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Physiologisches Institut, Tierärztliche Hochschule Hannover, D-30173 Hannover, Germany
Address all correspondence and requests for reprints to: PD Dr. B. Schröder, Physiologisches Institut, Tierärztliche Hochschule, Bischofsholer Damm 15/102, D-30173 Hannover, Germany. E-mail: bschroed{at}physiology.tiho-hannover.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Regardless of age, plasma calcitriol concentrations in def piglets were
unphysiologically low (16–21 pg/ml) and differed significantly from
those in respective con animals (60–69 pg/ml) and vitamin
D3-treated def piglets (50–56 pg/ml). However, newborn and
suckling def piglets had normal Ca (
3.0 mmol/liter) and
Pi (2.8 mmol/liter) plasma levels. Def piglets became
hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter)
between 4–6 weeks pp. Treatment with vitamin D3
significantly increased plasma Ca (3.2 mmol/liter) and Pi
(2.7 mmol/liter) levels in weaned def animals.
Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170–224 nmol/(cm2·h)] and decreased by approximately 80% during the first week of life before remaining constant [30–50 nmol/(cm2·h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9–2.2 nmol/(mg protein·10 sec); suckling piglets, 0.4–0.6 nmol/(mg protein·10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein·10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport. At weaning, Na+/Pi transport across the intestinal brush-border membrane is clearly stimulated by calcitriol, but no significant effects of age or calcitriol on the Km values (0.5–0.7 mmol/liter) were observed.
In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption does not occur until weaning.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Simultaneous actions of calcitriol on intestinal Ca2+ and Pi transport have also been demonstrated in weaned and adult pigs (11, 12, 13). Furthermore, the involvement of active mechanisms for Ca2+ and Pi absorption from the upper small intestines could be shown in neonatal piglets by in vitro measurements of unidirectional flux rates in Ussing chambers (14, 15, 16). These studies demonstrated adaptational processes in intestinal Ca2+ and Pi absorption during early postnatal development and, in contrast to the findings in weaned and adult animals, revealed some evidence for calcitriol-independent mechanisms regulating active Ca2+ absorption. Therefore, the present studies aimed at determining to what extent calcitriol may influence 1) the transepithelial active Pi absorption in the upper small intestines during postnatal development and 2) the kinetic parameters of Na+-dependent Pi uptake across the BBM of the enterocytes. For the studies we used normal piglets as control animals and calcitriol-deficient piglets suffering from an inherited disorder of renal production of calcitriol, known as pseudovitamin D deficiency rickets type I (15). In addition, a group of calcitriol-deficient animals was calcitriol repleted by treatment with pharmacological doses of vitamin D3. In each group, newborn, suckling, and weaned animals were examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Exp I (in vitro Pi flux rate measurements in
Ussing chambers), normal piglets of both sexes served as controls (con
piglets). Piglets with inherited calcitriol deficiency (def piglets)
were used as previously described (15). The ages of the piglets were
between at least 11 h and 6 days postpartum (pp; newborn), 21–28
days pp (suckling), or 6–8 weeks pp (weaned). In addition, a group of
def piglets was treated with single im injections of vitamin
D3 (def-D3 piglets). In detail, newborn def
piglets were treated 2 days before the experiments with 50,000 IU
vitamin D3 (cholecalciferol in aqueous solution; WDT,
Hannover, Germany). Suckling and weaned def animals were treated 3 or 6
days before the experiments with 100,000 or 200,000 IU, respectively.
This form of application was used because it has been shown previously
that treatment of def piglets with massive doses of vitamin
D3 resulted in a transient 4-fold increase in plasma
calcitriol concentrations 2–3 days postinjection (17). Furthermore, it
has been found that no substantial differences occurred in stimulation
of intestinal Ca2+ uptake mechanisms in weaned def piglets
when they received either six sequential iv injections of
physiologically adequate doses of calcitriol at 12-h intervals or a
single pharmacological dose of vitamin D3 (12). Newborn
piglets were only suckled before death, whereas suckling piglets were
allowed gradually to wean, with access to the normal pig diet of their
mothers. At an age of 4 to 6 weeks pp, the piglets were weaned and then
kept on a commercial pig starter diet (HG-Mast FN, Raiffeisen,
Hannover, Germany) ad libitum, with 0.9% (wt/vol) calcium,
0.65% (wt/vol) phosphate, and 50 µg vitamin D/kg. Water was
available at all times. Table 1
summarizes the different animal groups used in Exp I with their
respective treatments, age pp (status), and body weights.
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Blood
Blood samples were taken by venipuncture from the cranial vena
cava with ammonium heparinate-covered syringes and were centrifuged at
3500 x g at 4 C for 10 min. Plasma was stored at -70
C.
Intestinal segments
For all experiments, piglets were killed by being stunned with a
commercial abattoir shooting apparatus followed by exsanguination from
the carotid arteries. For Exp I, segments, about 20–40 cm in length,
of the proximal small intestines were removed within 5 min after death
from the abdominal cavity beginning 5 cm distal from the entry of the
pancreatic duct. Intestinal segments were immediately rinsed with
ice-cold saline (0.9% NaCl, wt/vol) and kept in a modified
glucose-containing Krebs-Henseleit buffer solution at 4 C while
continuously gassed with carbogen (95% O2-5%
CO2) until mounting in Ussing chambers. In newborn piglets,
this segment consisted of duodenal tissue and a minor part of the
proximal jejunum.
To obtain intestinal material for preparation of BBMV in Exp II, at least one segment, 60 cm long, from the proximal part of the small intestines beginning about 20 cm distal from the entry of the pancreatic duct was removed from the abdominal cavity within 5–8 min after death. The tissues were immediately rinsed with ice-cold saline (0.9% NaCl, wt/vol), cut into 15-g pieces, frozen in liquid nitrogen, and stored at -70 C until preparation of BBMV.
Flux rate measurements
Flux rate measurements of Pi across intact
epithelial tissues from stripped or partially stripped duodenal and
proximal jejunal segments were made using the in vitro
Ussing chamber technique as described in detail previously (15). The
unidirectional mucosal to serosal (Jms) and serosal to
mucosal (Jsm) flux rates of Pi were measured in
Ussing-type chambers with an exposed serosal area of either 0.5
cm2 (with tissues from newborn animals) or 1
cm2 (with tissues obtained from suckling and weaned
piglets). Tissues were incubated on both sides with 13 ml modified
Krebs-Henseleit buffer solution (pH 7.4) containing 125.4 mmol/liter
NaCl, 5.4 mmol/liter KCl, 1.2 mmol/liter CaCl2, 21
mmol/liter NaHCO3, 0.3 mmol/liter
Na2HPO4, 1.2 mmol/liter
NaH2PO4, and 0.01 mmol/liter indomethacin. In
addition, the serosal solution contained 10 mmol/liter glucose, and the
mucosal solution contained 10 mmol/liter mannitol. Buffers were
continuously circulated and gassed with carbogen at 39 C. As earlier
studies with small intestinal tissues from rabbits and our own
experiments with pigs provided substantial evidence for the involvement
of PGs in the modulation of electrogenic sodium and chloride
absorption, which may interfere with Pi transport, we used
indomethacin to reduce cyclooxygenase activity and to assure consistent
electrical properties of the duodenal epithelia from different animal
groups (18, 19).
About 20 min after mounting the tissues, 185 kilobecquerels [32P]orthophosphate (370 megabecquerels/ml; Amersham Buchler, Braunschweig, Germany) were added to one side of the tissue. After an initial equilibrium period of 20 min, flux rates were calculated from the rate of tracer appearance on the unlabeled side. Therefore, samples were obtained at three 10-min intervals at least.
Unidirectional Pi flux rates were calculated by using standard equations after counting respective tracer radioactivity in 4.5 ml scintillation fluid (Rotiszint Eco-Plus, Roth, Karlsruhe, Germany) in a Packard Tricarb liquid scintillation counter (United Technologies Packard, Downers Grove, IL) (15). To avoid possible influence of transepithelial electrical gradients, all flux measurements were carried out under short circuit current conditions. Net flux rates (Jnet) were calculated as differences between Jms and Jsm of paired tissues whose conductances did not differ by more than 25%.
Preparation of BBMV
The BBMV were prepared by a modification the
Mg2+-EGTA precipitation method originally described for
renal and ileal BBM from rats (20, 21), respectively, and (with minor
modifications) as has been adopted for preparations of BBMV from weaned
pig jejunum (12). Frozen intestines of 10–20 g wet weight from newborn
animals and 35–60 g wet weight from suckling and weaned piglets were
thawed on ice, and all of the following procedures were carried out at
4 C. Epithelial cells were harvested after 10-min vibration with a
Vibro mixer type E1 (Chemap, Volketswil, Switzerland) in either 40 ml
(newborn piglets) or 70 ml (older animals) 12 mmol/liter Tris-HCl
buffer (pH 7.1) with 300 mmol/liter mannitol and 5 mmol/liter EGTA. The
cell suspension was diluted with a 4-fold amount of ice-cold distilled
water and homogenized for 3 min with a blender (type 530.02, Moulinex,
Germany). After removal of foam, MgCl2 was added to a final
concentration of 10 mmol/liter. The solution was kept on ice for 15 min
and centrifuged at 3,300 x g for 15 min. The
supernatant was centrifuged at 27,500 x g for 30 min,
and the pellet was resuspended in 35 ml 2 mmol/liter Tris-HCl buffer
(pH 7.4) with 100 mmol/liter mannitol with 10 strokes (2580 rpm) in a
glass-Teflon Elvehjem-Potter (Braun, Melsungen, Germany). Precipitation
of membranes by MgCl2 addition and the two-step
centrifugation procedure were repeated. The resulting pellet was
resuspended with a glass-Teflon Potter, as described above, in 35 ml
vesicle buffer containing 10 mmol/liter HEPES-Tris, 100 mmol/liter KCl,
and 100 mmol/liter mannitol (pH 7.4). This suspension was centrifuged
at 30,000 x g for 40 min, and the final pellet was
resuspended in 1–3 ml of the same buffer by passing the mixture 10
times through a 26-gauge needle (0.45 x 25 mm). Final BBMV
suspensions were immediately frozen in liquid nitrogen and stored at
-80 C until determinations, which were performed within 4 weeks.
During this period, the properties of BBM Pi transport
remained constant. The functional integrity of the vesicles, such as
leakiness, was controlled by measuring substantial
Na+-dependent overshoot of glucose uptake into BBMV before
each experiment with Pi (12).
Uptake of Pi into BBMV
Uptake of 32P into BBMV was quantified using the
rapid filtration technique with cellulose nitrate filters of 0.65-µm
pore size (Sartorius, Gottingen, Germany). In general, 20-µl portions
of vesicle suspensions containing 160–300 µg protein were mixed with
80 µl transport buffer containing 37 kilobecquerels 32P
and different concentrations of unlabeled Pi (0.05–3
mmol/liter) in 10 mmol/liter HEPES-Tris, 100 mmol/liter NaCl or KCl,
and 100 mmol/liter mannitol (pH 7.4). Samples were incubated at 37 C,
then were mixed with 4 ml ice-cold stop buffer (10 mmol/liter
HEPES-Tris with 1 mmol/liter KH2PO4, 99
mmol/liter KCl, and 100 mmol/liter mannitol, pH 7.4) and immediately
filtered by vacuum suction. The filters were washed twice with 5 ml of
the stop buffer to remove extravesicular radioactivity. During this
procedure, protein loss was less than 3%. Finally, washed filters were
transferred into vials with 4 ml scintillation liquid (Rotiszint
Eco-Plus) for radioactivity measurements in a 2000 CA Tri-Carb liquid
scintillation analyzer (United Technologies Packard) with an accuracy
of 2% SD. Appropriate blanks were obtained from
incubations of 32P in transport buffer without vesicular
membranes, since in preliminary experiments no significant differences
had been observed measuring the blanks in the absence or presence of
BBMV. At the end of the experiments, total radioactivity was counted in
80 µl transport buffer.
Enzyme assays and protein determination
The activity of alkaline phosphatase (AP; EC 3.1.3.1) as a
marker for the BBM was calculated from photometric measurements of the
rate of hydrolysis of p-nitrophenyl phosphate according to
an assay kit from Boehringer Mannheim (Mannheim, Germany). The activity
of the Na+/K+-adenosine triphosphatase (EC
3.6.1.37) as a marker for the basolateral membranes (BLM) was
determined by the method of Mircheff and Wright (22). Protein was
determined with a Coomassie blue kit from Bio-Rad (Munich, Germany),
using 
-globulin as the standard protein. The activities of AP in
BBMV preparations were not significantly affected by age or calcitriol
conditions and ranged from 2010–6780 U/g protein in newborn piglets,
from 1900–4900 U/g protein in suckling piglets, and from 2080–4520
U/g protein in weaned animals. The relative enrichments of intestinal
BBM, expressed as the ratio of enzyme-based BBM to BLM enrichments
(BBM/BLM ratio), were not significantly affected by the different
calcitriol conditions, but tended to be higher in preparations of
suckling (6- to 9-fold) and weaned piglets (5- to 7-fold) compared with
those in newborn animals (4- to 6-fold). However, these differences
were not significant, indicating adequate enrichments of BBMV fractions
in all groups.
Ca, Pi, and calcitriol in
plasma
Plasma calcium and Pi were measured using test kits
provided by Boehringer Mannheim. Calcitriol concentrations were
determined using a commercial nonequilibrium competitive receptor
binding assay provided by Immundiagnostik (Bensheim, Germany),
involving C18-OH single cartridge extraction and separation
of calcitriol from plasma and the high specific calcitriol receptor of
calf thymus.
Chemicals
The chemicals for preparing the buffer solutions, chloroquine,
and colchicine were purchased from Sigma Chemie (Deisenhofen,
Germany). All other compounds were of analytical grade and were
commercially available.
Statistics
Statistical calculations were performed using the BMDP-92
software program (23). All values are given as arithmetic means
(
) with their SEMs for the appropriate number of
animals. P < 0.05 was considered significant.
Significant differences of Jnet from zero were determined
by unpaired Student’s t test (BMDP3D). Two-way ANOVA was
performed using the BMDP7D program, and for a significant result,
individual mean values were checked in pairs for significant
differences using the conventional Tukey’s test. Where mean values
were found to have a nonconstant variance, analyses were performed on
log-transformed data. Nonlinear regression curves were fitted by the
BMDP3R procedure, which is based on a modified Gauss-Newton algorithm.
This curve fitting provides estimations of the minimum (initial) and
maximum (final) values as well as half-time values (t1/2)
with respective asymptotic SDs. The kinetic values
[maximum velocity (Vmax) and Km] for
Na+ dependent uptake of Pi into BBMV were
calculated by fitting Pi uptake rates vs.
Pi concentrations in transport buffer to a rectangular
hyperbola relationship using a computer-aided curve-fitting program
(Graph Pad Software, San Diego, CA). The algorithm was obtained from
the law of mass action with regard to the nonspecific binding. Details
of the calculation modus have been described previously (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Regardless of postnatal age,
plasma calcitriol levels were significantly lower by 65–75% in def
piglets compared with those in con animals. Treating def piglets with
vitamin D3 resulted in significant increases in plasma
calcitriol concentrations, which reached more than 80% of respective
control values.
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Electrical properties of porcine duodenal epithelia during
postnatal development
Short circuit currents (Isc) and respective tissue
conductances (GT) of duodenal epithelia in Exp I are
presented in Table 3. Regardless of the
calcitriol conditions, Isc values were highest in newborn
animals and decreased gradually by approximately 40–70% in suckling
and by 55–75% in weaned pigs. This indicates substantial net
electrogenic ion transport across the duodenum in neonatal piglets
compared with that in the older piglets. Similarly, in all groups,
GT values were highest in newborn animals and decreased
during postnatal development. This effect was most pronounced in the
con group compared with def and def-D3 piglets (18%, 8%,
and 15%, respectively). Regardless of age, treatment of def piglets
with vitamin D3 caused lower GT values compared
with those in respective untreated animals of the same age.
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. Regardless
of age and plasma calcitriol levels, the Jms of
Pi exceeded the Jsm. This resulted in
significant net flux rates of Pi from the mucosal to the
serosal compartment. The Jsm were not significantly
affected by age or calcitriol status; therefore, changes in
Jnet are directly related to respective changes in
Jms. Regardless of calcitriol status, the rate of
Pi net flux rates in newborn animals was 6- to 7-fold
higher than that in suckling piglets. The Jnet of
Pi ranged between 169–224 nmol/(cm2·h) in
newborn animals and between 29–34 nmol/(cm2·h) in
suckling piglets.
To get more detailed information about this marked decrease,
Pi flux rates of newborn con and def piglets as a function
of early postnatal life are compared in Fig. 1. Time courses can be explained by
approximations to nonlinear regression curves, resulting in
t1/2 values of 0.62 ± 0.24 and 1.04 ± 0.31 days
for con and def animals, respectively. Based on these approximations,
the Jnet of Pi decreased from 414
nmol/(cm2·h) at parturition to 70
nmol/(cm2·h) at the end of first week pp in con animals
and from 370 nmol/(cm2·h) to 60
nmol/(cm2·h) in def animals. Time courses of
Pi net flux rates during the first week pp did not differ
significantly between the two groups, and this was also found during
the first 4 weeks of postnatal life.
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). In
contrast, Pi net flux rates in weaned def piglets were
reduced by about 80% compared with those in con piglets. Treating def
animals with vitamin D3 had no significant effect on
Pi net flux rates in newborn as well as in suckling
animals, but caused 5-fold increases after weaning.
Kinetics of Na+-dependent
Pi uptake into small intestinal BBMV as
affected by postnatal development and/or treatment with vitamin
Di
Initial Na+-dependent Pi uptake as a
function of the extravesicular Pi concentration was
saturable in all animal groups and was evaluated according to
Michaelis-Menten kinetics. Kinetic parameters of the
Na+-dependent Pi transporter as measured in the
presence of an inwardly directed Na+ gradient of 100
mmol/liter are presented in Table 4. The
Pi concentration that yielded half-maximal transport
(apparent Km) was estimated between 0.48–0.65 mmol/liter
and was not significantly affected by different calcitriol conditions
or age. Regardless of the calcitriol concentrations in plasma, the
apparent Vmax in newborn animals was 4- to 6-fold higher
than that in suckling piglets. The Vmax ranged between
1.90–2.15 nmol/(mg protein·10 sec) in newborn animals and between
0.36–0.57 nmol/(mg protein·10 sec) in suckling piglets. In con
piglets, the apparent Vmax remained relatively constant
during the weaning period until 6–7 weeks pp, whereas the
Vmax in weaned def piglets was decreased by more than 60%
compared with that in respective con animals. Treating def animals with
vitamin D3 had no significant effect on the
Vmax of Na+-dependent Pi uptake
into small intestinal BBMV of newborn as well as suckling animals, but
caused a 2.7-fold increase in Vmax compared with that in
untreated animals after weaning.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Active Pi absorption in newborn and
suckling piglets is calcitriol independent
Plasma calcitriol concentrations were already unphysiologically
low in newborn def piglets and did not change during further postnatal
development. Despite this defect and in contrast to weaned animals,
affected piglets showed constant active Ca2+ absorption
from the upper small intestines during the first 4 weeks of life. This
was not different from control conditions and could not be modulated by
treatment with vitamin D3 (31). From these results the
presence of calcitriol-independent mechanisms for active
Ca2+ absorption during postnatal development of pigs was
postulated. On the basis of the present data, this assumption also
holds for small intestinal active Pi absorption.
Furthermore, the presence of active mechanisms for Ca2+ and
Pi absorption from the upper small intestines could at
least partially contribute to normocalcemia and normophosphatemia
during the first 4 weeks pp, which was observed despite
unphysiologically low plasma calcitriol levels. In contrast, in weaned
def piglets, hypocalcemia and hypophosphatemia developed, which
correlated with low calcitriol concentrations in plasma (32).
In contrast to the relatively constant net flux rates of Ca2+ during early postnatal development and the weaning period, respective Pi transport showed remarkable adaptational processes, as active Pi absorption declined to only one fifth of the parturition level within the first week of life. A similar age-related change in Pi net absorption was found by Radde et al. (14) comparing duodenal epithelia from 1- to 14-day-old and 15- to 35-day-old piglets. Somewhat analogous findings to the rapid decrease in duodenal active Pi absorption were the decreases in monosaccharide (i.e. glucose and galactose), amino acid (i.e. leucine and proline), and Na+ and Cl- transport (16).
As mentioned above, analysis of the basic characteristics of the Na+-dependent Pi uptake into pig small intestinal BBM clearly demonstrated the calcitriol dependence of Pi transport capacities (Vmax) in weaned animals and the calcitriol independence but age-related adaptations in newborn and suckling piglets, whereas Pi transport affinities (Km) were not affected by either calcitriol or developmental factors. With respect to the weaning situation, similar results have been reported by others (8), and postnatal decreases in Na+- dependent Pi uptake into intestinal BBMV without significant effects on the Km values were also found in rats and rabbits (33, 34).
Regardless of the calcitriol concentrations in plasma Vmax values in suckling piglets were 4- to 6-fold lower than those in newborn animals. It should be noted that the Vmax values are usually expressed on a per unit protein basis; therefore, the present findings would also be explained by an increased BBM protein content, whereas Pi transport capacity remains unchanged. This assumption, however, appears unlikely because the AP levels, as a dominant enzyme of the intestinal BBM, were not significantly affected by age (see Materials and Methods).
Age-related adaptations of nutrient and electrolyte transport systems have often been attributed to the abrupt shift from placental nutrition to external feeding, including feeding changes from milk to the adult diet (35). Different models exist concerning the regulation of developmental changes in transport capacities. They may be induced by changes in the diet, such as carbohydrate and protein contents (36, 37, 38), or may be controlled by "hard-wired" nuclear events (39). There is also evidence for hormonal regulation of transport activities during the postnatal period (40, 41). From the present data we cannot decide which factors might be involved in Pi transport development, but a significant function of calcitriol appears rather unlikely. Further investigations should elucidate the effects of individual milk components on the development of intestinal Pi absorption. As Ca2+ and Pi metabolisms are intimately linked, potential candidates could include PTH-related peptide, which has been shown to enhance intestinal and placental Ca2+ transport (42, 43). In contrast, PTH-related peptide inhibited renal Na+/Pi cotransport, as reflected by decreased Vmax values (44). With respect to intestinal Ca2+ transport and renal Pi reabsorption, similar results were obtained with PRL and calcitonin, respectively (45, 46). Finally, casein phosphopeptides have been found to increase Ca2+ uptake into isolated piglet enterocytes by specific mechanisms (47).
The present Vmax values of Na+-dependent Pi uptake across the small intestinal BBM are quite well correlated with respective transepithelial Pi net flux rates. Assuming an average yield of 1.0 mg BBM protein from a mucosal sheet of 5 cm2 (30) enables a rough estimation of maximal apical Pi transport rates in nanomoles per cm2/h on the basis of Vmax. According to this assumption, Vmax values of 2.14, 0.36, and 0.27 nmol/(mg protein·10 sec), as found in newborn, suckling, and weaned def piglets, correspond to uptake rates of 154, 26, and 19 nmol/(cm2·h). These rough estimates are in good agreement with respective transepithelial Pi net flux rates obtained from Ussing chamber experiments. From this, it may be concluded that the apical Pi uptake represents the major rate-limiting step of the overall transepithelial Pi transport as has been discussed previously (9, 13).
Studies in vitamin D-deficient rats have shown that a single physiological dose of calcitriol enhanced intestinal Ca2+ absorption for up to 1 week (48), and this could also be true for Pi absorption. Thus, it might be possible that calcitriol derived from maternal circulation or from colostral origin could be sufficient for enhancement of neonatal intestinal Ca2+ absorption. However, active Pi absorption was present in proximal small intestines of def piglets during the first 4 weeks pp, which is much more than the normal enterocyte lifespan of 2–3 days (49) and could not be affected by treatment with vitamin D3. Furthermore, the def sows that were used had rather low plasma calcitriol levels (at least <30 pg/ml) at all stages of pregnancy and at parturition (50, 51), and calcitriol levels in the colostrum were below the detection limit (Schröder, B., unpublished data). Therefore, it appears unlikely that either uterine exposure to maternal calcitriol or dietary intake could account for active Pi transport during early postnatal development.
If receptor-mediated genomic action of calcitriol on intestinal Pi transport would be assumed, as has been shown for the human renal Na+/Pi cotransporter (52), it has to be kept in mind that the physiological effect of a hormone may be exerted not only by its circulating concentration but also by changes in the binding properties of the specific receptors in target tissues. Thus, it could be argued that low plasma calcitriol concentrations in def animals could be compensated by respective VDR adaptations to stimulate active Pi absorption in the neonatal gut. This possibility, however, can be excluded, because previous studies on VDR properties could not demonstrate any differences between con and def piglets (15). Some of the intestinal segments examined in that study were also used for Pi flux rate measurements. Interestingly, normal active Pi absorption was still present when VDR levels were beyond the detection limit (data not shown), indicating VDR-independent stimulation of the duodenal BBM Na+/Pi transport capacity in early postnatal life. This, however, has yet to be proven by experimental evidence.
In conclusion, our findings reveal calcitriol-independent mechanisms for active intestinal Pi absorption during the neonatal and suckling periods. The onset of the classical calcitriol-dependent mechanism for active intestinal Pi absorption occurs during weaning. Further studies of the neonatal active Pi transport, its development, and its regulation will focus on the mode of transcellular Pi movement and basolateral extrusion.
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Footnotes |
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Received August 26, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3-occupied vitamin
D receptors with cellular membrane acceptance sites. Endocrinology 137:3649–3658[Abstract]
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def; n = 10; r2 = 0.87)
during the first week pp. Nonlinear regression curves were calculated
with the BMDP3R software algorithm (for more details, see
Materials and Methods). The parameter values given in
the box represent estimated mean values with their
respective asymptotic SDs.