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Mechanisms involved in increased iron uptake across rat duodenal brush-border membrane during hypoxia Donagh K. O'Riordan *, Edward S. Debnam *t, Paul A. Sharp *t, Robert J. Simpson §, Eve M. Taylor § and Surjit K. S. Srai t Departments of *Physiology and t Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, London NW3 2PF and § Department of Clinical Biochemistry, King's College School of Medicine and Dentistry, London SE5 9PJ, UK 1.
2.
Chronic hypoxia enhances intestinal iron transport but the cellular processes involved are poorly understood. In order to assess the effects of 3 days of hypoxia on iron uptake across the duodenal brush-border membrane, we have measured the membrane potential difference (Vm) of villus-attached enterocytes by direct microelectrode impalement and have used semiquantitative autoradiography to study changes in expression of iron uptake during enterocyte maturation. Hypoxia increased duodenal Vm (-57-7 vs. -49-3 mV, P < 0X001). Ion substitution experiments revealed that hyperpolarization was due, at least in part, to a reduction in
brush-border Nae permeability. 3. Autoradiography revealed that hypoxia increased by 6-fold the rate of iron accumulation during enterocyte transit along the lower villus and enhanced by 3-fold the maximal accumulation of iron. Depolarization of the brush border, using a high-K+-containing buffer, caused a proportionally greater reduction in iron uptake in control compared with hypoxic tissue suggesting that the raised iron uptake is only partly driven by brush-border hyperpolarization. 4. We conclude that hypoxia increases the expression of iron transport in duodenal brushborder membrane and an enhanced electrical driving force may be involved in this response.
Very little is known about the cellular mechanism and control of duodenal iron absorption despite the fact that enterocyte handling of iron is of crucial importance in determining body iron status. Results from previous studies have suggested that iron uptake across the brush-border membrane (BBM) (Cox & Peters, 1980; Cox & O'Donnell, 1981), iron binding to cytosolic proteins (Conrad, Umbreit & Moore, 1993; Kozma, Chowrimootoo, Debnam, Epstein & Srai, 1994) or transfer of the metal across the basolateral membrane (BLM) into the portal blood (Snape, Simpson & Peters, 1990; Chowrimootoo, Debnam, Srai & Epstein, 1992a) are all important in controlling lumen-to-blood iron movement. Hypoxia promotes duodenal iron uptake in both rat (Osterloh, Simpson, Snape & Peters, 1987; Taylor, Raja, Simpson & Peters, 1997) and human (Reynafarje & Ramos, 1961) but the cellular basis of this adaptive response is
unknown. Enterocytes undergo structural and functional maturation during their transit along the villus (Smith, 1985) and maximal expression of BBM iron uptake does not normally occur until cells reach the upper villus (Debnam,
Srai, Chowrimootoo & Epstein, 1991; O'Riordan, Sharp, Sykes, Srai, Epstein & Debnam, 1995b). In this present work we have used quantitative autoradiography to study the effects of 50 5 kPa (0 5 atm) pressure, equivalent to an altitude of 5000 m above sea level, on the profile of BBM iron uptake during enterocyte transit along the villus. Finally, we have assessed the effects of hypoxia on the potential difference across the brush border (Vm), since Vm is a driving force for transporter-mediated iron entry (Raja, Simpson & Peters, 1989). Preliminary reports of this work have been published (O'Riordan, Simpson, Taylor, Sharp, Debnam & Srai, 1995c; O'Riordan, Simpson, Sharp, Debnain & Srai, 1996).
t To whom correspondence should be addressed.
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METHODS Animals and anaesthesia Experiments used male Wistar rats (230-250 g) bred at King's College School of Medicine and Dentistry and fed a standard maintenance diet (Diet CRM, Special Diets Service, Witham, Essex, UK). Hypoxia was induced by exposing animals to 50 5 kPa (0 5 atm) pressure for 3 days in a hypobaric chamber during which time animals had unlimited access to food and water. Less food was consumed during the first day of hypoxia and thereafter similar amounts of food were ingested by each animal group. Uptake experiments were carried out within 4 h of removing animals from the chamber. Anaesthesia prior to the removal of intestinal segments was achieved with sodium pentobarbitone (90 mg kg-', i.p.). After the removal of tissue, rats were killed by pneumothorax. All procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986. Autoradiography of iron uptake Duodenal segments, 1 cm long and taken 5 cm proximal to the ligament of Treitz, were washed through with cold NaCl (154 mM), rapidly everted and finally stretched over and secured to the end of a Perspex rod. The tissue was immediately immersed in oxygenated pre-incubation buffer at 37 °C containing (mM): Hepes, 16; glucose, 10; KCl, 3'5; MgSO4, 10; CaCl2, 1; and NaCl, 125 (pH 7'4). A rotating stir bar ensured the adequate exposure of tissue surface to the solution. After 5 min the tissue was transferred to fresh buffer containing 200 ZM 59Fe3+ complexed with nitrilotriacetate (NTA). 59FeCl3 was present at a specific activity of 14 1uCi ml-'. Some experiments used a high [K+] solution (110 mM), achieved by the partial replacement of NaCl with KCl. All incubations were terminated after 5 min by washing in 10-fold excess nonradioactive Fe(NTA)2 at 20 °C to displace surface-bound iron and the tissue was then washed twice with phosphate buffer (mM: NaCl, 137; KCl, 3-5; KH2PO4, 1 5; and Na2HPO4, 8 1; pH 7 3) and fixed in phosphate buffer containing 2 % (w/v) glutaraldehyde. Processing for autoradiography was carried out as described previously (Chowrimootoo et al. 1992a). Counterstaining with either Fast Green or Haematoxylin allowed visualization of the underlying villus structure. Microdensitometry Quantification of silver grains on developed emulsion was carried out on Fast Green-stained slides, since this agent does not stain nuclei or absorb light at the frequency used for scanning microdensitometry. The stain does, however, give enough detail of the underlying morphology to allow the identification of the crypt-villus junction. Scanning was carried out sequentially from the base to the tip of straight villi at 490 nm using a magnification of x 1000 and a 12 #um x 6 ,um scanning frame fitted to a Vickers M-85 microdensitometer. At least two villi from three to four separate animals in each animal group were used. Results are given in arbitrary units. Iron uptake by mucosal fragments The method used has been described previously (Taylor, Raja, Simpson & Peters, 1997). In brief, a 2 cm duodenal segment was cut open to form a sheet and this was cut into fragments of 3-15 mg weight. Washed fragments were pre-incubated for 1 min at 37 °C in oxygenated Hepes buffer and then exposed for 5 min to the same buffer but containing 100 ,UM 59Fe3+ and 200 uM nitrilotriacetate together with [57Co]cyanocobalamin (5 nM) as an extracellular marker. Fragments were than washed in cold Hepes buffer and their 59Fe3+ and 57Co2+ activity was measured by counting gamma radiation (LKB-Wallac 1282 Compugamma, Helsinki, Finland).
Intracellular iron uptake was expressed in picomoles per milligram of wet weight per minute (pmol (mg wet weight)-' min-').
Brush-border potential difference (Vm) A 1 cm section of duodenum was washed through with NaCl (154 mM), cut longitudinally and mounted as a flat sheet on a Perspex disc using cyanoacrylate adhesive applied to the muscle side. The disc formed the base of a tissue bath and the preparation was superfused at 32 °C with bicarbonate saline (mM: NaCl, 118&5; KCl, 4-6; CaCl2, 1'9; KH2PO4, 1P2; MgSO4, 1P2; and NaHCO3, 25), gassed with 95% 02-5% CO2 (v/v) and fed by gravity at a flow rate of 2 ml min-. For the low-Na+ buffer, NaCl was replaced with choline chloride whilst for the high-K+ buffer, NaCl was replaced with KCl. Each tissue section was used for a maximum of 30 min. Vm was measured as described previously (Debnam & Thompson, 1984), using borosilicate glass electrodes (o.d. 1-5 mm) filled with filtered 1 M KCl. Electrodes had a tip resistance of 15-40 MQ2 and a tip potential of less than 5 mV and the latter was subtracted from all recordings. A 1 M KCl-agar bridge in contact with the bathing solution was used as the reference electrode. The criteria for an acceptable impalement were: (a) an immediate negative deflection; (b) the maintenance of a stable potential difference for at least 15s; (c) an abrupt return to baseline upon withdrawal of the microelectrode; and (d) a similar electrode resistance (± 10%) and tip potential before and after impalement. The negative deflection upon impalement occurred in one phase and within the same time course in both normal and hypoxic conditions. All results represent values obtained in tissue from at least three animals per group. Statistics Values are expressed as means + S.E.M. Differences between groups were assessed by Student's unpaired t test and considered significant at P < 0'05. Chemicals 59FeCl3 was obtained from Amersham International (Amersham, Buckinghamshire, UK). [57Co]cyanocobalamin was from Amerlite Diagnostic Services (Amersham, Buckinghamshire, UK) and cyanocobalamin was from Duncan Flockhart and Co. Ltd (Uxbridge, Middlesex, UK). All other chemicals were purchased from Sigma (Poole, Dorset, UK) or Merck Ltd (Poole, Dorset, UK) and were of analytical grade.
RESULTS
Haematology Blood samples were removed by cardiac puncture from anaesthetized animals immediately before the removal of intestinal sections for uptake studies. Blood from hypoxic animals had increased haemoglobin concentrations (16'3 + 0 5 vs. 13-0 + 0 4 g (100 ml)-', P < o0005) and haematocrit values (0 53 + 0.01 vs. 0-41 + 0-01, P < 0 005). Methylene Blue staining of blood smears indicated reticulocytosis in hypoxic animals. Brush-border membrane potential difference (Vm) At normal,mucosal concentrations of Na+ and K+, duodenal Vm was increased significantly by hypoxia (Table 1). Ion substitution experiments, utilizing high-K+ and low-Na+ buffers, were carried out in order to define the membrane events underlying the hypoxia-induced hyperpolarization. The reduction of mucosal [Na+] from 143 to 25 mM increased Vm in control (P < 0 05) but not in hypoxic intestine. In
Hypoxia and intest'inal iron uptake
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no
differences
in
the
electrical
responses
to
increased [K+] were apparent in the two animal groups.
Autoradiography of iron uptake Autoradiographs showed the characteristic villus gradient of iron
uptake
with
no
iron accumulation
seen
in
crypt and
381
lower villus regions (Fig. lB and D). Light-field photographs indicated that absorbed iron was largely accumulated at the basolateral side of the cell (Fig. 1A and C). Densitometry studies of duodenal sections indicated that hypoxia enhanced the rate of accumulation of iron along the lower 200 ,m section of villus by some 6-fold (slope, 0-693 + 0-008 vs.
C
I..
200 stm
Figure 1. Effects of hypoxia on the villus distribution of brush-border iron uptake A and C, light-field microscopy of rat duodenum from control and hypoxic animals, respectively, showing dark-coloured silver grains indicating the region of iron uptake. The tissue has been stained with Haematoxylin to emphasize the underlying villus structure. The arrow in C indicates the basolateral accumulation of radioactive iron. B and D, dark-field micrographs of control and hypoxic villi, respectively, to show more clearly the gradient of brush-border 59Fe3+ uptake. The distribution of silver grains indicates that iron uptake increases towards the upper villus.
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Table 1. Effects of altering mucosal [Na+] or [K+] on duodenal brush-border potential difference (Vm) Vm (mV)
Concentration (mM)
Na
K+
CF-
143 0
5-8 5-8 125-0
125-5
25f0 25'0
125f5 125'5
Hypoxia
Control -49 3 + 1P2 (43) -55 5 + 0 9 (18)* -8-4 + 0-6 (14)
-57 7 + 0 9 (35)* -58-0 + 1P2 (37) -9 4 + 0 7 (14)
Substituting agents were choline chloride for NaCl (low [Na+], normal [K+]) and KCl for NaCl (low [Na+] and high [K+]). Results are given as means + S.E.M. with the number of impalements in parentheses. * P < 0-001 compared with control.
0 117 + 0-002 absorbance units (#m villus)-f, P < 0 001; Fig. 2A and B). Uptake in control villi continued at the same rate along the remaining 150 ,um villus length whilst uptake in hypoxic villi decreased progressively towards the villus tip. Hypoxia increased maximal iron accumulation by some 3-fold (129-6 + 8-7 vs. 42-3 + 12 6 a.u., P < 0 001).
In order to assess the contribution of brush-border hyperpolarization to enhanced iron uptake in hypoxic duodenum, autoradiography was carried out using tissue exposed to a high [K+] in the superfusion buffer, a procedure which depolarized the BBM (Table 1). Depolarization decreased iron accumulation by both normal and hypoxic villi, there
A 140 120 C 100C3 .-
80-
.?co 60Q C 0
4020 -
00
300 100 200 Distance from crypt-villus junction (um)
400
B 140 Co
120 -
C
a3 100 Cu
80 60 -
0)
40 20 -
1I
00
200 300 Distance from crypt-villus junction (um) 100
400
Figure 2. Quantification of autoradiographs showing effects of BBM depolarization on iron uptake along normal and hypoxic duodenal villi 59Fe3+ uptake along normal (A) and hypoxic (B) duodenal villi was quantified after incubation of tissue in either normal mucosal buffer (5 mm K+, *) or one containing 110 mm K+ (0), achieved by the partial replacment of NaCl in the buffer with KCl. Microdensitometry was carried out using Fast Greenstained slides. Measurement of grain density began at the crypt-villus junction (arrow) and progressed towards the villus tip. Results are given as the means + S.E.M. of 5-11 villi from 3-4 tissue sections in each group.
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being a proportionally greater reduction in maximal iron uptake in control (-68-9 %) compared with hypoxic (-35 4%) duodenum (Fig. 2A and B). Iron uptake by mucosal fragments In keeping with the above autoradiographic data, iron uptake by duodenal mucosal fragments was increased by 46% in hypoxic tissue (4-73 + 0O26 vs. 3-25 + 0O44 pmol (mg wet weight)-' min-', P < 0 05).
DISCUSSION The mechanisms controlling enterocyte iron transport are poorly understood. Transporters for iron at the brushborder and basolateral membranes have been postulated (Cox & O'Donnell, 1981; Snape et al. 1990) and intracellular iron binding proteins, which may regulate the availability of cytosolic iron for transport across the basolateral membrane, have been identified (Conrad et al. 1993; Kozma et al. 1994). The overall control of enterocyte iron transport will involve regulation at some or all three stages of the cellular process but at the present time the situation is confused by the fact that the rate-limiting step for the transcellular process is unknown. Hypoxia has been shown to increase accumulation of iron by the duodenal mucosa (Osterloh et al. 1987) and this implies adaptation at the brush border. The present experiments were designed to characterize the nature of this response, in particular to examine the relationship between enterocyte maturation and expression of iron uptake. Although the potential difference across the brush-border membrane is known to drive Na+-dependent uptake of many organic nutrients, for example glucose (Kimmich, Carter-Su & Randles, 1977), the concept that Vm plays a role in intestinal iron uptake is a relatively recent one. Previous studies have demonstrated increased iron uptake by mucosal fragments following an imposed potential difference across the BBM (Raja et al. 1989) and work using other experimental models of increased iron uptake, namely iron deficiency anaemia (O'Riordan, Sharp, Epstein, Srai & Debnam, 1995a) and haemolytic anaemia (O'Riordan et al. 1995b) provides circumstantial evidence that an enhanced brush-border electrical gradient powers the increased iron uptake required to satisfy a greater body demand for iron in these conditions. Our present study supports this notion by showing that hyperpolarization of the brush-border membrane accompanies increased duodenal iron uptake in hypoxia. Previous studies have revealed that the duodenal brushborder membrane of normal rats is permeable to Na, albeit less so than to K+ (Okada, Sato & Inouye, 1975; Okada, Irimajiri & Inouye, 1976). Results from ion substitution experiments imply that hyperpolarization arises from a reduced BBM permeability to Nae as is the case for the increased iron uptake in iron deficiency (O'Riordan et al. 1995a) and haemolytic anaemia (O'Riordan et al. 1995 b).
383
Taken together, it appears that hyperpolarization of the BBM, resulting from a reduced Na+ conductance, is an important adaptive process for upregulation of iron uptake in a wide variety of experimental conditions. Autoradiographs confirmed the characteristic villus gradient of duodenal iron uptake evident in our earlier work (Chowrimootoo et al. 1992a; O'Riordan et al. 1995b). Rapid stirring of the mucosal buffer in these experiments implies that the uptake profile was not caused by poor accessibility of the lower villus to iron. The hypoxia-induced stimulation of iron uptake along the villus is in keeping with the observation of enhanced iron accumulation by mucosal fragments. Since experimental depolarization of the brush border, achieved with a high-K+-containing buffer, only partially reduced iron uptake along the villus, it can be concluded that the mechanisms involved in adaptation to hypoxia are a combination of BBM hyperpolarization and enhanced expression or activity of membrane proteins responsible for the uptake process. The submaximal inhibition of duodenal iron uptake following exposure to increased buffer [K+], although differing from previous studies in mice (Raja et al. 1989), are consistent with other findings in the rat (Taylor et al. 1997). Since the level of food intake influences brush-border potential difference (Debnam & Thompson, 1984), the clear species difference in the response to depolarization may reflect an altered pattern of food intake during hypoxia in the two species (Taylor et al. 1997). It should be noted that enhanced brush-border uptake seen in this present work is unlikely to be the only explanation for the increased iron transport from lumen to blood noted in previous work using hypoxic rats (Osterloh et al. 1987). Alterations in the handling of iron at the basolateral membrane or within the cytosol should also be considered but these aspects have not been addressed in this present study. It is, however, of interest that increased basolateral transfer, particularly evident in the lower small intestine, is partly responsible for the high iron absorption in early postnatal life (Chowrimootoo, Gillett, Debnam, Srai & Epstein, 1992b). Whether alterations in basolateral transfer of iron occur in hypoxia is unknown. In summary, increased expression of BBM iron uptake during maturation of duodenal enterocytes is an important response to chronic hypoxia and will provide an elevated level of body iron to satisfy the requirements for increased erythropoietic activity. Adaptation of iron uptake during hypoxia involves an enhanced BBM electrical gradient resulting from a reduced Nae permeability. Cytosolic ironbinding proteins which have been previously implicated in the adaptation of enterocyte iron transport may also be involved in the upregulated iron transport. Alterations in the expression of these proteins in response to hypoxia may be relevant to both the increased membrane iron uptake seen in this present work and to the greater lumen to blood iron transfer noted previously.
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Acknowledgements WA'e are grateful to the Aiedical Research Council for supporting this -work, to Dr M. W. Smith for advice and facilities regarding quantitative autoradiography, and to Mr Ml. Adams for technical assistance.
Author's email address E. S. Debnam:
[email protected]
Received 5 September 1996; accepted 10 January 1997.
