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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 1alpha -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.

                              
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Table 1.   Descriptive characteristics of subjects in experiment I

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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

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

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.

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.

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

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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