Histochemical characterization of glycoproteins present in jejunal and colonic goblet cells of pigs on different diets. A biopsy study using chemical methods and peroxidase-labelled lectins

We examined the glycoprotein composition of intestinal goblet cells in jejunal and colonic biopsies obtained from pigs on different diets. Paraffin sections were stained both chemically and with the following horseradish-peroxidase conjugated lectins: Canavalia ensiformis (Con-A), Limulus polyphemus (LPA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA1), Glycine max (SBA) and Triticum vulgaris (WGA). Using chemical staining procedures, only small quantitative differences were noted between the two organs. With respect to lectin staining, the mucus of the jejunum was characterized by the absence of Con-A binding sites, and colonic mucus consistently exhibited an absence of SBA affinity. After dietary modifications, O-acetyl sialic acid reactivity was lowered in the jejunum but was enhanced in the colon. In the jejunum, the glycoproteins became neuraminidase susceptible, whereas the colon became characterized by the absence of neutral mucins. The affinity for the tested lectins after the different diets was variable, but the most striking effects were observed after the fibreless diet (milk alone). Our data suggest the existence of marked regional variations in goblet-cell mucus and indicate significant differences between the glycoprotein components of the jejunal and colonic mucosa. Furthermore, the biosynthesis of mucins in both regions was altered by even only short-term feeding modifications.     

Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit

Summary Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundantO-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars withvicinal diols whose periodate oxidation is prevented by anO-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisablevicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have anO-acyl substituent located at position C-8 or C-9 (or which have two or three side chainO-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated fromhuman colon, and differential interference contrast microscopydistinguished goblet and columnar cells. Activation with carbachol(CCh, 100 µM) or histamine (10 µM) released contents from gobletgranules. Stimulation with prostaglandinE₂(PGE₂, 5 µM) or adenosine (10 µM) did not release goblet granules but caused the apical margin ofcolumnar cells to recede. Goblet volume was lost during stimulationwith CCh or histamine (~160 fl/cell), but not withPGE₂ or adenosine. Three-quartersof goblet cells were responsive to CCh but released only 30% of gobletvolume. Half-time for goblet volume release was 3.7 min.PGE₂ stimulated a prolonged fluidsecretion that attained a rate of ~350 pl/min. Columnar cells lost~50% of apical volume during maximalPGE₂ stimulation, with a half-timeof 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. Theseresults support separate regulation for mucus secretions from gobletcells and from columnar cells, with control mechanisms restrictingtotal release of mucus stores.

     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E(2) (PGE(2), 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine ( approximately 160 fl/cell), but not with PGE(2) or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE(2) stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE(2) stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.     

Histochemical studies of intestinal epithelial goblet cell glycoproteins during the development of the human foetus

Summary Histochemical studies performed on specimens of intestine from 12 to 37-week human foetuses showed that the epithelial glycoproteins of the goblet cells of the small intestine are non-sulphated sialoglycoproteins containing neutral sugar (hexose, 6-deoxy hexose or N-acetyl hexosamine residues with Periodic acid-Schiff (PAS) reactive vicinal diols), sialic acids without O-acyl substituents, smaller and variable quantities of sialic acids with O-acyl substituents at positions C8 or C9 (or with two or three side chain substituents) and O-acyl sugars (neutral sugars with an ester substituent blocking PAS reactivity). In the lower small intestine glycoproteins containing 8 (or 9)-O-acyl sialic acids are first observed in goblet cells at the tips of the villi. As the foetus matures their quantity increases and they are found in goblet cells located along the length of the villi. Smaller quantities of O-acyl sialic acids and traces of O-acyl sugars occur in the goblet cells of the upper small intestine. The colonic goblet cells contain sulphosialoglycoproteins of two types. The first type, found in the majority of specimens, contains O-sulphate ester, neutral sugar, O-acyl sugars and 8 (or 9)-O-acyl sialic acids. The second type contains O-sulphate ester, neutral sugars, and sialic acids which are either without side chain O-acyl substituents or are a mixture of such acids and 8 (or 9)-O-acyl sialic acids; O-acyl sugars are reduced or absent. The degree of sulphation of the foetal colonic goblet cell epithelial glycoproteins differs with the region of the colon, the level of the crypt and the gestational age of the foetus in a manner consistent with that described by Lev & Orlic (1974). The detection of O-acyl sugars in foetal intestinal glycoproteins adds to the known examples of such sugars and strengthens the suggestion that they are a normal constituent of colonic epithelial glycoproteins.Part of this work was presented at the 32nd meeting of the Canadian Federation of Biological Sciences, Calgary, Alberta, June 1989 (abstract # 336).     

Secretagogue response of goblet cells and columnar cells in human colonic crypts1

Crypts of Lieberkühn were isolatedfrom human colon, and differential interference contrast microscopydistinguished goblet and columnar cells. Activation with carbachol(CCh, 100 µM) or histamine (10 µM) released contents from gobletgranules. Stimulation with prostaglandinE₂(PGE₂, 5 µM) or adenosine (10 µM) did not release goblet granules but caused the apical margin ofcolumnar cells to recede. Goblet volume was lost during stimulationwith CCh or histamine (~160 fl/cell), but not withPGE₂ or adenosine. Three-quartersof goblet cells were responsive to CCh but released only 30% of gobletvolume. Half-time for goblet volume release was 3.7 min.PGE₂ stimulated a prolonged fluidsecretion that attained a rate of ~350 pl/min. Columnar cells lost~50% of apical volume during maximalPGE₂ stimulation, with a half-timeof 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. Theseresults support separate regulation for mucus secretions from gobletcells and from columnar cells, with control mechanisms restrictingtotal release of mucus stores.

     

Exocytosis in colonic goblet cells visualized by video-enhanced light microscopy

In order to develop a method to quantify the mucus secretion, we observed the mucus epithelium of the rabbit colon under a video-enhanced differential interference contrast microscope. Upon stimulation with muscarinic agonists, secretory granules in individual goblet cells were found to undergo a rapid light intensity change. Simultaneously, the lumen was widened and filled with a cloudy material. In each cell, many of such responses were followed by formation of a large cavity which could be recovered after removal of the stimulant. We infer that the light intensity change of a granule arises from exocytosis. Direct counting of the frequency of these quantal responses would be very useful to monitor the secretory activity of single cell in real time at a high sensitivity.     

Cytoarchitectural reorganization of rabbit colonic goblet cells during baseline secretion

Light and electron microscopy were coupled with point counting methods to quantitate shape and volume changes of goblet cells during their migration and maturation from the base of the crypt to the colonic surface epithelium in the rabbit. After differentiation, goblet cells attain a broad pyramidal configuration in the basal third of the crypt. The cells elongate and dramatically decrease in volume as they move into the surface epithelium. The distributions and volume fractions of organelles were found to vary considerably, depending on the location of the goblet cell in the epithelium. Mucin granules are initially synthesized throughout the cytoplasm, but become increasingly concentrated as the cell matures. Organelles involved in synthesis such as the Golgi apparatus and rough endoplasmic reticulum (RER) similarly attain a more concentrated arrangement as the cell moves up in the crypt. The mean cell volume decreases from 1,228.8 microns3 for cells in the basal third of the crypt to 541.3 microns3 for goblet cells on the surface. Most organelles decrease in proportion to this decrease, although a disproportionately large decrease in the RER was measured. When actual subcellular volumes are calculated, a net decrease in several subcellular compartments is detected. This loss of granules and organelles is accomplished by the continual synthesis and secretion of mucin granules. Cytoplasm and organelles become entrapped in the upward movement of granules towards the cell apex, become irretrievably isolated, and are sloughed into the crypt lumen. This process accounts for the decrease in cell volume and contributes to the altered cytoarchitecture of the cell.     

Fast renewal of the distal colonic mucus layers by the surface goblet cells as measured by in vivo labeling of mucin glycoproteins

The enormous bacterial load and mechanical forces in colon create a special requirement for protection of the epithelium. In the distal colon, this problem is largely solved by separation of the bacteria from the epithelium by a firmly attached inner mucus layer. In addition, an outer mucus layer entraps bacteria to be cleared by distal transport. The mucus layers contain a network of Muc2 mucins as the main structural component. Here, the renewal rate of the inner protective mucus layer was studied as well as the production and secretion of Muc2 mucin in the distal colon. This was performed by intraperitoneal injection of N-azidoacetyl-galactosamine (GalNAz) that was in vivo incorporated during biosynthesis of O-glycosylated glycoproteins. The only gel-forming mucin produced in the colon is the Muc2 mucin and as it carries numerous O-glycans, the granulae of the goblet cells producing Muc2 mucin were intensely stained. The GalNAz-labeled glycoproteins were first observed in the Golgi apparatus of most cells. Goblet cells in the luminal surface epithelium had the fastest biosynthesis of Muc2 and secreted material already three hours after labeling. This secreted GalNAz-labeled Muc2 mucin formed the inner mucus layer. The goblet cells along the crypt epithelium accumulated labeled mucin vesicles for a longer period and secretion of labeled Muc2 mucin was first observed after 6 to 8 h. This study reveals a fast turnover (1 h) of the inner mucus layer in the distal colon mediated by goblet cells of the luminal surface epithelium.     

Histochemical study on the intestine goblet cells in cichlid and poecilid species (Teleostei)

Histochemical properties of intestine goblet cells in firemouth cichlid, zebra mbuna, freshwater angelfish and platyfish are described. Goblet cells occurred regularly in the epithelial cell layer throughout the entire intestine, they were strongly coloured by alcian blue at pH 2.5. This colour got gradually weaker when the pH was reduced, but still after alcian blue at pH 0.2 these cells displayed a distinct blue colour. When the goblet cells were treated with periodic acid-Schiff (PAS), they displayed a strong purple-magenta colour. The findings that a number of goblet cells displayed various colours between blue and purple-magenta when acidic alcian blue was followed by PAS, and between blue and red-brown when acidic alcian blue was followed by neutral red, may reflect different ages or stages of development and differentiation for these cells. However, such results may also suggest a true cellular heterogeneity in the present population of goblet cells, reflecting that the intestine mucus layer has a number of roles in teleosts like lubrication, protection, immunological defence, digestion and absorption.In the ferritin injected specimens of firemouth cichlid and platyfish, a number of macrophage-like cells in intestine wall displayed Prussian blue precipitations in tissue treated with acid ferrocyanide, suggesting that these cells play a cleansing role in the intestinal wall. No ferritin uptake was seen in the intestine goblet cells and eosinophilic granule cells.     

The colonic groove of the plains viscacha (Lagostomus maximus): Histochemical evidence of an abrupt change in the glycosylation pattern of goblet cells

The ascending colon of most rodent species shows a longitudinal colonic groove that works as a retrograde transport pathway for a mixture of bacteria and mucus toward the cecum. We describe the morphology and glycosylation pattern of the colonic groove of Lagostomus maximus to analyze the role of mucins in this anatomical feature. We also studied the distribution pattern of the interstitial cells of Cajal (ICC) to evaluate their regulatory influence on gut motility. The groove originated near the cecocolic junction and extended along the mesenteric side of the ascending colon, limited at both ends by nonpapillated ridges. These ridges divided the lumen of the ascending colon into two compartments: a narrow channel and a large channel, called the groove lumen and the main lumen, respectively. The histochemical analysis showed differences in the glycosylation pattern of the goblet cells inside and outside the groove. Unlike the mucosa lining the main lumen of the colon, the groove was rich in goblet cells that secrete sulfomucins. The PA/Bh/KOH/PAS technique evidenced an abrupt change in the histochemical profile of goblet cells, which presented a negative reaction in the groove and a strongly positive one in the rest of the colonic mucosa. The anti‐c‐kit immunohistochemical analysis showed different ICC subpopulations in the ascending colon of L. maximus . Of all types identified, the ICC‐SM were the only cells located solely within the colonic groove.     

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.     

Endothelin-3 stimulates survival of goblet cells in organotypic cultures of fetal human colonic epithelium

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.     

Distribution and histochemical characterization of goblet cells in the nasal cavity of piglets

The present study was designed to compare the distribution of goblet cells and the histochemical composition of mucosubstances produced by these cells in the nasal cavity of piglets aged from 1 to 28 days. Serial transverse sections were stained to demonstrate neutral, acidic, and sulfated mucosubstances. Sections located at eight reference levels rostrocaudally in the nasal cavity and defined regions on these sections were used for goblet-cell counting. There was a nonhomogeneous distribution of goblet cells in the nasal cavity of piglets. A rostrocaudal increase in goblet-cell density was observed with the highest densities found in the ventral meatus and on the septum. There was no difference in this pattern of distribution according to age of the piglets. However, age-related differences were observed in the prevalence of goblet cells containing sialomucins, sulfomucins, or both. While sialomucins were prevalent at 1 and 14 days, sulfomucins predominated in the rostral half of the cavity at 28 days. Our results indicate a maturation of the products of secretion with aging in piglets. The affinity of infectious agents for sialylated glycoconjugates and the predominance of sialomucins in the nasal cavity of newborn piglets could account for their greater susceptibility to bacterial infection.     

Turnover of the amino sugars of erythrocyte and thrombocyte glycoproteins in rats maintained on different carbohydrate diets

The turnover of erythrocyte and platelet glycoprotein amino sugars in rats fed carbohydrates has been studied. Partial replacement of starch by sucrose results in an almost 3-fold increase in the half-life of erythrocyte glycoprotein amino sugars and in a 2-fold increase in that of platelets as early as 14 days after keeping the animals on carbohydrates. It is concluded that study of dynamic parameters of cell amino sugars can be used for evaluation of the role played by food in metabolic and adaptive reactions both in experimental animals and man.     

Histochemical analysis of goblet cell mucins in the chick embryo colon

The histochemical characteristics of colonic epithelial mucins were investigated in the chick embryo. At the 14th day of incubation it was possible to demonstrate the presence of glycogen. At the 15th day a few epithelial cells showed the presence of neutral and sialylated mucins. On the 16th day, also sulfated secretory material was detectable together with neutral and sialylated mucins in cells with the typical shape of goblet cells. From the 17th day to the 20th day of incubation the two types of acid mucins appeared in some cells to be placed in distinct zones of the supranuclear cytoplasm. At the 21st day, neutral, sialylated and sulfated mucins were all present in the majority of goblet cells, which were found mainly in the epithelium lining the crypts.     

Stimulation of colonic goblet cells by cecal filtrates from rabbits with experimental mucoid enteropathy

Mucoid enteropathy was induced experimentally by ligation of the cecum, and sterile filtrates were prepared from cecal contents of sick and control rabbits. Explants were prepared from the colons of normal rabbits and were maintained in a short-term organ culture system. Cecal filtrates from rabbits with experimental mucoid enteropathy when added to the culture medium, stimulated mucus secretion and discharge from goblet cells. This was exhibited by an increase in number of visible goblet cells, presence of mucus in crypt lumen and/or presence of a significant amount of mucus blanketing the surface epithelium. The results indicated that a mucus-stimulating substance, a goblet cell secretagogue, was produced in the cecum of affected rabbits.