3H-L-histidine and 65Zn2+ are cotransported by a dipeptide transport system in intestine of lobster Homarus americanus
Department of Biology, 4567 St Johns Bluff Road, South, University of North Florida, Jacksonville, FL 32224, USA
* Author for correspondence (e-mail: gahearn{at}unf.edu)
Accepted 22 November 2004
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Summary |
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Key words: L-histidine, zinc, bis-complex, dipeptide, PEPT-1, glycyl-sarcosine, copper, heavy metal, Homarus americanus, intestine, transmural transport, epithelium, L-leucine, cotransport
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Introduction |
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Dietary metals, at low concentrations, also have an important role in
protein function and act as cofactors in many cellular reactions. It is
important, therefore, to characterize the membrane transport mechanisms by
which luminal metals are transferred into gastrointestinal absorptive cells
where they can help regulate a variety of cellular processes. Zinc is a
dietary metal that has a vital role in the operation of several hundred
proteins and its deficiency leads to impairments in growth and development as
well as in immune reactions and reproductive status of many animals
(Hambridge, 2000;
Bury et al., 2003
). Zinc
enters cells by a variety of known transport systems belonging to the ZTL and
ZIP gene families (Cragg et al.,
2002
; Gaither and Eide,
2001a
,b
),
through relatively unspecific DMT-1 transporters characterized for iron
(Gunshin et al., 1997
), or
through putative calcium channels (Bury et
al., 2003
). An additional zinc transport process that has received
attention in recent years is the apparent coupling of the metal with specific
amino acids such as L-histidine and L-cysteine
(Horn et al., 1995
;
Horn and Thomas, 1996
; Glover
and Hogstrand,
2002a
,b
;
Glover et al., 2003
). These
latter studies have suggested processes whereby luminal zinc complexes with
two amino acids in solution in a bis-complex (Zn-[His]2) and the
combination is transported as a unit across the cellular membrane. Other
mechanisms accounting for the transfer of both metal and amino acid across a
given membrane may also be possible and to date the identity of this amino
acid-dependent zinc transport system is unclear.
The present investigation is a study of transmural
3H-L-histidine and 65Zn2+
transport across the isolated and perfused intestine of the American lobster
Homarus americanus. Results show that the metal and amino acid may
cross this organ from lumen to blood via a dipeptide transporter that
has recently been reported to occur in lobster hepatopancreas
(Thamotharan and Ahearn, 1996)
and may be similar to PEPT-1 described for vertebrates. In addition, both
substrates may also cross the tissue by way of separate carrier processes that
both show a high degree of specificity for their respective solutes.
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Materials and methods |
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A physiological saline solution was developed in conjunction with the salt composition and osmolarity of lobster hemolymph. This medium included the following salt concentrations (in mmol l1): NaCl, 415; CaCl2, 25; KCl, 10.0; NaH2PO4.2H2O, 1.0; NaHCO3, 4.0; Na2SO4, 8.4; Hepes, 30. The osmotic pressure of this incubation medium was approximately 950 mosmol kg1 and the pH of the solution was adjusted to 7.1 for experimental conditions.
In vitro transmural transports of L-histidine,
Zn2+ and glycyl-sarcosine were examined using a simple perfusion
apparatus as described in detail previously (Ahearn and Hadley,
1977a,b
;
Ahearn and Maginniss, 1977
;
Brick and Ahearn, 1978
;
Wyban et al., 1980
;
Chu, 1986
). Briefly, intact
intestines were flushed of contents and mounted with surgical thread on
blunted 1820 gauge stainless steel needles in a lucite chamber
containing the incubation medium (10 ml), which served as the serosal medium.
This solution was also perfused through the intestines as the mucosal medium
using a peristaltic pump (Instech Laboratories, Inc., Plymouth Meeting, PA,
USA) at a flow rate of 380 µl min1 for periods of time up
to 180 min. Previous studies using other crustacean species have shown
intestinal viability under the conditions used in the present work for up to 5
h of continuous perfusion (Ahearn and Hadley,
1977a
,b
;
Ahearn and Maginniss, 1977
;
Chu, 1986
). Variable
concentrations of L-histidine, Zn2+ or glycyl-sarcosine
were added to the mucosal medium as needed. Experiments were conducted at
23°C.
An intestine was perfused with an unlabelled mucosal medium for 1020 min for tissue stabilization before a 60 min control flux interval with radiolabelled uptake medium. This control flux period was followed by one or two additional 60 min experimental flux periods using labeled perfusate of various compositions. Control experiments showed that a steady state appearance of isotope in the serosal compartment occurred after only 10 min of perfusion. All unidirectional flux measurements reported in this paper were conducted on intestines after they had reached the steady state. Experimental mucosal solutions containing L[2,5]3H-histidine (Amersham Biosciences Corp., Piscataway, NJ, USA), 65ZnCl2 (Oak Ridge National Laboratory, Oakridge, TN, USA), or glycyl-1,2-3H-sarcosine (Moravek Biochemicals, Brea, CA, USA) were next perfused through the intestine at pH 7.1. Triplicate 200 µl samples were removed from the serosal bath, added to scintillation cocktail, and counted for radioactivity in a Beckman LS6500 scintillation counter. Upon removal of the sample, an equal volume of saline solution was added back into the bath to maintain the volume of the surrounding medium. Subsequent corrections for isotope removal and bath dilution were made during transmural flux calculations. Samples of serosal media were taken every 510 min during a 90180 min time-course experiment. Unidirectional transmural flux rates (mucosa to serosa) were determined over 30 min periods with bath samples taken every 5 min. The mucosal test solutions consisted of varying 3H-L-histidine (150 µmol l1), 65Zn2+ (11000 µmol l1), 3H-glycyl-sarcosine (100 µmol l1), L-leucine (100 µmol l1), and CuCl or CuCl2 (50 µmol l1) concentrations. pH experiments were conducted in a similar fashion where the first flux interval was measured with a pH 7.0 saline delivered from one perfusion tube and this was followed immediately by exchanging perfusion tubes with a pH 6.0 saline. A bubble introduced between the two salines took less than 30 s to pass through a perfused gut, suggesting that minimal time occurred between tissue exposures to the two pH treatments. Specific conditions for each experiment are outlined in the figure legends. The rate of radioactivity increase in the serosal bathing medium was used to calculate the transmural mucosal-to-serosal transport rate of the isotope under the conditions of each experiment.
3H-glycyl-sarcosine was used as a representative substrate for
the dipeptide transport system previously identified for this lobster species
(Thamotharan and Ahearn, 1996)
and for the vertebrate PEPT-1 carrier system
(Adibi, 1997
).
L-leucine was used in the present study as a potential inhibitor of
L-histidine transport since both amino acids are known to be
transported by the L-lysine transport protein, and in lobster
hepatopancreas this carrier process is strongly inhibited by
L-leucine (Ahearn and Clay,
1987a
). Copper was used in the present investigation as a
potential inhibitor of zinc transport as a result of published competitive
interactions between these two metals at the hepatopancreatic brush border
membrane (Chavez-Crooker et al.,
2001
).
Each of the experiments was subjected to statistical tests with analysis of variance (ANOVA). Both 3H-L-histidine and 65Zn2+ transmural transport kinetics were fitted to MichaelisMenten functions using Sigma Plot software (Systat Software Inc., Point Richmond, CA, USA). Slopes of time-course curves were determined with linear regression analysis functions (first point of treatment to last point of treatment) using Sigma Plot software. Results are reported as representative experiments that were repeated three times producing qualitatively similar results. Data points on individual figures represent mean values from three replicates ±1 S.E.M.
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Results |
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To ensure that the results reported in Fig. 1, and other time-course experiments, represented substrate-induced changes in transmural transfer of radiolabelled solutes, control experiments were performed with both 3H-L-histidine and 65Zn2+. Both labeled substrates were perfused separately through the intestinal lumen for 180 min without the addition of other interacting luminal molecules and the appearance rate of the respective isotope in the serosal medium monitored. In both cases linear rates of isotope appearance in the serosal medium were observed over the entire incubation interval with no tendency toward isotope equilibration between the media on both intestinal surfaces (data not shown).
Kinetics of transmural 3H-L-histidine transport in the presence and absence of luminal zinc and L-leucine
Because at least a portion of the transmural transport rate of
3H-L-histidine was significantly affected by both zinc
and the amino acid L-leucine, the involvement of a carrier-mediated
transport system appeared likely in the transfer of this amino acid across
intestinal tissues. Fig. 2
illustrates the effects of varying luminal
3H-L-histidine concentration on the rate of
mucosal-to-serosal transmural transport of the amino acid in the absence of
either zinc or L-leucine. As shown in
Fig. 2, the movement of this
amino acid across lobster intestine was a hyperbolic function of luminal amino
acid concentration (2.550 µmol l1) and followed
the MichaelisMenten equation:
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|
As shown in Fig. 1, there
was a marked increase in transmural 3H-L-histidine
transport when 20 µmol l1 zinc was added to the luminal
perfusate. To assess the nature of this stimulatory action of the metal on
amino acid transport, an experiment was conducted to determine the effects of
variable luminal zinc concentrations (2.550 µmol
l1) on the mucosal-to-serosal transmural transport rate of
20 µmol l1 3H-L-histidine.
Fig. 3 shows that a hyperbolic
relationship occurred between the transmural amino acid transport rate and
luminal zinc concentration and followed a modified MichaelisMenten
equation given below:
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|
L-Leucine (100 µmol l1) inhibited the stimulation of transmural 3H-L-histidine transport by luminal zinc (Fig. 1), suggesting that both amino acids may interact with a common membrane agency. An experiment was conducted examining the transmural transport kinetics of 3H-L-histidine in the presence and absence of both 20 µmol l1 zinc and 100 µmol l1 L-leucine. Fig. 4 shows that a hyperbolic, MichaelisMenten type, response occurred between 3H-L-histidine transport and luminal histidine concentration in the absence of either metal or L-leucine. Addition of 20 µmol l1 zinc to the mucosal perfusate increased the mucosal-to-serosal flux of the radiolabelled amino acid as before. However, addition of both 20 µmol l1 zinc and 100 µmol l1 L-leucine simultaneously to the perfusing mucosal medium abolished the stimulatory action of zinc on 3H-L-histidine transport. These data suggest that zinc stimulates carrier-mediated 3H-L-histidine transport by a system that is markedly inhibited by the presence of 100 µmol l1 L-leucine. In a separate control experiment, carrier-mediated, 20 µmol l1 3H-L-histidine transport in the absence of luminal zinc (control flux=0.037±0.01 pmol cm2 min1; N=3) was unaffected by the presence of 50 µmol l1 L-leucine (flux with inhibitor=0.044±0.003 pmol cm2 min1; N=3) or 100 µmol l1 L-leucine (flux with inhibitor=0.043±0.007 pmol cm2 min1; N=3) (data not shown).
|
Data presented in Table 1 summarize the effects of both 20 µmol l1 zinc and 100 µmol l1 L-leucine on the mucosa-to-serosa transport kinetic constants of 3H-L-histidine in lobster intestine. The data in this table indicate that the apparent binding affinity of the carrier mechanism involved in transport of 3H-L-histidine across the intestine was significantly (P<0.01) reduced by the presence of zinc (control=6.2±0.8; test=19±3 µmol l1), while the apparent affinity constant was not significantly different (P>0.05) when both zinc and L-leucine were present together in the mucosal medium (control=6.2±0.8; test=4.5±1.7 µmol l1). Similarly, the apparent maximal transport rate was significantly (P<0.01) increased (control=0.09±0.004; test=0.28± 0.02 pmol cm2 min1) by a factor of three when zinc alone was present in the mucosal medium, but no significant (P>0.05) increase in maximal transport rate (control=0.09±0.004; test=0.08±0.01 pmol cm2 min1) was observed when both zinc and L-leucine were added together in the perfusate.
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Kinetics of transmural 65Zn2+ transport in the presence and absence of luminal L-histidine and copper
To assess whether the transmural transport of 65Zn2+
across lobster intestine was influenced by L-histidine or copper
ions, an experiment was conducted examining the time course of
mucosal-to-serosal 20 µmol l1 65Zn2+ transport
in the presence or absence of luminal 20 µmol l1
L-histidine or 50 µmol l1 Cu+
(cuprous ions). Fig. 5
indicates that the transmural transport rate of 65Zn2+
across lobster intestine in the absence of either amino acid or copper was 0.1
pmol cm2 min1. This rate was increased
twofold to 0.19 pmol cm2 min1 when 20
µmol l1 L-histidine was perfused through the
intestinal lumen with the radiolabelled ion. When 50 µmol
l1 Cu+ was perfused through the intestine with 20
µmol l1 L-histidine and 20 µmol
l1 65Zn2+, the transmural transport
rate of the radiolabelled ion was reduced to 0.1 pmol cm2
min1, a value that was half that of the stimulated condition
in the presence of L-histidine alone, and equal to the transport
rate of 65Zn2+ in the absence of either amino acid or
copper.
|
Fig. 6 indicates that the
mucosal-to-serosal transport rate of 65Zn2+ across
lobster intestine was a hyperbolic function of luminal L-histidine
concentration following the Michaelis-Menten relationship:
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|
Fig. 7 shows the result of an experiment varying luminal zinc concentration on the kinetics of transmural transport of 65Zn2+ across lobster intestine in the presence and absence of 20 µmol l1 L-histidine, 50 µmol l1 cuprous ions (Cu+) and 50 µmol l1 cupric ions (Cu2+). In the absence of either L-histidine or copper ions, the transmural transport rate of 65Zn2+ followed the MichaelisMenten equation, as given in Equation 1. Addition of 20 µmol l1 L-histidine to the luminal perfusate doubled the apparent maximal transport rate of zinc and adding both 20 µmol l1 L-histidine and 50 µmol l1 Cu+ together abolished the stimulation by the amino acid alone. Addition of 20 µmol l1 L-histidine and 50 µmol l1 Cu2+ reduced 65Zn2+ transport to values significantly below those observed under control conditions. While a straight line function could be fitted to these data, Sigma Plot software indicated a better fit to the results with a hyperbolic relationship. The kinetic constants for zinc transport under each of these four conditions are displayed in Table 2. These results show that addition of L-histidine to the luminal solution doubled the apparent maximal transport rate of 65Zn2+ across the tissue without affecting the apparent binding affinity of the transport system for zinc itself. Addition of Cu+ ions to the mucosal surface of the intestine increased the apparent binding affinity of the transport system to 65Zn2+ and abolished the stimulation of apparent maximal 65Zn2+ transport rate in the presence of the L-histidine. Addition of Cu2+ ions to the perfusate blocked both L-histidine-stimulated 65Zn2+ transport as well as a portion of the L-histidine-independent 65Zn2+ transport.
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|
In order to assess whether differences in 65Zn2+ transport in the absence of L-histidine could be observed with cupric and cuprous ions, an experiment was conducted to see the effect of copper valence on zinc transport. In the absence of L-histidine the control transport of 20 µmol l1 65Zn2+ across the intestine was 0.056±0.007 pmol cm2 min1 (N=3; data not shown). Addition of 100 µmol l1 cuprous ions (Cu+) to the perfusate had no effect on transmural 20 µmol l1 65Zn2+ transport (0.064±0.007 pmol cm2 min1, N=3; data not shown), but 100 µmol l1 cupric ions (Cu2+) significantly (P<0.01) reduced the transfer of 20 µmol l1 65Zn2+ across the tissue (0.032±0.002 pmol cm2 min1, N=3; data not shown). These results show that the cuprous ion inhibited zinc transport when the latter was cotransported with L-histidine, but only cupric ion appeared to inhibit the transfer of 65Zn2+ in the absence of the amino acid.
Effects of the dipeptide, glycyl-sarcosine, on transmural 3H-L-histidine transport in the presence and absence of zinc
To test the hypothesis that zinc and L-histidine were complexing
in solution and being transported together across the lobster intestine as a
binary complex containing 2 L-histidine/1 zinc ion, the effects of
the dipeptide, glycyl-sarcosine, on the transfer of
3H-L-histidine were examined in the presence and absence
of luminal zinc. Time-course experiments showed that in the absence of either
zinc or dipeptide the transmural transport rate of
3H-L-histidine across lobster intestine was very slow
(e.g. 0.02 pmol cm2 min1; data not shown).
Addition of 20 µmol l1 zinc to the luminal solution
increased the transmural transport rate of labeled amino acid by a factor of 3
(0.06 pmol cm2 min1). When 20 µmol
l1 zinc and 100 µmol l1
glycyl-sarcosine were added together to the luminal solution, the transport
rate of 3H-L-histidine across the intestine dropped to
0.03 pmol cm2 min1), a value that was
approximately half that of the stimulated rate induced by zinc alone, but
still higher than the value observed in the control condition without either
zinc or dipeptide.
In order to determine the specific effect that the dipeptide,
glycyl-sarcosine, was having on the transmural transport rate of
3H-L-histidine across lobster intestine, the kinetics of
zinc-stimulated radiolabelled amino acid transport were observed in the
presence and absence of the dipeptide. Fig.
8 indicates that addition of 100 µmol l1
dipeptide to the luminal medium during transit of
3H-L-histidine resulted in changes in
KH (control=18.9±2.9; test=7.9±2.3 µmol
l1) and Jmax (control=0.3±0.02;
test=0.13±0.01 pmol cm2 min1).
These results suggest that the dipeptide inhibited the transmural transport of
L-histidine by a mixed type of inhibitor response
(Segel, 1975).
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Effect of luminal pH on transmural 3H-L-histidine transport in the presence of zinc
In order to further characterize the transport of
3H-L-histidine in the presence of zinc across perfused
intestine, the transmural transport of the amino acid was measured at two
luminal pH conditions: control pH (pH 7.1) and acidic pH (pH 6.1). As shown in
Fig. 9, addition of luminal
zinc significantly increased the transmural transport of the radiolabelled
amino acid and this transfer rate was further elevated when the perfusate pH
was lowered from 7.1 to 6.1. These results suggest that zinc-dependent
3H-L-histidine transport was pH sensitive.
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Effects of L-histidine, L-leucine and zinc on transmural transport of 3H-glycyl-sarcosine
Figs 10 and
11 describe transmural
transport of 100 µmol l1 3H-glycyl-sarcosine
in the presence and absence of two L-amino acids and zinc. Addition
of either L-histidine or L-leucine to the luminal
perfusate simultaneously with 3H-glycyl-sarcosine had no effect on
the transmural transport of the dipeptide (P>0.05). Furthermore,
transmural 3H-glycyl-sarcosine transport was not affected by the
presence (0.006±0.0004 pmol cm2
min1; data not shown) or absence (0.007±0.0006 pmol
cm2 min1; data not shown) of luminal zinc
in the absence of amino acids. However, addition of zinc and either amino acid
together to the luminal solution with the radiolabelled dipeptide, resulted in
highly significant reduction in dipeptide transport across the tissue
(P<0.01). These effects suggest that a bis-complex between two
amino acids and the metal ion competes with 3H-glycyl-sarcosine for
transport by the peptide transport protein.
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Discussion |
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In contrast to the apparent high specificity of the transport systems
accommodating the amino acid or ion alone, the shared transporter that
simultaneously transferred both L-histidine and zinc across the
intestine was affected by both L-leucine and copper.
Fig. 4 and
Table 1 show that addition of
100 µmol l1 L-leucine significantly
(P<0.01) reduced 3H-L-histidine transport in
the presence of 20 µmol l1 zinc by lowering both the
apparent Km and Jmax of the carrier
process. In the presence of L-leucine the kinetic constants were
not significantly different (P>0.05) than those under control
conditions lacking both zinc and L-leucine. Such results show that
L-leucine exerted a mixed inhibitory effect (modification in both
Km and Jmax) on zinc-stimulated
3H-L-histidine transport
(Segel, 1975). Data in
Fig. 7 and
Table 2 show a similar pattern
of effect. In this example, copper significantly (P<0.01) reduced
both apparent Km and Jmax of
65Zn2+ transport by the cotransport carrier process by
acting as a mixed inhibitor of L-histidine-stimulated
65Zn2+ transport.
Fig. 8 provides strong
evidence that the cotransport system in lobster intestine, accommodating
simultaneous L-histidine and zinc transport, is the dipeptide
transporter previously described for the hepatopancreas of this animal
(Thamotharan and Ahearn,
1996), which may be related to the vertebrate PEPT-1 gene system.
Additional support for this notion is provided in
Fig. 9, showing that an acidic
luminal condition stimulates transmural 3H-L-histidine
transport in the presence of zinc. The PEPT-1 transport system is
proton-stimulated and the transport of any substrates by that carrier system
would likely be enhanced by acidic conditions. The dipeptide glycyl-sarcosine
is a substrate of this transport mechanism in both invertebrates
(Thamotharan and Ahearn, 1996
)
and vertebrates (Adibi, 1997
;
Fei et al., 1994
; Thamotharan
et al.,
1996a
,b
).
Fig. 8 indicates a significant
(P<0.01) and mixed inhibitory effect of the dipeptide on
zinc-stimulated 3H-L-histidine transport, suggesting
that the dipeptide and amino acid interact with each other for the cotransport
process with zinc. If this is the case, then the cotransport process
transferring both L-histidine and zinc across lobster intestine
likely accommodates two L-histidine amino acids linked to the zinc
cation in a bis-complex, as described by Horn et al.
(1995
) and Horn and Thomas
(1996
), and in this
configuration sufficiently resembles dipeptides in solution to utilize a
transport system that normally would accommodate two amino acids linked by a
peptide bond. The dipeptide transporter (e.g. PEPT-1) has a very broad
specificity for peptides, accepting a wide range of amino acid substrates. The
role of the peptide bond between two amino acids in a peptide being
transported on PEPT-1 has not been examined, but this study suggests that it
may not be critical for the successful transfer of the peptide components to
the trans side of the membrane. All that may be needed for PEPT-1 to
transport two amino acids across a membrane may be that they are associated in
solution with either a peptide bond or as a bis-complex with a metal cation.
If this is the case, the dipeptide transport system may be a significant means
by which cells are able to accumulate essential metals from their
environment.
Supporting evidence for the role of PEPT-1-like transporters as the responsive agents for transmural transport of L-histidine or L-leucine across the lobster intestine in the presence of luminal zinc is shown in Figs 10 and 11. Neither L-histidine nor L-leucine added alone to the luminal perfusate were able to influence the transmural transport of 3H-glycyl-sarcosine, but when the complexing ion, zinc, was included in the luminal solution, a marked reduction in the transfer of dipeptide across the gut was observed. These data suggest that when zinc was present, bis complexes between the metal and either amino acid were able to occur in solution and that, once formed, these complexes were able to compete with the dipeptide for transport by the peptide carrier system.
The model shown in Fig. 12
illustrates the results of the present investigation and suggests a mechanism
that would allow the independent transport of both L-histidine and
zinc on highly specific carrier proteins and allow the shared transport of
both substrates on a PEPT-1-like dipeptide transport protein. The model
suggests an apical location of all three carrier proteins. The vertebrate
PEPT-1 dipeptide transporter has been localized to the brush border membrane
in vertebrate intestine (Adibi,
1997), and physiological studies with other animals such as
lobsters (Thamotharan and Ahearn,
1996
) and fish (Thamotharan et
al., 1996a
; Verri et al.,
2000
) have also confirmed this location for the analogous
transporter. The model shows that zinc likely occupies a separate binding site
on the cotransport protein than occurs for either amino acid in the bis
complex, since Cu+ or Cu2+ may inhibit zinc-stimulated
L-histidine transport by competing with zinc for this site
(Fig. 7). Similar inhibitory
interactions between metal components have been reported for cadmium and zinc
stimulation of L-histidine transport in human erythrocytes
(Horn and Thomas, 1996
).
Alternatively, Zn2+ and either Cu+ or Cu2+
may interact in solution and compete with each other as bis-forming substrates
with amino acids. L-Leucine and Gly-Sar are shown to inhibit
L-histidine binding to the amino acid binding sites in
Fig. 12, but not
L-histidine transport by the high specificity amino acid
transporter occurring on the same membrane, because the same amount of
L-histidine transport under control conditions (e.g. no zinc, no
L-leucine) occurred when both zinc and L-leucine were
present together (Fig. 4).
Lastly, zinc is shown to be transferred across the apical membrane by a highly
specific carrier protein that was not apparently inhibited by cuprous ions,
but was inhibited by cupric ions (Fig.
7; time-course data reported in the text). The model shows that
once inside the intestinal epithelial cell, the exit processes to the blood
for either L-histidine or zinc are unclear at the present time.
|
The nature of zinc-independent 3H-L-histidine
transport in lobster intestine is not known. As shown in Figs
2 and
4, in the absence of zinc
stimulation, 3H-L-histidine transport occurred by a
saturable mechanism that had a high apparent binding affinity of about 6
µmol l1. Previous studies with lobster hepatopancreatic
brush border membrane vesicles have characterized a number of amino acid
transport proteins that occur on the luminal membrane of this absorptive organ
and are responsible for the transapical transfer of L-alanine
(Ahearn et al., 1986),
L-lysine (Ahearn and Clay,
1987a
), L-glutamate
(Ahearn and Clay, 1987b
),
L-leucine (Ahearn and Clay,
1988b
), and L-proline
(Monteilh-Zoller et al.,
1999
). To date there has not been any specific study of a
carrier-mediated transport process for L-histidine in either
lobster hepatopancreas or intestine. The L-lysine transport system
described for hepatopancreas (Ahearn and
Clay, 1987a
) would be a likely candidate for
L-histidine transport, but this transporter is strongly inhibited
by L-leucine, and since Fig.
4 and the results of a control experiment involving
3H-L-histidine transport in the presence of 100 µmol
l1 L-leucine (reported in the text) both suggest
a minimal effect of L-leucine on
3H-L-histidine transport in the present study of lobster
intestine, it is unlikely that L-histidine was using the
hepatopancreatic L-leucine-inhibited L-lysine
transporter. Clearly, further studies are needed to clarify the mechanism by
which L-histidine is transported across lobster intestine in the
absence of metals.
The nature of L-histidine-independent
65Zn2+ transport suggested in
Fig. 12 and experimentally
described in Fig. 7 is
similarly unclear at the present time. Experimental data presented in this
report indicated that cuprous ions (Cu+) had negligible effects on
the transport of 65Zn2+ in the absence of
L-histidine, while cupric ions (Cu2+) were effective
inhibitors of zinc transport under these conditions. These results suggest
that L-histidine-independent 65Zn2+ transport may take
place by a divalent cation transport system that does not recognize ions of
other valences. Previous work supports the suggestion of a divalent
cation-specific brush border antiporter in crustacean hepatopancreas
(Aslamkhan and Ahearn, 2003).
This study found that both calcium and cadmium acted as strong
trans-stimulators of lobster hepatopancreatic brush border transport
of both 55Fe2+ and 59Fe2+,
implying a tight coupling between the divalent cation fluxes across this cell
border. Other studies with lobster hepatopancreas have also shown a coupling
between the uptakes of copper and zinc from dietary constituents and
intracellular calcium activities
(Chavez-Crooker et al., 2001
).
Interactions between the transport of zinc and calcium have also been recorded
for gastrointestinal epithelial cells of asteroid echinoderms
(Zhuang et al., 1995
). These
studies, and the present investigation, provide strong support for the
occurrence of an invertebrate gastrointestinal brush border transport system
that accepts a wide range of divalent cations, including both metals and
calcium, but the nature of this mechanism is still unclear, as is its
relationship to other metal-transporting membrane proteins from vertebrate
cells.
A number of membrane-bound transport proteins that transport zinc into or
out of cells have been cloned in mammalian tissues, and include members of the
ZIP or ZTL families (Gaither and Eide,
2001a,b
;
Cragg et al., 2002
) of zinc
uptake proteins, the ZnT transport group for zinc efflux from cells
(McMahon and Cousins, 1998
),
and the more generic DMT-1 heavy metal transporter that accepts a wide variety
of metal ions (Gunshin et al.,
1997
). It therefore appears that metals such as zinc may enter
cells in the absence of organic solutes by binding to either zinc-specific
transporters (e.g. ZIP or ZnT proteins), or transporters that are mainly used
by other cations (Zuang and Ahearn, 1996) but accept metals when they are
present (e.g. DMT-1, calcium transport proteins). At present it is not known
which of these mechanisms is responsible for zinc entry into lobster
intestinal epithelial cells in the absence of L-histidine as
defined in the present investigation. Future studies may help to clarify this
situation.
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Acknowledgments |
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References |
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