Lysosomes of the Liver and the Duodenal Enterocytes,a Site of Handling of Two Rare Earths. Ultrastructural and Microanalytical Study

The frequent use of some trace elements such as gadolinium and terbium in medicine and modern industries make us worry about their behavior in the organism. In this work, we study the intracellular localization in the liver and in the intestinal mucosa of two rare earths, gadolinium and terbium, after intraperitoneal and intragastric administration. Three methods of observation and microanalysis were used: conventional transmission electron microscopy, secondary ion mass spectrometry, and electron probe microanalysis. After intraperitoneal administration, gadolinium and terbium were detected with phosphorus in lysosomes of hepatocytes and Küppfer cells and in territories near to biliary canalicule. One hour after intragastric administration, gadolinium and terbium were concentrated in lysosomes of the apical part of duodenal enterocytes. No gadolinium or terbium was detected in duodenum 4 days after administration. After intragastric administration, the microanalytical techniques failed to detect gadolinium or terbium in liver whatever the time of sampling. This mechanism of concentration-precipitation in the lysosomes of enterocytes limits the diffusion through the digestive barrier of foreign elements and then permits their elimination with apoptotic cells in the intestinal lumen. Some of these elements may be toxic, and none of them have a recognized physiological function. The intestinal mucosa plays an important role in the protection of the organism against the invasion of foreign elements.     

Role of the duodenal and hepatic cells lysosomes in the phenomenon of gadolinium accumulation

The behaviour of the intestinal mucosa and of the liver after an administration of a gadolinium salt has been studied in the Wistar rat using transmission electron microscopy, ion mass spectrometry, and electron probe microanalysis. Six hours after parenteral administration, gadolinium is concentrated with phosphorus in the lysosomes of hepatocytes and Küppfer cells. Six hours after its oral administration, gadolinium is detected in the duodenal enterocytes lysosomes, but never in those of the liver cells. It is suggested that this mechanism of local concentration limits the diffusion through the digestive barrier of foreign elements, some of them being toxic and none of them having a physiological function.     

Bioaccumulation of lanthanum by the mussels Mytilus edulis collected from French coasts. Microanalysis by X-ray spectrography and secondary ion emission

The common marine mussel Mytilus edulis collected from French coastal waters of the Channel, Atlantic Ocean and Mediterranean Sea was shown to contain lanthanum; higher levels were found in the samples collected from the Eastern Channel and more particularly from the Baie de Seine. 139La+ was detected within lysosomes of digestive gland, labial palp and gill epithelium, macrophage hemocytes and chitinous tissue. Lanthanum was always associated with high phosphorus contents in the lysosomes. Thus, lanthanum which exists in sea water at trace level is taken up by the Mussel, via gill and digestive tractus, in a soluble form and then concentrated in the form of an insoluble phosphate in the storage organelles. A comparison is made between the behaviour of lanthanides and actinides in the biological systems.     

Iron regulatory protein as an endogenous sensor of iron in rat intestinal mucosa. Possible implications for the regulation of iron absorption.

Duodenal enterocytes adjust intestinal iron absorption to the body's state of iron repletion. Here we tested how iron supply from the blood modulates the RNA-binding activity of iron regulatory proteins (IRP-1 and IRP-2) in immature duodenal rat enterocytes, and whether the modulation is compatible with the hypothesis that IRPs, in turn, may regulate the expression of iron transport proteins in maturating enterocytes during migration to the villus tips. Tissue uptake of parenterally applied 59Fe along the duodenal crypt-villus axis was compared to local IRP-1 and IRP-2 activity and to duodenal 59Fe transport capacity 12 h, 48 h, and 72 h after intravenous iron administration to iron-deficient rats. IRP-1 and IRP-2 activity was significantly increased in iron-deficiency. 59Fe administrated from the blood side was almost exclusively taken up by crypt enterocytes. Accordingly, the activity of IRP-1 decreased at this site 12 h after parenteral iron administration, but remained high at the villus tips. After 48 h the bulk of 59Fe containing enterocytes had migrated to the villus tips. Correspondingly, IRP-1 activity was decreased at duodenal villus tips after 48 h. IRP-2 activity also tended to decrease, though the change was statistically not significant. IRP-2 activity remained significantly higher at duodenal villus tips than in crypts, even after 72 h. Intestinal iron absorption capacity decreased with the same delay as IRP-1 activity after intravenous iron administration. In the ileum 59Fe uptake from the blood and IRP activity showed no significant difference between crypt and villus region. Luminal administration of iron decreased duodenal IRP-1 and IRP-2 activity at tips and crypts within 2 h. Thus, recently absorbed iron becomes available to cytosolic IRP during its passage through the enterocyte. Our results are compatible with a role of IRPs in gearing the expression of intestinal iron transporters in the duodenal brushborder to the body's state of iron repletion.     

Localization of fucosyl residues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells

We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.     

The role of enterocytes in the intestinal barrier function and antigen uptake

The intestinal epithelium is a critical interface between the organism and its environment. The cell polarity and structural properties of the enterocytes, limiting the amount of antigen reaching the epithelial surface, form the basis of the integrity of the epithelium. However, apart from their participation in digestive processes, the enterocytes perform more than just a passive barrier function. The resistance of the tight junctions regulates the paracellular transport of antigens. Furthermore, the enterocytes take up and process antigens, involving two functional pathways. In the major pathway, enzymes in the lysosomes degrade the antigens. In the minor direct transcytotic pathway, the antigens are not degraded and are released into the interstitial space. Moreover, the enterocytes can present processed antigens directly to T cells and are often directly involved in immune processes. In inflammatory conditions, the properties of the epithelial barrier and the outcome of the immune response to luminal antigens can be changed.     

Histological and ultrastructural study of the digestive tract of rice field eel, Monopterus albus

The histological characteristics of the digestive tract and the ultrastructure of mucosal cells of the stomach and intestine of rice field eel, Monopterus albus, are described to provide a basis for future studies on its digestive physiology. The digestive tract of the rice field eel is a long and coiled tube composed of four layers: mucosa, lamina propria‐submucosa, muscularis and serosa. The pharynx and oesophagus mucosa is lined with a stratified epithelium. The stomach includes the cardiac and pyloric portions and the fundus. Many gastric pits are formed by invaginations of the mucosal layer and tubular gastric glands formed by the columnar cells in the fundus. The intestine is separated from the stomach by a loop valve and divided into a proximal portion and a distal portion. The proximal intestinal epithelium consists of columnar cells with microvilli towards the lumen and goblet cells. The enterocytes are joined at the apical surface by the junctional complex, including the evident desmosomas. Numerous lysosomes and some vesicles are evident in the upper cytoplasm of the cells, and a moderate amount of endoplasmic reticulum and lysosomes are scattered in the supranuclear cytoplasm. The epithelium becomes progressively thicker and the folds containing large numbers of goblet cells are fewer and shorter in the distal portion of the intestine. At the ultrastuctural level, the columnar cells of the tubular gastric glands have numerous clear vacuoles and channels. A moderate amount of pepsinogen granules are present in the stomach. The enterocytes of the intestinal mucosa display a moderate amount of endoplasmic reticulum and lysosomes, and long and regular microvilli.     

Identification of an intestinal heme transporter

Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.     

Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications

Summary Two protein antigens, horseradish peroxidase (HRP) and ferritin, have been administered to the digestive tract of carp. Electron-microscopical observations reveal considerable absorption of both antigens in the second segment of the gut (from 70 to 95% of the total length) and also, although to a lesser extent, in the first segment (from 0 to 70% of the total length). Even when administered physiologically with food, a large amount of ferritin is absorbed by enterocytes in the second gut segment.HRP and ferritin are processed by enterocytes in different ways. HRP seems to adhere to the apical cell membrane, probably by binding to receptors, and is transported in vesicles to branched endings of lamellar infoldings of the lateral and basal cell membrane. Consequently, most of the HRP is released in the intercellular space where it contacts intra-epithelial lymphoid cells. Only small amounts of HRP become localized in secondary lysosomes of enterocytes. Ferritin does not bind to the apical cell membrane; after uptake by pinocytosis, it is present in small vesicles or vacuoles that appear to fuse with lysosome-like-bodies. In the second segment, intact ferritin ends up in the large supranuclear vacuoles (after 8 h), where it is digested slowly. Although no ferritin is found in the intercellular space, ferritin-containing macrophages are present between the epithelial cells, in the lamina propria and also to a small extent in the spleen. The transport of antigens from the intestinal lumen, through enterocytes, to intra-epithelial lymphoid cells or macrophages may have immunological implications, such as induction of a local immune response and prospectives for oral vaccination.     

Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa

Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

Alterations and reversibility of digestive and absorptive functions of rat intestine following medroxyprogesterone acetate administration

The long term (90 days) effects of medroxyprogesterone acetate (MPA) administration on the digestive and absorptive functions of the small gut have been investigated in female albino rats. The uptake of glucose and amino acids was found to be significantly increased while Ca++ uptake decreased following MPA treatment (35 mg/kg body weight). The observed increase in glucose uptake might be due to carrier mediated transport in enterocytes and not to a change in cell number. The Michaelis constant for glucose uptake was not altered by MPA. Activities of brush border membrane disaccharidases, leucine aminopeptidase and basolateral membrane enzyme Na+, K+-ATPase were significantly increased in response to MPA treatment. It was observed that these biochemical alterations caused by MPA in intestinal digestive and absorptive functions were reversible by 5 weeks after termination of the drug treatment. The action of the drug appears to closely resemble that of known effects of glucocorticoids on intestinal mucosa.     

Transcytosis of gastric leptin through the rat duodenal mucosa

Leptin is secreted into the gastric juice by epithelial Chief cells and reaches the duodenum in a biologically intact active form. We assessed the possibility that this gastric leptin crosses the intestinal mucosa by transcytosis through enterocytes to reach blood circulation. Endogenous gastric leptin secretion was triggered by cholinergic stimulation. In another set of experiments, recombinant leptin was inserted in vivo into the duodenal lumen. Plasma levels of leptin were assessed by enzyme immunoassay and Western blot, and duodenal tissue was processed for immunocytochemistry. We first observed that leptin was found inside duodenal enterocytes from fed rats but not inside those from fasted ones. Stimulation of gastric secretion by a cholinergic agent led to rapid increases in plasma leptin levels (202 +/- 39%) except when the pylorus was clamped. Insertion of recombinant leptin into the duodenal lumen raised plasma leptin concentrations (558 +/- 34%) quite rapidly, whereas carrier solution without leptin had no effect. The use of FITC-tagged leptin reinforced these results. Light and electron microscopy revealed the cellular compartments involved in its transcytosis, namely, the enterocyte microvilli, the endocytotic vesicles, the Golgi complex, and the basolateral interdigitations. Leptin was also present in the lamina propria, in capillary endothelial cell plasmalemmal vesicles, and in capillary lumina. These results demonstrate that gastric exocrine leptin is internalized by duodenal enterocytes and delivered to the lamina propria and blood circulation. It may thus be able to play important paracrine and endocrine functions for the control of gastric emptying and nutrient absorption.     

Internalization and transcytosis of pancreatic enzymes by the intestinal mucosa.

As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.     

Immunocytochemical localization of cathepsins B,H, and L in the rat gastro-duodenal mucosa

Summary Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

Mucolipin Co-deficiency Causes Accelerated Endolysosomal Vacuolation of Enterocytes and Failure-to-Thrive from Birth to Weaning

During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3^−/−;Trpml1^−/−) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3^−/−;Trpml1^−/− mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns with lysosomal storage disorders. Finally, we conclude that mucolipin-endowed lysosomes in the young play an evolutionarily-conserved role in the intracellular digestion of maternally-provided nutrients, whether milk in mammals or yolk in oviparous species.     

Fat absorptive processes in the intestine of the Antarctic fish Notothenia coriiceps (Richardson, 1844)

Absorption of lipids by the enterocytes of Notothenia coriiceps, an omnivorous Antarctic and subAntarctic fish, was studied by light and electron microscopy. The lipids are absorbed by the anterior and middle intestine segments. They appear as fat droplets that measure from 0.5 to 7 μm of diameter and which accumulate in the apical cytoplasm within the first 24 h and seem to be the main fat storage form in the enterocytes. Fat is also observed as lipid particles with 60–300 nm inside the rough endoplasmic reticulum and cytoplasmic smooth vesicles. The epithelial intercellular space and the mucosal inner lamina contain lipid particles, which probably are the fat transport form. Our observations show that an intense lipid absorptive process takes place in N. coriiceps digestive system, due to the great extension of the intestine involved and due to the great lipid accumulation found in the epithelial compartment.     

Fat intestinal absorption in the catfish--a histochemical study in glycol methacrylate embedded tissue

The intestinal absorption of lipids was investigated in plastic sections from glycol methacrylate embedded intestine after fat administration. In the catfish, the lipids are absorbed by the enterocytes of the proximal intestinal segment, thus forming fat cytoplasmic inclusions that were demonstrated by Sudan black B staining. The histochemical characterization of lipids by the Nile blue sulphate test revealed the neutral or triglyceride nature of the cytoplasmic droplets, both after the corn oil and oleic acid feeding. There is lipid accumulation in the lamina propria and lymphatic vessels.     

Phthalate esters immunolocalized in the gastrointestinal tract of shi drum Umbrina cirrosa (L.) and rainbow trout, Oncorhynchus mykiss (W.)

The occurrence of phthalate esters in freshwater and marine aquacultural species like rainbow trout Oncorhynchus mykiss and shi drum Umbrina cirrosa, respectively, were determined by immunohistochemical approach. The results showed a similar distribution in the gastrointestinal tract of both species. In particular, intense immunoreactivity was found at gastric gland level. In the intestinal tract, goblet cells failed to stain, whereas enterocytes showed the highest binding of phthalates restricted to the apical cytoplasm. This distribution of phthalate esters at gastric gland and enterocyte level may have implications for the physiology of the digestive process and intestinal biotransformation. Phthalates are confirmed to be widely diffused contaminants, absorbed via the alimentary canal; thus a multidisciplinary approach could be useful to examine sea and freshwater environments.     

Microanalysis and image processing of stable and radioactive elements in ecotoxicology. Current developments using SIMS microscope and electron microprobe

Ecotoxicological investigations were performed on two sets of biological models. The first one concerns marine pollution and was composed of invertebrates (molluscs and crustaceans) contaminated by stable or radioactive elements originating from wastes discharged into sea water. The second one concerns freshwater pollution and was composed of vertebrates (fish) contaminated by aluminium which was dissolved in rivers, as a consequence of an atmospheric pollution by acid rain. Mechanisms involved in the uptake, storage and elimination processes of these toxicants were studied, with a special emphasis on cellular and subcellular aspects of concentration sites. Two microanalytical methods were employed: secondary ion mass spectrometry (SIMS), using the ion microscope and the ion microprobe, and X-ray spectrometry using the electron microprobe (EMP). SIMS, which enables the visualization of trace elements, was associated with an image processing system using a highly sensitive television camera connected to an image computer. Polychromatic images were obtained, allowing to establish the cellular distribution of metal contaminants. In marine organisms, the target organs and tissues of Al, rare earth elements (Tm and La) and radionuclides (U, Pu, Am) were shown to be mainly digestive gland and exoskeleton. The target organelles were shown to be spherocrystals and lysosomes where the enzymatic lysosomal coprecipitation with phosphorus was observed. Amoebocytes, which are enzymatically equipped with lysosomal phosphatase, were involved in the phagocytic clearance of metal pollutants. In trout, two processes appeared to be involved in Al accumulation. The first one corresponds to the well known insolubilisation of Al phosphate, within lysosomes of organs devoted to uptake and excretion such as gill and kidney. The second one demonstrates that organs and tissues which cannot eliminate, such as bone, heart and brain, retain Al, exhibiting a high intracellular metal concentration; moreover, large Al deposits inducing nervous tissue destruction have been observed. Data have been discussed in connection with the relationship between man and his environment.