Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation.

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.

Identification of R-GRAMP, a membrane glycoprotein of regulated secretory granules in primate cells.

Investigation of membrane assembly and traffic in the regulated secretory pathway may be facilitated by identification of membrane components that are unique to regulated secretory granules. To identify such markers, we isolated integral membrane proteins by Triton X-114 extraction from well-differentiated monolayers of an exocrine cell line, the goblet cell subclone (18N2) of the human colon carcinoma cell line HT29, and used the extracts as immunogens to produce monoclonal antibodies (mAbs). Immunofluorescence microscopy of HT29 goblet cell monolayers identified one mAb (MG-1) that labeled a component of mucin granule membranes. Immunofluorescence of frozen semithin sections of normal intestine, and various other human and monkey tissues, showed that this antigen is present in regulated secretory granule membranes of primate exocrine cells, endocrine cells, and tissue granulocytes. EM immunogold labeling of goblet cells, enteroendocrine cells and eosinophils confirmed that the antigen is associated with secretory granule membranes and not with plasma membranes. The antigen was identified by SDS-PAGE autoradiography of immunoprecipitates from HT29 goblet cells metabolically labeled with [35S]methionine and [35S]cysteine or [3H]glucosamine, as a glycoprotein with an apparent molecular mass ranging from 23 to 37 kDa. Digestion of immunoprecipitates with N-glycosidase F reduced the apparent mass to 16 to 19 kDa. This small, highly-glycosylated protein was named "R-GRAMP" (for regulated granule-associated membrane protein) to reflect its wide distribution in secretory granule membranes of regulated exocrine, endocrine and granulocytic cell types. This distribution suggests that it may play a common functional role in regulated secretion.

The development of corticotrophs in the fetal sheep pars distalis: the effect of adrenalectomy or cortisol infusion.

At 90 days gestation a uniquely fetal-type and an adult-type corticotroph have been observed in the fetal sheep pars distalis (term approximately 147 days). Between 90 and 130 days gestation the fetal type is predominant, and its numbers decline toward term. In this study the effect of the endogenous cortisol surge on the change in the population of corticotrophs in the pars distalis was investigated in sheep fetuses after bilateral adrenalectomy at 120 days gestation or after an infusion of 2 mg cortisol/day between 109 and 115 days gestation. The total proportion of corticotrophs, expressed as a percentage, decreased significantly (P less than 0.01) from 115 days in saline-infused controls (21.09 +/- 1.10%) and 135 days in intact controls (14.59 +/- 1.12%). The percentage of adult-type corticotrophs increased significantly (P less than 0.01) from 5.65 +/- 0.77 at 115 days, to 11.93 +/- 1.41 at 135 days. The percentage of fetal-type corticotrophs decreased significantly (P less than 0.001) from 14.91 +/- 0.35 at 115 days to 2.33 +/- 0.48 at 135 days. A small proportion of ACTH-immunoreactive cells could not be defined as either adult- or fetal-type corticotrophs. These changes in the corticotroph population had not occurred at 135 days in fetuses that had been adrenalectomized at 120 days; the percentage of corticotrophs relative to unstained cells (21.70 +/- 0.46%), the percentage of adult-type corticotrophs (6.42 +/- 0.29%), and the percentage of fetal-type corticotrophs (14.65 +/- 0.49%) were similar to those in 115-day-old fetuses, indicating that the normal change in the corticotroph population between 115 and 135 days gestation was dependent upon the presence of the fetal adrenal. In fetuses exposed to exogenous cortisol between 109 and 115 days gestation, the percentage of corticotrophs relative to unstained cells (16.53 +/- 1.68%), the percentage of adult-type corticotrophs (12.40 +/- 1.34%), and the percentage of fetal-type corticotrophs (3.78 +/- 0.58%) were similar to those at 135 days. This indicates that a short period of increased fetal plasma cortisol can bring about premature maturation of the corticotrophs in the fetal sheep pars distalis. We have also described an ACTH-immunoreactive cell which has characteristics of both an adult- and a fetal-type corticotroph. Its morphological appearance suggests that it may be a transitional stage from the fetal- to the adult-type corticotroph.(ABSTRACT TRUNCATED AT 400 WORDS)

Co-localization of neuron-specific enolase-like and kallikrein-like immunoreactivity in ductal and tubular epithelium of sheep salivary gland and kidney.

Neuron-specific enolase-like and kallikrein-like immunoreactivity was found to be co-localized in the ductal elements of the submandibular gland and in the more distal portions of the nephron in the kidney of newborn lambs. Some glomerular peripolar cells in the kidney were immunopositive for neuron-specific enolase without detectable kallikrein-like immunoreactivity.

Regulation of gastrointestinal growth in fetal sheep by luminally administered insulin-like growth factor-I.

Fetuses swallow large volumes of amniotic fluid. Absence of swallowing results in gastrointestinal tract (GIT) growth deficits. While it is not yet known to what extent the growth factors present in amniotic fluid are involved in GIT ontogeny, milk-derived growth factors are considered to be important for neonatal growth. Our experiment tested the hypothesis that a luminal growth factor (insulin-like growth factor-I, IGF-I) can sustain or promote GIT growth in utero in a model of gastrointestinal tract growth retardation. Ten-day infusion of either human recombinant IGF-I or vehicle into twin fetal sheep at 80 days gestation via an indwelling esophageal catheter resulted in altered GIT growth. Weight of the forestomach and small intestine increased. Significant histological changes were noted in the proximal small intestine, i.e. the region most exposed to the luminal infusion. Mucosal tissues were reduced in size. While the enterocytes in the proximal small intestine were generally more mature with regard to the ontogeny of the apical endocytic complex (which is responsible for uptake and transport of whole peptides), there were also many abnormal cytological features present. These included the development of large lysosomal-like inclusion bodies and many surfactant-like particles within the apical cytoplasm. Plasma IGF-I levels were on average 20% higher in treated siblings, suggesting that luminal IGF-I crossed the fetal gut and entered blood. IGF-II levels were not significantly affected. These observations are consistent with the suggestion that growth factors, which are present in swallowed amniotic fluid, influence fetal ontogeny.

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis.

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

Is fetal enteral nutrition important for normal gastrointestinal growth?: a discussion.

Long-term total parenteral nutrition results in atrophy of small intestinal structure and function. Maintenance or re-establishment of enteral nutrition can prevent or redress this loss. Paradoxically, the fetus develops in a total parenteral nutrition environment, but at the same time must achieve appropriate levels of gastrointestinal maturation in readiness for enteral feeding soon after birth. The fetus swallows large amounts of fluid during life in utero and growth is arrested if fetal ingestion is impaired. It is possible therefore that enteral nutrition provided by fetal swallowing is just as important in ensuring normal gastrointestinal homeostasis and growth in the fetus as it is in the adult.

Fetal organ growth in response to oesophageal infusion of amniotic fluid, colostrum, milk or gastrin-releasing peptide: a study in fetal sheep.

The hypothesis of the present study was that the infusion of the biological fluids to which the developing gut is normally exposed (i.e. amniotic fluid, colostrum, milk) and a single growth factor (gastrin-releasing peptide), which is found in high concentrations in fetal fluids and milk, could ameliorate the altered growth induced by the elimination of swallowed input secondary to ligation of the oesophagus. At 108-110 days of gestation the fetal oesophagus was ligated and a catheter inserted towards the stomach (32 fetuses). At 117-119 days of gestation saline (n = 5), amniotic fluid (n = 5), colostral whey (n = 5), milk whey (n = 5) or gastrin-releasing peptide (3.6 nmol day(-1), n = 6), was infused for 7 days (4 x 20 mL day(-1)), or no infusion was given (ligated group, n = 6). A further 15 fetuses were not ligated (normal group, n = 15). All fetuses had carotid artery and/or jugular vein catheters implanted. At 124-126 days of gestation the fetus was delivered and fetal body and organ weights recorded. Analysing the results by ANOVA, there were no effects of either ligation alone or infusion after ligation on fetal weight, crown-rump length, or weight relative to bodyweight of heart, adrenal, pancreas, large intestine and cecum. There were significant differences between the infusion groups for lungs, kidney, pancreas, total gut, abomasum, small intestine, spleen, chest and neck thymus, and mesenteric lymph nodes. Ligation alone significantly reduced small intestinal growth and increased kidney and spleen growth. Colostrum infusion enhanced growth of most organs. Gastrin-releasing peptide significantly increased growth of all the immune organs studied. It was concluded that at an age when premature delivery could be encountered, the fetal gut is capable of significant adaptive growth, to varying degrees, depending on the enteral diet. Growth effects in organs distant to the gut suggest that either gastrointestinal uptake and transport of growth factors or altered nutrient uptake and/or availability can affect the growth of other major fetal organs.

Failure of short-term luminal IGF-I to protect against atrophy in a model of fetal esophageal atresia.

Short-term luminal infusion in utero (3 days) of insulin-like growth factor I (IGF-I) failed to protect the fetal small intestine against atrophy induced by ablation of swallowing. Human recombinant IGF-1 (or vehicle) was infused into the duodenum of fetal sheep at 125 days' gestation for 3 days (day 1, 0.025 mg; day 2, 0.25 mg: day 3, 2.5 mg). Fetal swallowing was prevented by esophageal ligation, and a carotid catheter was implanted for blood sampling. There were no changes in body growth of in major organ growth. Small intestinal (SI) weight (corrected for body weight) was significantly lower for IGF-I treated fetuses. Villus height decreased significantly in proximal regions. Villus enterocyte cellularity was reduced significantly in the proximal regions. The percentage of crypt cells labeled with a 4-hour pulse of tritiated thymidine (as assessed by autoradiography) decreased significantly in the proximal SI only, from 16.14% (1.06% SEM) to 13.28% (1.05% SEM) (P < .05). Plasma levels of IGF-1 increased in the treated fetuses by an average of 76%. IGF-1 immunoreactivity was detected in the apical endocytic complex of enterocytes from proximal SI. This study shows that wasting of fetal intestinal tissues in the absence of enteral input cannot be prevented by IGF-1 delivered luminally.

Remodeling of the rat small intestinal mucosa during the suckling period.

The development of the suckling rat small intestine has been reexamined to investigate changes to cell kinetics and morphology more fully. In this period of rapid growth and reorganization, villi and crypts steadily increase in size in proximal regions; however, in distal regions a dramatic shortening of villi occurs at about 15 days. The result of this remodeling is that the normal adult proximodistal gradient of villus height is established At 15 days, migration rates in both regions increase dramatically. In proximal regions, the extent of 24-h migration of thymidine-labeled enterocytes is dependent on villus height. In distal regions, the increase in migration rate can be explained by the shortening of the villi. The extent of 24-h migration was not related to villus height. On the basis of these results, it is suggested that care must be taken in drawing conclusions relating any parameter to assumed distal region cell renewal patterns. This is because changes in the relative proportions of cell types present apparently can be brought about by villus deletion, independent of cell renewal.

Restitution of swallowing in the fetal sheep restores intestinal growth after midgestation esophageal obstruction.

We have shown that, in the fetal sheep, abolition of fluid ingestion early in gestation results in a profound gastrointestinal tract (GIT)-specific growth retardation and that these effects can be reversed if fetal swallowing is restored, even for relatively short periods (15 days). The fetal esophagus was ligated at 60-65 days of gestation in 11 fetal sheep (term is 145-148 days). At 136 days of gestation, body and tissue growth of six fetuses were compared to eight age-matched control fetuses. There were no effects on body growth, but the growth of the GIT was significantly retarded. The small intestine was the most severely affected region; villi were smaller in both proximal and distal regions, and villus density was increased and crypt density decreased. The growth-retarding effects are progressive such that they become more pronounced as the period of absence of swallowed input to the GIT is increased. Thus the effects observed in our study (ingestion abolished for approximately 80 days) are much more marked than those in our earlier short-term studies (40-50 days). Five of the fetuses with esophageal ligations underwent further surgery at approximately 120 days' gestation to correct the esophageal obstruction so as to allow the resumption of fluid ingestion. By 136 days, the values of most intestinal morphological parameters had begun to move toward control values.(ABSTRACT TRUNCATED AT 250 WORDS)

An integrated model for intestinal development in the fetal sheep.

The interrelationships between 22 parameters relating to small intestinal morphology and enterocyte cell kinetics were investigated. It was found that all significant correlations could be related to changes in intestinal diameter. The area of accessory and mucosal coats in cross section was computed and a constancy of relative composition, independent of experimental perturbation, was noted. It appears likely that despite seemingly trophic responses in specific tissue components, an overall balance of these components is maintained in the developing intestine. The need to develop integrated models of gut structure and function to allow adequate interpretation of results is discussed.

Perinatal development of the small intestine of the sheep.

Soon after birth, the villi in distal regions of the small intestine of the sheep rapidly decrease in size so that by day 6 the normal proximal-distal size gradient (tallest villi proximally) is established. This is the reverse of the gradient found throughout fetal life, where distal villi are taller than proximal villi. While crypt depth increases in both regions, the increase is more marked in proximal regions. The proportion of crypt cells labelled with a pulse of (3H) thymidine increases dramatically during day 1. Vacuolated cells are present in distal regions at day 2, but are absent by day 5. It is possible that the rapid loss of villus tip cells in distal regions is the means by which closure is achieved in this species. There is evidence to suggest that endogenous cortisol plays an important role in these changes.

Immunohistochemical identification of plasma proteins in cytoplasmic granules of peripolar cells of the sheep.

The cytoplasmic granules of glomerular periopolar cells of the newborn lamb and sheep have been shown to contain immunoreactive albumin and immunoglobulin using immunohistochemistry and ultrastructural immunocytochemistry. It is most likely that the granule contents include material derived from filtered plasma proteins, although the basis for such uptake, localised to glomerular epithelial cells in the peripolar region, remains unexplained.

Enterocyte ultrastructure and uptake of immunoglobulins in the small intestine of the neonatal lamb.

Although the small intestine of the sheep is relatively mature at birth, there are still vacuolated enterocytes present for at least 2 days in distal regions. In the distal regions, vacuolated cells possess a range of vesicle morphology which might be indicative of at least 2 separate routes for enterocyte handling of proteins taken up from the lumen. The localisation of immunoreactive immunoglobulins within the enterocytes, presumably of colostral or milk origin, in both proximal (non-vacuolated) and distal (vacuolated) regions, does not follow patterns which suggest orderly renewal at closure. It is suggested that closure is not solely brought about by epithelial cell replacement.

Systemic and luminal influences on the perinatal development of the gut.

At birth, the mammalian gastrointestinal tract (GIT) must be able to support a shift from mainly parenteral nutrition in the fetus (via the placenta) to enteral nutrition in the neonate. In the perinatal period the GIT therefore undergoes enhanced growth as well as morphological and functional differentiation, and this maturational programme is influenced by a complex interplay of local, systemic and luminal factors. This review shows how systemic and luminal factors may influence GIT development in the perinatal period of the pig and sheep, two long-gestation species. Adrenocortical hormones play a pivotal role in the prepartum maturation of the GIT in addition to their better known effects on the development of many other tissues and body systems. More particularly, in the fetal pig and sheep, the prenatal development of gastric acid and gastrin secretion, and of GIT hydrolase activities (chymosin, pepsin, amylase, lactase, aminopeptidases) is influenced by cortisol. Additionally, glucocorticoids exert effects throughout the GIT by influencing morphological, cytological, and functional differentiation. Since the GIT epithelial cells comprise a renewing cell population there are also changes in cell kinetics. In addition to systemic factors, the presence of growth factors, hormones and nutrients from swallowed amniotic fluid (fetus) and colostrum (neonate) may influence GIT development. In utero, fetal fluid ingestion has been shown to modulate tissue growth, macromolecule and immunoglobulin transport, enterocyte differentiation, cell turnover and activity of brush-border hydrolases. These effects may be mediated via regulatory peptides (e.g. insulin-like growth factor I, gastrin-releasing peptides, insulin, epidermal growth factor, gastrin). A physiological role of luminally derived growth factors is supported by a number of unique structural and functional adaptations of the GIT in the fetus and neonate (low luminal proteolysis, intestinal macromolecule transport). Thus, in the pig and sheep, both systemic and luminal factors appear to play critical roles in GIT development in the perinatal period.

Ultrastructural anomalies in the fetal small intestine indicate that fetal swallowing is important for normal development: an experimental study.

Fetal swallowing is established early in development and if fetal ingestion is prevented, the gastrointestinal (GI) tract fails to grow normally. In this article we describe the ultrastructural features of GI tissues developing in the absence of swallowing, in the fetal sheep. We have noted a number of defects in enterocyte morphology. These include abnormal or absence of microvilli, inappropriate cell extrusion, glycogen accumulation and altered lysosomal morphology. Many of these changes resemble those seen in malnourished infants. It is possible that fetal ingestion provides a significant source of nutrients, ensuring adequate GI tract growth in utero, in addition to specific growth factors which may be present in ingested fluid.

Identification of a marker antigen for the endocytic stage of intestinal development in rat, sheep, and human.

Vacuolated enterocytes are highly endocytic epithelial cells present in intestines of diverse mammalian species during neonatal and/or fetal development. Using monoclonal antibodies raised against membrane fractions, we previously identified a 55-61 kd membrane glycoprotein that is restricted to apical endosomal tubules of vacuolated enterocytes in fetal and suckling rats. To determine whether this cell-specific antigen is present in vacuolated enterocytes of fetal sheep or humans, the endosomal antigen was immuno-affinity purifed from rats and used to generate and purify specific rabbit polyclonal antibodies. Light microcopic and electron microscopic immunocytochemistry showed that antigens cross-reactive with the rat endosomal antigen are present in vacuolated enterocytes of fetal sheep and fetal human small intestine and are restricted to apical endosomal tubules in these cells. Immunoblot analysis of tissue extracts from fetal human intestines showed antigen(s) at 55 and 60 kd, as well as a major form at 130 kd. Cross-reactive antigen(s) from fetal sheep intestines appeared as 42- and 50-kd bands. Although the molecular identities of the sheep and human antigens are not yet established, these results show that these antigens can serve as markers for the endocytic state of intestinal developmemt in humans as well as other mammals.

Administration of insulin-like growth factor-I (IGF-I) peptides for three days stimulates proliferation of the small intestinal epithelium in rats.

It has previously been shown that longterm administration of insulin-like growth factor-I (IGF-I) or the analogue Long R3 IGF-I (LR3IGF-I) selectively stimulate growth of the gastrointestinal tract in gut resected, dexamethasone treated, and normal rats. In this study, the short-term effects of IGF-I administration on intestinal proliferation have been investigated. Female rats (110 g, five-six/group) were infused for three days with 2.5 mg/kg/day of either IGF-I or LR3IGF-I and compared with vehicle treated or untreated control rats. LR3IGF-I but not IGF-I increased body weight and wet tissue weight of the small and large intestine (+20%), compared with controls. Tissue weight responses were independent of food intake and were reflected in the histology of the tissue. In LR3IGF-I treated animals, duodenal and ileal crypts length were increased by 13 and 22%, respectively, associated with an increase in crypt cell number. No such histological changes were seen in IGF-I treated rats. Tritiated thymidine labelling indices were significantly increased after administration of either IGF-I or LR3IGF-I (up to 14%) in both the duodenum and ileum. In IGF-I treated rats, increased nuclear labelling was not associated with an increase in the crypt compartment. In contrast, LR3IGF-I induced proportional increments in thymidine labelling and crypt size, suggesting that LR3IGF-I is not only more potent than the native peptide but also induced proliferative events more rapidly. In the colon, the thymidine labelling index was low, however, a non-significant increase in the number of cells labelled with thymidine was seen. These results suggest that within a three day treatment period intestinal mitogenesis is more advanced in animals treated with LR3IGF-I. The differences in proliferative response between the two peptides may be accounted for by variations in pharmacokinetics, clearance rates, and interactions with circulating and tissue specific binding proteins.

Growth of the fetal sheep small intestine.

Body growth of the sheep fetus was studied over the period 60 to 145 days gestation (term is approximately 147 days). Fetal length increased more rapidly than did weight. Growth was also assessed in the component tissues of the intestinal wall. In all cases, growth was more rapid late in gestation. Comparison of the mean size or density of intestinal components at two ages (115 and 136 days gestation) revealed significant increases in proximal regions for the following parameters: intestinal diameter, total wall thickness, mucosal thickness, villus height, crypt depth, villus base width and crypt density. In distal regions significant increases were noted in all of the above with the exception of villus base width and crypt density. The magnitude of age related changes was most often greater in proximal regions. This suggests that morphological maturation proceeded at a faster rate in proximal portions of the small intestine.