Contributions of different NaPi cotransporter isoforms to dietary regulation of P transport in the pyloric caeca and intestine of rainbow trout
New Jersey Medical School, Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA
* Author for correspondence (e-mail: Ferraris{at}umdnj.edu)
Accepted 10 March 2004
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Summary |
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Key words: NaPi cotransport, phosphorus, rainbow trout, pyloric caeca, intestine
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Introduction |
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In mammals, the main site of dietary P absorption is the proximal small
intestine (Danisi and Murer,
1991). In fish, however, little is known about the site of dietary
P absorption. In brook trout, serum 32P concentrations increased
rapidly in the first few hours following consumption of a calcium-free
synthetic diet containing 32P
(Phillips et al., 1961
). The
rapid increase of 32P in the blood was thought to be due to
transmural transport in vivo of Pi through the stomach. In carp, Pi
transport in vitro from mucosa to serosa occurred against a
concentration gradient, and this active uptake was greatest in the middle
intestine (Nakamura, 1985
).
Active Pi transport in rainbow trout was also higher in the proximal than
distal intestine (Avila et al.,
2000
). We have previously detected NaPi cotransporter mRNA and
protein in trout PC (Sugiura et al.,
2003
), but no functional study of the role of PC in dietary P
absorption and transport has been reported.
Despite the absence of knowledge on the caecal role of dietary P
absorption, the absorption of some other nutrients in PC is known.
Digestibility in vivo of 18 amino acids in trout PC was 6080%
in fish fed a fishmeal-based diet
(Dabrowski and Dabrowska,
1981). The contribution of PC to the total uptake capacity (of the
entire gut) for glucose, the dipeptide carnosine and nine amino acids was
6881%, which corresponded to the contribution of PC to the gut total
surface area in trout (
70%; Buddington
and Diamond, 1987
). These studies suggest that uptake per unit
surface area is similar in PC and intestine and that PC plays a dominant role
in total amino acid absorption in trout.
Dietary regulation of Pi transport and the NaPi-II transporter mRNA/protein
expression in the intestine has been studied in mammals
(Hattenhauer et al., 1999;
Huber et al., 2000
,
2002
;
Katai et al., 1999
) and fish
(Coloso et al., 2003
), but
nothing was known regarding Pi transport, NaPi-II expression or their dietary
regulation in PC. Since PC is a primary site of dietary nutrient absorption in
trout (Buddington and Diamond,
1987
), it is crucial to study its regulatory mechanism.
Rainbow trout is an important aquaculture species in many parts of the
world, and fed intensively with pellet feeds. Unfortunately, the absorption of
dietary P in contemporary commercial feeds appears to be only about
4050% (Hardy and Gatlin,
2002), meaning that the majority of dietary P is excreted into the
aqueous environment where P is the limiting nutrient for eutrophication.
Excessive discharge of P from aquaculture facilities, therefore, causes
excessive algal blooms, eventually leading to the destruction of ecosystems.
Knowledge of the mechanisms underlying absorption of dietary P may help
improve absorption of dietary P by the fish, and alleviate the environmental
burden of aquaculture.
We initially measured Pi and H+ concentrations in the lumen of the gastrointestinal (GI) tract, and determined the kinetics and energy-, Na- and pH-dependence of Pi transport in PC. We then compared partial sequences and sites of expression of NaPi cotransporter in the GI tract, and dietary regulation of P uptake between PC and intestine. Finally, we correlated NaPi cotransporter gene expression and function to traditional indices of dietary P deficiency. We found that PC is the major site of dietary P absorption, and that the absorption is modulated by several factors, including temperature, luminal pH, luminal (dietary) Pi concentration and fish P status. We also found that the nucleotide sequence and dietary regulation of NaPi cotransporter in PC differ from those in the intestine.
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Materials and methods |
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Fish were killed 6 h after feeding by severing the spinal cord immediately posterior to the cranium. PC, intestine and blood samples were collected immediately. PC and intestine were gently perfused with an ice-cold Krebs Ringer buffer, kept in the ice-cold buffer, and used for the uptake assay within 1 h, or immediately stored in the RNAlater solution (Ambion, Inc., Austin, TX, USA) for later measurements of NaPi cotransporter mRNA abundance. Blood samples were collected from the caudal peduncle, and the serum was separated within 1 h by centrifugation (12 000 g, 5 min) and stored (20°C) until analysis. Bone samples (anterior 1/3 of the vertebral column) were collected from whole fish stored at 20°C. Fish were briefly heated to 8090°C, and skeletal muscle was removed. The vertebral column was washed in warm tapwater, dried, defatted (methanol:chloroform=1:1, v/v), and redried to constant mass.
Diet composition
Commercial trout feed used in the uptake study had the following analytical
composition (%, dry basis): crude protein 46.4; fat 21.8; crude fiber 2.6; ash
10.39; Ca 2.45; total P 1.50; available P 0.716. Experimental LP and HP diets
used in the regulation study contained the following ingredients (%, dry
basis): commercial trout feed (acid-washed) 70; wheat gluten 5; egg albumin 5;
wheat flour 10; soybean oil 10. To this mixture were added vitamin and mineral
premix 25 g kg1, CaCO3 30 g
kg1, NaH2PO4·H2O 38
g kg1 (HP diet only), and NaCl 16.4 g kg1
(LP diet only). The dough was cold-extruded, air-dried and crumbled to make
pellets. The diets had the following analytical compositions (%, dry basis):
total P 0.27 (LP) or 1.30 (HP); available P 0.07 (LP) or 0.84 (HP); total Ca
1.82 (LP) or 1.83 (HP). The available P contents were previously determined in
a separate experiment, following a standard method using chromic oxide as a
marker (Bolin et al.,
1952).
Pi uptake assay
Tissue Pi uptake rates were determined in vitro according to
Karasov and Diamond (1983)
modified by Avila et al.
(2000
). Briefly, each sleeve of
PC (1 cm long) was carefully everted and tied to a stainless steel rod of an
appropriate diameter. The sleeve was then pre-incubated for 5 min at 15°C
in a Ringer solution gassed with 99%O2/1%CO2. The Ringer
solution had the following composition (mmol l1): NaCl
136.6; KCl 4.83; CaCl2 1.53; Hepes 5. The final pH was adjusted to
7.37.4 with HCl or KOH (15°C). To this,
NaH2PO4 was added as needed, replacing a portion of NaCl
on an iso-osmotic basis. The sleeve was incubated at 15°C in the Ringer
solution (10 ml) for 5 min with vigorous stirring (1200 r.p.m.) to reduce
unstirred layer effects. All the incubation (Ringer) solutions contained
H332PO4 (Perkin Elmer Life Sciences, Boston,
MA, USA) and 1,2-3H polyethylene glycol (3H-PEG),
(Perkin Elmer). The 3H-PEG was added as a non-absorbable inert
polymer to correct adhering fluid on the tissue surface. The tissue
radioactivity was determined for 32P and 3H using a
liquid scintillation counter (LS 7800, Beckman Instruments, Irvine, CA, USA).
We chose a 5 min incubation period since, in a preliminary assay, this
duration was long enough to measure Pi uptake accurately and uptake was linear
within this incubation duration. The initial and final radioactivity
(32P and 3H) of the incubation media was the same in all
experiments.
The inhibition assay was conducted twice
(Table 1). All the incubation
media, except that containing excess unlabelled Pi (P++), contained only
tracer Pi (32P as H3PO4) in order to measure
primarily the active component of Pi transport. The tracer 32P
concentration was 0.284 nmol l1 (experiment 1) and 0.170
nmol l1 (experiment 2). Two Na+-free media were
prepared in which choline chloride or KCl replaced all NaCl in the Ringer on
an iso-osmotic basis. The pH was adjusted to 7.37.4 with KOH. Three
potential inhibitors of Pi uptake were also tested: (i) an inhibitor of
Na+/K+ ATPase, ouabain, 5 mmol l1
(Lucu and Flik, 1999), to
determine dependence of Pi uptake on Na+ gradient across the apical
membrane; (ii) a known specific inhibitor of mammalian NaPi, phosphonoformic
acid (PFA), 5 mmol l1
(Loghman-Adham, 1996
), to
determine involvement of NaPi in Pi uptake; and (iii) an inhibitor of
cytochrome c oxidase in the mitochondrial respiratory chain, sodium
azide (NaN3), 10 mmol l1
(Utoguchi et al., 1999
), to
determine ATP-dependence of Pi uptake. All the inhibitors were purchased from
Sigma-Aldrich Co. (St Louis, MO, USA). Excess P (P++) was also added to
determine whether tracer P uptake by trout PC is carrier-mediated. Trout can
survive at freezing temperature, hence the effect of a colder temperature was
assessed by determining uptake in an ice-cold (2°C) medium. We then
estimated the temperature coefficient Q10 from the following
equation:
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Luminal pH and Pi concentration
Luminal contents were collected immediately after killing fish (6 h after
feeding with commercial trout feed). Surgical clamps were used to separate the
gut sections before dissecting the gut. Luminal contents were immediately
centrifuged after collection (12 000 g, 5 min) in microfuge
tubes (1.5 ml) to obtain luminal fluid. The pH of the fluid was measured
immediately after centrifugation using pH strip paper (colorpHast, EM Science,
Gibbstown, NJ, USA) calibrated to standard buffer solutions. The pH of the
adherent fluid (fluid remaining on the tissue after luminal fluid was
collected) was determined by applying the pH strip paper directly onto the
luminal surface of the dissected tissue. The remaining luminal fluid was
stored (20°C) in microfuge tubes. The Pi concentration was
determined (Taussky and Shorr,
1953) on the frozen-stored samples after they had been thawed,
acidified (5% TCA) and completely solubilized. Fish used for luminal pH and
[Pi] measurements were not used for other experiments.
Serum Pi and bone P concentrations
Serum Pi and bone P concentrations of fish fed LP or HP diets were
determined to verify the P status of the fish. The serum Pi was determined on
the frozen-stored samples (Taussky and
Shorr, 1953). Bone samples (defatted and dried) were ashed at
550°C overnight, acid-solubilized (hydrochloric:nitric acid, 1:1),
partially neutralized, diluted and analyzed for P
(Taussky and Shorr, 1953
).
Degenerate RT-PCR and subcloning
Total RNA was extracted from RNAlater-stabilized tissues using Trizol
reagent (Invitrogen, Carlsbad, CA, USA), and reverse-transcribed using
NaPi-specific degenerate primers designed from consensus sequences of other
species (forward: 5'-GCTGGIGAYATCTTCMAGG-3', reverse:
5'-AAGTGRCASARIGCAATCTG-3', corresponding to nt positions
344362 and 14031422, respectively, in the flounder sequence;
GenBank accession number U13963). The PCR product of the expected size (1079
bp) was gel-purified, subcloned and sequenced (GenBank accession number
AY500241).
RT-PCRSouthern blot
Specific primers were designed from a unique region where the two closely
related NaPi cotransporters have different nucleotide base sequences. Thus,
the primers amplified either intestine-type NaPi (I-NaPi) or PC-type NaPi
(PC-NaPi) cotransporters, but not both. This specificity was verified by
amplifying plasmid clones containing either the I-NaPi or PC-NaPi insert
(result not shown). The forward primer was common
(5'-GTCTTCTGGATTGCTGGAGGTC-3', corresponding to nt positions
469490 in the flounder sequence), and only the reverse primers
differed, although they were derived from the same corresponding position
(I-NaPi: 5'-GGTCAGGCAGGTTAGCATAGG-3', PC-NaPi:
5'-GGTCAGGCAGGTTAGCAAAAAC-3', corresponding to nt positions
10111031 and 10101031, respectively, in the flounder sequence).
ß-actin primers were as follows: forward:
5'-ACATCAAGGAGAAGCTGTGCTAC-3', reverse:
5'-TACGGATGTCCACGTCACAC-3', nt positions 683705 and
903922, respectively, in rainbow trout ß-actin sequence (GenBank
accession number AF157514).
Total RNA (0.5 µg) from PC and the proximal intestine was
reverse-transcribed (RT) using oligo-dT20 to make cDNA from mRNA,
and 1.0 µl of this solution was PCR-amplified using either the
I-NaPi-specific primer or the PC-NaPi-specific primer for 14 cycles at
94°C 30 s, 63°C 30 s, 72°C 60 s, per cycle. ß-actin served as
the control housekeeping transcript, being independent of dietary P intake or
NaPi cotransporter distribution, and was amplified for 7 cycles at 94°C 30
s, 58°C 30 s, 72°C 60 s, per cycle. I-NaPi, PC-NaPi and ß-actin
mRNA abundances were determined from the same RT preparations. Possible
genomic DNA contamination was negligible since the number of PCR cycles was
small. PCR products were separated on a 1% TAE-agarose gel, and
Southern-transferred onto Hybond-NX nylon membranes (Amersham Biosciences,
Piscataway, NJ, USA) according to a standard protocol
(Brown, 1995). A mixture of
pure cDNAs (I-NaPi, PC-NaPi and ß-actin, on an equimolar basis) was
labeled with 32P-dCTP using Rediprime II DNA labeling system
(Amersham). The membranes were pre-hybridized in FPH solution (5x SSC,
5x Denhardt solution, 50% formamide, 1% SDS, and 100 µg yeast tRNA
ml1) for 2 h at 42°C, to which the labeled probes were
added and hybridized overnight at 42°C. The membranes were washed twice at
42°C with 2x SSC0.1% SDS for 30 min each, then once at
50°C and 23 times at 60°C with 0.1 x SSC0.1% SDS
for 30 min each, and exposed to BioMax MR film (Kodak, Rochester, NY, USA) for
612 h at 80°C.
The RT-PCR-Southern blotting procedure was chosen to quantitate I-NaPi and PC-NaPi abundance because northern blots might not clearly distinguish between the two closely related transcripts. Since the number of PCR amplification cycles was very low, there was no risk of reaching the asymptote of the amplification plateau indicating the end of PCR-induced exponential increases in product concentration. To validate this procedure, I-NaPi mRNA abundance was also quantified by northern blot in the proximal intestine where PC-NaPi was almost absent, and the result was almost identical to that of RT-PCR-Southern blot (data not shown).
Statistical methods
Values are means ± S.E.M. (N). The treatment
means were compared by a two-tailed t-test (for two treatments) or a
one-way ANOVA followed by the NewmanKeuls multiple comparison test (for
three or more treatments). The population variances of uptake and
concentration data were tested by Bartlett's test, and when the variances were
heterogeneous at P<0.05, the data were log-transformed before
analysis of variance (ANOVA). The kinetic parameters of Pi uptake were
determined by nonlinear regression using the MichaelisMenten equation:
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Results |
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Total Pi transport continued to increase as the Pi concentration in the
incubation medium increased (Fig.
2A). The increase was linear at Pi concentrations higher than 1
mmol l1. The slope of the linear component
(Kd=0.039±0.002 min1) was similar
to the slope (0.042±0.008) of uptakes determined in the presence of the
NaPi-competitive inhibitor PFA; both slopes were assumed to represent passive
transport. When the linear component was subtracted from the total Pi
transport, the remainder was the saturable component of Pi transport. The
saturable component, with a
max=64.6±7.6 nmol g
tissue1 min1 and a
Km=0.474±0.199 mmol l1, was
assumed to represent the carrier-mediated component of transport. These
kinetic parameters calculated by nonlinear regression correlated well with
those estimated by the EadieHofstee plot
(Fig. 2B).
|
Tracer Pi uptake was not inhibited by replacement of NaCl by choline chloride or KCl in the preincubation and incubation media (Fig. 3A). Preincubation and incubation of the tissue in ouabain also had no effect on Pi uptake. Addition of excess non-labeled Pi (10 mmol l1) dramatically reduced but did not abolish tracer Pi uptake. Addition of PFA and, to a lesser extent, NaN3 also significantly reduced Pi uptake. These observations were confirmed in another experiment with slightly different conditions and using a different batch of fish (Fig. 3B).
|
Tissues incubated at 2°C had lower uptake rates (54.5%, P=0.04) compared with those incubated at 15°C. The Q10 of carrier-mediated Pi uptake was estimated to be 1.630±0.144 (N=8). Pi uptake in Na+-containing medium increased markedly between pH 7 and 9 (Fig. 4). Pi uptake in Na+-free medium was significant but did not change significantly between pH 4 and 9.
|
The nucleotide sequence of NaPi cotransporter mRNA isolated from trout PC
(GenBank accession number AY500241) was different from those of all previously
known NaPi cotransporter mRNAs, including that of the trout intestinal NaPi
cotransporter mRNA. The PC-NaPi cotransporter mRNA, however, was most closely
related to trout I-NaPi mRNA (92% identity in nucleotide base sequence),
followed by flounder kidney/intestine NaPi cotransporter mRNA (80%), and carp
kidney NaPi-IIb2 transporter mRNA (77%). The identity of PC-NaPi mRNA with
trout renal NaPi cotransporter mRNA was low (67%). The unique bases were
evenly distributed over the sequenced stretch (1.08 kb), and were mostly
conservative substitutions.
In the stomach, neither I-NaPi nor PC-NaPi mRNA was detected (Fig. 5). In PC, PC-NaPi mRNA was generally more abundant than I-NaPi mRNA. The total abundance of these two isoforms, however, was lower in PC than in the intestine. In the proximal intestine, I-NaPi mRNA was very abundant, whereas only a trace amount of PC-NaPi mRNA was detected. In the distal intestine, I-NaPi mRNA was abundant, whereas PC-NaPi mRNA was not detected.
|
In both the intestine and PC, total Pi-uptake was independent of dietary P levels at days 2 and 5 (Fig. 6). By day 20, however, the uptake was markedly higher in LP than HP fish in the intestine (P=0.002) but not in PC (P=0.35). Pi uptake in the intestine increased in LP (day 2 < day 5 < day 20, P=0.0003) and decreased in HP (day 2 > day 5 > day 20, P=0.007) over time, whereas in PC, the uptake increased only modestly in LP (P=0.14) and did not change significantly (P=0.76) in HP. Pi uptake per wet mass of tissue, however, was always higher in PC than in the proximal intestine.
|
At day 20, serum Pi concentration was much less (P=0.0005) in fish fed LP (2.44±0.26 mmol l1, N=5) than in fish fed HP (4.86±0.35 mmol l1, N=5). Bone P levels were also lower (P=0.001) in fish fed LP (10.12±0.12%, N=5) than in fish fed HP (10.75±0.05%, N=5). Serum Pi and bone P concentrations correlated well (P=0.00010.0048) to I-NaPi mRNA abundance in both the proximal intestine and PC (Fig. 7A,B), but not to PC-NaPi mRNA abundance in PC (P=0.04 and 0.15, respectively) (Fig. 7A,B). Serum Pi and bone P concentrations also correlated to the Pi uptake rate in the intestine (P=0.001 and 0.007, respectively), but not in PC (P=0.4 and 0.7, respectively) (Fig. 7C,D). In the proximal intestine (P=0.0003), but not in PC (P=0.4), the abundance of I-NaPi mRNA was positively correlated with the rate of Pi uptake. In PC, the abundance of PC-NaPi mRNA was weakly correlated to the rate of Pi uptake (P=0.048).
|
In the proximal intestine, only trace amounts of PC-NaPi mRNA could be
detected in both LP and HP fish. In the proximal intestine, I-NaPi mRNA
abundance was higher (P=0.0002) in LP than in HP fish (1.5-fold)
(Fig. 7A,B). In PC, I-NaPi mRNA
abundance was markedly higher (P=0.0006) in LP than in HP fish
(49-fold), whereas that of PC-NaPi was only slightly higher
(P=0.01) in LP than in HP fish (
2-fold)
(Fig. 7A,B). Total NaPi
cotransporter mRNA abundance (relative to beta-actin mRNA) was higher in the
intestine than in PC, especially in HP fish
(Fig. 7A,B). Pi uptake rate
(g1 tissue), however, was higher in PC than in the
intestine, especially in HP fish (Fig.
7C,D).
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Discussion |
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Kinetics of Pi transport in trout PC
The present study shows that brush border Pi uptake in trout PC, as in
trout intestine, is a carrier-mediated active process. Excess `unlabeled' Pi
reduced tracer Pi uptake by 85% in PC
(Fig. 3) and
95% in the
intestine (Avila et al., 2000
).
PFA reduced both PC and intestinal Pi uptake by
70%. These findings
directly demonstrate the general similarity of these transporters to those of
mammalian intestinal and renal carrier-mediated Pi uptake known to be
inhibitable by PFA. Passive Pi uptake estimated from the slope was almost
identical to that estimated from PFA incubation at low Pi concentrations
(<1 mmol l1) (Fig.
2), suggesting that most of the carrier-mediated Pi transport in
PC is PFA-sensitive or NaPi-mediated transport. Slightly higher passive Pi
uptake estimated from PFA than that estimated from the slope at higher Pi
concentrations (510 mmol l1;
Fig. 2) suggests that the
inhibition of active Pi transport by PFA was incomplete.
Sodium azide clearly reduced Pi uptake by 60%, indicating the energy
(ATP) dependence of Pi uptake in PC. The Q10 of tracer Pi transport
was 1.6, indicating, as with all energy-dependent processes, its sensitivity
to changes in temperature. None of these inhibitors or ice-cold incubation,
however, inhibited Pi uptake completely.
There are modest differences in Pi transport kinetics between intestine, as
reported previously (Avila et al.,
2000), and PC from the present study. The Km
and
max values are,
respectively, 2.5 and 3.4 times higher in the intestine (1.2 mmol
l1, 220 nmol g1 min1)
than in PC (0.474 mmol l1, 64.6 nmol g1
min1). These differences may be due to differences in the
transporters (see below) or to developmental stages, as the intestinal Pi
transport (Avila et al., 2000
)
was determined using much smaller fish (11 g body mass). The
Km of PC, however, is similar to the values reported for
mammalian intestine (Danisi and Murer,
1991
).
As shown by RT-PCR-Southern blotting, both PC-NaPi and I-NaPi cotransporters are present in PC, and thus the carrier-mediated active Pi uptake must be considered as the sum of both transporters. Although, in PC, PC-NaPi is generally more abundant than I-NaPi, the contribution of I-NaPi to active Pi uptake can be significant, especially under dietary P restriction where I-NaPi mRNA abundance in PC dramatically increases (Fig. 7A,B).
Na+-independence
Incubation in Na+-free medium decreased intestinal Pi uptake by
>90% (Avila et al., 2000)
but did not decrease Pi uptake in PC. The Pi uptake in PC was the same whether
extracellular Na+ was substituted by either extracellular
K+ or choline, or whether intracellular Na+ was
increased by ouabain, thereby eradicating the Na+ gradient required
for Pi entry into the cell. Pi uptake increased dramatically at high pH in
Na+-containing, but not in Na+-free, medium. Hence, the
Na+-dependency is pH dependent.
The molecular basis of the difference in Na+-dependency must be
the difference in amino acid sequence between PC-NaPi and I-NaPi. Putative
amino acids responsible for the difference in Na+-affinity (though
not Na+ dependency) between renal and intestinal isoforms in
mammalian NaPi-II (de la Horra et al.,
2001), however, differ from those corresponding amino acids in
trout PC and intestine. In zebrafish and flounder kidneys
(Graham et al., 2003
), the
corresponding amino acids in the NaPi cotransporter sequence that are thought
to confer Na+-affinity are the same as those in trout. However,
both zebrafish and flounder NaPi-IIb are Na+-dependent. Also, trout
intestinal NaPi-II is Na+-dependent
(Avila et al., 2000
).
Na+-independent, active (carrier-mediated) Pi transport has been
reported in rat and chick intestinal basolateral membrane, and in dog renal
basolateral membrane (Danisi and Murer,
1991
). Thus, PC-NaPi may be related to the mammalian and avian
basolateral Pi transporter.
pH-dependence
When Na+ is not required to transport Pi in trout PC, what is
the alternative cation in the transport system? In ruminant duodenum, where
the pH of the digesta is quite acidic, H+ rather than
Na+ drives brush border Pi uptake
(Shirazi-Beechey et al.,
1996). In goat jejunum, Pi uptake was stimulated
60% when pH
was decreased from 7.4 to 5.4, but this effect was abolished in the absence of
Na+ (Schroder and Breves,
1996
). In frog and rabbit intestine, H+ stimulated
glucose transport in Na+-free medium
(Hoshi et al., 1986
). When
expressed in Xenopus laevis oocytes, the intestinal sodium-glucose
cotransporter (SGLT1) is capable of acting both as a low affinity/high
capacity transporter and a high affinity/low capacity transporter, depending
on the driver cation (Hirayama et al.,
1994
). Thus, in the duodenum and proximal jejunum where the pH of
chyme is quite acidic and the glucose concentration high, SGLT1 can act as a
low affinity/high capacity glucose transporter using the proton gradient.
These studies suggest that H+ can substitute for Na+ as
the driver cation under Na+-free or Na+-limiting
conditions.
Pi uptake in PC markedly increased at alkaline pH, and this increase was
strictly Na+-dependent. This pH dependency is similar to that of
the mammalian renal NaPi cotransporter isoform that is
Na+-dependent and has higher transport rates at alkaline pH
(Hilfiker et al., 1998;
Murer et al., 2001
). It is
different from that of the mammalian intestinal NaPi cotransporter isoform,
which is also Na+-dependent but has higher transport rates at
neutral to acidic pH (Berner et al.,
1976
; Borowitz and Ghishan,
1989
; Danisi et al.,
1984
; Lee et al.,
1986
; Tenenhouse,
1999
; Xu et al.,
2002
). In chick, pig and sheep intestine, however, Pi transport
rate is higher at alkaline pH than acidic pH
(Danisi and Murer, 1991
), which
is in agreement with our data in trout PC and also with data from the
intestinal and renal NaPi cotransporter isoforms of zebrafish and flounder
(Forster et al., 1997
;
Graham et al., 2003
;
Kohl et al., 1996
;
Nalbant et al., 1999
). The
amino acid motifs that apparently confer the pH dependency in the NaPi-II
intestinal and renal transporter isoforms of mammals (de la Horra et al.,
2000) are different from the corresponding sequence in trout PC and intestinal
NaPi cotransporter and in zebrafish NaPi cotransporter isoform found in both
kidney and intestine. Since at pH levels higher than 7.2, divalent ions
(HPO42) will be the dominant species over
monovalent ions (H2PO4), the preferred
ions for the fish NaPi cotransporter isoforms and mammalian renal NaPi
cotransporter isoform may be the divalent form, whereas for mammalian
intestinal transporter isoform, the preferred species may be the monovalent
form. Further study is needed to clarify the pH and Na+ dependency
of Pi transport systems.
One feature of trout PC is that the luminal pH is fairly high, and in the intestine it becomes even higher toward the distal intestine. The differences in luminal pH and Pi concentration between PC and the intestine might have led to the development of two functionally different Pi transporters. It is unclear why the caecal pH of trout is well below the optimal pH for the transporter. Since in PC, both PC-NaPi and I-NaPi are present, it is possible that only I-NaPi is pH sensitive, whereas PC-NaPi is not. Our experimental procedure did not distinguish this.
Differences in patterns of regulation
The PC-NaPi mRNA abundance is only weakly regulated by dietary P. In
contrast, I-NaPi mRNA abundance is tightly regulated by dietary P, and the
diet-induced difference is inversely but linearly proportional to diet-induced
changes in serum Pi and bone P levels. Hence, an abundance of I-NaPi, but not
PC-NaPi, indicates the P status (or adequacy of dietary P intake) of fish.
Also, an abundance of I-NaPi and PC-NaPi mRNA in the same fish (using the same RT preparation) were only weakly correlated with one another. These differences in regulation of mRNA abundance lead to differences in regulation of function. In the intestine, where I-NaPi is predominant, saturable Pi uptake is diet-dependent, whereas in the PC, where PC-NaPi is predominant, saturable Pi uptake is mostly independent of diet (Fig. 6).
At the physiological luminal Pi concentration of 20 mmol
l1, when trout are fed P-sufficient diets, the contribution
of active, carrier-mediated Pi transport to the total Pi transport in PC is
only about 7.7%. However, under conditions of chronic dietary P restriction,
there can be a higher max
with lower luminal P concentrations, which increases the significance of
active Pi transport in PC. Although this kind of adaptive upregulation was
remarkable at a molecular level (i.e. I-NaPi mRNA abundance in PC increased
eightfold at day 20 in LP fish), the functional difference between LP and
HP fish in PC was not pronounced compared with that in the intestine
(Fig. 7), suggesting that Pi
uptake in PC is mediated mainly by PC-NaPi cotransporter.
Because the diffusive component predominates at high luminal Pi
concentrations, the absorption of Pi in PC is poorly regulated, and there
might be little functional significance of PC-NaPi cotransporter in fish
consuming commercial fish feeds of normal-to-high P content. The predominant
passive Pi transport also explains our previous finding of high in
vivo fractional P absorption even at excess dietary P intakes
(Sugiura et al., 2003), and
our previous observation of acute tetany (typical sign of hypocalcemia) when
trout were fed a low-calcium high-P diet (S. H. Sugiura, unpublished
observation).
Role of PC in dietary P absorption
Substituting the prevailing luminal Pi concentration
(Fig. 1) and kinetic constants
of Pi uptake in PC (Fig. 2) and
in intestine (Avila et al.,
2000) into Equation 2, one can calculate that the total rate of
transport g1 PC is about 850 nmol min1,
whereas transport g1 intestine is 240 nmol
min1. Since mass is directly proportional to mucosal surface
area (Ferraris and Diamond,
1989
), and PC represents 70% of surface area
(Buddington and Diamond, 1987
),
approximately 89% of total Pi absorption takes place in PC. Clearly, Pi
absorption through the PC is more significant than that through the intestine.
About 92% of PC uptake is diffusive and cannot be regulated, which suggests
that there are limitations to physiological or endocrinological approaches
towards enhancing intestinal or caecal Pi uptake in trout, since only
carrier-mediated transport can be physiologically regulated.
In the present study, localization of NaPi mRNA within a caecum was not studied. The I-NaPi and PC-NaPi cotransporters may be uniquely distributed within a caecum, and the distribution of I-NaPi, but not PC-NaPi, could be modulated by dietary P intake or P status of fish. The distribution of PC-NaPi among PC in the same fish may also be different, since some PC are located adjacent to the pyloric sphincter of the stomach, which could receive more acidic chyme than the PC that are located almost in the middle part of the proximal intestine. The large within-treatment variance in the uptake assay might be partly explained by the possibly unique adaptation of each caecum, depending on the anatomical location.
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