Duodenal Ca2+ absorption is
not stimulated by calcitriol during early postnatal development of
pigs
Bernd
Schroeder1,
Maria Regina
Dahl2, and
Gerhard
Breves1
1 Department of Physiology,
School of Veterinary Medicine, D-30173 Hannover; and
2 Department of Veterinary
Physiology, Justus Liebig University, 35392 Giessen, Germany
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ABSTRACT |
The role of calcitriol in stimulating intestinal
active Ca2+ absorption during
postnatal life was studied in newborn, suckling, and weaned control
(Con) piglets and piglets suffering from inherited calcitriol
deficiency (Def piglets). In addition, a group of Def piglets was
treated with vitamin D3
(Def-D3 piglets), which normalized plasma calcitriol levels. Regardless of age, duodenal
calbindin-D9k concentrations
ranged between 1,839 and 2,846 µg/g mucosa in Con piglets, between
821 and 1,219 µg/g mucosa in Def piglets, and between 2,960 and 3,692 µg/g mucosa in Def-D3 animals.
In weaned animals, active Ca2+
absorption as calculated from in vitro
45Ca2+
flux rate measurements in Ussing chambers could be related to calbindin-D9k levels. Thus active
Ca2+ absorption was completely
absent in Def animals but was reconstituted in
Def-D3 animals. In contrast, in
newborn Def piglets active Ca2+
absorption functioned normally despite the low plasma calcitriol and
mucosal calbindin-D9k levels and
could not be affected by treatment with vitamin
D3. Similar results were obtained
from suckling Def piglets. The microtubule-disrupting agent colchicine caused significant inhibition of transepithelial net
Ca2+ absorption in duodenal
epithelia from newborn piglets without exerting an effect in suckling
and weaned animals. Colchicine had no effect on
Ca2+ uptake across the brush
border membrane of mucosal enterocytes or on glucose-dependent
electrogenic net ion flux rates in duodenal preparations from newborn
Con piglets. In conclusion, our findings reveal intestinal active
Ca2+ absorption during early
postnatal life of pigs that involves calcitriol-independent mechanisms
and that may include intact microtubule actions.
phosphate absorption; calbindin-D9k; colchicine
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INTRODUCTION |
IT IS GENERALLY ACCEPTED that
the upper small intestine is the major site for active
Ca2+ absorption, which is
stimulated by calcitriol in most vertebrate species so far investigated
(1, 3, 10, 21, 23, 25). According to the present concept, calcitriol
exerts at least one of its effects on the intestinal mucosa by vitamin
D receptor (VDR)-mediated genomic actions (6). This process results in an increased production of
calbindin-D9k, a cytosolic
Ca2+-binding protein that has been
proposed to facilitate the movement of
Ca2+ across the cytosol from the
apical to the basolateral side of the enterocytes (2, 11, 25, 40).
Evidence for this cellular process has also been found in pigs (14,
46). In chick intestine, transcytotic vesicular
Ca2+ transport that depends on
adequate function of certain elements of the cytoskeleton such as
microtubules has been proposed (34, 37). However, it must be kept in
mind that the exact mechanisms that are involved in transcellular
active Ca2+ transport have still
not been definitively clarified. This includes the role of calcitriol
in modulating Ca2+ uptake across
the brush-border membrane (BBM) of the enterocytes as well as the
extrusion process at the basolateral side via
Ca2+ pumps (21, 54). In addition
to its well-known long-term genomic effects, vitamin D hormone also
initiates an acute enhancement of intestinal
Ca2+ transport, at least in chicks
(38). This rapid, nongenomic calcitriol effect has been called
"transcaltachia" and could be attributed to a plasma membrane
receptor-mediated event (35). However, the physiological relevance of
the transcaltachia process for the in vivo situation is still under
discussion (25).
In contrast to the calcitriol-dependent active
Ca2+ absorption in weaned and
adult pigs, earlier studies with newborn piglets have provided indirect
evidence for calcitriol-independent mechanisms during the first week
postpartum (46). This is mainly based on the observation that newborn
piglets suffering from inherited calcitriol deficiency (pseudovitamin D
deficiency rickets type I) showed normal active
Ca2+ absorption during this period
of life despite low plasma calcitriol and low mucosal VDR levels,
whereas at weaning active Ca2+
absorption in these piglets was completely absent under the same conditions.
This study (46), however, could not exclude the possibility that during
early postnatal life even low endogenous plasma calcitriol
concentrations or exogenous calcitriol from the maternal circulation
could have influenced the cytosolic VDR system and thereby might have
stimulated intestinal Ca2+
absorption. Such an effect had already been described in a
study with vitamin D-deficient rats that showed enhanced intestinal Ca2+ transport up to 1 wk after
administration of a single physiological amount of calcitriol (19).
To obtain more detailed knowledge of the role of calcitriol in active
Ca2+ absorption in the neonatal
gut, high doses of vitamin D3 were administered to Def piglets and the resulting duodenal
calbindin-D9k concentrations were
compared with unidirectional Ca2+
flux rates in newborn, suckling, and weaned animals. In addition, the
effect of the microtubule-disrupting agent colchicine on active Ca2+ absorption during postnatal
development was evaluated (7).
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MATERIALS AND METHODS |
Animals and feeding.
The protocol of the animal treatment was approved and its conduct
supervised by the animal welfare officers of the School of Veterinary
Medicine, Hannover, and the Justus Liebig University, Giessen, Germany.
In experiment I normal piglets of both
sexes served as controls (Con piglets). Piglets with pseudovitamin D
deficiency rickets type I were used as described previously (46, 48).
Affected animals suffer from an inherited defect of renal
25-hydroxyvitamin D3
1
-hydroxylation that leads to calcitriol deficiency (Def piglets). The ages of the piglets were between 11 h and 6 days postpartum (newborn), 21-28 days postpartum (suckling), or 6-8 wk
postpartum (weaned). In addition, a group of Def piglets was treated
with single intramuscular injections of vitamin
D3
(Def-D3 piglets). In detail,
newborn Def piglets were treated 2 days before the experiments with
50,000 IU of 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 of vitamin
D3, respectively. All newborn
piglets were only suckled before death, whereas suckling piglets were
allowed gradually to wean, with access to the normal diet of their
mothers. At the age of 4-6 wk postpartum, 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) Ca2+,
0.65% (wt/vol) phosphate, and 50 µg vitamin D/kg. Water was available at all times. Table 1 summarizes
the different animal groups of experiment
I with their respective treatments, age postpartum, and
body weight.
For practical reasons the small intestinal segments of the weaned group
in experiment II were obtained from 6- to 7-mo-old porkers of ~100-kg body weight that had been slaughtered
at the local abattoir. In experiment
III newborn Con piglets of both sexes were used to
study intestinal mannitol flux rates and the effects of colchicine on
glucose-stimulated electrogenic net ion flux rates and to study the
time courses of Ca2+ and glucose
uptake into isolated duodenal BBM vesicles (BBMV).
Blood.
Blood samples were taken by venipuncture from the cranial vena cava
with ammonium heparinate-covered syringes and were centrifuged at 3,500 g at 4°C for 10 min. Plasma was
stored at
70°C.
Intestinal segments.
On the day of the experiment, piglets were stunned with a commercial
abattoir shooting apparatus and then killed by exsanguination from the
carotid arteries. Within 5 min after slaughter, segments (~20- to
40-cm length) of the proximal small intestine were removed 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 at 4°C in the same
glucose-containing buffer solution that was later used in the flux rate
studies. The solution was continuously gassed with carbogen (95%
O2-5%
CO2) until the tissues were
mounted in Ussing chambers. In newborn piglets, the tissues were
primarily duodenal epithelia, with a minor portion obtained from the
proximal jejunum. Furthermore, rinsed segments of the proximal jejunum
~60 cm in length were frozen in liquid nitrogen and stored at
80°C until preparation of mucosal scrapings for
determination of calbindin-D9k concentrations and until preparation of BBMV.
Flux rate measurements.
Studies of Ca2+,
Pi, and mannitol flux rates across
intact epithelial tissues from stripped duodenum were made using the in vitro Ussing chamber technique as described in detail previously (43,
46, 47). The unidirectional mucosal-to-serosal
(Jms) and
serosal-to-mucosal
(Jsm) flux
rates of single Ca2+
(experiment I), simultaneous
Ca2+ and
Pi (experiment
II), or mannitol (experiment
III) were measured in Ussing-type chambers with an
exposed serosal area of either 0.5 cm2 (tissues from
newborn animals) or 1 cm2 (tissues from suckling and weaned
piglets). The tissues were incubated on both sides with 13 ml of a
buffer solution (pH 7.4) containing (in mmol/l) 125.4 NaCl, 5.4 KCl,
1.2 CaCl2, 21 NaHCO3, 0.3 Na2HPO4,
1.2 NaH2PO4,
and 0.01 indomethacin. In addition, the serosal solution contained 10 mmol/l glucose and the mucosal solution contained 10 mmol/l mannitol.
Because earlier studies with small intestinal tissues from rabbits and
our own experiments with pigs provided substantial evidence for the
involvement of prostaglandins in the modulation of electrogenic
Na+ and
Cl
absorption that may
interfere with Ca2+ and/or
Pi transport, we used indomethacin
to reduce cyclooxygenase activity and to ensure consistent electrical
properties of the duodenal epithelia from different animal groups (45,
51). In the flux rate studies of mannitol, both buffer solutions
contained 5 mmol/l glucose and 1 mmol/l mannitol. All buffers were
continuously circulated and gassed with carbogen at 39°C.
About 20 min after the tissues were mounted, in
experiment I 185 kBq of
45CaCl2
(1.14 MBq/mg; DuPont NEN, Bad Homburg, Germany) were added to either
side of the mucosa. In experiment II
Ca2+ tracer and 185 kBq of
[32P]orthophosphate
(370 MBq/ml, Amersham Buchler, Braunschweig, Germany) were used
simultaneously. A double-isotope labeling technique was applied to
control the specificity of the inhibitory effect of colchicine on
Ca2+ flux rates. In
experiment III 185 kBq of
[3H]mannitol (832.5 GBq/mmol, DuPont NEN) were used.
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 taken in three 10-min intervals (basal
conditions). After basal flux rate measurements in
experiment II, 1 mmol/l colchicine was
given into the mucosal compartment. After a 10-min exposure, sampling
was continued to obtain Ca2+ and
Pi flux rates for an additional
three 10-min intervals.
Unidirectional Ca2+ and mannitol
flux rates were calculated by using standard equations after counting
respective tracer radioactivity in 4.5 ml of scintillation fluid in a
Packard Tricarb liquid scintillation counter (46). In
experiment II, unidirectional flux
rates of Ca2+ and
Pi were calculated by the same
procedure after corrections of the overlap of the
32P tracer radioactivity into the
45Ca channel.
To avoid transepithelial electrical gradients, all flux measurements
were performed under short-circuit current
(Isc)
conditions. Net flux rates
(Jnet) were
calculated as differences between
Jms and
Jsm of paired
tissues whose conductances did not differ by >25%.
Mucosal calbindin-D9k concentrations.
The measurements of mucosal
calbindin-D9k concentrations in
intestinal tissues were performed by standard ELISA (48). Porcine calbindin-D9k purified from upper
small intestinal mucosa and antisera raised in New Zealand White
rabbits with antibodies against calbindin-D9k were kindly provided
by Dr. J. A. H. Timmermans (Dept. of Physiology, Univ. of Nijmegen, The
Netherlands). In general, preparation and heat treatment of mucosal
scrapings and gel filtration chromatography to isolate pig
calbindin-D9k from samples were
carried out according to the method of Gleason and Lankford (16).
Purification was monitored by SDS-PAGE (28) and fluorescence
spectrometry (excitation, 276 nm; emission, 306 nm) (4). The
competitive ELISA procedure has been described elsewhere (52).
Calbindin-D9k concentrations are
expressed as micrograms of
calbindin-D9k per gram of mucosal
scrapings.
Preparation, enrichment, and functional integrity of BBMV.
BBMV from the proximal small intestines of three newborn Con piglets
were prepared by a differential centrifugation method involving two
Mg2+-EGTA precipitation steps as
described recently (42). Final BBMV suspensions were immediately frozen
in liquid nitrogen and stored at
80°C until determinations
that were performed within 4 wk. During this period the properties of
initial Ca2+ uptake into BBMV
remained constant. To control the enrichment of the BBM, the activity
of alkaline phosphatase (EC 3.1.3.1) as a marker for the BBM fraction
was calculated from photometric measurements of the rate of hydrolysis
of p-nitrophenyl phosphate according
to an assay kit from Boehringer (Mannheim, Germany), and the activity
of the
Na+-K+-ATPase
(EC 3.6.1.37) was determined as a marker for the basolateral membranes
(BLM) (42). The relative enrichments of small intestinal BBM from
newborn Con piglets, expressed as the ratio of enzyme-based BBM-to-BLM
enrichments (BBM/BLM) were four- to sixfold, which was not
significantly different from respective values in weaned animals (42).
This indicates adequate enrichments of BBMV fractions. The functional
integrity of the vesicles, e.g., leakiness, was excluded by
demonstrating substantial overshoot of
Na+-dependent glucose uptake into
BBMV (see Fig. 2). Time-dependent glucose uptake was determined at 0.5, 1, 2, 3, and 60 min with 0.05 mmol/l
[3H]glucose-D-glucose
{specific activity of
D-[1-3H(N)]glucose
370-1,100 GBq/mmol; DuPont NEN}. Time-dependent
Ca2+ uptake into BBMV (1, 2, 5, 10, and 60 min) was studied in the presence of 0.25 mmol/l
45Ca2+/Ca2+
(specific activity of
45CaCl2
28 GBq/mmol; Amersham Buchler) after 15-min pretreatment with 1 mmol/l
colchicine by using the rapid filtration technique with cellulose
nitrate filters of 0.65-µm pore size (Sartorius, Göttingen,
Germany). Details have been described elsewhere (21, 42).
Calcitriol and
Ca2+ in plasma.
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 highly specific
calcitriol receptor of calf thymus. Total plasma
Ca2+ concentrations were measured
using a Boehringer test kit.
Chemicals.
The chemicals for preparing the buffer solutions, colchicine, and
ouabain were purchased from Sigma (Deisenhofen, Germany). All other
compounds were of analytical grade and were commercially available.
Statistics.
Statistical calculations were performed using the BMDP-92 software
program (9). All values are arithmetic means ± SE of the appropriate
number of animals. P values <0.05 were
considered significant. Significant differences of
Jnet from zero
were tested 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.
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RESULTS |
Calcitriol and
Ca2+
concentrations in plasma and calbindin-D9k
in duodenal mucosa as affected by postnatal development and treatment
with vitamin D3.
Concentrations of calcitriol and total
Ca2+ in plasma as well as
calbindin-D9k levels in duodenal
mucosa are presented in Table 2. Regardless
of postnatal age, plasma calcitriol levels were significantly lower by
60-80% in Def piglets compared with those in Con animals of the
same age. Treating Def piglets with vitamin D3 resulted in significant
increases in plasma calcitriol concentrations compared with those in
untreated Def animals, and final calcitriol concentrations in
Def-D3 piglets reached at least
80% of respective control values.
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Table 2.
Calcitriol and plasma Ca2+ concentrations and
calbindin-D9k levels in porcine duodenal mucosa as
affected by postnatal development and vitamin D3
treatment
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Plasma Ca2+ concentrations were
not affected by nonphysiologically low plasma calcitriol levels in
newborn and suckling Def piglets. However, plasma
Ca2+ levels in weaned Def piglets
were significantly decreased compared with those in Con animals.
Because of this hypocalcemia the animals developed typical clinical
symptoms of rickets (46). Plasma Ca2+ concentrations were increased
in all Def-D3 piglets compared with respective data of Con or Def animals. In suckling and weaned Def-D3 piglets this effect was
significant in comparison with Con and Def animals, respectively, and
it was shown in previous studies that enhancement of plasma
Ca2+ levels is related to the
prompt healing of rachitic symptoms in vitamin
D3-treated animals (22).
Duodenal calbindin-D9k levels were
related to plasma calcitriol concentrations and were significantly
lower in Def piglets compared with those in respective Con piglets of
the same age. Treating Def animals with vitamin
D3 led to significant increases of
calbindin-D9k levels compared with
respective untreated Def piglets and even resulted in higher
concentrations than found in respective Con piglets. This difference,
however, was not significant.
Electrical properties of porcine duodenal epithelia as affected by
postnatal development and treatment with vitamin
D3.
Isc and
respective tissue conductances
(GT) of
duodenal epithelia in experiment I are
presented in Table 3. Regardless of different calcitriol conditions,
Isc values were
highest in newborn animals and decreased gradually by ~30-60%
in suckling animals and 60-90% in weaned pigs. This indicates
substantial net electrogenic ion transport across the duodenum of
neonatal piglets compared with 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 that in Def and Def-D3
piglets (24, 12, and 15%, respectively). Regardless of age, treatment
of Def piglets with vitamin D3
caused lower GT
values compared with those in untreated animals of the same age. In
general, Isc and
GT data of
experiment I were confirmed in
experiment II using duodenal epithelia
from Con animals (Table 4). However, a
significant decrease of
Isc by 30% was
observed in duodenal tissues from newborn piglets in the presence of 1 mmol/l colchicine in the mucosal bathing solution. This effect on
Isc was absent in
older animals.
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Table 3.
Electrical parameters and unidirectional and net Ca2+
flux rates in porcine duodenal epithelia as affected by postnatal
development and vitamin D3 treatment
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Table 4.
Electrical parameters and unidirectional and net Ca2+
and Pi flux rates in duodenal mucosa during postnatal
development of Con piglets as affected by 1 mmol/l colchicine
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Ca2+ flux
rates across duodenal mucosa as affected by postnatal development and
treatment with vitamin D3.
Unidirectional and net Ca2+ flux
rates across duodenal epithelia of experiment
I are presented in Table 3. Regardless of age and
calcitriol condition,
Jms of
Ca2+ exceeded
Jsm. Except in
weaned Def piglets, this resulted in significant net flux rates of
Ca2+ from the mucosal to the
serosal compartment. Because the fluxes were measured in the absence of
electrochemical gradients, the significant
Jnet values
indicate the presence of active mechanisms for duodenal
Ca2+ absorption in all groups and
ages except in weaned Def piglets. Jsm values 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 amount of the
Ca2+ net absorption in newborn
animals was similar and ranged between 58 and 74 nmol · cm
2 · h
1.
In Con piglets the Ca2+ net
absorption remained relatively stable during postnatal development until 6-7 wk postpartum (Table 3) and decreased during a further fattening period (Table 4). In contrast, active
Ca2+ absorption in the Def group
was decreased by ~50% in suckling animals and was completely absent
in weaned piglets. Similar decreases during the first 4 wk of life were
found in Def-D3 piglets; however, at the weaning period
Jnet values were
significantly increased by treatment with vitamin
D3 compared with those in
untreated Def animals.
Ca2+ flux
rates across duodenal mucosa and BBM as affected by colchicine.
The results indicate that colchicine at a concentration of 1 mmol/l
produced ~35% inhibition of net
Ca2+ absorption in duodenal
epithelia of newborn Con piglets (Table 4). Similar results were
obtained from studies with newborn Def piglets, in which the
Jnet of
Ca2+ was reduced by the
application of colchicine from 54.3 ± 7.6 to 39.8 ± 10.5 nmol · cm
2 · h
1
(mean ± SE, n = 3 pigs). The
inhibition of the net Ca2+
absorption was only related to respective changes of
Jms. No effect of
colchicine on active Ca2+
absorption was observed in duodenal tissues of suckling and weaned pigs.
The unidirectional and net flux rates of
Pi in duodenum were not affected
in newborn, suckling, and fattened pigs (Table 4). Furthermore, the
time courses of Ca2+ uptake (Fig.
1) and
Na+-dependent glucose uptake (Fig.
2) into isolated BBMV from small intestinal
mucosa of newborn Con piglets were not affected by 15-min preincubation
with 1 mmol/l colchicine.

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Fig. 1.
Time course of Ca2+ uptake into
duodenal brush-border membrane vesicles (BBMV) from newborn control
(Con) piglets as affected by colchicine. For details of BBMV
preparation and Ca2+ transport
assay see MATERIALS AND METHODS.
Vesicles were filled and incubated with (in mmol/l) 10 HEPES-Tris, 100 KCl, and 100 mannitol (pH 7.4) at 21°C in absence or presence of 1 mmol/l colchicine and 0.25 mmol/l labeled/nonlabeled
Ca2+. Each assay was performed in
triplicate. Each point is mean ± SE of 3 experiments with different
animals.
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Fig. 2.
Time course of glucose uptake into duodenal BBMV from newborn Con
piglets as affected by colchicine. For details of BBMV preparation and
glucose transport assay see MATERIALS AND
METHODS. Vesicles were filled with (in mmol/l) 10 HEPES-Tris, 100 KCl, and 100 mannitol (pH 7.4) and incubated in same
buffer system with 100 mmol/l KCl (K) or NaCl (Na) at 21°C in
absence or presence of 1 mmol/l colchicine and 0.05 mmol/l
labeled/nonlabeled glucose. Each assay was performed in triplicate.
Each point is mean ± SE of 3 experiments with different animals.
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In addition, glucose-dependent electrogenic net ion flux rates in
duodenal tissues from newborn piglets as indicated by respective Isc values were
not affected by the application of colchicine (Fig.
3).

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Fig. 3.
Electrogenic net ion transport (short-circuit current,
Isc) across
stripped duodenal epithelia from newborn Con piglets as affected by
glucose and colchicine. Colchicine (1 mmol/l) was added to mucosal
compartment 70 min after tissues were mounted. Tissues were incubated
either in presence of glucose (experiment
I, mucosal glucose 10 mmol/l) or mannitol
(experiment 2, mucosal mannitol 10 mmol/l). In experiment 2, after 110 min of incubation 10 mmol/l glucose and mannitol were added to mucosal
and serosal compartments, respectively. In both experiments,
10 4 mol/l ouabain was added
to serosal side of epithelia at 140 min to block activity of
basolateral
Na+-K+-ATPase.
Each point represents mean ± SE of 3 experiments with 6- to
8-day-old animals.
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Mannitol flux rates across duodenal mucosa from newborn Con piglets.
In Table 5 the unidirectional and net flux
rates for mannitol in pig duodenum are shown. Because of the presence
of mucosal glucose (5 mmol/l), the
Isc values were
five- to sevenfold higher compared with respective values under
glucose-free conditions in experiments
I and II. The
Jms of mannitol
exceeded the flux rate in the opposite direction, resulting in a
mannitol net absorption of ~17
nmol · cm
2 · h
1.
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Table 5.
Electrical parameters and unidirectional and net mannitol flux
rates in duodenal epithelia from newborn Con piglets
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DISCUSSION |
Intestinal active
Ca2+ absorption
in weaned piglets.
The well-established concept of calcitriol-dependent active
Ca2+ absorption in the proximal
small intestines in weaned and adult pigs (14, 21, 44, 46) could be
confirmed by the present experiments and therefore needs no further
discussion. In contrast, our studies with newborn and suckling animals
suggest the prevalence of calcitriol-independent mechanisms or factors
synergistic to calcitriol mediation of active
Ca2+ absorption during early
postnatal life.
Active Ca2+
absorption in newborn and suckling piglets.
Plasma calcitriol concentrations were already nonphysiologically low in
newborn Def piglets and remained unchanged during the first 4 wk of
postnatal life. On the target cell level, these plasma calcitriol
concentrations were reflected by at least 50% lower mucosal
calbindin-D9k contents compared
with those at normal plasma calcitriol concentrations. However, in
contrast to weaned animals this reduction had no significant effect on
active Ca2+ absorption in newborn
Def piglets. This at least indicates a different role of calbindin in
neonatal active Ca2+ transport
compared with its role after weaning. This assumption is supported by
the finding that a threefold higher
calbindin-D9k level as induced by
vitamin D3 treatment of Def
piglets did not benefit active
Ca2+ absorption.
Because present studies with Def piglets could not be performed in the
total absence of calcitriol, it cannot be decided whether the observed
neonatal active Ca2+ absorption
could exclusively be mediated by calcitriol-independent mechanisms or
could also be explained by factors synergistic to calcitriol or even
overriding its effect. Further investigations should elucidate the
potential regulatory effects of certain milk components or endocrine
factors on the postnatal development of intestinal
Ca2+ absorption. Potential
candidates could include prolactin, which has been shown to stimulate
intestinal Ca2+ absorption in
vitamin D-deficient rats (39), parathyroid hormone-related peptide,
which enhances intestinal and placental
Ca2+ transport (31, 55), and
calcitonin (24). Casein phosphopeptides should also be considered
because they have been found to increase Ca2+ uptake into isolated piglet
enterocytes by specific mechanisms (29). Finally, growth factors are
also known to be synergistic with the effects of calcitriol (8, 13,
20).
Regardless of differing calcitriol status or intestinal
calbindin-D9k levels, active
Ca2+ absorption decreased by
30-60% during the first 4 wk of life. In general, 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 a solid diet,
as well as morphological changes (5). Different models exist concerning
the regulation of developmental changes in transport capacities. These
changes may be induced by changes in the diet, such as carbohydrate and
protein contents (15, 18, 49), or may be controlled by
"hard-wired" nuclear events (53). Reduction of net
Ca2+ absorption from the
gastrointestinal tract because of decreases in growth rates appears to
be rather unlikely, because pigs of this age are still characterized by
high bone accretion of Ca2+ (12).
From the present data we cannot decide which factors might be involved
in Ca2+ transport development, but
a significant function of calcitriol appears rather unlikely because
the calcitriol levels in Def plasma were persistently low during
postnatal development. The effects, however, could be explained by a
respective decrease of plasma growth hormone levels. However, instead
of this, significant increases of growth hormone and insulin-like
growth factor I were observed in 10- to 45-day postpartum piglets (30).
With respect to the onset of classical calcitriol-dependent intestinal
Ca2+ absorption, it is noteworthy
that regardless of plasma calcitriol levels, 30-50% of the
suckling piglets (3-4 wk postpartum) showed intestinal net
Ca2+ flux rates that were rather
low or even zero, whereas the rest of the animals showed active
Ca2+ absorption as found in
newborn piglets. Also, in contrast to the situation in weaned Def
piglets, net Ca2+ flux rates in
suckling Def animals could not be stimulated by treatment with vitamin
D3. Taken together, these findings
could be interpreted as a consecutive replacement of apparent
calcitriol-independent mechanisms of intestinal active
Ca2+ transport by
calcitriol-dependent processes during weaning.
Electrogenic net ion flux rates as indicated by
Isc in intestinal
tissues from newborn piglets were significantly higher than in weaned
animals. Therefore, it must be determined whether the net flux of
Ca2+ could also be explained by
paracellular net transport of water ("solvent drag") because of
other active ion-transport processes. These experiments were performed
by measuring unidirectional flux rates of mannitol to estimate net
water absorption. In the presence of mucosal glucose (5 mmol/l)
electrogenic Na+ transport was
stimulated at a maximum. Under these conditions Isc values
increased up to 7.6 µEq · cm
2 · h
1,
which is almost fivefold higher than in the absence of mucosal glucose,
and estimated net water absorption could account for net
Ca2+ transport of ~20
nmol · cm
2 · h
1.
Under glucose-free conditions water transport would be accordingly lower and thus could not explain present net
Ca2+ flux rates.
Studies in vitamin D-deficient rats have shown that a single
physiological dose of calcitriol enhanced intestinal
Ca2+ absorption for up to 1 wk
(19). Thus it might be possible that calcitriol derived from maternal
circulation or from colostral origin could be sufficient to satisfy the
intestinal VDR requirement for enhancement of neonatal intestinal
Ca2+ absorption. However,
significant active Ca2+ absorption
was present in proximal intestines from Def piglets during the first
3-4 wk after birth, which is much more than the normal enterocyte
life span of 2-3 days (50), despite nonphysiologically low mucosal
calbindin-D9k contents, and could
not be affected by the treatment with vitamin
D3. Furthermore, plasma calcitriol levels were significantly lower in Def sows at all stages of pregnancy and at parturition (57 ± 7 pmol/l) compared with calcitriol levels in normal sows (185 ± 30 pmol/l) (26, 27). Thus the calcitriol levels in Def sows did not differ significantly from those determined in newborn Def piglets. In addition, calcitriol levels of colostrum were below the detection limit (B. Schroeder, unpublished data). Therefore, it appears rather unlikely that either in utero exposure to
maternal calcitriol or dietary calcitriol intake can account for
Ca2+ absorption during early
postnatal development.
Because the physiological effect of a hormone may not only be exerted
by its circulating concentration but also by changes of the binding
properties of the specific receptors in target tissues, it could be
argued that low plasma calcitriol concentrations in Def animals could
be compensated by respective VDR adaptations to stimulate active
Ca2+ absorption in the neonatal
gut. This possibility, however, can be excluded, because a previous
study could not demonstrate any differences between Con and Def piglets
concerning VDR development or its binding properties (46).
Interestingly, some of the intestinal segments examined in that study
showed no detectable VDR levels, although normal active
Ca2+ absorption was present.
Role of calbindin-D9k in neonatal gut.
Regardless of postnatal age, treatment with vitamin
D3 caused significant increases of
intestinal calbindin-D9k levels.
This observation indicates a "functional" VDR system in
intestinal target cells, although the VDR concentrations were rather
low in neonatal piglets (~15% compared with weaned piglets; Ref.
46). However, it must be noted that the animals were treated with
pharmacological doses of vitamin
D3. This strategy not only
increases plasma calcitriol concentrations but also produces
long-lasting increases of 25-hydroxyvitamin D3 levels that could exert
VDR actions (22). The physiological role of
calbindin-D9k during this period
of life is not clear. It has been proposed that cells (such as those of
the intestinal epithelium) that are exposed to high concentrations of
Ca2+ because of their absorptive
activities may require protective buffers to minimize the toxicity of
free Ca2+ (34). Thus
calbindin-D9k could function in
such a context. Experimental support for this assumption comes from
experiments measuring intracellular free
Ca2+ concentrations in isolated
porcine duodenal enterocytes from Con, Def, and
Def-D3 piglets using a fura 2 fluorescence imaging system (48). Different increases of cytosolic free
Ca2+ concentrations after
incubation of cells with the Ca2+ ionophore ionomycin could
be directly related to respective
calbindin-D9k levels in different
animal groups. On the other hand, experimental evidence was given that
the calbindin-D28k molecule of the
chick intestinum might be a microtubule- or endocytic
vesicle-associated protein that could be involved in vesicular
Ca2+ transport processes (37).
Effect of colchicine on active
Ca2+ absorption.
Because our data do not support the model of
"calbindin-D9k-facilitated
transcellular Ca2+ movement"
(25) during early postnatal life, we used colchicine as an antagonist
of cytosolic microtubule functions in order to examine the possible
role of "vesicular- and microtubule-associated Ca2+ transport" as it has been
proposed for chicks (36, 37). This model includes apical
internalization of Ca2+ in
endocytic vesicles, fusion of the vesicles with lysosomes, and movement
of the lysosomes (along microtubules) to the basolateral membrane,
where exocytosis of the contents completes the transport process. At
least one of these steps appears to be generated by a calcitriol
membrane receptor-mediated, "rapid" nongenomic process (35). In
the present study we used colchicine at a concentration of 1 mmol/l in
the mucosal compartment. This concentration has already been shown in
earlier studies to produce an effect of 34-46% inhibition of
Ca2+ absorption in duodenum of
mature rats (32, 33), and this is in the same order of magnitude as the
decrease of net Ca2+ absorption in
small intestinal epithelia from neonatal piglets. In suckling piglets
we only found a small decrease of active
Ca2+ absorption of 6%. This
effect, however, was not significant. No effect could be observed with
weaned animals. We obtained similar results from recent studies using
latrunculin B as an actin filament-disrupting agent (B. Schroeder,
unpublished data). The data suggest that in contrast to the pig the rat
may maintain the newborn phenotype of transport.
From the present data it is difficult to decide whether the inhibitory
action of colchicine was through microtubules alone or could also
include plasma membrane events (17, 41). On one hand, the effect of
colchicine on transepithelial Ca2+
movement appears to be rather specific because simultaneously measured
net absorption of Pi was not
affected. On the other hand, the
Isc values
obtained from colchicine-treated duodenal epithelia of newborn piglets
from experiment II were decreased by
~30%. This could be explained by inhibitory effects of the substance on net electrogenic ion transport, such as secondary active
Na+-glucose symport. However, as
shown in experiment III, such an effect can most likely be excluded, because the effect of colchicine on
Isc was only
transient and could not influence the glucose-induced Isc stimulation
that was reflected by electrogenic
Na+ transport, as indicated by
ouabain inhibition of the basolateral Na+-K+
pump (Fig. 3). A significant effect of colchicine, whether direct or
indirect, at the enterocyte apical membrane outer surface that could
inhibit Ca2+ uptake can be
excluded because neither Ca2+- nor
Na+-dependent glucose uptake into
isolated duodenal BBMV was affected. However, it must be kept in mind
that microtubules are depolymerized by cold temperatures as commonly
used during the BBMV preparation procedure and therefore were probably
not present in the uptake studies. This means that inhibitory effects
of colchicine located at the inner surface of the cell membrane cannot
be excluded from the present results. In summary, it can be assumed
that the action of colchicine on
Ca2+ transport was restricted to
the cytosolic compartment of the enterocytes.
In conclusion, our findings reveal intestinal active
Ca2+ absorption during the early
postnatal life of pigs that involves calcitriol-independent mechanisms
and that may include microtubule actions. It appears that the onset of
the classical calcitriol-dependent and calbindin-mediated mechanisms occurs during weaning, with corresponding decreases of
transport features from the neonatal period.
 |
ACKNOWLEDGEMENTS |
The financial support of the Deutsche Forschungsgemeinschaft
(Schr342/2-1/2) is gratefully acknowledged.
 |
FOOTNOTES |
Address for reprint requests: B. Schroeder, Dept. of Physiology, School
of Veterinary Medicine, Bischofsholer Damm 15/102, D-30173 Hannover,
Germany.
Received 7 August 1997; accepted in final form 19 March 1998.
 |
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