Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

Summary The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas

The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.     

Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.     

Freeze-fracture and morphometric analysis of occluding junctions in rectal glands of elasmobranch fish

Summary The structure of occluding junctions in secretory and ductal epithelium of salt-secreting rectal glands from two species of elasmobranch fish, the spiny dogfishSqualus acanthias and the stingrayDasyatis sabina, was examined by thin-section and freeze-fracture electron microscopy. In both species, occluding junctions between secretory cells are shallow in their apical to basal extent and are characterized by closely juxtaposed parallel strands. Average strand number in the dogfish was 3.5±0.2 with a mean depth of 56±5 nm; in the stingray a mean of 2.0±0.2 strands encompassed an average depth of 18±3 nm. In contrast, the linear extent of these junctions was remarkably large due to the intermeshing of the narrow apices of the secretory cells to form the tubular lumen. Morphometric analysis gave values of 66.8±2.5 and 74.9±4.6 m/cm² for the length of junction per unit of luminal surface area in the dogfish and stingray, respectively. This junctional morphology is similar to that generally described for leaky epithelia. In comparison, the stratified ductal epithelium which carries the NaCl-rich secretion to the intestine is characterized by extensive occluding junctions which extend 0.6–0.8 m in depth and consist of a mean of 12 strands arranged in an anastomosing network, an architectural pattern typical of tight epithelia. The length density of these junctions in the dogfish rectal gland was 7.6±0.1 m/cm².The junctional architecture of the rectal gland secretory epithelium (few strands, large junctional length densities) is similar to that described for several other hypertonic secretory epithelia [20, 34] and is compatible with the recent model for salt secretion in rectal glands [39] and in other Cl^– secretory epithelia which posits a conductive paracellular pathway for transepithelial Na⁺ secretion from intercellular space to the lumen to form the NaCl-rich secretory product.     

Partitioning of paracellular conductance along the ileal crypt-villus axis: A hypothesis based on structural analysis with detailed consideration of tight junction structure-function relationships

Summary Current models of intestinal transport suggest cells which absorb ions are located on the villus while secretory cells are located in the crypt and putatively have paracellular pathways which are highly conductive to Na⁺. One approach to assess possible variation in small intestinal paracellular conductance along the crypt-villus axis is to morphometrically analyze the structural aspects of crypt and villus tight junctions (TJs) which relate to paracellular resistance. Such detailed analysis of junctional structure in this heterogeneous epithelium would permit one to compare intestinal TJ structure-function relationships with those in a structurally simpler epithelium such as that of toad urinary bladder. This comparison would also be of considerable interest since previous similar comparisons have failed to consider in detail the geometric dissimilarity between these two epithelia. We applied light, electron microscopic, and freezefracture morphometric techniques to guinea pig ileal mucosa to quantitatively assess, for both crypts and villi, linear TJ density, relative surface contributions, and TJ strand counts. Mean linear TJ densities were 76.8 m/cm² for crypt cells and 21.8 m/cm² for villus absorptive cells. Mean TJ strand counts were 4.45 for undifferentiated crypt cell TJs and 6.03 for villus absorptive cell TJs. The villus constituted 87% and the crypt 13% of total surface. We utilized these data to predict paracellular conductance of cryptsvs. villi based on equations derived from those of Claude (P. Claude,J. Membrane Biol. 39:219–232, 1978). Such analysis predicts that 73% of ileal paracellular conductance is attributable to the crypt. Furthermore, we obtained literature values for paracellular resistance in mammalian ileum and toad urinary bladder and for toad bladder TJ structure and linear density and constructed a relationship which would allow us to more accurately compare TJ structure-function correlates between these two epithelia. Such a comparison, which considers both surface amplification and TJ structure and distribution in these epithelia, shows that one would predictin vitro measured values for paracellular resistance should be approximately two orders of magnitude less in mammalian ileum than in toad urinary bladder. This predicted discrepancy (115-fold) correlates well with the observed difference (100-fold). These findings suggest that highly similar TJ structure-function relationships apply to these geometrically dissimilar tissues and that, in mammalian ileum, the crypt compartment may be responsible for the majority of net ileal paracellular conductance. We speculate that high crypt linear TJ density and low crypt TJ strand counts may serve as the structural basis of massive paracellular Na⁺ movement which is coupled to active Cl^– secretion and appears to originate from the crypt following exposure to intestinal secretagogues.     

Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: Physiological rearrangement of tight junctions

Summary All epithelia slough dying cells but the consequences of this physiological process to epithelial barrier functions is unknown. In mammalian small intestine absorptive cells are known to migrate from the villus base to the villus tip from which they slough. These villus tip extrusion zones are often envisioned as sites at which macromolecules could leak across the epithelium. However, only trace amounts of macromolecules cross this epithelium even though, based on known epithelial turnover rates, extrusion events occur millions of times daily. Here, we examine the characteristics of the epithelial barrier to macromolecular permeation at villus tip extrusion zones in rats and hamsters. Freeze-fracture, light and electron microscope studies reveal that extruding cells do not leave transient holes behind as they lift from the epithelium. Rather, as cells extrude, processes of adjacent cells extend under them. Moreover, tight junction elements proliferate between extruding cells and their neighbors and appear to move down the lateral margin of the extruding cell as it extends into the lumen. These observations suggest that newly formed junctional elements zipper the epithelium closed as extrusion proceeds thus preventing epithelial discontinuities from occurring. Correlative in vivo perfusion experiments using horseradish peroxidase as a macromolecular-tracershow that the above described dynamic alterations in tight junctions at extrusion sites are generally sufficient to prevent transepithelial leaks of macromolecules.     

Acute and Subacute Effects of Thiamine Deficiency On the Morphometry and Cell Kinetics of Jejunal and Ileal Epithelial Cells

Abstract. The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (I_p) and crypt cell cycle time (T_c) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. the jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, I_p and T_c.     

Sulphur uptake in the mucosa adjacent to carcinoma of the large intestine

Synopsis A histochemical study of the epithelial mucosubstances was performed on surgical specimens from cases of carcinoma of the large intestine. Thein vitro uptake of sulphur was investigated in the same material by organ culture and autoradiography.Pieces of normal mucosa at a distance from the tumour area and of apparently normal mucosa surrounding the tumour were taken in every specimen and the findings compared.The distribution of mucosubstances in normal colon and rectum, called here the normal mucous pattern, shows a predominance of sulphated mucosubstances occupying from the lower half to the whole of the crypt. Non-sulphated acid mucosubstances are usually present in the upper part of the crypt. In the surface epithelium both types of acid mucin are usually present.This normal mucous pattern changes both qualitatively and quantitatively in the mucosa adjacent to the tumours, despite its being morphologically normal. This area may be termed the transitional mucosa; in it, a gradual decrease of sulphated material was observed together with an increased amount of a non-sulphated acid mucosubstance, most probably a sialic acidcontaining one.Studies with sulphur show an uptake of the isotope along the crypt and surface epithelium in the normal, compared with the findings in transitional mucosa where either the isotope is present only in the surface epithelium, or no uptake is observed either in surface epithelium or in crypt cells.An interpretation and practical application of these findings are discussed.     

Acute and subacute effects of thiamine deficiency on the morphometry and cell kinetics of jejunal and ileal epithelial cells

The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (Ip) and crypt cell cycle time (Tc) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. The jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, Ip and Tc.     

The effect of atrial natriuretic peptide on intestinal electrolyte transport.

The effect of atrial natriuretic peptide (ANP) on rat small intestinal electrolyte transport was examined. In vivo, intravenous administration of rat ANP(99-126) induced diuresis and natriuresis in conjunction with a significant decrease in intestinal water (basal, 37.1 +/- 5.7 versus ANP 28.5 +/- 6.0 microliters/cm per 20 min, P less than 0.05) and Na+ (4.0 +/- 0.7 versus 2.8 +/- 0.9 mumol/cm per 20 min, P less than 0.05) absorption (n = 9). In vitro, in Ussing chambers, in both jejunum and ileum, addition of 1.0 microM ANP to short circuited, stripped tissue produced a maximal increase in short circuit current and stimulated net Cl- secretion due to a significant increase in the unidirectional serosal to mucosal flux (JCl-sm: jejunum 17.4 +/- 1.3 versus 19.8 +/- 1.3 microEq/cm2 per h, P less than 0.01, n = 6; ileum 13.4 +/- 0.5 versus 17.2 +/- 0.6, P less than 0.01, n = 6) which was inhibited by the calcium channel antagonist verapamil (82 +/- 26%, P less than 0.05) and by the 5-HT2 receptor antagonist cinanserin (72 +/- 44%, P less than 0.05). Guanylate cyclase activity was stimulated by ANP in intact epithelium, but not in isolated crypt and villus enterocytes.     

Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras

The cell migration pathway in the intestinal epithelium of DDK in equilibrium C57BL/6JLac mouse chimeras is demonstrated using Dolichos biflorus agglutinin-peroxidase as strain-specific marker. Cell sheets of one genotype extend in relatively straight lines from crypt to villus apex. Narrow sheets are mostly interrupted in the distal two-thirds of duodenal but not ileal villi, suggesting that in the duodenum cell loss occurs below the apical extrusion zone. These differences between duodenum and ileum correspond to differences in villus shape. The pattern of cell migration in Peyer's patch epithelium is consistent with that of the duodenum. In chimeric colon, sharply demarcated territories of crypts with a narrow cuff of surface epithelium represent the counterpart of the villus/crypt unit of the small intestine.     

Variations in capillary permeability from apex and crypt in the villus of the ileo-jejunum

Summary The permeability of fenestrated capillaries in an organ is believed to be homogeneous. However, the permeability of fenestrated capillaries in different organs and to various exogenous tracers varies from a complete restriction, as found in the eye (Pino and Essner 1980, 1981; Pino 1985a) to the freely permeable peritubular capillaries of the kidney (Venkatachalam and Karnovsky 1972). In the present report we demonstrate that within any single intestinal villus from the ileo-jejunum of the rat, the permeability of fenestrated capillaries is not uniform. Exogenous hemoglobin (Einstein-Stokes radius [ESR] = 3.2 nm) exits all capillaries at any villar level in less than 5 min. In contrast, all villar capillaries restrict catalase (ESR = 5.2 nm) at 5 min, but by 60 min the tracer is present extravascularly in crypt and lower villar regions. Apical capillaries are slightly permeable to catalase at 2 h, but the bulk of the tracer remains in the lumina. The particulate tracer ferritin (ESR = 6.1 nm) is restricted 3–10 times more by apical capillaries than basal ones and is found in increasing concentration extravascularly at lower villar and crypt levels after 20 min. Following an 18-h circulation, a second dosage of ferritin is restricted by the endothelium at all villar levels. Immunocytochemical localizations of the plasma proteins albumin (ESR = 3.5 nm) and IgG (ESR = 5.5 nm) revealed an apparent lack of restriction at all villar levels. These results demonstrate that apical villar capillaries in the ileojejunum are more restrictive to exogenous molecules with ESR5.2nm. Also, the passage of tracer molecules out of an endothelium alters the subsequent permeability of that vessel.     

Experimental modulation of occluding junctions in a cultured transporting epithelium

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.     

THE EFFECT OF TRANSPOSITION TO JEJUNUM ON EPITHELIAL CELL KINETICS IN AN ILEAL SEGMENT

Epithelial cell kinetics were studied in an ileal segment after transposition to proximal jejunum. The number of cells per villus column in the transposed ileum increased after 4-7 days to reach values normal for jejunum after 14-30 days. This increase was accompanied by a simultaneous increase in the number of cells per crypt column up to 130% of values in jejunum and ileum in situ. The percentage of labelled crypt cells, after labelling with ³H-thymidine, and the relative size of the proliferative cell compartment in the crypt in the transposed ileum did not differ from values in the ileum in situ at any time interval after surgery. The total proliferative activity per crypt, which was determined by scintillation counting of isolated crypts after ³H-thymidine labelling, increased two-fold from 7 days after surgery. Cell migration studies showed that the increase in the number of villus cells was probably not caused by a change in the life span of the epithelial cells. It seems that the increase in the number of villus cells in ileal epithelium after transposition to proximal jejunum is brought about by an enlargement of the crypt, while the relative size of the proliferative cell compartment in the crypt remains unchanged.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

Changes in [3H]galactose uptake in human colonic mucosa with carcinoma: An ultrastructural study

Summary As part of our investigation on glycoprotein synthesis in pre-malignant colonic epithelium, changes in the uptake of [³H]galactose were studied at the ultrastructural level. Normal control mucosa from rectal biopsies of patients with no known gastro-intestinal disease and mucosa adjacent to carcinoma (transitional mucosa) from specimens resected for colo-rectal cancer were compared. These tissues were incubated in TC 199 medium containing [³H]galactose for various intervals of time and for pulse labelling. Silver grain distribution was statistically analysed. The results showed a reduction in the incorporation of the galactose by transitional mucosa. The uptake by this mucosa was less uniform than normal and showed considerable peaking in the endoplasmic reticulum of the goblet cells and in the Golgi of the absorptive cells, suggesting a blockage or alteration in glycoprotein synthesis. The differences were most marked in the middle crypt (the region of differentiation) and in the upper crypt (the region of maturation).     

Storage of glycogen in rat colonic epithelium during induction of secretion and absorption in vitro

Summary Changes induced in the ultrastructure of the epithelium of the rat colon descendens by long-term electric field stimulation (EFS) in an Ussing chamber were investigated. The anion secretion, which was induced by EFS and was measured by the short-circuit current, fell continuously during a 5 h stimulation. At the end of the stimulation period, small particles were observed in the epithelium; these did not appear in unstimulated control tissue. They were localized predominantly in the apical part of the cell. By staining with periodic acidthiosemicarbazide-silver proteinate and because of their sensitivity to -amylase, they were identified as glycogen deposits. This storage of glycogen was time-dependent and was first visible after an EFS of 2 h. It did not appear if glucose was substituted in the bathing solution by sodium butyrate. Glycogen particles were also observed after addition of forskolin, which in contrast to EFS causes a high secretory activity that is stable over several hours. The surface cells contained significantly more glycogen than the crypt cells when secretion was stimulated by EFS or forskolin. The formation of glycogen during EFS was not prevented by tetrodotoxin (TTX). In contrast, TTX itself, which causes maximal absorptive activity by blocking secretomotor neurons, induced the appearance of glycogen in the enterocytes without EFS. However, in the presence of TTX, the amount of glycogen was the same in surface and crypt cells. The results demonstrate that the capacity to synthesize and store glycogen, which has up to now only been observed in embryonic or tumor epithelial cells, is still present in adult colonic mucosa. Procedures carried out to change the functional state of the epithelium seem to induce, at least in vitro, a disinhibition of this capacity.     

Carbonic anhydrase is present in human oesophageal epithelium and submucosal glands

Summary Carbonic anhydrase (EC 4.2.1.1) activity was investigated in normal human oesophageal mucosa using the Hansson and Ridderstråle catalytic cobalt methods. The enzyme was detected in the cell membranes and nuclei and, to a lesser extent, in the cytoplasm of the epithelial cells of the mucosa giving a chicken wire appearance. Activity decreased towards the lumen. Other stratified squamous epithelia - buccal mucosa, ectocervix and skin - gave a similar pattern. Acinar cells of oesophageal submucosal glands also exhibited activity for the enzyme, but the ducts did not. The formation of reaction product was prevented by acetazolamide and ethoxzolamide and by the omission of bicarbonate from the substrate medium. Carbonic anhydrase in oesophageal squamous epithelium may be involved in the control of intra- and extracellular pH, while that in the glands is more likely to be concerned with bicarbonate secretion.