrin is carried out by the placental copper oxidase. Expression of IREG1 was not altered by iron deficiency, whereas copper oxidase activity was increased. In BeWo cells made iron deficient by treatment with desferrioxamine ('deferioxamine'), iron accumulation from iron-transferrin increased, in parallel with increased expression of the transferrin receptor. At the same time, iron efflux also increased, showing a higher flux of iron from the apical to the basolateral side. The data show that expression of placental proteins of iron transport are up-regulated in maternal iron deficiency, resulting in an increased efficiency of iron flux and a consequent minimization of the severity of fetal anaemia.
Biochemical Journal 07/2001; 356(Pt 3):883-9. · 4.90 Impact Factor
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ABSTRACT: The mechanism of iron release from the placenta into the fetal circulation is not well understood. Ceruloplasmin, a plasma ferroxidase, has been implicated in iron efflux from a variety of cell types. The hypothesis is that circulating ceruloplasmin facilitates iron efflux by oxidizing the released Fe(II) to Fe(III) for incorporation into transferrin. We tested whether this mechanism mediates iron release from placental cells into the fetal circulation, using the BeWo cell line, a choriocarcinoma which can differentiate into a syncytium.(59)Fe release from undifferentiated or differentiated cells and from cells grown on porous filters was not stimulated by extracellular ceruloplasmin. Instead, we found that BeWo cells express an endogenous ferroxidase. The protein is membrane bound and cross-reacts with an anti-ceruloplasmin antibody, but has a different size; 100 and 140 kDa. Similar immunoreactivity was identified in first- and third-trimester human placentae. In BeWo cells, the protein has a perinuclear localization but does not entirely co-localize with markers for the endoplasmic reticulum or Golgi apparatus. We propose that this oxidase performs the same function as serum ceruloplasmin and is involved in iron release into the fetal circulation.
Placenta 12/2000; 21(8):805-12. · 3.69 Impact Factor
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ABSTRACT: The organometallic anticancer agent titanocene dichloride, Cp(2)TiCl(2), is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp(2)TiCl(2) with human serum transferrin (hTF) and that of Ti(2)-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp(2)TiCl(2) reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [(1)H, (13)C] NMR studies of epsilon-[(13)C]Met-hTF show that Ti(IV) loads the C-lobe first followed by the N-lobe and binds in the specific Fe(III) sites. The protein conformational changes induced by Ti(IV) appear to be similar to those induced by Fe(III). Carbonate can act as a synergistic anion in Ti(2)-hTF but does not appear to be essential. A specific Ti(IV)-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti(2)-hTF solutions is lowered, no Ti(IV) is released at the endosomal pH of ca. 5.0-5.5, but one Ti(IV) dissociates between pH 4.5-2.0. In contrast, in the presence of 1 mM ATP, all Ti(IV) is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, Fe(III) displaces Ti(IV) rapidly from the C-lobe of Ti(2)-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species Fe(C)Ti(N)-hTF might also provide a route for Ti(IV) entry into tumor cells via the transferrin receptor. Ti(2)-hTF effectively blocked cell uptake of radiolabeled (59)Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.
Biochemistry 08/2000; 39(33):10023-33. · 3.42 Impact Factor
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ABSTRACT: This paper has, given some idea of our concepts of the processes involved in the transport of Cu across cell membranes in the liver, which we have summarised in Fig 1. Cu(II)His2 is reduced to Cu(I). This is transported across the membrane, re-oxidised, either before or after binding to glutathione (Freedman et al., 1989) or HAH1 (Klomp et al., 1997), binds to SAHH, and donates Cu(II) to the ATPase. It is very interesting that cells which are very diverse from an evolutionary point of view still use very similar methods to handle the metal. Whether regulation of transport is also the sam remains to be seen. We would guess that, although there will be strong similarities, there will also be very significant differences, reflecting the different environments seen by different tissues in mammalian cells and given the different requirements of the tissues.
Advances in experimental medicine and biology 02/1999; 448:29-37. · 1.09 Impact Factor
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Biochemical Society Transactions 06/1998; 26(2):S191. · 3.71 Impact Factor
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Biochemical Society Transactions 06/1998; 26(2):S99. · 3.71 Impact Factor
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ABSTRACT: The genes responsible for Wilson disease and Menkes syndrome have been cloned and identified as copper ATPases. These enzymes form part of a large family of transporters, the P-type ATPases. Although copper ATPases share strong structural similarities with these other pumps, comparatively little is known about their physiologic function. In this review, we examine data relating to the Wilson disease protein, ATP7B, in the liver. We present evidence suggesting that ATP7B is located intracellularly, together with data suggesting that, at least in part, ATP7B may also be found on the canalicular membrane. We also examine the form of copper that the transporter recognizes. We then review data on the Long-Evans Cinnamon rat, a model for Wilson disease, and discuss what effect the Wilson disease mutation has on copper transport. Finally, we conclude that, although we have made major advances in our understanding of copper metabolism in the liver, there are still many questions awaiting answers.
American Journal of Clinical Nutrition 06/1998; 67(5 Suppl):982S-987S. · 6.67 Impact Factor
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ABSTRACT: When hepatocytes are incubated with the chelator diamsar, two pools can be identified, which we have termed extractable and nonextractable. On entering the hepatocyte, 67Cu first associates with the extractable pool and, after approximately 2 h, moves to the nonextractable pool. Both pools demonstrate saturation and are filled as a function of Cu concentration and incubation time. Using the Michaelis-Menten equation, we have estimated the size of the pools after incubation with 67Cu for 30 min and 4 h. During this period the extractable pool decreases in size from 200 +/- 27 to 116 +/- 5 pmol/microgram DNA, whereas the nonextractable pool increases from 28 +/- 9 to 77 +/- 11 pmol/microgram DNA. Movement of Cu from the nonextractable pool to the extractable pool is slow and incomplete. Using [3H]diamsar, we demonstrate that uptake of the chelator is not rate limiting and probably does not occur by pinocytosis. Incubation with diamsar does not affect the activity of superoxide dismutase or cytochrome-c oxidase, although it does prevent the incorporation of 67Cu into ceruloplasmin. Incubation with zinc, which induces metallothionein, results in an increase in 67Cu associated with the nonextractable pool, suggesting that 67Cu-metallothionein constitutes at least part of the nonextractable pool.
The American journal of physiology 07/1997; 272(6 Pt 1):G1400-7.
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ABSTRACT: The Wilson disease adenosinetriphosphatase (ATPase; ATP7B) is believed to bind copper as Cu(I). We provide evidence to suggest that the ATPase actually transports Cu as Cu(II). When the copper is presented to rat liver microsomes as Cu(I), virtually all uptake is ATP independent. If the copper is presented as copper oxalate [Cu(II)], total uptake is reduced to approximately 10% of Cu(I) levels, but ATP-dependent uptake rises, both as a proportion of total uptake and in absolute terms. The reducing agent vitamin C and the Cu(I) chelator bathocuproine both override the effect of oxalate. The data indicate that there are two transporters in the microsomes, an ATP-independent Cu(I) transporter and an ATP-dependent Cu(II) pump. The activity of the Cu(I) transporter correlates most strongly with alkaline phosphatase, suggesting that it is derived from plasma membrane contamination. Cu(II) ATP-dependent transport correlates only with beta-1, 4-galactosyltransferase, which indicates that it is located in the Golgi apparatus.
The American journal of physiology 12/1996; 271(5 Pt 1):G741-6.
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ABSTRACT: The influence of K+ and Ca2+ on Zn2+ transport into cultured human fibroblasts was investigated. Zn2+ uptake was markedly reduced in the presence of both valinomycin and nigericin (electrogenic and electroneutral K+ ionophores, respectively), and by reduction in the transmembrane K+ gradient produced by replacement of extracellular K+ with Na+, suggesting that Zn2+ may be driven by a Zn2+/K+ counter-transport system. To test the counter-transport hypothesis, we used 86Rb as an analog of K+ for efflux studies. The rate of Rb+ efflux was 3760 times that of Zn2+ uptake, thus the component of K+ involved in the Zn2+ counter-transport system was only a small proportion of the total K+ efflux. In investigating the effect of Ca2+ on Zn2+ uptake, we identified two components: (1) a basal Zn2+ uptake pathway, independent of hormonal or growth factors which does not require extracellular Ca2+ and (2) a Ca(2+)-dependent mechanism. The absence of Ca2+ decreased Zn2+ uptake, while increasing extracellular Ca2+ stimulated Zn2+ uptake. The effect was mediated by Ca2+ influx as the ionophores A23187 and ionomycin also stimulated Zn2+ uptake. We could not ascribe the Ca2+ effect to known Ca2+ influx pathways. We conclude that Zn2+ uptake occurs by a K(+)-dependent process, possibly by Zn2+/K+ counter-transport and that a component of this is also Ca(2+)-dependent.
BioMetals 02/1996; 9(1):29-37. · 2.82 Impact Factor
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ABSTRACT: This paper has examined copper uptake from CuHis2 complexes by cytotrophoblast cells isolated from term human placenta. Uptake is time-dependent, reaching equilibrium after about 90 min, and saturable, with a calculated apparent Km of 0.174 +/- 0.061 microM and Vmax, measured over 30 min, of 0.721 +/- 0.092 pmol/min/micrograms DNA. To determine whether ATP was required for uptake, cells were incubated with inhibitors of glycolysis (iodoacetate) and the TCA cycle (sodium azide and cyanide). Iodoacetate and sodium azide had no effect on uptake, but cyanide decreased the initial rate of uptake. This effect was due to copper binding to the inhibitor and decreasing the effective substrate concentration rather than inhibition of uptake through ATP depletion. Ouabain and monensin had no effect, showing that neither the Na+ gradient nor endocytosis were involved in uptake. The monovalent ion chelator, bathocuproine sulphonate, had no effect on uptake but buthionine sulfoximine, an inhibitor of glutathione synthesis, did decrease both the rate of uptake and equilibrium copper levels, suggesting that copper may bind to glutathione within the cell. The data show that copper is taken up by a passive carrier-mediated transporter and, following uptake, binds to glutathione within the cell.
Biochimica et Biophysica Acta 12/1995; 1269(3):233-6. · 4.66 Impact Factor
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ABSTRACT: In this paper we have studied copper (Cu) uptake by microvillar vesicles isolated from human term placenta. We have characterised Cu uptake from CuHis2 complexes and shown that ceruloplasmin (Cp) inhibits uptake. Inhibition is complex and variable; in one series of experiments, the Vmax for uptake drops from 31.3 +/- 1.2 nmol/min per mg vesicle protein without added Cp to 11.3 +/- 1 nmol/min per mg vesicle protein at 91 micrograms/ml Cp. Similarly, the K0.5 increases from 0.35 +/- 0.08 microM to 1.35 +/- 0.25 microM, while the n value (the Hill coefficient) falls from 1.9 +/- 0.23 in the absence of Cp to 1.1 +/- 0.13 In another series, Cp had no effect below concentrations of about 100 micrograms/ml and in a third series only increased K0.5. The variability in effect seems to be related to the specific activity of the ceruloplasmin, which in turn is related to the copper complexes of the protein. The effect is specific for Cp; apotransferrin and a2-macroglobulin have no effect. 67Cu-labelled ceruloplasmin binds specifically to vesicles of term placenta with an affinity of 2.8 microU/mg vesicle protein and a Bmax of 79 microU/mg vesicle protein. CuHis2, but not histidine alone, can block the uptake. The data can be reconciled by proposing that the binding site of the transporter is relatively small and recognises a Cu-dihistidine structure common to the low-molecular-weight complex and to the Type I and Type II coppers of ceruloplasmin. We have used these observations to develop an isolation method for the transporter and have identified it as a protein of M(r) 90,000 which is closely associated with alkaline phosphatase. There are also two proteins of M(r) 45,000 and 40,000 which may be breakdown products of the larger complex. Antibodies to the 45,000 protein block Cu binding and uptake from CuHis2 complexes, strongly implicating it as the copper transporter/ceruloplasmin receptor of human term placenta.
Biochimica et Biophysica Acta 11/1995; 1245(2):153-60. · 4.66 Impact Factor
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ABSTRACT: In freshly isolated fetal guinea pig type II pneumocytes, zinc uptake is time and temperature dependent. Two pathways of uptake exist, resulting in a rapid phase that reaches a steady state within 30 s and a slower linear phase that does not attain a steady state within 60 min. Both processes exhibit saturation kinetics. The rapid phase has a maximal zinc uptake of 60.7 +/- 9.3 pmol.10(6) cells-1.30 s-1 and an apparent affinity (Kt) of 13.7 +/- 5.4 microM. The maximum velocity of uptake (Vmax) of the slower phase is 24.6 +/- 1.9 pmol.10(6) cells-1.min-1 with a Kt of 22.0 +/- 3.6 microM. Epinephrine, terbutaline, dibutyryl adenosine 3',5'-cyclic monophosphate, and dexamethasone have no significant effect on zinc uptake, while arachidonic acid (AA) stimulates. Dose-response data of AA-stimulated zinc uptake gives an apparent K0.5 of 0.42 +/- 0.01 microM and a Hill coefficient of 1. The maximal uptake in the rapid phase is significantly increased to 146.8 +/- 12.4 pmol.10(6) cells-1.30 s-1 and in the slow phase, the Vmax for zinc uptake is also significantly increased to 33.0 +/- 1.8 pmol.10(6) cells-1.min-1 by 10 microM AA. However, the Kt values in both processes remain unchanged after AA stimulation. The effect is not mediated by either leukotrienes or prostaglandins but can be mimicked by other unsaturated fatty acids.
The American journal of physiology 08/1995; 269(1 Pt 1):L71-7.
