Rat Small Intestinal Epithelium Research Articles

Calmodulin in rat enterocyte: an immunogold electron-microscope study.

Immunogold labeling of ultrathin sections of the epithelium of rat small intestine has been used to obtain insights into the ultrastructural localization and possible function of calmodulin in the enterocyte. Calmodulin is found mainly overlying the periphery of the microvillous core, in agreement with the location of the 110-kDa calmodulin complex. Extremely small amounts of calmodulin can be detected along the interdigitating basolateral membrane. This immunogold electron-microscope study suggests that calmodulin plays an important role in regulating the mechanochemical activity of myosin I but not in processes associated with the basolateral membrane of rat enterocyte.

Cholesterol synthesis and high density lipoprotein uptake are regulated independently in rat small intestinal epithelium.

The rates of high density lipoprotein HDL uptake and cholesterol synthesis were compared in the normocholesterolaemic (SW) and genetically hypercholesterolaemic (RICO) rat intestine. The RICO rat has a hyperintestinal cholesterol synthesis. 14C sucrose, a marker which becomes irreversibly entrapped within the cells, was used to measure total rat HDL uptake over 24 hours in the various cells of the small intestinal mucosa. The rates of sterol synthesis were estimated in vivo with 1-14C acetate, as previously validated. The rates of HDL uptake in the upper villus cells were similar along the length of the small intestine in both types of rat, but the rates of sterol synthesis varied up to eightfold. When the mucosal epithelium was divided along the villus/crypt axis, HDL uptake increased two to threefold and cholesterol synthesis two to fivefold in the upper villus compared with the crypt cells in both SW and RICO rats. The high cholesterogenesis in the mucosal cells of the RICO rat is not related to a modified HDL cholesterol uptake. Thus, cholesterol synthesis and HDL uptake seem to be regulated independently in the rat small intestinal mucosa.

Generation of neomucosa in vivo by transplantation of dissociated rat postnatal small intestinal epithelium

Abstract A novel method to study the generation of rat small intestinal mucosa, by transplantation of disaggregated postnatal rat small intestinal epithelium is described. Cellular aggregates, comprised of epithelium with attached proliferative cells and closely associated stromal tissue, were isolated from postnatal rat small intestine by enzymatic digestion, then grafted immediately to the subcutaneous plane of adult recipients. On graft retrieval after 14 days, 39% of cellular transplants to nude mice, and 84% of cellular transplants to inbred rats had developed into small intestine-like structures. These structures were comprised of a circumferential layer of epithelium surrounding a central mucin filled lumen. This neomucosal layer exhibited well formed crypts and villi, and contained all epithelial stem cell lineages i.e. absorptive enterocytes, goblet cells, Paneth's cells and entero-endocrine cells. Proliferative activity within this neomucosa was confined to crypt regions as in normal postnatal small intestine. Developmental maturation within the regenerated neomucosa was demonstrated by organotypic morphogenesis, i.e. formation of mature crypts and villi, and progressive cytodifferentiation with increased numbers of goblet cells, entero-endocrine cells and Paneth's cells. Altered patterns of brush border enzyme expression further confirmed a temporal progression of development within neomucosal enterocytes. It is concluded that after “extensive” mucosal disaggregation, postnatal small intestinal epithelial progenitor cells retain the capacity for organotypic regeneration of neomucosa when transplanted to ectopic sites in adult recipients. These small aggregates of epithelium and stroma are capable of generating the topographical signals necessary for the three dimensional regeneration of this tissue. Furthermore, the multipotent generative potential of the stem cells within these cellular aggregates is maintained with production of all progeny.

Heparin stimulates the proliferation of intestinal epithelial cells in primary culture

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

Progressive morphogenesis in vivo after transplantation of cultured small bowel epithelium.

An experimental model for the primary culture and transplantation of late foetal rat small intestinal epithelium is described. Multicellular aggregates of mucosal epithelium containing pre-crypt proliferative cells were isolated from 20-day foetal rat intestine by enzymatic disaggregation. Cellular aggregates, which we refer to as "epithelial organoids," attached readily in culture, proliferated, and spread to produce coalescing colonies within 10 days. Enterocytes were maintained in culture for 3 days, removed as cell sheets, and incubated overnight with foetal mesenchyme. Fourteen recombinant preparations were then grafted to the renal subcapsular space of adult nude mice. Four of six grafts retrieved after 1 wk had developed. Histology demonstrated the formation of simple tubular structures lined by a polarized columnar epithelium. At 14 days, two of eight grafts had developed and demonstrated temporal progression of morphogenesis. Histology showed rudimentary crypts and villi lined by different epithelial cell types, including enterocytes and goblet cells. Small bowel proliferative cells within "epithelial organoids" from 20-day foetal intestine, may be maintained in primary culture for up to four days. After short term primary culture, these proliferative cells retain the capacity for progressive organotypic morphogenesis and pluripotent cytodifferentiation, after transplantation to adult recipients.

Spatial analysis of transcriptional activation in fetal rat jejunal and ileal gut epithelium.

In the week before birth (days 15-21 of the 22-day gestation period), the fetal rat small intestinal epithelium undergoes rapid proliferation and differentiation. The developing gut changes from a mesenchymal tube lined by endoderm to a complex absorptive surface composed of differentiated epithelium overlying nascent villi. To begin to characterize the molecular events that take place in nascent intestinal epithelial cells as cytodifferentiation proceeds from jejunum to ileum, we examined spatial, temporal, and cellular patterns of transcriptional activation of the rat liver fatty acid-binding protein (L-FABP) and apolipoprotein (apo) AIV genes. In situ hybridization analyses revealed that transcription of both genes is activated in the jejunum between fetal days 17 and 18, yet their expression is not initiated in the ileum until 1-2 days later. Transcriptional activation proceeds in a "wavelike" fashion along the horizontal length of the gut and coincides with villus morphogenesis. As nascent villi emerge, heterogeneous cellular accumulation of L-FABP mRNA occurs in both fetal jejunal and ileal epithelium, but persists only in the ileum. In contrast, cellular expression of apo AIV mRNA in nascent epithelium is predominantly homogeneous in both regions of the gut. These results indicate that transcriptional activation in the fetal gut epithelium is a complex, dynamic process that is spatially regulated along the horizontal axis of the intestine. Initiation of transcription in enterocytes is closely linked to villus morphogenesis and histological cytodifferentiation.

Intracellular variation of rat intestinal mucin granules localized by monoclonal antibodies

Monoclonal antibodies produced against rat small intestinal mucins were utilized to study variability of stored mucin granules within rat ileal goblet cells. Eleven antibody-secreting hybridoma cultures were produced; six of these uniformly labeled stored mucin granules in virtually all goblet cells, suggesting that some antigenic features are common to all granules. The other five stained goblet cells in the rat small intestinal epithelium nonuniformly. R803, R805, and R807 localized within almost all goblet cells but revealed differential labeling of centrally and peripherally located mucin granules. R804 uniformly labeled the mucin granules of most villous goblet cells; some of the crypt goblet cells were uniformly labeled, but the majority were only partially labeled, resulting in a mottled staining pattern. R808 stained only a small portion of crypt goblet cells; there is, however, an increase in both number of goblet cells labeled and in uniformity of staining of the stored granule mass from the base of the crypt to the surface, resulting in uniform labeling of virtually all goblet cells at the villus tip. This study demonstrates for the first time that rat small intestinal mucin granules are immunologically heterogeneous and nonuniformly distributed within the epithelium. Additionally, staining patterns within the stored granule mass suggest that structurally distinct subpopulations of mucin granules may exist within a single goblet cell.

Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody.

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.

Characterization of crystalline rat liver fatty acid binding protein produced in Escherichia coli.

The principal absorptive cell of the rat small intestinal epithelium contains two homologous cytosolic proteins that bind long chain fatty acids. These are known as intestinal and liver fatty acid binding proteins (FABP). While their precise physiological roles have not been defined, they are believed to represent a multifunctional cytosolic transport system that is involved in the trafficking of exogenous lipids to sites of metabolic processing. 13C NMR studies have revealed differences in their fatty acid binding stoichiometries, binding mechanisms, and the ionization properties of bound fatty acids. To understand the functional differences, liver FABP has been crystallized for eventual comparison with the known crystal structure of intestinal FABP. The lattice type is trigonal with unit cell dimensions of a = b = 84.1 A and c = 44.2 A. The space group as determined by examination of the Patterson symmetry is either P3(1)21 or P3(2)21.

Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villus-crypt axis

Rat small intestinal epithelium is equipped with peptide YY (PYY)-preferring receptors, which also recognize neuropeptide Y (NPY) with high affinity. We therefore examined the distribution of PYY-NPY receptors along the villus-crypt axis after separation of mature villus cells from proliferative crypt cells. Specific 125I-labeled PYY binding was nine times higher in crypt cells than in villus cells. This was not due to differential degradation of PYY or PYY binding sites by the two cell populations. Rather, Scatchard analysis of equilibrium binding data showed that binding capacity (Bmax) of receptors increased from villus to crypt. Bmax were 166 +/- 36 and 21 +/- 3 fmol/mg protein, and dissociation constants (Kd) were 0.10 +/- 0.02 and 0.05 +/- 0.02 nM in crude membranes prepared from crypt and villus cells, respectively. For all cell populations, NPY and rat pancreatic polypeptide were 8- and 1,800-fold less potent than PYY in inhibiting 125I-PYY binding, respectively. Therefore, receptors appear to be PYY preferring along the entire villus-crypt axis. Both peptides (at the maximally active concentration of 1 microM) reduced vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) by 50% in crypt cells. PYY was four to six times more potent than NPY in agreement with the expression of a PYY-preferring receptor. By contrast, neither PYY nor NPY altered VIP-stimulated cAMP levels in villus cells. These results indicate that PYY-preferring receptors, negatively coupled to the cAMP production system, are preferentially expressed in crypt cells where intestinal ionic secretion is believed to take place.

Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat

Epidermal growth factor may play an important part in postnatal gastrointestinal development. However, little is known of its role prenatally. The aim of this study was to detect epidermal growth factor in amniotic fluid and to study its uptake and transfer across the epithelium of fetal rat small intestine. Anesthetized 20-day gestation rats underwent caesarian section. Three fetuses were exteriorized, their abdomens were opened, and ligated loops of proximal and distal small intestine were infused with 100 μg epidermal growth factor. Infused segments were removed 30 min later and processed for electron microscopy, and tissue was embedded in LR gold resin. Sections were treated with rabbit anti-rat epidermal growth factor antibody, followed by 5 nm gold-labeled goat anti-rabbit immunoglobulin before staining. Epidermal growth factor was measured in amniotic fluid by radioimmunoassay. In the proximal and distal small intestine epidermal growth factor was found membrane-associated along the luminal surface of microvilli, within apical invaginations and endosomal compartments, free from the membrane in multivesicular bodies, within large clear vesicles, basal vesicles and in association with the basolateral membrane and beyond. Epidermal growth factor was found in amniotic fluid at a concentration of 0.38 ± 0.07 (SD) ng/ml. This study shows that epidermal growth factor is present in amniotic fluid and is transported across the epithelium of fetal rats by an endocytotic process in both the upper and lower small intestine. It is likely that amniotic fluid epidermal growth factor plays a part in intestinal mucosal development, and may be active systemically after transepithelial passage in utero.

Peptide-YY and Neuropeptide-Y Inhibit Vasoactive Intestinal Peptide-Stimulated Adenosine 3′,5′-Monophosphate Production in Rat Small Intestine: Structural Requirements of Peptides for Interacting with Peptide-YY-Preferring Receptors*

Previous binding studies indicated that peptide-YY (PYY) and neuropeptide-Y (NPY) shared a common PYY-preferring receptor site in rat small intestinal epithelium. We showed here that PYY and NPY inhibited vasoactive intestinal peptide (VIP)-stimulated cAMP production in epithelial cells isolated from rat small intestine and examined their structure-activity relationship. Inhibition of VIP-stimulated cAMP by PYY or NPY is time and dose dependent; half-maximal effects were observed for 10 and 107 nM, respectively. In contrast, the structurally related peptide, pancreatic polypeptide, was only active at 1 microM. PYY or NPY reduced the efficacy of VIP by about 50% without altering its potency. Both peptides also suppressed prostaglandin E1-, prostaglandin E2-, and forskolin-stimulated cAMP production and reduced basal cAMP levels. Their inhibitory effects were observed throughout the small intestine, including duodenum, jejunum, and ileum, but not in large intestine. PYY or NPY and epinephrine (through alpha 2-adrenergic receptors) did not exert additive inhibitory effects on intestinal cAMP production. Several fragments of PYY and NPY were used to characterize their structural requirement for inhibiting VIP-stimulated cAMP production and competing with [125I]PYY for binding to intestinal membranes. A highly significant correlation was observed between IC50 values measured in the two assays. No partial sequence of PYY retained the full activity of intact PYY, but the C-terminal portion of PYY was shown to be much more important than the N-terminal portion. Deletion of 21 amino acids from the N-terminus [PYY-(22-36)] only resulted in a 4- to 5-fold decrease in potency compared to that of PYY-(1-36). In contrast, PYY-(27-36) exhibited a drastic loss of potency. The N-terminal fragments PYY-(1-22) and PYY-(1-28) also had very low potencies. Similar results were obtained with NPY fragments. These results provide the first insight on the negative coupling of PYY-preferring receptors with the cAMP production system in small intestine and evidence of the crucial role of the C-terminal portion of PYY in interaction with these receptors.

Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression.

We have examined the pattern of differentiation of the small intestinal epithelium in fetal rats during the 17th through 21st days of gestation. Five genes expressed in late fetal, neonatal, and adult enterocytes were used as markers of differentiation. They encode three homologous small cytoplasmic hydrophobic ligand binding proteins--liver fatty acid binding protein (L-FABP), intestinal fatty acid binding protein (I-FABP), and cellular retinol binding protein II (CRBP II)--and two apolipoproteins--apoAI and apoAIV. RNA blot hybridization studies indicated that gradients in mRNA concentration from the proximal small intestine to colon appear coincident with the initiation of rapid epithelial cell proliferation and villus formation (days 17-19 of the 22-day gestation period). Immunocytochemical studies disclosed a remarkably heterogeneous pattern of cell-specific expression of the three hydrophobic ligand binding proteins that was not apparent with either apoAIV or apoAI. This "mosaic" staining pattern was observed in morphologically similar cells occupying identical topographic positions along nascent villi in 17- to 18-day fetuses. The onset and resolution of this mosaicism varies between I-FABP, L-FABP, and CRBP II in the proximal small bowel, although it completely resolves by the first postnatal day. The distal small intestine exhibits a developmental delay of 1-2 days in the appearance of this heterogeneous pattern of initial gene expression. Double-label immunofluorescent analyses using L-FABP and I-FABP antibodies indicated that on the 18th day of gestation the proximal small intestinal columnar epithelium contains several populations of enterocytes expressing neither, one, or both proteins. The potential significance of this mosaic pattern of intestinal epithelial differentiation is discussed in light of recent studies with transgenic and chimeric mice.

Identification of calcium‐dependent, phospholipid‐ and calmodulin‐activated protein kinases in rat small intestinal epithelium

The mechanisms by which increases in cytosolic calcium promote ion‐secretion in the intestinal epithelium may involve the phosphorylation of key transport proteins in the microvillus membrane. Using both direct and indirect methods evidence has been provided for two distinct calcium‐regulated protein kinase activities in rat small intestinal epithelium — one activated by phosphatidylserine (C‐kinase), the other by calmodulin (CDR‐kinase).C‐kinase activity was identified in cytosolic and particulate fractions including purified brush border membranes using a specific histone substrate. In the presence of phosphatidylserine and diacylglycerol, calcium markedly stimulated enzyme activity, half‐maximal effects occurring at 2.8X10‐7 mol/1 free calcium. Phorbol esters, known activators of C‐kinase, were also shown to stimulate the phosphorylation of an endogenous peptide (Mr = 94 000) in cell homogenates.The addition of exogenous calmodulin (10–100 μg/ml) stimulated a 50–75% increase in the labelling of peptide (Mr = 50 000–55 000). Trifluoperazine (0.1–10 μmol/l) inhibited basal phosphorylation of this and other peptides suggesting the likely involvement of a protein kinase activated by endogenous levels of calmodulin. Cytosolic fractions were also able to phosphorylate purified myosin light chains, a known substrate for CDR‐kinase, in the presence of submicromolar concentrations of free calcium (ED50= 2.4X10‐7 mol/l) although no direct stimulation of this activity by calmodulin could be demonstrated. MLC‐phosphorylated activity was not identified in particulate fractions.

Initial kinetic changes of prostaglandin E2-induced hyperplasia of the rat small intestinal epithelium occur in the villous compartments

This study was performed to further identify the sequence of cell kinetics that occurs in the development of gastric and intestinal epithelial hyperplasia after orally administered prostaglandins of the E series. A high-dose, short-treatment schedule was used to examine the initial effects on kinetic parameters in the rat small intestinal epithelium. Groups of rats were killed after a single dose of oral prostaglandin E2 at 1 h after in vivo labeling with [methyl-3H]thymidine and during continued treatment at 6, 12, 24, 48, 72, and 96 h. As evidenced by autoradiography, the earliest change produced by prostaglandin E2 was an increased cellularity of the villous compartment (p < 0.05 after 24 h). There was no change of labeling index of the villous compartment or of the leading edge of labeled cells within 24 h. At 48 h, the increased cellularity was accompanied by a significantly elevated labeling index of the villi. Throughout the study period no significant differences were observed between groups in the number of cells or labeling indices in the jejunal crypts, of in cellular input from the crypts to the villi. Epithelial turnover time in the placebo and treatment groups was 69 and 71 h, respectively. To exclude the possibility that prostaglandin E2 initially affects cell birth rate and mean cell cycle time, a metaphase blocker was given after 4 days of treatment in a second study. Animals were killed after 0, 0.5, 1.0, 1.5, and 2.5 h. The rate of entry into mitoses was 8.1% cells/h in controls compared with 8.2% cells/h in treated rats. The distribution of mitoses within crypts was identical in the two groups and the mean cell cycle time was 13.6 and 13.2 h, respectively. Also, in this study there were trophic changes of the villi. It is concluded that the hyperplasia produced by oral prostaglandin E2 starts in the villi of the small intestine and is initiated by reduced cell exfoliation from the villous tips. Previously recorded retention of cellular elements in villi and crypts, increased cellularity of the proliferative compartments, and reduced mitotic index are secondary events.

Immunohistochemical localization and characterization of a protein from the basolateral membrane of rat small intestine epithelium using monoclonal antibody GZ-1.

The proteins of the basolateral membrane (BLM) of small intestine epithelial cell in rat have been less precisely described than those of the microvillus membrane (MVM). To identify BLM-specific proteins, Balb/c mice were immunized with isolated intestinal epithelial cells and monoclonal antibodies (MAb) to their cell membrane, produced with the hybridoma technique. One of the MAb so obtained (GZ-1), a class 1 IgG, is specifically directed to a surface membrane protein of intestinal epithelium (GZ-1-Ag). The MAb served to characterize the protein as follows. Light microscopic immunohistochemical FITC labeling and, still more clearly, electron microscopic labeling with colloidal gold on Lowicryl sections of small intestinal tissue, show that the GZ-1-Ag occurs only in BLM of the absorptive cell and the goblet cell. It is not present in the MVM, the tight-junction area, and probably in the desmosomal sections of the membrane. The crypt cells are more markedly labeled with GZ-1 than are the villus cells; the villus cells are also more clearly labeled from the duodenum to the ileum. Gross analysis of the position of the gold marker on the BLM indicates that GZ-1-Ag is probably integrated into the lipid bilayer. With immunoblotting (with HRP as marker), a single band of MW 42,000 D can be identified as the corresponding GZ-1-Ag from the protein band pattern obtained with SDS-PAGE from BLM isolated in the presence of protease inhibitors (PI). In BLM fractions isolated without protease inhibition, a band of MW 30,000 D can be labeled with GZ-1. These results are interpreted as follows: GZ-1-Ag is a protein of MW 42,000 D. On isolation of the BLM without PI, a piece of this protein is broken off by proteolysis. The larger piece of the molecule (30,000 D) is not accessible to the proteolytic enzyme owing to its localization in the BLM, and therefore remains intact (and recognizable by the Ab). The preferred position of the gold marker on the BLM is in agreement with this explanation.

Interaction of Peptide YY with Rat Intestinal Epithelial Plasma Membranes: Binding of the Radioiodinated Peptide

High affinity binding sites for peptide YY (PYY) have been identified and characterized in plasma membranes prepared from rat jejunal epithelium by studying the kinetics, stoichiometry, and chemical specificity of the interaction of 125I-labeled PYY with membranes. Binding of [125I]PYY was rapid, saturable, reversible, specific, and depended on temperature, pH, and ionic strength. In optimized steady state conditions of binding (2 h of incubation at 15 C), the degradation of both [125I] PYY and binding sites did not exceed 20%. The concentration dependence of PYY binding, determined by adding increasing concentrations of [125I]PYY, indicated that specific binding saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 434 +/- (SE) 56 pM and a binding capacity of 336 +/- 41 fmol/mg protein (n = 11). Identical results were obtained when increasing concentrations of unlabeled PYY were added to a fixed concentration of [125I]PYY, indicating that the radioiodinated peptide has the same apparent affinity as native PYY. Peptides structurally unrelated to PYY, such as members of the vasoactive intestinal peptide family, insulin, or cholecystokinin octapeptide, were unable to compete with [125I]PYY for binding to membranes. Rat, human, and avian pancreatic polypeptides, which display, respectively, 42%, 47%, and 53% homology with PYY, did inhibit [125I]PYY binding but with an approximate or equal to 100,000-fold lower potency than PYY, indicating the strict structural requirement for recognition by PYY binding sites. In contrast, natural or synthetic neuropeptide Y, which has 25 out of 36 amino acids in common with PYY, retained a high affinity for PYY binding sites [only 4.7 +/- 1.2 (n = 5) times lower than that of PYY]. Specific [125I]PYY binding was particularly high in the upper small intestine and could not be detected in stomach, large intestine, or liver. These findings indicate that rat small intestinal epithelium expresses specific binding sites for the candidate gut hormone PYY that also binds the neuropeptide Y with high affinity, suggesting that the two peptides may regulate the function of small intestinal epithelium, through interaction with a common receptor site.

Ontogenic Timing Mechanism Initiates the Expression of Rat Intestinal Sucrase Activity

Morphologic and enzymic differentiation occurs in rat small intestinal epithelium during 16-20 days of postnatal life. This change is considered to be initiated by an ontogenic timing mechanism and is modulated by extrinsic systemic and luminal factors. The importance of the ontogenic timing was tested directly using a transplantation technique in which jejunal isografts from newborn (day 0) and 5-day-old (day 5) rats were implanted under the skin of newborn (day 0) hosts. Isografts showing cryptvillus architecture were obtained in 44% and 21% of transplants, respectively. Day 0 isografts and host intestine expressed sucrase activity at about 16-18 days of age and showed similar crypt cell labeling and epithelial migration after [3H]thymidine injection. Day 5 isografts expressed sucrase activity when the hosts were 13 days of age, whereas host intestine showed no detectable sucrase activity. Isograft lactase activities in both experimental transplant models were significantly higher than host intestinal lactase up to 28 days of age, suggesting that luminal factors are important in modulating lactase activity during the first 4 wk of postnatal life. It is concluded that (a) no systemic factors at day 13 inhibit the expression of sucrase activity and (b) an ontogenic timing mechanism in the jejunum initiates the expression of sucrase activity.

Chronic effects of alcohol on the epithelium of the small intestine using two experimental models.

The effects of chronic alcohol ingestion on the rat small intestinal epithelium have been measured using two different experimental models, viz. either a solid diet plus alcohol or a liquid diet plus alcohol. All experimental animals consumed adequate quantities of food and exhibited a normal growth rate; thus the recorded effects were unlikely to be associated with malnutrition. After a period of 24 days, there were significant decreases in the jejunal villus cell population in both groups of experimental animals and corresponding increases in the crypt cell populations; no such changes were found in the ileum. Alcohol had no significant effect on the crypt cell production rate and the growth fraction at either of these two sites in the intestine, and thus the major effect of alcohol was to cause a lengthening of the cell cycle time in the jejunal crypts.

Function and regulation of hepatic and intestinal fatty acid binding proteins

Two structurally different fatty acid binding proteins (FABP) have been isolated from rat liver and small intestinal epithelium. hFABP is a 14 184 Da protein found in abundance in both liver and small intestine, whereas gFABP (15 063 Da) is abundantly present only in small intestine. This review discusses studies which have provided insight into the physiological functions of these proteins. These include analyses of endogenous and exogenous ligand binding to FABP in vitro; examination of the modulating effect of FABP preparations on enzyme activities in vitro; exploration of relationships between alterations in cytosolic FABP content in response to hormonal, pharmacological, and dietary manipulations and changes in the rates of cellular fatty acid uptake and utilization; and studies of hFABP turnover and the mechanisms of FABP regulation. These experiments provide compelling evidence for a broad role of the FABPs in the transport, utilization and cellular economy of free fatty acids in the liver and small intestine, and also in protecting several aspects of cellular function against the modulatory effects of fatty acids, fatty acyl-CoA esters, and other ligands. Studies of FABP regulation also suggest a role in long-term rather than short-term modulation .of hepatic fatty acid metabolism and indicate that hFABP and gFABP may perform different functions in the small intestine.