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Am J Physiol Regul Integr Comp Physiol 283: R296-R302, 2002; doi:10.1152/ajpregu.00760.2001
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Vol. 283, Issue 2, R296-R302, August 2002

Phosphate transport in the duodenum and jejunum of goats and its adaptation by dietary phosphate and calcium

Korinna Huber, Christina Walter, Bernd Schröder, and Gerhard Breves

Department of Physiology, School of Veterinary Medicine, D-30173 Hannover, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endogenous Pi recycling is a characteristic feature of the P homeostasis in ruminants. A pronounced salivary Pi secretion into the rumen is balanced by a high intestinal Pi absorption and an almost complete renal Pi reabsorption. In monogastric animals, the major Pi transport mechanism across the apical membrane of the enterocyte is an Na+-dependent transport mediated by NaPi cotransporter type IIb. In ruminants, an Na+-, as well as an H+-dependent, Pi transport system seems to exist in the small intestines. Therefore, morphological localization, type of ionic dependence, and ability to adapt to dietary P or Ca restriction of duodenal and jejunal Pi transport were characterized in goats. In the duodenum, there was an H+-dependent, Na+-sensitive Pi transport system that did not belong to the NaPi type II family and was not influenced by dietary P or Ca restriction. In contrast, in the jejunum, there was an Na+-dependent, H+-sensitive Pi transport mainly mediated by NaPi IIb. P restriction stimulated the NaPi IIb protein expression, resulting in higher Pi transport capacity.

NaPi cotransporter type IIb; proton-sensitive phosphate transport; ruminants


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN RUMINANTS, PHOSPHATE (Pi) is essential for both the host animal and the rumen microbes. A substantial proportion of microbial requirement is met by a substantial endogenous recycling of Pi, which is maintained by salivary Pi secretion and respective intestinal Pi absorption. In addition, an almost complete renal Pi reabsorption prevents urinary Pi loss.

In the animal kingdom, membrane transport systems for Pi are key elements in maintaining Pi homeostasis in the organisms. Two Na+-Pi cotransporter families are well described in their functional and structural properties for mammalians (16). Pi absorption in the small intestines of monogastric animals is mainly achieved by a secondary active Na+-coupled Pi cotransporter located in the brush-border membrane of enterocytes. This apical Pi transport is stimulated by dietary P restriction and calcitriol (3). In ruminants, the mechanism of intestinal Pi transport is obviously more complex than in monogastric species. In the duodenum of sheep, an Na+-independent, proton-driven Pi transport has been described (13). Destruction of the inwardly directed H+ gradient into duodenal brush-border membrane vesicles (BBMV) inhibited Pi uptake, suggesting the presence of an H+-Pi cotransport system. On the other hand, for sheep and goat jejunum, an Na+-dependent Pi transport system has been reported (14, 15). Stoichiometry of this Na+-Pi transport suggested an interaction of two or more Na ions with one inorganic P ion at pH 7.4. The structural identification of the jejunal Na+-Pi transporter in goats was achieved by RT-PCR cloning (5) with primers homologous to the NaPi IIb nucleotide sequence cloned from mouse duodenum (4). In the jejunum of goats, an Na+-Pi cotransporter of type IIb was expressed with high homology to murine intestinal NaPi IIb, because mouse-specific NaPi IIb antibody was able to detect goat NaPi IIb protein in jejunal brush-border membranes (5). Goat transporter affinity for Pi (Km 0.03 ± 0.08 mM) was in the same range as that described for murine NaPi IIb (Km 0.05 mM), but the affinity for Na+ was quite different: Km 5.9 ± 0.7 mM in goat NaPi IIb and ~30 mM in mouse NaPi IIb. The higher sodium affinity of goat NaPi IIb can partly explain the higher Pi absorption capacity in goat jejunum compared with that of monogastric animals.

From these functional and structural studies it can be assumed that Pi transport in goat jejunum is almost completely Na+ dependent, but it is still unclear to what extent this Na+-dependent transport is mediated by NaPi IIb cotransporters.

Low dietary P significantly increased the Pi transport capacity in goat and sheep small intestines in both the H+-Pi transport system in the duodenum (13) as well as the Na+-Pi transport system in the jejunum (15). The upregulation of Na+-Pi cotransport in monogastric animals was based on an increase in the specific NaPi IIb protein without increasing amounts of NaPi IIb mRNA, indicating a posttranscriptional process for adaptation to dietary P restriction (3).

It is still unknown how Pi transport systems in small ruminants adapt to dietary P restriction. For caprine NaPi IIb cotransporter, the molecular biological tools are now available to study its expression at the mRNA and at the protein level. Therefore, it can be clarified whether the process of adaptation to dietary P restriction in goats is mediated by NaPi IIb.

It was the aim of the present study to characterize the properties of duodenal and of jejunal Pi transport mechanisms in goats influenced by dietary P or Ca restriction. In contrast to monogastric animals, intestinal Pi transport is mediated in goats by two different transport systems. These transporters are distinct in morphological localization, in their type of ion dependency, and in their ability to adapt to dietary P restriction.


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Feeding

The protocol of the animal treatment was approved and its execution supervised by the animal welfare officer of the School of Veterinary Medicine, Hannover.

Male White Saanen goats (4-5 mo old) were subdivided into three groups with different feeding regimens with adequate or reduced P or Ca supply. Each group of up to 12 animals was housed separately. Water was available at all times. In the control group, daily P and Ca intake was 1.8 g P and 6.1 g Ca; in the P-restricted group (-P), the intake was 0.8 g P and 5.9 g Ca; and in the Ca-restricted group (-Ca), the intake was 1.9 g P and 2.9 g Ca. Reduction of P and Ca supply was maintained for at least 6-8 wk. Plasma samples were obtained to define P status of the animals. In general, the experimental design was equivalent to previous studies (11, 14).

Animals were killed by stunning using a captive bolt pistol and bled from the carotid arteries. Duodenal and mid-jejunal segments were obtained within 3-5 min after slaughtering. After being rinsed with ice-cold saline (0.9% NaCl wt/vol), the mucosa was stripped off underlying muscle layers, frozen in liquid nitrogen, and stored at -80°C for structural and functional analyses (preparation of tissues for immunohistochemistry, see below). For Ussing chamber experiments, duodenal segments were rinsed with ice-cold saline and kept in a glucose-containing buffer solution at 4°C continuously gassed with O2-CO2 (95:5 vol/vol). The slaughtering and tissue sampling procedure has been described in detail by Schröder and Breves (14).

Northern Blot Analyses

Poly(A)+RNA from duodenal and jejunal mucosa was fractionated in 1.0% formamide-agarose gels and transferred by capillary blotting onto nitrocellulose membranes. After baking the membranes in a vacuum oven for 2 h at 80°C, prehybridization/hybridization was performed in a solution containing 5 × standard saline citrate (SSC), 5 × Denhardt's solution, 0.1% SDS, and 40% formamide. For blocking unspecific binding sites of membranes, denatured herring sperm DNA (20 µg/cm2) was added. Radioactively labeled NaPi IIb and beta -actin-specific probes (50 µCi [32P]dCTP for each probe, specific activity 3,000 Ci/mmol, NEN Life Science Products, Boston, MA) were created by using a random prime labeling system (AmershamPharmaciaBiotech, Rediprime II, Buckinghamshire, UK). Hybridization was performed at 42°C overnight. The membranes were washed three times for 10 min with 2 × SSC-0.1% SDS first at room temperature, then with 0.5 × SSC-0.1% SDS at 37°C and, finally, with 0.2 × SSC-0.1% SDS at 42°C. The membranes were analyzed after exposure to a phosphor imager screen for 2-4 h with a phosphor imager system (BioRad). Relative abundance of specific mRNA was quantified by reference to beta -actin as the internal standard using the quantification software Quantity One (BioRad).

Western Blot Analyses

BBMV that had been isolated for uptake studies were fractionated by SDS-PAGE in an 8.3% discontinuous polyacrylamide gel according to Laemmli (9) (for the preparation of BBMV, see below). Probes were not heat denatured before electrophoresis because of the heat instability of NaPi IIb protein. The separated proteins were transferred onto nitrocellulose membranes by tank blotting (blotting time 2 h). After being blocked with 2% fat-free milk solution overnight, membranes were incubated for 2 h with the mouse-specific anti-NaPi type IIb antibody (1:2,000, derived against a synthetic oligopeptide of the NH2 terminus of the transporter). The antibody was kindly provided by Prof. Dr. J. Biber and Prof. Dr. H. Murer, Institute of Physiology, University of Zürich-Irchel, Zürich, Switzerland. Specificity of the murine antibody in goats was controlled by preincubation of the antibody with the antigenic peptide (data not shown). Immunolocalization of the primary antibody was performed using an anti-rabbit secondary antibody (1:1,500) coupled with horseradish peroxidase. Bands were detected by the enhanced chemiluminescence system (AmershamPharmaciaBiotech) according to the manufacturer's protocol. To quantify relative protein expression levels, beta -actin-specific immunodetection was performed on the same blots. Bands were analyzed quantitatively using the Quantity One software.

Immunohistochemistry

Small pieces of jejunal and duodenal gut wall of five goats were sliced and fixed by immersion in 3% paraformaldehyde (wt/vol), 100 mM cacodylate, and 100 mM sucrose (pH 7.4) overnight. Subsequently, tissues were frozen in liquid propane and stored at -80°C. For immunohistochemical analysis, cryosections of ~8-µm thickness were prepared on poly-L-lysine-coated slides. After permeabilization and blocking in PBS (100 mM) containing 0.5% Triton X, 4% goat serum, and 0.1% NaN3 1 h at room temperature, the preparations were incubated with anti-NaPi type IIb antibody (1:500 diluted in PBS) overnight in a moisture chamber. Subsequently, the tissues were washed three times for 10 min with PBS at room temperature and were incubated for 2 h with an anti-rabbit secondary antibody labeled with the fluorophore dichlorotriazinyl aminofluorescin (1:200, Jacksons Labs, purchased from Dianova). A second slide was incubated only with labeled secondary antibody to determine the background fluorescence. The preparations were examined with an epifluorescence microscope (IX70, Olympus) using a modified U-MNIBA filter block with the following specifications: beam splitter DM 505 (505 nm), excitation filter BP 470-490 (470-490 nm), barrier filter D 520/20(510-530 nm). Pictures were made with a black and white video camera (model 4910, Cohu, San Diego, CA) connected to a Macintosh Computer and controlled by IPLab Spectrum 3.0 software (Signal Analytics, Vienna, VA). Frame integration and contrast enhancement were employed for image processing. The pictures were colored by means of the IPLab software.

Preparation of BBMV and Uptake Measurements

BBMV were prepared from duodenal and jejunal epithelia with a modified Mg2+-EGTA precipitation method, and Pi uptake into BBMV was quantified using the rapid filtration technique as described by Schröder and Breves (14). Pi uptake was determined as total Pi uptake regardless of concentrations of mono- and divalent Pi anions because there is no experimental evidence for a preferential transport of one of the ionic forms of Pi (unpublished results). Duodenal vesicular Pi transport of each feeding group was analyzed in the presence of an inwardly directed gradient of K+ (extravesicular buffer: 100 mM KCl, 100 mM mannitol, 10 mM HEPES-Tris) at an extravesicular 5.4 with pH 7.4 intravesicularly (intravesicular buffer: 100 mM mannitol, 100 mM KCl, 10 mM HEPES-Tris). Pi uptake as a function of time was performed with either an inwardly directed Na+ (extravesicular: 100 mM; intravesicular: 0 mM) or K+ gradient at both pH 5.4 and 7.4 extravesicularly with pH 7.4 intravesicularly. Jejunal Pi transport across the apical membrane was analyzed in the presence of an inwardly directed Na+ gradient (extravesicular: 100 mM; intravesicular: 0 mM) at an extravesicular pH 7.4 and 5.4, respectively, to determine the H+ sensitivity of the Na+-dependent Pi transport system. Kinetic parameters Vmax (nmol · mg protein-1 · 10 s-1) and Km (mM) for Na+- and K+-dependent Pi uptake in each gut segment were calculated from the Michaelis-Menten kinetic of Pi uptake into the BBMV (14).

Measurement of Duodenal Transepithelial Pi Flux Rates

Duodenal transepithelial Pi flux rates were determined using the Ussing chamber technique as described in detail by Schröder et al. (15). Mucosal Na+ was present or absent with pH 7.4 or 5.4, and unidirectional Pi flux rates [mucosal to serosal (Jms), serosal to mucosal (Jsm)] were measured in the absence of a transepithelial electrical gradient in all experimental groups allowing to calculate Pi net flux rates (Jnet = Jms - Jsm). Under those conditions, significant positive Jnet values have to be interpreted as active Pi absorption.

Determination of Plasma Pi and Ca

Plasma Pi was determined colorimetrically using the vanadate-molybdate method (8) and plasma Ca by the standard o-cresolphthalein complex method (12)

Statistics

Values were given as means ± SE/SD, with n = number of animals. Significance of differences was tested by one-way ANOVA and as a posttest by Tukey's t-test assuming a Gaussian distribution of data (software Graphpad prism 2.01, San Diego, CA; www.graphpad.com). P values <0.05 were set to be significant. Linear regression (see Fig. 4) was also calculated with Graphpad prism 2.01. All immunodetection and hybridization experiments were performed at least in duplicate. The significance of the effects of group, Ca, pH, and the interaction of Na × pH on duodenal transepithelial Pi flux rates were tested by three-way ANOVA [BMDP-92 software program (2)].


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

P Status of the Animals

Dietary P restriction resulted in hypophosphatemia associated with hypercalcemia, which was used as an indicator of a shortage in Pi (Table 1). Dietary Ca restriction led to a slight increase in Pi concentrations at unchanged Ca levels.

                              
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  		Table 1.   Plasma concentrations of Pi and Ca in control, P-, or Ca-restricted goats

Pi Transport in the Duodenum of Goats

Structural and functional characteristics of duodenal Pi transport. In Northern and Western analyses, neither NaPi IIb transcript nor NaPi IIb protein was found in the duodenum of goats of each feeding group (Fig. 1).


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  		Fig. 1.   NaPi IIb gene expression in the small intestines of goats. Northern analysis (A) of jejunal and duodenal poly(A)+RNA of 1 animal of each feeding group (con, control animal; -P, P-restricted animal; -Ca, Ca-restricted animal) was performed. Hybridization with a goat-specific NaPi IIb probe revealed strong bands in the jejunum, but not in the duodenum. The lack of NaPi IIb expression in goat duodenum was confirmed by Western analysis (B). NaPi IIb protein was immunodetected with a mouse-specific NaPi IIb antibody only in the jejunum. Integrity of poly(A)+RNA and proteins was proven by beta -actin detection.

Using the Ussing chamber technique, duodenal transepithelial Pi flux rates were measured either in the presence or in the absence of mucosal Na+ at pH 7.4 and 5.4, respectively. At a mucosal pH of 7.4, Jnet of Pi ranged between 11 and 22 nmol · cm-2 · h-1 irrespective of mucosal Na+ concentrations and different P or Ca supply (Table 2). Reducing the mucosal pH to 5.4 increased Jnet of Pi to ~200 nmol · cm-2 · h-1 at high mucosal Na+ concentrations and to ~50 nmol · cm-2 · h-1 under Na+-free conditions. Enhancement of transepithelial flux rates was due to respective increases Jms at unchanged Jsm. Na affected Jnet of Pi only at low pH as indicated by significant interactions between Na and pH in the ANOVA. Again, dietary regimens had no significant effects on Pi flux rates.

                              
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  		Table 2.   Unidirectional and net flux rates of Pi of duodenal epithelia as affected by mucosal Na+ and H+ concentrations. Influence of dietary P or Ca restriction.

Kinetic parameters of proton-dependent Pi transport in the duodenum. Time-dependent Pi uptake in duodenal BBMV of each feeding group exhibited an overshoot phenomenon if an inwardly directed H+ gradient was established irrespective of the presence of an inwardly directed Na+ gradient (Fig. 2). In each animal, extravesicular Na+ slightly stimulated H+-dependent Pi uptake. Pi uptake in the absence of an inwardly directed H+ gradient was linear representing the diffusional part of Pi passover. The kinetic parameters of the duodenal H+-dependent Pi transport are presented in Table 3. Neither in the P- nor in the Ca-restriction experiment were any significant differences in Vmax or Km values observed. Km values were similar in all groups and ~10-fold higher (0.4 mM) compared with jejunal NaPi IIb transporter (0.04 mM) (see below).


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  		Fig. 2.   Pi uptake in duodenal brush-border membrane vesicles (BBMV) as a function of time. The experiments were performed for 2 animals of each feeding group, resulting in identical overshoot phenomenon. Due to the high interindividual variation, data are given exemplarily for 1 -P goat. +Na/-Na means the presence and the absence of an inwardly directed Na+ gradient, respectively; pHo 5.4/pHo 7.4 means the presence and the absence of an inwardly directed H+ gradient, respectively. Pi overshoot was only observed in the presence of an inwardly directed H+ gradient. The slight stimulation of H+-dependent Pi uptake by Na+ was seen in each animal.


                              
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  		Table 3.   Kinetic parameters of H+-dependent Pi transport in goat duodenal BBMV

Pi Transport in Goat Jejunum

Immunohistochemical detection of NaPi type IIb in the jejunum of goats. Na+-dependent Pi transporters of type IIb were found in the apical membranes of the mature jejunal enterocytes of control goats by immunohistochemical analysis (Fig. 3B). In the apical membranes of duodenal enterocytes, no NaPi IIb-specific signal could be detected (Fig. 3A).


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  		Fig. 3.   Immunohistochemical detection of NaPi IIb in the apical membrane of goat midjejunum (B; control animal) but not of goat duodenum (A; control animal). NaPi IIb staining was performed in paraformaldehyde-fixed cryosections with mouse-specific anti-NaPi IIb antibody (1:500). Specific signal was generated using a secondary antibody labeled with DTAF (1:200). Incubation of jejunal tissues with secondary antibody only did not reveal any signal (C).

Influence of dietary P or Ca restriction on Na+-dependent Pi transport and NaPi IIb expression. The influence of the dietary P and Ca supply on the NaPi IIb transcription level was measured for each feeding group by performing quantitative Northern analyses; the ratio of NaPi IIb-mRNA to beta -actin for the control group was 1.26 ± 0.24, for the -P group 3.41 ± 2.20, and for the -Ca group 0.78 ± 0.21 (mean ± SD, n = 3). The high variance in the -P group was due to a single animal with a transcript level equivalent to that of the control animals. P restriction did not significantly increase the amount of NaPi IIb transcript. Reduced Ca supply did not influence the transcript level.

The relative abundance of specific NaPi IIb protein (ratio NaPi IIb to beta -actin) as a function of transport capacity (Vmax) of Na+-dependent Pi transport into jejunal BBMV is shown in Fig. 4 for control and P-restricted goats. Higher Pi transport rates into BBMV were associated with increased amounts of specific NaPi IIb protein (r2 = 0.63). P restriction (Fig. 4 and Fig. 5, top left) stimulated this NaPi IIb-mediated Na+-Pi transport. The transport affinity (Km) of ~0.038 mM was not changed by P restriction (Fig. 5, bottom left). In Ca-restricted animals, NaPi IIb protein levels were not affected (data not shown).


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  		Fig. 4.   Relative amounts of NaPi IIb protein in brush-border membrane of goat jejunum (given as ratio of NaPi IIb to beta -actin) as a function of Vmax of jejunal Na+-dependent Pi transport (given as nmol Pi · mg protein-1 · 10 s-1). Higher capacity of Na+-Pi transport was correlated with an increased abundance of NaPi IIb protein, indicating that the major extent of Na+-Pi transport was mediated by NaPi IIb.



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  		Fig. 5.   Influence of dietary P restriction and pH on kinetic parameters of Na+-dependent Pi transport in goat jejunum. Data are given as means ± SE, n = 8/group, significance level for the effect of P restriction was P < 0.05 (*). Vmax values of Na+-Pi transport into BBMV were significantly increased in P restriction (* P < 0.05) independent of an inwardly directed H+ gradient. Reducing the extravesicular pH to 5.4, Vmax as well as Km(Pi) values increased significantly (P < 0.001 and P < 0.05, respectively). Km(Pi) was not affected by P restriction. Interaction between P restriction and pH was not significant.

pH sensitivity of jejunal Na+-Pi transport. The proton sensitivity of Na+-Pi transport was studied in jejunal BBMV. Lowering the extravesicular pH from 7.4 to 5.4 at an intravesicular pH of 7.4 stimulated Vmax values significantly in both the control group (7.4/7.4: 0.132 ± 0.015; 5.4/7.4: 0.343 ± 0.037 nmol · mg protein-1 · 10 s-1) and the -P group (7.4/7.4: 0.290 ± 0.049; 5.4/7.4: 0.544 ± 0.065 nmol · mg protein-1 · 10 s-1) (Fig. 5, top). The increases in transport capacities were associated with corresponding increases in Km. Thus the affinities for Pi were reduced in response to an inwardly directed proton gradient. P restriction did not affect the pH-dependent changes in affinity (Fig. 5, bottom).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pi transport was characterized structurally and functionally in goat duodenal and jejunal epithelia and in isolated BBMV from duodenal and jejunal enterocytes. Ion dependency, kinetic parameters, and adaptation to dietary P and Ca restriction were studied.

Pi Transport in the Duodenum of Goats

For sheep, H+-Pi transport in the upper small intestines has been described (13). This is in contrast to monogastric animals, in which duodenal Pi transport has been identfied as a secondary active Na+-coupled process (1). Na+-Pi cotransporter type IIb gene expression was found in the duodenum of mice (4) but not in goats (Fig. 1). Therefore, Pi transport in goat duodenum was studied to permit characterization of transepithelial Pi transport as affected by H+ and Na+. The kinetic parameters of H+-Pi transport across brush-border membranes and influences of dietary P and Ca restriction were investigated.

Functional properties of duodenal Pi transport system. The significantly higher Na+-independent Pi net transport at pH 5.4 compared with pH 7.4 on the mucosal side indicates an H+-dependent Pi transport process in the duodenum of goats (Table 2). In the presence of high luminal Na+ concentrations, this transport is effectively stimulated (Table 2). These findings are confirmed by Pi uptake studies in duodenal BBMV. In the absence of extravesicular Na+, Pi uptake as a function of time exhibited an overshoot phenomenon only at low pH. This indicates a carrier-mediated Pi transport that is dependent of H+. The presence of extravesicular Na+ at low pH had a slight stimulating effect on Pi uptake (Fig. 2). This might have been due to a direct positive interaction of Na+ ions with the Pi carrier in analogy to that which has been described for the H+ ion as "competitive inhibition" interaction with the Na+ binding site of renal NaPi IIa (10). The affinity of the duodenal H+-dependent Pi transporter is substantially lower than that of Na+-Pi cotransporter type IIb. The lack of NaPi IIb expression, the difference in affinity, and the H+ dependency of Pi transport in the goat duodenum indicate a novel kind of Pi transporter for which molecular structure still has to be identified.

Influence of dietary P or Ca restriction on duodenal Pi transport. Dietary P and Ca restriction did not change transepithelial net Pi flux rates (Table 2) or Pi transport capacities across brush-border membranes (Table 3) in goat duodenum. The order of magnitude of duodenal H+-Pi transport capacity (Vmax: 2.1 nmol · mg protein-1 · 10 s-1) as well as transporter affinity (Km: 0.4 mM) in goats correspond to those reported for sheep [Vmax: 3.6 nmol · mg protein-1 · 10 s-1; Km: 0.6 mM (13)]. Unlike in goats, in sheep, dietary P restriction (low-P diet) resulted in a higher duodenal Pi transport capacity compared with control (high-P diet) (13). However, because P intake of the sheep was not precisely defined in that study, this discrepancy cannot be explained. A surplus of dietary P could effect a decrease of Pi transporter expression as observed for jejunal NaPi IIb in goats getting twice as much dietary P as required (unpublished results). This would also result in differences of transport capacity between low- and high-P groups.

Pi Transport in the Jejunum of Goats

Na+ dependency and kinetic parameters of jejunal Pi transport in goats are well defined (14). Structurally, an Na+-Pi transporter could be identified that belongs to the NaPi IIb family (5), but it is still not clear whether Na+-Pi transport is exclusively mediated by NaPi IIb transporters. There is now strong evidence for the importance of NaPi IIb for Na+-Pi transport for the following reasons. First, NaPi IIb is located in the apical membrane of goat enterocytes (Fig. 3). Second, the affinity of goat jejunal Na+-Pi transport as determined by studies of Pi uptake into isolated BBMV [Km ~0.03 mM; Fig. 5 (14)] is in the same range as that described for the murine NaPi IIb (4). Third, the transport capacity of goat jejunal Na+-Pi transport is positively correlated to the amounts of specific NaPi IIb protein in the brush-border membranes (Fig. 4). Therefore, it is concluded that NaPi IIb mediates the major part of Na+-dependent Pi transport in goat jejunum.

Influence of dietary P and Ca restriction on the NaPi IIb expression. Dietary P restriction in mice resulted in increased intestinal NaPi IIb protein levels at unchanged NaPi IIb mRNA levels, indicating a posttranscriptional process for adaptation (3). Until recently, adaptation of jejunal Pi transport to dietary P and Ca restriction in ruminants has been studied at the functional level only. P restriction in goats increased transepithelial net Pi flux rates (15); it increased transport rates into BBMV without influencing Pi affinity of the transport system (Fig. 5). These processes are now being studied at the molecular basis in ruminants. Dietary P restriction did not induce significant increases in specific expression of NaPi IIb at the mRNA level. However, the ability of individual animals to adapt to low dietary P intake cannot be excluded. The expression of specific NaPi IIb protein was stimulated significantly (Fig. 4). Therefore, it is suggested that molecular adaptation to dietary P restriction in ruminants is mediated by an increased specific transporter protein level without increased corresponding gene transcription rate. However, an unaffected amount of NaPi IIb mRNA might also have been the result of an increased transcription rate with a corresponding increase in mRNA turnover rate. Ca restriction enhanced neither mRNA nor protein level, although in earlier experiments, an increase in Vmax of Pi uptake into jejunal BBMV of Ca-restricted goats was observed (14). Jejunal Pi transport of Ca-restricted goats may be upregulated by changing the sodium affinity of the transport system.

Influence of protons on the function of the Na+-dependent Pi transport. H+ strongly stimulated goat jejunal Na+-Pi transport in both control and P-restricted groups (Fig. 5, top), whereas transporter Pi affinity was significantly lowered by H+ (Fig. 5, bottom). Na+ affinity was not examined in this study, but the changes in transport rates observed here could be due to an increase in Na+ affinity resulting in a higher Pi transport across apical membranes. From studies on renal Pi transport, it is already known that H+ can influence Na+ binding at the NaPi II transporters. For renal NaPi type IIa, another member of the Na+-Pi cotransporter family that is expressed only in the kidney, a significant but opposite pH sensitivity has been described (6, 10). NaPi IIa activity decreases with increased proton concentrations by the competition of H+ with Na+ for interaction with the carrier. Preferential transport of divalent Pi also contributes to the pH dependency observed here (10). Three charged amino acids in the third extracellular loop (REK) were identified as the molecular determinants for pH sensitivity by site-directed mutagenesis (6). In intestinal murine NaPi IIb, three neutral amino acids (GNT) corresponded to this position, resulting in no or a slight increase in Pi transport activity at higher luminal proton concentrations (4). Unlike in mice, the stimulating effect was more pronounced in goats (where the pH was lowered to 5.4 as opposed to 6.0 in mice studies); however, no molecular mechanism of a direct H+-Na+ "interaction" on the transporter is as yet known. A preferential transport of monovalent Pi ions could also contribute to intestinal pH sensitivity. Furthermore, the pH sensitivity could be due to the coexpression of the duodenal H+-Pi transport system and the jejunal NaPi IIb transporter in the goat jejunum. Therefore, Pi uptake studies into jejunal BBMV were performed in the absence of Na+ in the extravesicular fluid at pH 7.4 and 5.4, respectively. Under these conditions, Pi uptake seemed to be linearily dependent on Pi concentration at both pH levels and was ~10-fold lower than in the presence of Na+ (unpublished observations), indicating that the H+-Pi transport system is apparently not expressed in goat jejunum.

The physiological relevance of this marked H+ responsiveness of intestinal Pi transport in goats could be reflected by the lower pH values in the digesta of the upper small intestines. The comparably low bicarbonate secretions into the upper small intestines of sheep are responsible for the relatively acid pH levels of ~2-4 up to the upper jejunum, whereas in nonruminant animals the pH of digesta rises rapidly to values above 6 at the beginning of the duodenum (7).

The present study has shown clear evidence that at least two different mechanisms are involved in goat intestinal Pi absorption. In the duodenum, there is a proton-dependent, sodium-sensitive system that is not influenced by dietary P restriction. In the jejunum, Pi transport is mediated by a sodium-dependent, proton-sensitive system, mainly by NaPi cotransporter type IIb. This Pi transport system adapts to dietary P restriction by increasing the transporter capacity due to higher transporter protein expression.


    ACKNOWLEDGEMENTS

We thank M. Burmester and G. Becker for excellent technical assistance. A special thanks goes to W. V. Engelhardt for critical reading and correction of the manuscript.


    FOOTNOTES

The financial support of the Deutsche Forschungsgemeinschaft (SFB 280) is gratefully acknowledged.

Address for reprint requests and other correspondence: K. Huber, Dept. of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15/102, D-30173 Hannover, Germany (E-mail: korinna.huber{at}tiho-hannover.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpregu.00760.2001

Received 26 December 2001; accepted in final form 28 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 283(2):R296-R302
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