I. INTRODUCTION

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Ontogenetic development of the mammalian intestine is a topologically and temporally highly organized process. One of the crucial results of this process is the formation of a specialized intestinal epithelium that fulfills digestive and absorptive functions including certain endocrine and immunological roles. The early ontogenetic development can be divided into five phases (159, 254): 1) morphogenesis, 2) cytodifferentiation and fetal development including preparation of the epithelium for absorption of colostrum and milk, 3) birth and early suckling period (the shift from an intra- to an extrauterine environment), 4) suckling period, and 5) weaning of the offsprings from mother's milk to a solid diet. The first two phases occur during gestation and prepare the intestine for the postnatal life when the intestine begins to be fully responsible for providing nutrients. During the suckling period, the immature gastrointestinal tract is offered a monotonous diet, mother's milk, which exerts high demands on the nutritional adequacy of the diet. The dietary transition from maternal milk to eating the adult diet, which occurs at weaning, is accompanied by profound modifications of digestive and transport functions when neonatal properties are lost and mature ones are acquired.

The development of the gastrointestinal tract in utero is characterized by extensive structural and functional changes of the intestinal epithelium. However, there are marked differences in the stages of intestinal maturation in various mammalian species at birth. The variations in the timing and extent of intestinal maturation reflect the duration of the gestational period. The altricial species such as the mouse and rat, which are born after a short gestation, closely depend on their dams for nutrition, thermoregulation, locomotion, and evacuation of the bowels. They do not achieve independence until after weaning (159). In contrast, intestinal development in the precocial species that have a long gestation period, such as the guinea pig, sheep, and pig, occurs earlier in utero (13, 372, 426). These species are more representative of human development in the fetal and neonatal periods (202).

In this review, a brief description of the pattern of growth and development of the intestinal mucosa is first presented, with emphasis on the mechanisms of the developmental changes. After this, the ontogenetic patterns of intestinal absorption of nutrients as well as ion and water absorption and secretion are examined together with the developmental control of these processes. Finally, the bioenergetics of intestinal transport are discussed. Although some results have been obtained in humans and other species, most studies have used rodents as a model of gastrointestinal development. Consequently, this review is predominantly focused on the rat, but comparison is also made with the gastrointestinal development in other species and humans.

	II. GROWTH AND DEVELOPMENT OF INTESTINAL MUCOSA

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The processes that accompany the morphogenesis and cytodifferentiation of the intestinal mucosa are temporally and topologically highly organized when temporal changes superimpose on the spatial diversity of gene expression found along the crypt-villus and duodenal-colonic axis (406). The primitive gut tube, comprised of the endoderm surrounded by mesenchyme, is established early in ontogeny. Later, the stratified endoderm formed of undifferentiated cells undergoes a cranial to caudal transition into a simple columnar epithelium with nascent villi. Cellular proliferation is detectable along the nascent villi as well as in the intervillus region but later becomes confined to the intervillus region, and crypts begin to develop. Simultaneously with these morphological changes, the expression of intestine-specific genes can be first detected (323). Even if there are timing differences between various species, the developmental pattern is basically similar.

As the mesenchyme invaginates into the undifferentiated endoderm, nascent villi begin to emerge. It seems that the formation of mesenchyme invaginations initiates the formation of villi (23). Injections of [³H]thymidine have been demonstrated to gradually confine cellular proliferation to the intervillus region of primordial crypts (10). Later on, this intervillus epithelium undergoes reshaping to form crypts by the penetration of epithelial cells into the underlying mesenchyme. Analysis of chimeric mice during the perinatal period revealed that at the early postnatal life the crypts contain cells of the mixed genotype (i.e., they are polyclonal). In contrast, the crypts in the mature intestine are composed of cells of one genotype derived from a single progenitor (342). These crypts contain only a small group of proliferating, undifferentiated stem cells that give rise to several cell phenotypes that migrate onto several adjacent villi (341). The mechanism of purification leading to monoclonality remains unknown. Early crypt development is typical for humans (268) and species with a long gestation period such as sheep (407), but not rodents, where the formation of crypts is observed only after birth (168). In addition to crypts, the proximal colon of the human fetus or neonatal rodents has transient villuslike structures that are not true villi but tall longitudinal folds (255). These villuslike transient structures have properties similar to enterocytes of the small intestine, such as the capability of endocytosis (285) and expression of brush-border enzymes (118, 209, 452) or transporters (see sect. IIIC1).

The cell proliferation pattern after birth differs from species to species. It is generally accepted that crypt cell proliferation in the small intestine is low in suckling rodents until the beginning of weaning (202). The redifferentiation of the intestine during weaning is associated with accelerated proliferation of enterocytes that contributes to crypt depth and villus height enlargement (202). It has been suggested, however, that there are differences in the pattern of mucosal remodeling. In the rat colon, for example, the epithelial labeling index during suckling and weaning periods was found to be relatively constant (246) or was even decreasing (65), whereas it was increasing in the mouse colon (255). The rapid reduction of villus height and increase of crypt depth was observed during suckling and weaning periods in species with longer gestation, e.g., lamb, piglet, and guinea pig (13, 148, 426).

The conversion of stratified epithelium into a simple epithelium involves distinguishing of the cytoplasmic membrane into two domains: the apical and basolateral membrane (5) separated by tight junctions (236). This process determines the polarity of epithelial cells, because only the basolateral membrane seems to recognize the basement membrane of the extracellular matrix and adhere to it. However, the basement membrane is not only the base for epithelial cells that is formed by the complementary deposition of epithelial and mesenchymal products (367) but also a structure that is able to control the ontogeny of epithelial cells (297). There is increasing evidence that morphogenetic movements, differentiation, and migration of epithelial cells are influenced by alterations in the composition of the extracellular matrix via cell surface receptors of epithelial cells that interact with proteins of the extracellular matrix (178). Developmental changes of these receptors, E-cadherins and integrins, have recently been described (20, 313, 365). One candidate for mediating mesenchymal-epithelial crosstalk is the Shh protein that is expressed throughout the embryonic intestinal endoderm (6). Although Shh appears to be involved in signaling from the endoderm to the mesoderm, signaling in the opposite direction may involve the scatter factor (325), the Fkh6 factor present in the embryonic gut mesoderm (190) or smads proteins which are involved in transforming growth factor- (TGF-) transduction pathway (369).

Concomitantly with the formation of villi and crypts, various epithelial lineages emerge from the immature primitive cells: absorption cells (enterocytes), mucus-secreting goblet cells, a variety of enteroendocrine cells, and Paneth cells synthesizing antibacterial peptides and enzymes. The developing intestine is able to establish and maintain differences in the differentiation programs of each lineage as a function of their location along the proximo-to-distal and crypt-to-villus gradient (326, 366). The capacity of the intestinal epithelium for establishing and maintaining these differences in expression are already evident in late fetal life (326). The complex interaction between luminal microbial flora and lymphoid cells with epithelial cells leads to differentiation of the follicle-associated epithelium and specialized epithelial M cells, which deliver luminal macromolecules or other foreign material from the lumen directly to intraepithelial lymphocytes (279).

In summary, intercellular communications represent one of the key processes in the differentiation and proliferation of the intestinal epithelium. The epithelial and subepithelial cells produce extracellular molecules that create a specialized local environment that can initiate and regulate genomic expression. The control of protein expression requires time- and space-specific transcriptional, posttranscriptional, and posttranslational mechanisms that follow precise spatial differentiation from the crypt to the villus tip and from the duodenum to the rectum. The basal differentiation program is encoded in the fetal endoderm and mesenchyme, yet extrinsic endogenous or exogenous substances play an important modulatory role in generating spatial differentiation during development (see sect. IV).

	III. ONTOGENY OF INTESTINAL TRANSPORT

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There is a close relation between the degree of maturation and absorptive functions of the intestine. This means that the intestinal transport of solutes is not constant, but specific developmental patterns can be observed despite species differences. Some of the developmental changes are specific, i.e., transport of one or several similar solutes is changed, or nonspecific when transport of many different solutes is altered. Whereas the changes are mainly reversible in adult animals, the developmental changes are mostly irreversible (107). The nonspecific adaptive changes reflect 1) changes in mucosal surface area, 2) changes in proliferation and migration of enterocytes, 3) changes in phospholipid composition and fluidity of the plasma membrane, and 4) changes in paracellular permeability. The specific changes are restricted to changes in turnover number, site density of transporters, the affinity constant, and/or expression of different isoforms of transporters.

When considering the nonspecific effects, dramatic changes in intestinal morphology and in the rate of cell proliferation and migration are two very important mechanisms that determine the ontogenetic changes of mucosal weight and surface area and thus of the total transport capacity. As reviewed in section IIA, the intestinal development is associated not only with the growth of the intestine but also with marked changes of intestinal villi in the small intestine and a transient appearance of villuslike structure in the proximal colon. Furthermore, postnatal development of enterocytes is associated with amplification of the surface of microvilli (apical membrane) and of the basolateral membrane (265, 413). The developmental changes of surface area are influenced by rapid growth of intestinal mucosa that is predetermined in altricial species by decreased cell turnover (202). Because of this decreased turnover rate, the lifetime of enterocytes is longer in the immature intestine than in adulthood and slower cell kinetics influence the presence of enterocytes of different phenotypes. The cells expressing one transport phenotype are replaced in these animals more slowly by cells exhibiting other transport properties. In addition, there are differences in the distribution of transporters along the crypt-to-villus axis in the immature and adult intestine. Nutrient transport in the immature intestine is localized not only in the upper villus as in the mature intestine but also along the whole villus down into the crypts (371).

Transport capacity can also be influenced by ontogenetic changes of paracellular permeability (see sect. IIII) and by remodeling of membrane phospholipids. The membrane fluidity of intestinal cell membranes is age dependent due to increasing cholesterol-to-phospholipids and protein-to-phospholipids ratios and changes in the spectrum of fatty acids (4, 102, 347, 348). As the changes of phospholipid environment alter the activity of various transporters (39, 40), the developmental changes of membrane phospholipids become a potential regulator of intestinal transport. However, this hypothesis has not been confirmed conclusively.

The specific developmental changes (density of transporters, their affinity, and turnover number) are discussed in the following sections.

B.  Interaction of Intestine With Ingested Colostrum and Milk

1.  Uptake of macromolecules: nonspecific and receptor-mediated endocytosis

Transfer of macromolecules across the intestinal wall represents an important transport mode that facilitates the uptake of a number of protein molecules such as immunoglobulins, growth factors, and many antigens including microorganisms. This transport is localized in enterocytes and specialized cells called M cells that are involved in the nonreceptor passage of intact macromolecules/antigens (337). From the physiological point of view, the transport of macromolecules is particularly important during early postnatal life when it facilitates the absorption of growth factors and immunoglobulin (Ig) G from maternal colostrum and milk. This is crucial for ungulates such as piglets or calves that are born nearly agammaglobulinemic but are capable of transferring intact immunoglobulins from ingested colostrum to the circulation during the first postnatal days (430). Nevertheless, other mammals, which are born more or less hypoglobulinemic such as the rat, mouse, or human, also receive IgG passively from the maternal milk through the proximal small intestine absorption (181, 412).

Transport of macromolecules follows two different pathways: 1) specific receptor-mediated transcytosis and 2) nonspecific transcytosis (Fig. 1). Specific transport macromolecules bind to specific receptors that shuttle them across the intestinal epithelium. On the other hand, nonselective transport is ensured by vesicular transport of macromolecules that adhere to the surface membrane or are transported in the fluid-phase compartment of the vesicles. The functional ability of the intestinal epithelium to take up macromolecules seems to be related to the presence of the apical tubular system and large supranuclear vacuoles whose distribution and ontogenetic patterns vary dramatically in different species. The intestine of altricial species contains cells with tubular systems and endocytotic complexes until weaning when the immature vacuolated enterocytes are replaced by mature nonvacuolated cells (202). In ungulates, massive nonselective endocytosis and transport of all intraluminal macromolecules take place during the first 2 postnatal days, and immunoglobulins compete with other proteins (30, 430, 431).

View larger version (32K): [in this window] [in a new window]   Fig. 1. Transepithelial transport of immunoglobulins. A: immunoglobulins IgA that are present in colostrum or milk bind to its receptor in the apical membrane; the complex is endocytosed, transported through the cell within transport vesicles, and secreted into the contraluminal compartment. B: in some mammals, one can see massive absorption via a nonselective endocytotic pathway when all intraluminal macromolecules are endocytosed, partially destroyed, and partially transported across the enterocytes. C: secretory immunoglobulins are transported across the enterocytes also by endocytosis. IgA dimers bind to receptors on the basolateral membrane, and the IgA-receptor complex is endocytosed and transported through the cell within transport vesicles. Cleavage of the complex at the apical membrane releases the secretory component attached to the IgA molecules.

The effective transport of ingested proteins is facilitated by decreased proteolytic degradation of proteins due to the presence of colostral protease inhibitors (428, 429). In contrast to ungulates, rat and rabbit milk has a relatively low protease inhibitor capacity (412), and IgG could escape the degradation by binding to the receptors. Rodent immunoglobulins are endocytosed and selectively transported across the intestinal mucosa due to binding to specific receptors for the Fc portion of IgG (182). The expression of Fc receptors is transcriptionally regulated, is maximal in proximal duodenum, and declines progressively in the distal bowel (245). The receptors bind with IgG at acidotic pH, are endocytosed via coated pits (169, 182), and transported to the basal surface within the vesicles that are protected against fusion with lysosomes (153). In human infants, IgG is transferred by the placenta during late gestation; nevertheless, the intestinal epithelium also expresses the receptors in the fetal and neonatal period, and macromolecular transport persists until after birth when it gradually decreases (14, 181).

Macromolecules that bind to the apical membrane in a nonspecific manner and nonadherent soluble molecules enter the vesicular system; however, only the adhered macromolecules cross the epithelial layer, whereas the soluble molecules are destroyed (133, 165, 392). The reason why macromolecules adhere more to the surface of neonatal than to that of adult enterocytes (380) is not clear. The relative contribution of enterocytes and M cells in nonspecific macromolecular transport is uncertain, even though some data indicate selective adherence of milk immunoglobulins to M cells (328), which appear early in embryogenesis (268, 375).

The relatively high permeability of the intestinal epithelium for macromolecules declines after birth, but the time, when their transport ceases, is species dependent. In newborn pigs, guinea pigs, and hamsters, the transport capacity decreases rapidly within the first postnatal days, whereas the transfer is terminated ~21 days after birth in rats and rabbits (213, 390, 430, 431). The process leading to decreased uptake of macromolecules is called gut closure and depends on factors such as epithelial maturation or increased intraluminal proteolysis (244, 245, 391).

In summary, the neonatal intestine is capable to absorb macromolecules from colostrum and milk including immunoglobulins, growth factors, and food antigens. The extent of this transfer is related to the importance of passive immunity for the newborns and differs between species. The specific transport of macromolecules is achieved in many cases by binding of luminal factors to specific receptors that shuttle them across the intestinal mucosa without intracellular hydrolysis. Intact food proteins cross the epithelium predominantly through specialized M cells.

Several lines of mostly indirect evidence indicate that milk-borne hormones and growth factors might be absorbed in the intestine (Table 1). Because gastric secretion is attenuated and proteolytic digestion is incomplete in newborns and sucklings, the peptidic substances such as epidermal growth factor (EGF) and insulin-like growth factor (IGF) can avoid digestion and reach the small intestine. In suckling animals, the intestinal degradation is low but increases in weanling animals (302, 316, 317, 352), and this low degradation facilitates the absorption of intact biologically active substances from the intestine.

The cellular transport mechanisms of milk-borne hormones and factors across the intestinal epithelium are not quite clear. Intestinal cells have receptors for some of these substances that are not only localized in the basolateral but also the apical membrane (62, 197, 395; for details, see Table 2). This transport might therefore be analogous to that demonstrated for IgG. Data concerning the rat ileum show that enterocytes of suckling rats take up the nerve growth factor (NGF) and EGF by both fluid-phase and receptor-mediated endocytosis and that a small fraction of NGF and EGF is transported across the epithelium into the circulation (134, 364). As the EGF receptor/tyrosine kinase is localized on the basolateral membrane only and not on the apical membrane (396), the putative receptors for transepithelial transport (134) seem to be different from EGF receptor/tyrosine kinase.

In conclusion, hormones and growth factors may survive digestion and may interact with enterocytes. The principal target tissue of these substances seems to be the intestine where they influence proliferation and differentiation (56, 58, 176, 301, 302), even though conflicting results have been obtained recently (57). A fraction of the substances that had been taken up may survive intracellular degradation and become absorbed into the neonatal circulation. However, the physiological importance of this absorption has yet to be established.

C.  Ontogeny of Nutrient Absorption

1.  Prenatal development

In view of the fact that fetuses extract nutrients from the placenta, it might be expected that the intestinal absorptive capacity of nutrients is negligible. However, as far as this question has been studied, it appears that some active transport pathways are present long before birth. Although premature transport activity was observed in various species, there are differences in the time of onset and the spectrum of transporters that are expressed. In precocious animals and in humans, the transporters can be detected earlier than in altricial species (17, 49, 51, 303). The prenatal expression of transporters during the third trimester allows the fetus to obtain carbohydrates, amino acids, and proteins from swallowed amniotic fluid (45, 304).

Active electrogenic transport of glucose is already present in the human fetal small intestine (220), and the duodenum-to-ileum gradient of glucose absorption is established between 17 and 30 wk of gestation (241). A detailed analysis of glucose uptake across the apical membrane of fetal enterocytes demonstrated two Na⁺-dependent pathways differing in their affinities, whereas a single system has been found in adulthood (238, 242). Similarly, two Na⁺-dependent glucose transport systems have recently been found in porcine proximal small intestine at a comparable gestational age, which was followed by a shift to a single high-affinity transport system at birth (53).

Although the issue of two distinct apical carriers with different affinities for glucose is still controversial, the high-affinity Na⁺-dependent system seems to be represented by the SGLT1 contransporter that actively transports glucose and galactose across the apical membrane and the mRNA levels of which increase during fetal development (91, 422). The developmental expression of facilitated glucose transporter isoform GLUT2, which is responsible for extrusion of glucose across the basolateral membrane, follows that of SGLT1 (91). In contrast to the adult intestine, which possesses only one facilitated glucose transporter (GLUT2), the fetal small intestine expresses two systems, GLUT2 and GLUT1, which are known from adult erythrocytes (91, 250). The expression of GLUT1 appears earlier than GLUT2 (before the formation of rat intestinal villi) and decreases gradually during fetal life. In parallel with the formation of villi, the enterocytes begin to express GLUT2 and to transfer it to the basolateral membrane (250). The mechanism of this developmental regulation is unknown, but the early expression of GLUT1 may play a role in early cell proliferation and growth. The apically localized fructose transporter GLUT5 is also expressed in fetal enterocytes and is developmentally modulated (91, 237).

The transporters of amino acids also develop prenatally (239); however, the developmental pattern of uptake is not identical for all amino acids (303). It thus seems reasonable to consider the differences in developmental regulation of individual systems. Some elements of the complex "machinery" of lipid absorption are also expressed in fetal small intestine. It has the ability to synthesize lipids, phospholipids, and apolipoproteins as well as to elaborate and secrete lipoproteins (21, 221, 223, 329). Because dietary fat in the milk is fundamental for proper growth and development during the suckling period, the functional system of lipid absorption should be considered to operate immediately after birth.

Prenatal expression of the nutrient transport pathway(s) is not localized only in the small intestine but is also situated in the colon, where it disappears soon after birth. For example, Na⁺-dependent glucose absorption and alanine transport were observed in fetal rat colon (306, 309) and methionine transport in fetal lamb colon (374).

With a few exceptions, the total intestinal transport capacity increases with age due to the increase of intestinal mass, but the transport for most carbohydrates and amino acids studied decreases in relation to intestinal weight (50, 52, 311, 403). In some species such as the pig, the intestine of which undergoes dramatic structural and functional changes during the first 24 h after birth, this decline may reach 50% of initial values (311, 451). The reason for this developmental decrease expressed in relation to tissue weight or surface area is not fully understood. In principle, there are three possible mechanisms responsible for the developmental decrease: 1) changes in the density of transporters, 2) replacement of one isoform by another, and 3) changes in turnover rate of the transporter. First, the decline cannot be attributed to the decrease of mucosa at the expense of muscle (52) but seems to be, at least partially, caused by a dilution of fetal enterocytes with new cells that have not yet had adequate time to express active transporters. In the neonatal intestine, nutrient transport occurs along the whole crypt-villus axis, whereas in the adult intestine the absorption of nutrients is shifted to the upper part of villi (371). Second, the existence of multiple carbohydrate transporters has been proposed on the basis of kinetic analysis (53, 238), but molecular biological proof is lacking. Third, although developmental changes of the Na⁺-K⁺-ATPase turnover number has been reported (295), no information is available for nutrient transporters.

As far as glucose absorption is concerned, the lactose in milk is hydrolyzed in the small intestine by lactase into glucose and galactose, and these sugars are absorbed by a Na⁺-dependent transport pathway (84, 162, 319, 357). This pathway reflects the SGLT1 cotransporter whose mRNA abundance is developmentally regulated (91, 121, 218, 261). The activity of this transport pathway exhibits a proximal-to-distal gradient with differences between activities in proximal and middle segments that disappear with age and a significant decrease of transport activities during weaning (282, 403). The cotransporter also translocates galactose so that the developmental pattern of galactose for this reason resembles that of glucose (52, 403). Fructose absorption follows quite a different developmental pattern, since it increases in the small intestine during weaning in the rat and rabbit, but a smaller increase is found in pigs (52, 311, 403). However, fructose utilizes a different transporter, GLUT5 (91, 360), for entering into enterocytes. Its expression is enhanced during weaning, and it is prematurely induced by dietary fructose (360). Although absorption of glucose, galactose, and fructose across the apical membrane is mediated by two different proteins, their transport across the basolateral membrane may be mediated by a single protein, the facilitated glucose transporter GLUT2, that decreases during the late suckling period (261).

The developmental decrease of transport relative to intestinal weight was also observed for some amino acids (52, 403). The developmental changes are consistent with a greater activity and/or number of transporters in enterocytes. A developmental decrease was not only observed in the epithelium but also in isolated enterocytes or apical membrane vesicles (269, 351). To my knowledge, information about the developmental regulation and contribution of individual amino acid transporters for the transport of different amino acids is absent. Adult intestine transports amino acids via Na⁺-dependent and Na⁺-independent pathways, and single amino acid can utilize several transporters. The analysis of amino acid transport in the suckling small intestine has proven Na⁺-independent lysine and alanine absorption as well as Na⁺-dependent alanine, taurine, and glutamine absorption (84, 184, 269, 319).

Na⁺-dependent amino acid and glucose transport systems also operate in neonatal colon during the first postnatal days and rapidly disappear later (156, 306, 350, 373, 374). The physiological significance of this phenomenon remains a matter of speculation; however, the transport ability of the colon to absorb carbohydrates and amino acids can compensate for the temporary low capacity of the small intestine to absorb nutrients. This hypothesis is supported by the finding of lactose absorption in the proximal colon of newborn sucklings (272, 273).

The principal source of energy during the suckling period is lipids. Although pinocytosis of lipids in intact globules of milk seems to be an important pathway for lipid absorption after birth (28), the immature intestine is also able to absorb fatty acids and cholesterol (253, 300). Triglycerides, which form the largest fraction of milk lipids, are digested in the absence of bile acids by gastric and lingual lipases (147) predominantly in fatty acids and 2-monoacylglycerols, whose uptake is significantly higher in the immature intestine than in adulthood (116, 253, 300). The uptake is probably a passive process (253) even if the existence of brush-border fatty acid binding protein in adult enterocytes indicates that absorption could be mediated by both diffusional and carrier-mediated processes (382). In addition, intestinal absorption of carnitine, a cofactor for lipid utilization, is increased in the neonatal small intestine (224). Little is known regarding the further fate of lipolytic products. Nevertheless, we can assume that they are easily resynthesized into triglycerides, phospholipids, and cholesteryl esters because the activity of reesterification enzymes in the immature intestine is as high as it is in adulthood (355). Apolipoproteins that are associated with lipids to form macromolecular complexes of lipoproteins are also developmentally regulated and present already in the fetal intestine (35, 377).

In summary, most nutrient transporters appear already prenatally and might thus constitute preparation against the risk of premature birth. The postnatal ontogeny of nutrient transporters reflects the need to absorb developmentally increasing quantities of nutrients that are required for growth and metabolism. The postnatal changes in transport capacities reflect age-dependent changes in intestinal mass, physicochemical properties of the cell membranes, types of transporters, and their kinetics. The colonic absorption of nutrients in sucklings seems to represent the additional salvage that compensates the loss of nutrients from the small intestine and less developed colonic bacterial fermentation.

D.  Ontogeny of Salt and Water Absorption

1.  Prenatal development

With the evidence about prenatal absorption of nutrients reviewed in section IIIC1, the absorption of ions and water should also be mentioned. Unfortunately, the data are too restricted to make definite conclusions about the significance of ion and water absorption of amniotic fluid. Analyses of luminal samples of ovine fetuses showed that osmolarity significantly increases from the rumen to the ileum and returns to near isotonic values in colon (354). This means that either colonic absorption is suppressed/immature near term or that there is a considerable colonic secretion. Some data indicate that at least three types of transport systems (Na⁺ channels, Na⁺/H⁺ exchanger, and Na⁺-nutrient cotransport) may be operating at the apical membrane. The presence of the Na⁺/H⁺ exchanger and Na⁺-glucose cotransporter have been demonstrated indirectly in the rat fetal colon (306) and presence of -subunit of Na⁺ channels in the mouse and rat fetal colon (86, 424). As was mentioned in section IIIC, fetal small and large intestine are able to absorb some nutrients via the Na⁺-dependent pathway (53, 306, 309). The basolateral Na⁺ pump (Na⁺-K⁺-ATPase) that produces the electrochemical gradient for Na⁺ that is used as an energy source for many cotransports or countertransports is present in fetal intestine and increases until birth (125, 174, 418).

A) WATER ABSORPTION. Water absorption is a passive process by which water passes across paracellular and transcellular pathways in response to osmotic gradients created by transcellular absorption of Na⁺ and other solutes. Similarly to other cells, some water channels of the aquaporin family (AQP) were recently identified in enterocytes, predominantly in the colon. Whereas AQP1 is expressed only in colonic crypts (154), other AQP are notably absent in certain parts of gastrointestinal tract (122, 180). In contrast, the recently cloned AQP8 has been found to be strongly expressed in enterocytes (204, 232). However, further studies are required to elucidate the cellular and subcellular localization and function of individual AQP in the intestine. Nothing is known about the developmental pattern of AQP expression in the intestine, even though remarkable changes in AQP expression have been reported in the developing kidney (440).

In situ perfusion experiments in suckling rats demonstrated a fourfold lower water absorption in the small intestine than in the colon (258) and a decrease of colonic water absorption during early postnatal development (111, 447). The high water absorption measured in these in vivo experiments may reflect leaky paracellular pathways. This conclusion is indirectly supported by several findings. First, an imposed osmotic gradient results in water secretion, but the relationship between water movement and the osmotic gradient is nonlinear in the adult intestine and linear in sucklings (448). Second, hydrostatic hydraulic conductance is two times higher in suckling rats than in adult animals (433). Finally, no developmental changes were observed in the mucosal colonic surface (113) and apical membrane surface of colonic enterocytes (413).

B) SODIUM CHLORIDE ABSORPTION. The findings of increased water absorption during early postnatal life suggest that intestinal epithelia share increased NaCl absorption. High Na⁺ absorption, which decreases during postnatal life, was observed in the rat, rabbit, and human colon (111, 187, 284) as well as in the rat small intestine (447). The Na⁺ pathway that dominates in the colon is electrogenic Na⁺ transport via amiloride-sensitive Na⁺ channels (284, 290, 292). The channels are formed by three subunits from which gradually increase in abundance from fetal life to the end of weaning (86, 424, 425) and correlate with the development of electrogenic Na⁺ transport. The capacity of electrogenic Na⁺ transport increases during suckling period and decreases during weaning. Although it persists until adulthood in rabbits (31, 284), it disappears in rats after weaning (290, 292) and is replaced by Na⁺/H⁺ exchange in the adult colon (31). In the pig, Na⁺ absorption is electroneutral at birth, but this rapidly changes to an electrogenic one (85).

The developmental profile of colonic Na⁺/H⁺ exchange and the mechanism of the replacement of the electrogenic Na⁺ absorption by the electroneutral pathway is unknown. In the small intestine, the ratio of two isoforms of Na⁺/H⁺ exchanger, NHE2 and NHE3, is age dependent, and the capacity of the Na⁺/H⁺ exchange increases during development and reaches the highest values after weaning (81, 82, 199). Even if the functional coupling of Na⁺/H⁺ and Cl/HCO₃ exchangers is considered for the villus enterocytes in the small intestine, the developmental pattern of Cl/HCO₃ exchanger is inverse to that of Na⁺/H⁺ exchanger (322), and its expression is regulated at the level of transcription (73). The uncoupling of Na⁺ and Cl absorption is supported by the presence of Cl transport that is insensitive to Na⁺ absence in the rat and rabbit colon (284, 306). The existence of Cl absorption that does not follow the developmental decrease of Cl/HCO₃ exchanger capacity indicates the existence of other chloride transport pathways in immature intestine, Cl channels, and Na⁺-2Cl-K⁺ cotransporter (93). The serosa-electropositive potential difference generated by active ion transport, usually Na⁺ absorption, promotes electrodiffusion of Cl through "shunt" pathways that have both a cellular and a paracellular component. As Cl is usually above electrochemical equilibrium in various epithelia in adulthood; this cellular shunt pathway could cause a "backleak" of Cl. Hence, the paracellular pathway would seem to be more desirable.

Na⁺ is extruded across the basolateral membrane by Na⁺-K⁺-ATPase (Na⁺ pump) that increases during the suckling and weaning period (295, 353, 449, 450). The developmental increase in Na⁺-K⁺-ATPase activity follows the mRNA expression for - and -subunits of this protein, and their expression reaches adult levels at the end of weaning (125, 450). Although the activity of Na⁺-K⁺-ATPase [measured as the hydrolysis of ATP under maximum velocity (V_max) conditions] and the number of pump molecules increase during development, the maximum pumping activity of Na⁺-K⁺-ATPase remains constant. This means that the turnover rate per single pump is either higher during the suckling and weaning period than in adulthood or that there is a considerable latent pool of individual pumps that increases during development (295).

C) POTASSIUM ABSORPTION. The maintenance of potassium homeostasis represents a vital function of renal tubules and the intestinal epithelium. Intestinal K⁺ transport is bidirectional, and therefore, perfusion studies performed on the immature intestine demonstrated both absorption and secretion. K⁺ secretion was observed in the rat jejunum and ileum (447) and in the pig and human colon (27, 187), whereas absorption was found by others (110, 257). These contradictory results seem to reflect methodological differences. In vitro preparations of the intestine usually express very high K⁺ secretion due to activation of neurohumoral secretory pathways, whereas secretion is absent in vivo. After solving these methodological problems, it was recently possible to demonstrate active K⁺ absorption via H⁺-K⁺-ATPase in both the immature small (434) and large intestine (2, 3). In agreement with the need of infants to retain K⁺ for normal growth, active K⁺ absorption and activity of H⁺-K⁺- ATPase are higher during early postnatal life than in adulthood (2, 3).

D) CALCIUM ABSORPTION. Intestinal Ca²⁺ absorption in mammals involves two processes: paracellular movement and transcellular transport, which involves both nonsaturable and saturable vitamin D-dependent pathways. In newborn rats, the absorption is a nonsaturable diffusional process via paracellular and maybe also transcellular routes which are present in all intestinal segments. This absorption, which is insensitive to vitamin D, is later replaced by a combination of a saturable vitamin D-dependent and -independent absorption (346, 404). The nonsaturable diffusional pathway operates also as a single Ca²⁺ absorptive pathway in preterm low-birth-weight infants (42). The appearance of saturable active Ca²⁺ absorption is associated with a substantial increase in the capacity for other steps involved in transcellular Ca²⁺ absorption: 1) entry along a steep electrochemical gradient (129), 2) translocation through the cell (123, 249), and 3) extrusion at the basolateral membrane against a steep electrochemical gradient by Ca²⁺ pump, i.e., Ca²⁺- ATPase (130, 249).

E) PHOSPHATE ABSORPTION. Similarly to Ca²⁺, infants require a large amount of phosphate to mineralize the developing skeleton, but in contrast to Ca²⁺, the apical membrane potential severely limits passive entry of negatively charged phosphate ions, which are therefore absorbed predominantly by secondary active Na⁺-dependent transport in a stoichiometry of 2Na⁺:1P_i (37). In agreement with the increased requirements of immature animals for phosphate, the capacity of this transporter decreases during development, and its distribution is restricted from the duodenum, jejunum, and proximal ileum in suckling rats only to the duodenum and proximal jejunum in adult animals (37).

In conclusion, ion transport undergoes postnatal changes both in the small and large intestine. In infant mammals, the colon appears to have a significant role in absorption of water and ions and in maintenance of normal fluid and electrolyte homeostasis. This role may be extremely important during early postnatal growth when the small intestine absorption and renal reabsorption functions are not fully developed.

Suckling mammals have a high absorptive capacity for many trace minerals and vitamins. Together with the high bioavailability of these substances in milk (227), the increased absorptive capacity during the suckling period may be an important factor in ensuring sufficient amounts of trace minerals and vitamins during accelerated growth and development.

Kinetic studies have shown that both passive-linear and carrier-mediated processes are involved in the absorption of trace minerals in the immature intestine. Specific carriers have been postulated for iron (376), copper (416), and zinc (423) but not for manganese (26). Copper, which is absorbed by a nonsaturable and later also by a saturable pathway, displays elevated absorption localized predominantly in the ileum (172, 416). The onset of saturable components correlates with the increased expression of metallothionein, which has been suggested as a regulator of copper and zinc homeostasis (416). Similarly as copper, iron absorption is also increased in the immature intestine even if neonatal animals are iron repleted (74, 277, 376). The increased iron absorption is due in part to enhanced transport in the immature ileum and jejunum.

Although intestinal iron absorption plays a pivotal role in the regulation of body iron status, knowledge of the underlying cellular mechanisms is still fragmentary even though one possible iron transporter has been isolated recently (114) and developmental changes of iron binding proteins occur in enterocytes (205). The high bioavailability of milk iron and the presence of iron-binding lactoferrin and transferrin in the milk and intestine and their resistance to proteolytic attack during transit through the digestive tract (227) suggest the role of facilitated uptake of iron from iron-binding proteins into the small intestine. Depending on the species, either transferrin or lactoferrin are the major iron-binding proteins in milk. For example, the rat has a high transferrin concentration, whereas humans or monkeys have a high content of lactoferrin while the pig has both proteins (227). Rhesus monkey lactoferrin receptors of the brush-border membrane are present in all age groups including fetuses, but the number of receptors is highest during the suckling period (90). Highly specific lactoferrin receptors have also been found in the small intestine of human fetuses and infants (193), and transferrin receptors have been reported in the rat small intestine (192). Despite the identification of receptors for iron-binding proteins in the brush-border membrane of enterocytes in immature and adult intestines, the quantitative significance of the receptor pathway in iron absorption remains to be elucidated. Even though bound lactoferrin is internalized (132), it is not certain whether this process is involved in the developmental changes of iron uptake (74).

Vitamins penetrate through the epithelium by the saturable nonlinear transport (observable at lower physiological concentrations) and by simple linear diffusion (observable at higher concentrations). The saturable transport that occurs through the action of specific transporters represents both facilitated diffusion or ATP-driven active transport (324). The development of vitamin absorption was investigated only for a few vitamins, but it has shown that the developmental patterns are not identical for the absorption of all vitamins. The transport of water-soluble vitamins such as folate and riboflavin are carrier-mediated, Na⁺-dependent processes the capacity of which decreases during postnatal life (330, 335). Folate in milk is largely bound to a high-affinity folate-binding protein that strongly enhances the folate uptake probably via a specific binding receptor (335). In contrast to unbound folate, the bound fraction seems to be absorbed more avidly. This absorption represents an endocytotic process that has higher capacity in the ileum than jejunum (247). Riboflavin absorption is also enhanced by milk factors (368). Biotin absorption follows different developmental pattern. Unlike adult rats, in which biotin is absorbed preferentially in the jejunum, suckling rats absorb biotin predominantly in the ileum. Whereas the nonsaturable pathway decreases in both segments during development, the capacity of saturated carrier-mediated transport increases in the jejunum and decreases in the ileum (333, 334).

Milk also strongly promotes the transport of cobalamin (409). Cobalamin absorption operates in all segments of the small intestine and exhibits saturating kinetics. The intrinsic factor, which is known to promote cobalamin absorption in the adult intestine, does not compete with the milk factor. It promotes absorption only in the ileum and is more effective in weanling animals. In contrast, the milk factor is more effective in suckling rats where the intrinsic factor/cobalamin receptor activity is very low (312, 409). Postnatal development of the intrinsic factor and the intrinsic factor/cobalamin receptor peak in the rat between days 20 and 24 after birth (312), and this correlates with the production of the intrinsic factor in the stomach (96). A different situation seems to occur in the human intestine, in which intrinsic factor/cobalamin receptors are already present in early gestation (344) and fetal parietal cells are able to produce the intrinsic factor (1). It cannot therefore be excluded that the intrinsic factor already stimulates absorption before birth.

Lipid-soluble vitamin absorption depends on bile acids. It is thus likely that the differences in vitamin absorption are largely attributable to differences in bile acid secretion during early postnatal development (171). In principal, the properties of lipid-soluble vitamin absorption are similar to those of water-soluble vitamins. This is most obvious in the development of retinol transport, which has been the most thoroughly studied lipid-soluble vitamin. The uptake of retinol into enterocytes is a passive carrier-mediated process whose maturation is associated with a decrease of transport capacity and increase of the affinity. The distribution of transport capacity along the intestine follows the same pattern as in adult animals, i.e., it is higher in the jejunum and lower in the ileum (94, 331). Milk lactoglobulins enhance absorption of retinol (332). Recent studies have conclusively demonstrated that the absorption of retinol from the complex of retinol with retinol binding protein is a saturable, receptor-mediated, and specific process that differs from the absorption of free retinol (95).

Altogether, the above results suggest that the transport of trace elements and vitamins undergoes clear developmental changes that usually include a decrease in the rate of nonsaturable transport processes and the changes in the capacity and affinity of the saturable pathways. Studies on the intestinal transport of trace minerals and vitamins in the mature and especially the immature intestine have so far been focused on the proof of saturated, carrier-mediated systems and other mechanistic aspects. Hence, little is known about the specific transport proteins and their regulation at the cellular level. Even less has been done to elucidate the mechanisms involved with exporting trace elements and vitamins from enterocytes. There is, therefore, a clear need for further data to understand the mechanisms of age-related changes in trace element and vitamin absorption.

It is known that term and preterm newborns have higher fecal fat loss compared with older infants and adults. This decreased ability to absorb lipids seems to be due to a lower activity of pancreatic and small intestinal lipases and intraluminal bile acid concentrations. Despite the high fat intake in milk during the suckling period, the enzyme activities and bile acid metabolism have been found to be decreased in newborn animals. The adult mode of micellar absorption appears later when the pancreatic lipase activity and intestinal bile acid concentration increase (398). Significant changes in bile acid metabolism, including the bile acid pool and the development of hepatic transport mechanisms, occur during weaning, and this appears to be responsible for the increase of bile flow (384). The depleted bile acid pool in newborn and suckling animals, compared with adults, appears to be partly due to the postnatal maturation of specific components of the enterohepatic circulation such as intestinal bile salt reabsorption. In the immature intestine, there is passive bile salt diffusion along the entire length (163, 226), with a diminishing absorption rate during development (378). Maturation increases bile acid transport capacity in the ileum. This increase is observed in altricial species during weaning (19, 226, 269) and early after birth in precocious animals (163). It reflects the expression of an active Na⁺-dependent carrier-mediated transport pathway across the apical membrane (19, 269, 358). It is not obvious whether this is a "hard-wired" preprogrammed system or whether it responds to expanding circulating bile acid pools and increased bile acid flow into the intestine (359).

The osmotic driving force for fluid accumulation within the lumen depends either on the inhibition of active ion absorption or on stimulation of active ion secretion, or both. Active intestinal secretion is driven predominantly by Cl secretion and less by HCO₃ and K⁺ transport from the serosal to the mucosal side. Cl enters the cell across the basolateral membrane through Na⁺-2Cl-K⁺ cotransporter and is extruded into the lumen through apical Cl channels. Na⁺ and K⁺ are recycled across the basolateral membrane via Na⁺-K⁺-ATPase and K⁺ channels. The mechanism of K⁺ secretion is similar to that of Cl except that K⁺ leaves the cell across the apical membrane through K⁺ channels (146, 293). The secretion of HCO₃ into the mucosal layers provides a first line of mucosal protection against acids arriving from the stomach and thus predominates in the duodenum. This secretion is both a metabolically dependent transcellular process and a process involving paracellular migration into the duodenal lumen (115).

Active colonic K⁺ secretion is high during the suckling period and declines later as a result of decreased plasma levels of aldosterone (293). In contrast to the developmental profile of K⁺ secretion, the profiles of Cl and HCO₃ secretion have not yet been fully investigated, even though acid secretion in the stomach is not fixed in adult form at birth (1, 438). Active Cl secretion was observed in the small intestine of suckling mice and pigs (252, 320) and in the distal colon of suckling rabbits (67, 308, 310). The product of cystic fibrosis transmembrane conductance regulator gene encoding a cAMP-regulated Cl channel has already been demonstrated in the human fetal small and large intestine, and its abundance was relatively stable during prenatal development (127, 402, 408). In addition, cAMP-dependent Cl channels have been recently demonstrated in colonocytes of newborn, suckling, and weaning rabbits (93)

The strongest evidence that the secretory machinery comes through age-related shifts was provided in studies of bacterial enterotoxin and bile acid secretion. In contrast to adulthood, the immature colon is challenged by entrance of bile acids that are potent secretagogues. As mentioned in section IIIF, bile acids are reabsorbed in the small intestine, but active ileal transport is not present at birth and develops gradually during the suckling period. In agreement with these developmental changes of bile acid transport, the colon of suckling rabbits does not respond to bile acids by water and chloride secretion, and the secretory response can be seen only after weaning (308). This lack of response is not a general phenomenon of colonic mucosa, because the colon secretion can be elicited by a variety of other cAMP-dependent secretagogues (308) and the colonocytes have developed cAMP- and protein kinase C-dependent signal transduction cascade from an earlier stage of development (93). The only observed difference between immature and mature colon is that the sensitivity of cAMP concentration response to bile acids is lower in the immature than in adult enterocytes (310).

The second evidence comes from human and veterinary medicine which shows that the incidence and severity of infectious diarrhea decreases with age. One of the important factors that is responsible for this fact is the developmental regulation of bacterial/enterotoxin-induced intestinal secretion. Bacterial enterotoxins such as choleratoxin of Vibrio cholerae (CT) or heat-stable toxin of Escherichia coli (STa) are more effective secretagogues in the immature than in the mature intestine (75, 80, 252). In the rat jejunum, there is 600-fold developmental decrease in STa sensitivity, whereas in the ileum, it undergoes no changes (80). Similarly, suckling rat small intestine is 50-fold more sensitive to CT than that of weaned animals (75).

The reason for the increased sensitivity has not been fully explained, even though the mechanism of secretion stimulated by enterotoxins is known. STa belongs to the family of heat-stable peptides that bind to specific receptors on the intestinal brush-border membrane and stimulate net fluid secretion by cGMP. The STa receptor has been identified as a transmembrane protein, guanylate cyclase C, whose extracellular domain includes the STa binding region, and the intracellular domain is the site of guanylate activity. CT enterotoxin is an oligomeric protein composed of five B subunits and one A subunit that interact with a specific glycolipid receptor in the brush-border membrane. The A subunit enters the cell, where it is cleaved into two proteins. One of them is an ADP-ribosylating protein that uses the G protein (G_s) as its substrate. As a result, adenylate cyclase localized on the basolateral membrane is permanently activated to produce cAMP. The accumulated cAMP opens Cl channels in crypts and inhibits Na⁺/H⁺ and Cl/HCO₃ exchangers in the villi (76).

The above-mentioned mechanisms of fluid secretion induced by enterotoxins indicate that there are several points at which the developmental changes of secretion might occur: 1) age-related changes in the affinity or number of membrane receptors, 2) developmental changes in receptor-effector coupling, 3) decreased effector capacity, and 4) changes in indirect activation via the enteric nervous system and immune system.

First, a developmental increase in the number of STa receptors was observed in the brush-border membrane of porcine, murine, rat, and human intestine without changes in their affinity (79, 80, 259, 387). The age-dependent changes in STa receptors are both under transcriptional and nontranscriptional control because the developmental profile of STa binding receptors is not identical to the expression of guanylate cyclase C mRNA (210). In contrast to STa receptors, the number of CT receptors is similar in the intestine of preweaned and postweaned animals. Even if the binding affinity is somewhat higher in the immature intestine, it cannot explain the developmental changes in the intestine (75).

Second, the developmental pattern of secretion may also reflect changes in transduction pathways. Adenylate cyclase in preweaned rats is nine times more sensitive to the interaction of CT with its receptors than those of weaned animals (349). The factor responsible for this increased sensitivity is the cholera toxin-sensitive subunit of adenylate cyclase, G_s protein, the expression of which decreases during development (338). In addition, some other factors may influence the secretion. Older pigs produce antisecretory proteins that are capable of inhibiting enterotoxin-induced secretion (211). The uptake of the A subunit of CT seems to be more rapid in suckling than in the adult small intestine due to its higher macromolecular transport capacity (217). As far as receptor-effector coupling is concerned, the sensitivity of guanylate cyclase to STa is similar during development (80, 259).

Third, the data summarized in previous sections indicate that the immature intestine possesses both cAMP- and cGMP-induced secretory pathways that do not exhibit an identical developmental pattern as enterotoxin-induced secretion. For example, the maximum secretory capacity of the pig jejunum is similar in the mature and immature intestine, although the secretory response to CT is age dependent (252). Thus the hypothesis of suppressed secretory capacity in adulthood does not seem to be justified.

Fourth, in addition to the direct effect of enterotoxins on enterocytes, some data indicate that enteric neurons and inflammatory responses are involved in intestinal secretion. The direct stimulatory effect of CT binding to enterocytes is estimated to cover ~25% of the secretory response in vivo, and the remainder is considered to be induced indirectly through stimulation of other intestinal cells. These release mediators activate the intestinal intramural nervous reflex and thus evoke fluid secretion or influence enterocytes directly (229, 263). The indirect effect of enterotoxins was observed in the mature intestine, but some recent data have also proven their indirect effect in immature intestine (119, 155). The data about possible developmental changes of nervous secretory reflexes are still missing.

In addition to ion and water secretion, the intestinal epithelium secretes macromolecules such as mucins and immunoglobulins that play an important role in intestinal defensive mechanisms. Mucins, high-molecular-weight glycoproteins secreted by goblet cells, contribute not only to the lubrication of mucosal surfaces but also to the protection against attaching particles of intestinal flora, or some molecules of dietary origin, to the intestinal epithelium as a result of its binding capacity for various lectins. However, little attention has been paid to the age- and diet-dependent changes in mucin secretion and composition. Mucin is already present in fetuses (54, 379); the mucin secretory capacity of enterocytes increases during early postnatal life (44), and its composition depends on the age and diet (362, 411). In newborn rats, mucin contains more protein and sulfate compared with that in adults (362). Similar developmental changes were recently observed in porcine mucin (411). Maybe the higher degree of sulfation is associated with secretory products of immature goblet cells. Mucosal IgA is secreted into the intestinal lumen by basolateral-to-apical transcytosis. IgA binds to the membrane poly-IgA receptor localized at the basolateral membrane, and the receptor-ligand complex undergoes internalization by endocytosis. The poly-IgA receptor is a precursor of the secretory component that is released into the lumen together with IgA (206). The potential influence of intestinal maturation on IgA secretion is not known; however, it has been noted that the production of the secretory component is very low in the intestine of suckling rats (64). Nevertheless, the antibody secretory system is able to respond to antigenic stimulus also at a time when this system is still incompletely mature (59).

Studies of intestinal morphology, enzymology, and transport have demonstrated the existence of both horizontal (proximo-to-distal) and vertical (crypt-to-villus) axes in the intestine. They are already to be found during gestation, but in altricial species they are not completed until after several weeks of postnatal life (70, 240, 241). The factors that regulate postnatal development of intestinal gradients and their interrelationship are not yet clear. The mesenchymal-endodermal recombination experiments and isografts of fetal intestine (see sect. IVA) support the hypothesis that a hard-wired genetically fixed program in the intestinal endoderm encodes the expression of intestinal gradients even though some other factors, such as luminal nutrients, may modify their expression (70, 121). The spatial differences in gene regulation seem to be temporally regulated by different regulatory elements in various intestinal segments (135).

The most dramatic developmental changes in the distribution of transport functions along the horizontal axis occur in the colon. The fetal and neonatal colon possesses many morphological and functional features that are distinct from those present in adulthood. In contrast to the adult colon, the fetal and neonatal colons have villuslike structures (83, 255) and significant capacity of active absorption of luminal carbohydrates and amino acids (see sect. III, C1 and C2). Furthermore, the shift in the mechanism of colonic Na⁺-absorptive pathways occurs during early postnatal development (see sect. IIID2). Less dramatic changes in the regional distribution of transport functions can be observed along the small intestine. Nevertheless, the development in this part of the gut is also associated with qualitative and quantitative changes along the duodenum-ileum axis. A typical example of such changes is the distribution of pinocytotic and endocytotic activity of vacuolated enterocytes, which disappears from the small intestine in early postnatal life (see sect. IIIB2), as well as the expression of saturable absorption of bile acids (see sect. IIIF) and some vitamins (see sect. IIIE).

Similar to the changes along the horizontal axis, the vertical distribution of transport functions along the crypts and villi is also not constant, and pronounced changes occur in their developmental profile (121, 371, 410). These developmental changes along the vertical axis may represent a gradient replacement of the cells of an immature phenotype with other cells that differ in transport functions. We can relate these changes to the rate of cell turnover in the intestinal epithelium because cell replacement is slow at birth, whereas weaning is associated with an increase in mitotic activity and migration together with a decrease of enterocyte life span (202). Studies involving the expression of intestinal genes along the crypt-villus axis in the adult intestine indicate that it is regulated by a combination of positive and negative elements (activators, repressors). The transcriptional machinery is present in crypt enterocytes, but the transcription of genes is normally repressed (406).

The regulation of intestinal transport functions is dependent, to a large extent, on the junctional complex connecting enterocytes together (Fig. 2). It determines the extent to which solutes and water are absorbed or secreted. As a barrier between the luminal and basolateral compartments, tight junctions selectively control the passive diffusion of ions and other small solutes through the paracellular pathway and thereby influence any gradient created by the activity of pathways associated with the transcellular route. The presence of paracellular pathways with high permeability, such as in the small intestine, permits rapid transepithelial diffusion and precludes the presence of a large transepithelial electrical potential and concentration gradient across the epithelium. On the other hand, these routes enable the absorption of some solutes coupled to fluid absorption via convection (solvent drag) without additional expenditure of energy and may indirectly influence the transcellular movement through an influence on transcellular Na⁺-dependent absorption of nutrients due to the paracellular backflux of Na⁺ (235). Pappenheimer and Reiss (296) calculated that the effective pore size of adult intestinal epithelium is able to admit passage of solutes of 5,000 Da at the tight junction. Thus even small macromolecules and not only ions, nutrients, and water could pass by this route under physiological conditions (12). In addition, tight junctions are dynamic structures that readily adopt to a variety of physiological conditions and may change the size of the effective pore (16).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Schematic diagram of intestinal epithelium. The tight junctions (1) define the boundary between apical (3) and basolateral (4) membrane and prevent free movement of water and solutes through the lateral intercellular space. The permeability of tight junctions significantly influences transepithelial transport (2, lateral intercellular space; 5, transcellular transport; 6, paracellular transport).

Although these observations are of considerable importance for the physiology of absorption of nutrients, the role of paracellular pathways in the developing intestine has not received sufficient attention. The questions of how large is the passive paracellular component of solute absorption and how the ratio of transcellular to paracellular absorption is altered during development have not yet been solved. Only some indirect evidence suggests that the properties of the paracellular pathway are changing during development. Permeability of the immature intestine is higher than in adults (433), and the response of the intestine to hypertonic perfusion is age dependent (448). These data indicate that paracellular pathways might change with ontogeny. What is now necessary to reveal is the importance of paracellular volume flow coupled with solvent drag for absorption processes in the developing intestine.

	IV. DEVELOPMENTAL REGULATION OF INTESTINAL TRANSPORT

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The survival of newborns and the maintenance of homeostasis require normal regulation of the intestinal mucosa operating as a functional unit. This unit is composed of several cell types from which only some have transport functions and other serve predominantly regulatory or immunologic roles. The complex regulation involves coordination of intestinal transport and digestive processes in various segments of the intestine with alterations of blood flow and intestinal motility. However, only sparse information about the regulatory processes is available from studies conducted largely on adult animals, and even less is known about their maturation.

Studies of the adult intestine have revealed that the regulation of intestinal transport depends not only on the intrinsic properties of enterocytes but also on systemic and local hormones, growth factors, neurotransmitters, and complex interactions between enteric nerves in the submucosa and immune cells in lamina propria (Fig. 3). These cells release a variety of mediators influencing intestinal transport either directly or indirectly. Enterocytes rarely encounter a single signal but are more likely to encounter a cocktail of several paracrine or endocrine signals simultaneously or sequentially (see Tables 2-4). Thus in vivo biological activity is a sum of synergistic and antagonistic actions of multiple signals of genetic, nervous, hormonal, and dietary origin. How these regulatory networks operate in the immature intestine and what are the triggers that cause these changes are discussed in detail in the following sections.

View larger version (80K): [in this window] [in a new window]   Fig. 3. Schematic diagram of interactions involved in the regulation of intestinal absorption and secretion. Several factors that regulate/modulate intestinal transport are shown. Luminal stimuli (1) interact with enterocytes and with enteroendocrine, immune, or M cells, the basolateral surface of which is modified to form an intraepithelial pocket into which lymphocytes and macrophages migrate. Enteroendocrine or immune cells respond to stimuli by secretion of various mediators that influence the enterocytes, immune cells, enteric neurons, or other cells of lamina propria through paracrine action (2, paracrine stimuli). The enteric nervous system (ENS) receives its input not only from paracrine stimuli but predominantly from sensory neurons and the central nervous system. This information is integrated in the enteric nervous plexus and initiates activation of motor neurons (3, nervous stimuli) that regulate various mucosal functions. The last factors that regulate mucosal functions and influence the cells of lamina propria are hormonal stimuli (4).

Transplantation of fetal intestinal isografts beneath the kidney capsule of adult rats or mice showed that the developmental and positional (along proximo-to-distal and crypt-to-villus axes) information for morphological and enzymatic patterns is fixed in the grafts and that the hormonal milieu can only modify this information. This means that much of epithelial control is likely to depend on autocrine factors and the surrounding mesenchymal cells that provide essential paracrine factors that have the potential to exert the regulatory role in proliferation, migration, and differentiation (97, 194, 441). Detailed studies of nutrient transport and especially of digestive enzymes in small rodents during the suckling and weaning period have also shown that age-related changes in nutrient transporters and enzymes are genetically programmed and less influenced by the diet or hormones (48, 158, 216, 276, 403, 417). The triggering signals for these developmental changes were denoted hard-wired by Toloza and Diamond (403). These signals are independent of external information and are to be switched on at the relevant time independently of external signals that normally also appear at that time. Such hard-wired genetically programmed mechanisms of developmental regulation have been demonstrated for galactose and fructose absorption (403). Whereas galactose absorption declines during early postnatal life, fructose absorption is turned on at the end of weaning, and neither of these processes is dependent on the dietary changes during weaning. The apical fructose transporter GLUT5 is an example of a transporter that possesses a hard-wired ontogenetic timing mechanism that induces the formation of the transporter in the intestine independently of dietary signals (91, 360).

Although the development of transporters can be reprogrammed by interactions of genetic determinants with other factors mentioned in the following sections, it is the regulation of transcription that ultimately controls the major of these changes. Studies performed in transgenic mice showed that transcription of intestinal genes is regulated by multiple transcriptional elements with an activatory or repressive role (406). The recent discovery that homeobox gene Cdx2 is an important transcriptional factor for the sucrase-isomaltase promotor in the intestinal cell line (385) raises the issue of whether mammalian genes related to the Drosophila segmentation gene caudal might play a role in the developmental regulation of intestinal transcription. Currently available data have demonstrated developmental changes in the expression of Cdx1 and Cdx2 genes in the intestine (98, 186, 383) that might indicate the important role of these genes in intestinal development. However, Cdx genes are clearly insufficient for developmental regulation of transcription because they are expressed not only in immature but also in adult intestine.

Plasma levels of many hormones exhibit dramatic changes during development, and it is therefore reasonable to consider their role in the regulation of intestinal transport. One of these is aldosterone, which seems to be the most important factor responsible for increased electrogenic Na⁺ absorption in the immature intestine. The developmental changes of electrogenic Na⁺ transport correlate well with the increased plasma concentrations of aldosterone both in animals (104, 109, 284, 290) and humans (187). The action of aldosterone is a genomic effect, and the expression of the mineralocorticoid receptor gene is activated before the developmental increase of electrogenic Na⁺ transport becomes detectable (124). High Na⁺ transport capacity reflects the adaptation to postnatal dietary Na⁺ intake and Na⁺ requirement for growth, because the growing animals need to accumulate Na⁺ in the expanding extracellular fluid volume. Because the sources of Na⁺ in the milk are limited, the animals are threatened by a relative Na⁺ deficiency, and therefore, Na⁺ absorption is stimulated (289, 290).

Aldosterone increases the entrance of Na⁺ across the apical membrane by induction of amiloride-sensitive Na⁺ channels (292, 425) and the extrusion of Na⁺ across the basolateral membrane by stimulating the sodium pump, i.e., Na⁺-K⁺-ATPase (109, 449). At present it is not clear whether the cellular effect of aldosterone is primarily a direct hormonal effect on the pump activity or whether the increased basolateral Na⁺ extrusion is the substrate effect due to higher apical Na⁺ permeability and higher intracellular Na⁺. Because aldosterone administration in doses adequate for inducing profound antinatriuresis did not alter the Na⁺-K⁺-ATPase gene expression (125), the second hypothesis seems to be more probable.

Despite the generally accepted hypothesis that aldosterone regulates Na⁺ channels, some evidence has raised the question regarding its validity. First, aldosterone produces in the adult colon increase in the abundance of - and -subunit but not the -subunit of Na⁺ channels. Nevertheless, the abundance of all three subunits increases in the immature intestine (425). Second, the effect of increasing plasma concentrations of glucocorticoids on the development of the -subunit can be excluded because exogenous glucocorticoids do not influence its abundance during the suckling period (424). Third, the regulatory role of aldosterone requires the permissive effect of thyroid hormones (78, 291), the developmental profile of which follows that of aldosterone (290, 291). The mechanism of this effect is unknown. Nevertheless, the interaction of aldosterone and thyroid hormones cannot be localized at the level of Na⁺ channel expression (270) but at the level of operating sodium channels (78). The effect of thyroid hormones could explain the developmental changes in sensitivity of the intestinal epithelium to aldosterone observed in the rat colon (294). The permissive effect of thyroid hormones has also been described in the maturation of other transporters (92) and of digestive enzymes (251, 444), and it is likely that the effect of thyroid hormones is targeted directly to the enterocytes. In addition, it has recently been shown that the developmental changes of responsiveness to 3,3',5-triiodothyronine (T₃) are induced by the changes in c-erbA-2 protein which are thought to inhibit T₃ action (170). The inactivation of the thyroid hormone receptor gene delays intestinal maturation (120).

The developmental profile of plasma corticosterone is very similar to that of aldosterone. However, its plasma concentration is higher by several orders of magnitude than for aldosterone (157, 290). Considering the well-known high promiscuity of steroid receptors, the manyfold higher glucocorticoid concentration could antagonize aldosterone regulation in the mineralocorticoid target segments especially in the colon. The exclusive occupancy of mineralocorticoid receptors by aldosterone in the face of much higher circulating levels of corticosterone or cortisol is attained by the enzyme 11-hydroxysteroid dehydrogenase (11-HSD). This enzyme converts glucocorticoids (cortisol, corticosterone) to biologically less active 11-oxoderivatives (cortisone, 11-dehydrocorticosterone) that have low affinity to glucocorticoid receptors. The activity of 11-HSD is not distributed homogeneously along the intestine, and its developmental profile is not the same in individual intestinal segments (288). Whereas the activity of colonic 11-HSD is relatively high since birth and does not change substantially until adulthood, ileal 11-HSD activity is low during the first postnatal week and increases during the weaning period. 11-HSD is important for the developmental determination of aldosterone responsiveness, and its inhibition accelerates maturation of colonic Na⁺-K⁺-ATPase (125, 305).

In contrast to mineralocorticoid receptors, the expression of glucocorticoid receptors in the immature intestine is higher during the suckling period and decreases to adult levels during weaning (124, 160). The presence of glucocorticoid receptors in the immature small and large intestine indicates that the glucocorticoids may have possible regulatory effects on the developmental regulation of intestinal transport not only postnatally but also prenatally (47). Many studies have proven a stimulatory effect of glucocorticoids on Na⁺-dependent absorption of Na⁺, alanine, glucose, or bile acids and on Na⁺-K⁺- ATPase (18, 141, 164, 184, 257, 321, 339, 418) and their inhibitory effect on saturable phosphate and nonsaturable Ca²⁺ absorption in the immature intestine (38, 131). However, the interpretation of these findings is difficult because exogenous glucocorticoids were usually administered in high pharmacological doses that did not discriminate between mineralocorticoid and glucocorticoid receptors. We also do not know whether glucocorticoids influence the intestinal transporters at the level of their transcription or posttranslation directly or if they act by altering cell kinetics. For example, glucocorticoids precociously induce sucrase activity by initiating the sucrose-isomaltase gene expression, whereas the later effect seen at the time of normal onset of the enzyme activity appears to be secondary to glucocorticoid-mediated changes in enterocyte kinetics (216, 276, 281). Some results indicate that glucocorticoids are able to operate both at the level of transcription (82) and posttranscription (32, 149).

The ambiguity of the glucocorticoid effect can be demonstrated on the mechanism by which glucocorticoids influence phosphate and Ca²⁺ transport which is controlled by vitamin D (128). One of the possibilities concerns the indirect influence on transport via parathyroid hormone and vitamin D (calcitriol) or by a direct effect on the developmental expression of vitamin D receptors (VDR). In a recent study, Lee et al. (215) proved that glucocorticoids can precociously induce intestinal VDR expression. In contrast to the effect of vitamin D, the effect of glucocorticoids was not specific and reflected a general effect of glucocorticoids on intestinal maturation. During the suckling period, although the Ca²⁺ and phosphate requirements are high, active Ca²⁺ transport, enterocyte calbindin-D_9k, and plasma calcitriol concentrations are low so that the small intestine is relatively refractory to calcitriol (77, 123, 144, 345, 346). The induction of VDR at weaning (77, 144) may be one of the essential factors involved in the development of vitamin D-dependent Ca²⁺ and phosphate absorption and its regulation.

Corticosteroids together with insulin may influence lipid absorption in the immature intestine through the regulation of apolipoprotein synthesis. Insulin reduces triglyceride and chylomicron production in human fetus explants and inhibits apo B-48 and apo B-100 secretion, without affecting apo A-1 (222). In contrast, jejunal explants from 2-day-old piglets respond to cortisol and insulin by stimulation of apo A-1 synthesis, whereas EGF plus insulin stimulates apo A-IV synthesis (33). Insulin, whose plasma levels increase similarly to glucocorticoids (36), is believed to be one of the key hormones, together with glucocorticoids, involved in the maturation especially at the onset of weaning (61). The maturation induced by insulin is at least partly triggered by binding of the hormone to the enterocyte insulin receptor (62, 63).

Furthermore, intestinal closure that reduces the absorption of macromolecules seems to be associated with the action of some hormones and growth factors, but the effect is species dependent. In rodents, the cessation of transport is accelerated by exogenous administration of glucocorticoids, thyroxine, secretin, EGF, and insulin (150-152). At least in the case of exogenous glucocorticoids and thyroxine, this decrease is associated with the inhibition of Fc receptor mRNA expression, but endogenous adrenal steroids do not appear to be entirely responsible for the closure (244, 245). In ungulates, the effect of hormones is less obvious. Whereas closure in pigs was related to plasma insulin (386), precocious gut closure after glucocorticoid administration was not detected in ruminants (175, 339).

In summary, there are several hormones that are able to modulate the postnatal changes in the intestinal transport despite the presence of local timing mechanisms. It seems likely that the hormones become of major importance when intestinal development needs to be accelerated and coordinated with developmental changes in the whole organism (e.g., weaning).

A large number of cytokines and other mediators that have been shown to affect intestinal water and solute transport (Table 3) are produced by epithelial cells, mesenchymal cells of lamina propria, and cells of the mucosal immune system. These substances act either directly at the level of enterocytes or indirectly through interactions with other cells such as enteric neurons or by influencing capillary blood flow or intestinal motor function. The integrative role of cells of the mucosal immune system, epithelium, mesenchyme, and enteric nervous system (ENS) is supported by the close anatomical association of these cells and the presence of receptors for gut hormones, neurotransmitters/neuropeptides, and cytokines among the aforementioned cells. Unfortunately, almost all information has been obtained in the adult intestine, and the data concerning maturation of paracrine regulatory network are very limited. The paracrine mediators summarized in Table 3 have both a proabsorptive and a prosecretory effect. Many of them have already been identified in the fetal intestine, and it was shown that their expression undergoes subsequent developmental changes (173, 207, 230, 271, 280, 421, 445). Some of them are likely candidates as signals for the regulation of intestinal transport in the immature intestine (Table 4). Recently, the endogenous ligands for the STa receptor (membrane-bound guanylate cyclase) with natriuretic and diuretic activity have been identified, called guanylin and uroguanylin and localized both in the kidney and intestine. It cannot be excluded that they play a role in the immature intestine. In suckling mice, both uroguanylin and guanylin bind to STa receptors, but only uroguanylin stimulates intestinal fluid secretion so that its action is similar to STa. The reason for this discrepancy seems to be due to the different effect of luminal fluid peptidases on guanylin and uroguanylin (69, 137).

In the last decade, it has become evident that complex interactions between ENS, intestinal immune system, and the epithelium governs the transport rates of ions and water across the epithelium (167). The effector cells responsible for immune-mediated intestinal secretion are cells of lamina propria such as mast cells, fibroblasts, macrophages, and granulocytes. Some of the immune cells, especially mast cells, are in close proximity to enteric nerves, and it is supposed that the mediators of the immune system alter the function of enterocytes either directly or indirectly via enteric neurons. Alternatively, the stimulation of enteric neurons influences the immune network and plays a role in the coordination between immune and nonimmune cells of the mucosa and submucosa. Such an interaction already operates in newborns (7). The strongest evidence for the influence of the immune system on intestinal ion and water transport emerged from experiments on allergic reactions after exposure of the intestine to various antigens such as lactoglobulin, ovalbumin, or various organisms (bacteria, parasites). The local sensitization of the intestine suggests that the immune system is reached by antigens from food or from parasites. Their entry into the mucosa induces inflammation, antigen processing, and local sensitization, whereas reinfection can lead to hypersensitivity reactions. Under these conditions, the intestinal type I hypersensitivity reaction is associated with a rapid decrease of NaCl absorption and enhanced secretion of Cl and water, or both (166).

The intestinal epithelium also interacts with other intestinal cells through growth factors such as TGF-, TGF-, and IGF (437). In the immature intestine, TGF- expression appears to dominate in the villus (183, 298), whereas TGF- expression is localized in the crypts (100, 299). The receptors for TGF- and its homolog, the EGF, are present both in fetal and neonatal small and large intestine (197, 256, 396, 405). Whereas the expression of TGF- mRNA is very marked in both the immature and mature intestine (100), the expression of EGF mRNA increases during development (99) even if the abundance of EGF receptors is not age dependent (396). This means that the low expression of EGF mRNA in sucklings corresponds to a high concentration of EGF in rat milk (139, 283), whereas the absence of TGF- in rat milk is accompanied by a high concentration of TGF- mRNA in enterocytes. Similarly, high levels of TGF- in early milk and the decrease during the weaning period correlate negatively with endogenous TGF- production in the small intestine that increases during the weaning period (298). The sensitivity of absorptive functions to EGF has recently been demonstrated both in the fetal and neonatal rabbit intestine. Transamniotic administration of EGF stimulated fetal intestinal proline and glucose transport (46). Intraperitoneal administration of EGF to newborn rabbits increased intestinal water, Na⁺, and glucose absorption by inducing specific transport processes (independently of mucosal hyperplasia), whereas the effect of orogastrically administered EGF appeared to be secondary to mucosal hyperplasia (286).

The fetal and neonatal intestines also express IGF-I and -II (24, 101, 287) and IGF receptors (343, 370, 446). IGF binding is considerably high in newborn pigs and rats and is followed by a progressive decrease later, even if the timing depends on the species (343, 370, 446). In contrast to EGF, IGF-I was recently shown to stimulate intestinal absorption of amino acids but not of glucose in the small intestine of piglets (108).

In summary, it is obvious that progress continues to be made in the understanding of paracrine signals in the regulation of intestinal transport. However, little is known from the studies mentioned above about the paracrine regulation of intestinal transport in the young mammalian intestine and the relative role of individual systems, even though such information would be important for the understanding of diarrheal and other gastrointestinal diseases in neonates.

Studies performed in postweaned animals have revealed that the regulation of intestinal water and electrolyte transport depends on the mutual interactions between extrinsic sympathetic and parasympathetic nerves and intrinsic (enteric) nervous system composed of the myenteric and submucous plexus and enteroendocrine cells. Sympathetic adrenergic nerves appear to have a tonic proabsorptive influence on basal transport functions by stimulating water and electrolyte absorption (with the exception of pigs). The parasympathetic cholinergic pathway enhances Cl secretion; however, its role in maintaining the basal tone of intestinal transport seems to be minimal. The ENS is formed from efferent and afferent neurons and interneurons that provide an intricate network for the reflex control of intestinal mucosa (43). The number of transmitters operating in this network is quite large (Table 3). In principle, the response of the intrinsic nervous system, which is predominantly antiabsorptive and secretory, is based on a cholinergic and a peptidergic network. The mediators released by neurons interact with enteroendocrine and immune cells that release paracrine modulators influencing both the neural system and enterocytes (43).

In comparison with adults, virtually nothing is known about the maturation of intestinal transport neuromodulation and possible involvement of the nervous system in mediating transport development (Table 4). Nevertheless, it has been suggested that the neuronal functions might be different during the early postnatal development than in adulthood. The functional/neurochemical differentiation of ENS is achieved during weaning in altricial species (103, 248) and even earlier in human (212) or pig (415). Evidence that the enteric nervous system is able to influence ion and water transport in the immature intestine has been provided both in the jejunum (67) and distal colon (15), and it was shown that the neural component of basal ion transport measured as a short-circuit current is high during the perinatal period and declines later (15). Electric field stimulation, which is used experimentally to release transmitters from enteric neurons, stimulates secretion in the fetal and suckling pig colon and in the murine jejunum (15, 67). Because the effect of stimulation is less potent in the presence of tetrodotoxin or of the muscarinic antagonist atropine, both cholinergic and noncholinergic regulations operate in the immature intestine. Serotonin and norepinephrine have been shown to stimulate Cl secretion in the colon of newborn, suckling, and weaning pigs (15). These mediators have a similar effect in the small intestine (138, 234, 318). Endogenous dopamine has prosecretory effects, and dopaminergic neurons or enterocytes producing dopamine seem to be an important component of Na⁺ homeostasis in young animals (112, 419). Higher -adrenergic tone seems to be responsible for increased absorption in the immature intestine (2).

The reason for the developmental decrease of nervous components of intestinal ion transport is not known. It may reflect 1) developmental changes in prosecretory and proabsorptive tone, 2) changes in the density of enteric nerves, 3) changes in the quantity of neural mediators, or 4) changes in the sensitivity to basal neurotransmitter release. Each of these mechanisms appears to be corroborated by various findings. First, the immature intestine is sensitive to norepinephrine and enkephalin and has a higher proabsorptive adrenergic tone than the mature intestine (112, 264). Second, ENS continues to undergo developmental changes after birth (103, 248). Third, postnatal changes in the neurotransmitter content have been reported (140, 271). Fourth, the mechanism(s) responsible for the age-dependent desensitization or sensitization could involve up- or downregulation of a number of receptors (see Table 2), changes in receptor sensitivity, and changes in transduction pathways (coupling) as has been reported for the cholera toxin response (see sect. IIIG). The ability of immature rabbit ileum to generate cGMP in response to the atrial natriuretic peptide without stimulation of Cl secretion and the absence of cAMP-stimulated Cl secretion in immature rabbit cecum (41, 307) indicates that young animals possess less developed secretory mechanisms or lack the ability to couple secretory stimuli effectively to the secretory responses. Because the rabbit enterocytes possess an effective cAMP-signal transduction cascade from early stages of development (93), the immature intestine seems to have a functional second messenger system.

In summary, the neurally mediated pathways that regulate intestinal transport appear to operate at an early age even though maturation of the intestine is associated with changes in the sensitivity to various neurotransmitters and perhaps with changes in receptor-effector coupling.

The ability of the intestine to regulate its luminal environment is well known; nevertheless, the last decade has provided evidence for the existence of a converse relation, i.e., the possibility that the epithelium responds to luminal molecules. These relations are more obvious during intestinal development when the diet exerts a distinct influence on the development of the intestine. The changing dietary input during ontogeny (amniotic fluid, maternal milk, weaning, postweaning diet) imposes different demands on the intestine and influences its morphology, enzyme equipment, and transport (49). In addition, the diet contains substances that do not play a nutritional role (e.g., polyamines), but they directly stimulate the growth of intestinal mucosa and may influence weaning (60). Similarly, maternal milk and colostrum (139, 283) or amniotic fluid contain many biologically active substances (427) that stimulate both intestinal growth and development (437), and this stimulatory effect can also be detected in various intestinal cell lines (71, 179). It can therefore be postulated that some macronutrients or micronutrients (hormones, growth factors) play an important role in the growth and development of neonates.

Although the list of the biologically active substances in the milk is rapidly increasing, the question of whether these substances are of any biological significance has not yet been reliably decided. Biologically active milk hormones or factors should meet several criteria. Any substance has at least to retain its biological activity in the offspring, its removal from milk has to affect the offspring, and its replenishment has to correct the effect of its removal. These criteria are best met for EGF, although some conflicting results have been reported. This substance is present in higher concentrations in milk than in plasma and persists during passage along the gastrointestinal tract of sucklings where it is absorbed (139, 437). Two findings have strongly suggested that luminal EGF is involved in the regulation of the postnatal development of intestinal mucosa. First, luminal EGF has a stimulatory influence on the proliferation and differentiation of enterocytes (29, 185, 437). Second, EGF receptors coupled with tyrosine kinase activity respond to luminal EGF only in the immature but not in the mature intestine (396). Like EGF, IGF-I, which is also present in the colostrum and milk (139, 283, 437), stimulates intestinal proliferation and differentiation (58, 176, 231, 283, 437). Some other hormones present in milk (glucocorticoids, insulin) also exert biological effects on the immature intestine, but these are less obvious (62, 363, 443). For example, early weaning of rats to artificial formulas containing cow milk antigens delays gut closure (11, 389), and this delay can be prevented by supplementation of the artificial formulas with corticosterone (390).

The mitogenic activity of milk declines gradually during lactation (179) so that the regulatory role of milk in the intestinal development may depend not only on the content of macronutrients and micronutrients, but also on the gradual changes in milk composition and availability during the suckling and weaning period (196). For example, the milk intake in the rat during the first week exceeds the demands of the pups, whereas milk availability during the second week of life does become the limiting factor (436). Later, with the initiation of weaning, milk is supplemented with a gradually increasing proportion of solid food until milk is no longer consumed.

The studies on the effect of dietary input on intestinal transport functions have shown the effect of diet (colostrum, milk) on absorption (49). Dietary input decreases the transport capacity for nutrients and electrolytes in piglets immediately after birth due to suckling the mother (161, 311), whereas prolonged suckling prevents the decline of Na⁺ and glucose absorption in the rat colon and lamb small intestine (110, 357). Similarly, early introduction of a high-fructose diet stimulates premature expression of the apical fructose transporter GLUT5 and increases fructose absorption (89, 360).

Studies of malnutrition have also provided strong evidence that diet influences intestinal transport. Although malnutrition or fasting during the suckling and weaning periods are known to be associated with dramatic changes in mucosal barrier functions, they also modulate intestinal transport of nutrients and ions. The permeability of the intestinal epithelium for macromolecules is increased (68, 327), and the content of mucosal IgA and the secretory component is markedly diminished (142). The decrease of the IgA barrier is due to a lower number of polymeric immunoglobulin receptors that translocate IgA across the epithelial monolayer (142). The maximum transport capacity of Na⁺-dependent glucose transport (66, 88) is increased similar as net Cl secretion that is enhanced due to stimulation of both Na⁺ and Cl secretory fluxes via activation of the Ca²⁺-, cAMP-, and cGMP-dependent secretory pathways (66, 68, 88).

The mechanisms by which the changes of dietary intake influence the intestinal development are only poorly understood, and the data indicate that both indirect and direct effects of dietary substrates may be involved. The direct effect of nutrients on the postnatal shift in gene expression and processing of gene products has been implicated in the postnatal disappearance of glucose absorption in the small intestine of lambs. As a consequence of the rumen maturation, which coincides with weaning, dietary carbohydrates are fermented into short-chain fatty acids (SCFA) by rumen microflora, the amount of hexoses entering the small intestine gradually decreases (22), and the capacity of Na⁺/glucose cotransport declines (357). The maintenance of glucose transport and SGLT1 protein requires the luminal presence of milk or glucose which primarily control SGLT1 expression at the posttranscriptional level (218). The sugar sensor seems to be localized in the intestinal epithelium, even if it is not clear whether it is localized in the crypt cells, whose programming to synthesize SGLT1 mRNA is very rapid, or if it is possibly localized in villi localization and the receptor is linked to crypts via a neural or a paracrine mechanism (356). Similarly, the effect of a high-fructose diet on the expression of GLUT5 but not SGLT1 (360) reflects luminal but not systemic endocrine regulation (262, 361). Recent findings indicate that luminal lipids also have a direct effect on lipid absorption, particularly on apolipoprotein synthesis (34, 420).

Relevance of the indirect effect of luminal molecules is due to the fact that epithelium acts as a mediator between the luminal content and target cells beyond the epithelium barrier such as the cells of the immune system. The epithelium is able to secrete cytokines and growth factors or may present antigens directly to T cells through the histocompatibility complex II molecules that are developmentally regulated (177, 336). On the other hand, activation of the immune system, especially T cells, seems to stimulate epithelial proliferation and growth during weaning (394).

The indirect postnatal effect of the diet on intestinal transport can be the result of developmental changes of the membrane lipid composition and fluidity (4, 102, 347, 348). This prediction seems to be supported by the following two findings. First, dietary fat influences membrane lipid composition and fluidity in the adult intestine and thereby the activities of Na⁺/H⁺ exchanger, Na⁺-glucose cotransporter, and water permeability (39, 40). Second, the short-term high-cholesterol diet during weaning prevents the normal decline of jejunal glucose transport and enhances the age-related transport of galactose and leucine (401). Because the fat content of milk and postweaning diet is different, the dietary changes could control the age-dependent changes in the activity of some transporters. However, the extent to which the developmental remodeling of the membrane phospholipid and cholesterol content is a direct consequence of dietary lipid composition is not clear and should be further studied.

Postnatal dietary changes are also associated with the development of intestinal, predominantly colonic and rumen microflora (388, 414). Neonates are born with a sterile gastrointestinal tract, and the development of the intestinal ecosystem requires a certain time (233). In the case of the pig colon, the anaerobic microflora that is responsible for fermentation of carbohydrates in SCFA is established approximately in the first 2 wk of life (274). Young rats begin to use large bowel fermentation during weaning (282). The fermentation of carbohydrates in newborn colon, together with the above-mentioned lactose colonic absorption (see sect. IIIC2), helps the rat to conserve energy of carbohydrates that have not been digested and absorbed in the small intestine and that are converted into SCFA (198). In addition, SCFA's stimulate colonic ion transport (188, 275), and some of them (n-butyrate) stimulate cytokines (126). The spectrum of SCFA produced by bacterial flora depends on both the type of bacteria and the substrate available for SCFA production. It has been shown that infants fed breast milk produce predominantly acetic acid (55), whereas casein-based milk formulas are substrate especially for butyrate and propionate (260).

Taken together, the above findings suggest that the dietary effects are not restricted only to modulation of intestinal transport which is genetically hard-wired, but in some cases dietary factors are primary signals for development of transporters. The influence of dietary factors is not restricted to the period of weaning, but the interactions between dietary factors and intestine begin already during fetal life when fetuses start swallowing amniotic fluid and continue during suckling when the newborns receive colostrum and milk.

Specific composition of the diet is an important modulator of structure and function of the gastrointestinal tract (106, 107), especially during weaning when dramatic dietary changes occur in the majority of mammals. For example, composition of the weaning diet is important for the determination of colonic bacterial metabolism and SCFA production in adulthood (9). Similarly, variations in dietary intake of cholesterol, lipids, proteins, and carbohydrates during suckling and weaning result in changes of intestinal size and transport in later life (191, 397, 400, 401). Refeeding a diet, which was given transiently during the suckling and weaning period, results in intestinal adaptive changes that differ from the situation when the diet is given for the first time in adulthood. Such late effects of early exposure to high-protein and high-carbohydrate diets were observed in mice. The animals were fed either a high-carbohydrate, maintenance-protein diet or carbohydrate-free, high-protein diet during gestation and early postnatal life. It was found in adulthood that early diet irreversibly affected the intestine and body size, but no specific effect on transport of glucose or amino acids was noted (191). Similarly, late effects were also demonstrated in adult rats after early feeding of diets supplemented with saturated or polyunsaturated fatty acids. Rats weaned on a polyunsaturated diet for 2 wk, switched to a saturated diet for 8 wk, and rechallenged with a polyunsaturated diet for 2 wk had decreased ileal glucose absorption when compared with animals weaned to a saturated diet for 10 wk and switched to a polyunsaturated diet for 2 wk (400). The refeeding of a diet supplemented with cholesterol in adulthood was associated with different changes of lipid and carbohydrate uptake as compared with feeding this diet for the first time in adult rats (399). The stimuli that regulate intestinal adaptation to the absorption of diets at weaning and in adulthood are only incompletely understood. For example, different changes in phospholipids of apical membrane may modify nutrient absorption (195).

The aforementioned findings demonstrate that dietary manipulations during the prenatal, suckling, or weaning periods have long-lasting and apparently irreversible effects on some transport mechanisms. The demonstration of such developmental windows (critical periods) for programming of irreversible switching on or off once during the individual lifetime in response to suitable environmental conditions (191) has a great significance. This means that not all developmental programming is absolutely predetermined because the developmental window enables irreversible modulation of the developmental pattern during a limited period of time, i.e., during weaning.

	V. COUPLING OF ENERGY TO TRANSPORT PROCESSES IN THE DEVELOPING INTESTINE

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The energy requirements of the intestinal mucosa are high not only due to energy consumption for the synthesis of proteins and proliferation, but also due to the high capacity of active intestinal absorption and secretion. These dissipative processes utilize large amounts of energy, especially free energy from ATP (243). ATP, which is produced by oxidative phosphorylation and partially also by glycolysis, is utilized by various ion pumps (ATPases) to cover the expenditure required for transport work. Even if the detailed mechanisms of coupling between ATP production and ATP consumption (hydrolysis) are not fully understood, both processes are coupled in the intestinal epithelium similarly as in other epithelia (8, 225, 320).

As was discussed in the previous sections, many pathways of intestinal transport of solutes are directly or indirectly linked to active Na⁺ transport that is occurring through the basolateral membrane via the sodium pump, i.e., Na⁺-K⁺-ATPase that is responsible for maintaining Na⁺ and K⁺ gradients across the plasma membrane. In the intestine, only one isoform has been identified during development, and it is unlikely that circumstances such as ontogeny, hormones, and nutritional conditions may alter the type of the Na⁺-K⁺-ATPase isoform (295, 450). The expression of this isotype, considered at the level of mRNA or at the level of Na⁺-K⁺-ATPase activity under V_max conditions (i.e., saturating concentrations of ATP, Na⁺, and K⁺), increases during development both in the small (353, 450) and large intestine (295). Because the intracellular Na⁺ concentration is much lower under physiological conditions than at V_max conditions, it is reasonable to suppose that Na⁺-K⁺-ATPase does not operate at maximum capacity and that the transport work performed (ATP hydrolysis) is much lower than its maximum. The question is whether the ATP content in enterocytes is rate limiting for the operation of Na⁺-K⁺-ATPase. Experiments performed in adult animals have indicated that it is not (136) and that Na⁺-K⁺-ATPase is fully activated by ATP concentrations known to exist in cells (189).

In addition to the considerable fraction of ATP consumption by Na⁺-K⁺-ATPase, several other transport ATPases have been identified in the mature intestine. These are as follows: 1) Ca²⁺-ATPase involved in active Ca²⁺ extrusion across the basolateral membrane and uptake of Ca²⁺ by intracellular organelles, 2) H⁺-K⁺-ATPase responsible for active K⁺ absorption, and 3) HCO₃- ATPase of unknown function, which may be involved in acid transport. With the use of molecular biology analysis, temporarily upregulated H⁺-K⁺-ATPase expression has been demonstrated in the intestine of suckling animals (424). Several lines of evidence suggest that at least two K⁺-absorptive ATPases, ouabain-insensitive H⁺-K⁺- ATPase and ouabain-sensitive Na⁺-independent K⁺- ATPase, are involved (3). A developmental increase in intestinal Ca²⁺-ATPase has also been reported during the weaning period (249).

Immediately after birth, the energy costs in mammals are covered by milk, which represents a high-fat and low-carbohydrate diet. This composition of the diet requires rapid development of fatty acid -oxidation and ketogenesis. As a result of the energy taken in the form of fatty acids, ketogenesis markedly increases not only in the liver but also in the intestinal mucosa, but in contrast to the liver, intestinal capacity disappears after weaning (143).

Although the immature intestine is ketogenic, and suckling rats have increased concentrations of blood ketone bodies, mucosal 3-hydroxybutyrate oxidation is lower in suckling rat jejunum than in weanling animals, being similar to glucose (201). In contrast, the ability of colonocytes to utilize glucose or butyrate is highest in newborns and declines later (208). The preferential fuel in the immature intestine is neither glucose nor ketone bodies but glutamine (201, 435) which is usually considered the major fuel source of energy for rapidly dividing cells. The actual mechanisms for developmental changes of fuel metabolism have not yet been elucidated. It cannot be excluded that milk-borne growth factors or developmental hormonal changes may regulate intestinal metabolism in a similar manner as do the dietary changes at weaning (117). The preferential use of glutamine indicates that the fuels enter the central pathways of oxidative metabolism preferentially in the form of -ketoglutarate and to a lesser extent in the form of acetyl CoA, such as glucose and fatty acids (200, 201). In addition to oxidative metabolism, glycolysis also seems to be an important energy-producing process in the immature intestine (87, 435).

In summary, the intestinal mucosa of immature intestine is metabolically more similar to adult liver (ketogenic, gluconeogenic) than to the adult mucosa. Although suckling diet consists of a high percentage of lipids, the preferred oxidative substrate in enterocytes of suckling and weanling rats is glutamine. The oxidation of glucose and fatty acids increases after weaning. The mechanism of these oxidation changes is not obvious, but changes in diet might be a significant factor in intestinal substrate oxidation.

	VI. CONCLUSIONS AND PERSPECTIVES FOR FUTURE RESEARCH

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In this review, current knowledge about the development of intestinal transport functions was reviewed in the context of intestinal mucosa ontogeny. Nevertheless, it is not possible to provide a uniform description of the intestinal ontogenetic development. Much work has been done in studying the development of intestinal transport functions and digestive enzymes, but much less attention has been paid to the ontogenetic regulation of these processes. The relative paucity of available experimental data and the predominantly used species, the rat, make it difficult to generalize on the basis of a factological mosaic and to give a comprehensive description in mammals including humans. Important gaps concern the regulatory mechanisms of irreversible adaptations and maturation of regulatory networks operating in reversible adaptations of intestinal transport functions during the perinatal period and during weaning. It is also not known whether the transport processes and their regulatory mechanisms mature in concert during ontogeny or if they follow different time sequences. The role of the enterocyte environment in determining the receptor-effector coupling with respect to the regulation of developmental processes is a new area that will be of great importance in the future. The solution to these questions will require an understanding of both molecular structure and the function of transport and regulatory proteins as well as the knowledge of the complex control of the developing intestinal epithelium. These questions will no doubt be at the forefront of contemporary molecular biology, biostructure, biochemistry, and biophysics.

In conclusion, despite the fact that we know a lot about intestinal biology and physiology, we still have a lot more to learn about the mechanisms and regulatory signals of ontogeny. Information about how they change relative to each other under various physiological conditions will provide a better understanding of the processes involved in intestinal transport. This knowledge will help to recognize the possible signals that are able to program intestinal systems permanently during developmental windows (critical periods of development) and will improve the treatment of preterm and full-term newborns and infants, many of whom suffer from diarrhea.