Keratinization of the esophageal epithelium of domesticated mammals.

We studied the esophageal epithelium for keratinization characteristics from samples of domesticated mammals of three nutrition groups (herbivores: horse, cattle, sheep; omnivores: pig, dog, rat; carnivores: cat) using histochemistry (keratins, disulfides), sulfur measurements, and cryo-SEM. Keratins were found in all esophageal layers of all species, except for the equine Stratum corneum. The positive reaction staining of Pan-keratin was remarkable, but decreased in intensity toward the outer layers, whereas in the pig and cat, staining was confined to the corneal layer. The herbivores revealed positive staining reactions in the upper Stratum spinosum, particularly in the sheep. Regarding single keratins, CK6 immunostating was found in most esophageal layers, but only weakly or negatively in the porcine and equine Stratum corneum. CK13 staining was restricted to the sheep and here was found in all layers. CK14 could be detected in the equine and feline Stratum basale, and upper vital layers of the dog and rat. CK17 appeared only in the Stratum spinosum and Stratum granulosum, but in all layers of the dog and cat. Disulfides reacted strongest in the Stratum corneum of the herbivores, as corroborated by the sulfur concentrations in the esophagus. Our study emphasized that keratins are very important for the mechanical stability of the epithelial cells and cell layers of the mammalian esophagus. The role of these keratins in the esophageal epithelia is of specific interest owing to the varying feed qualities and mechanical loads of different nutrition groups, which have to be countered.

Demonstration of free fatty acids in the integument of semi-aquatic and aquatic mammals.

The sensitive red fluorescence dye BODIPY® 665/676, and embedding in the water-soluble resin Technovit® 7100 were used to demonstrate free fatty acids in the epidermis of seven semi-aquatic and aquatic mammalian species with a sparse or dense hair coat. The staining generally marked lipid layers of varying thickness between the lamellae of the Stratum corneum, as found particularly in very densely haired species (otter), but also in rather sparsely haired animals (beaver, nutria), and especially in the seal. The very sparsely haired capybara contained no free fatty acids in the corneal layer system, but exhibited an accumulation of such substances in the vital epidermis. All haired species showed a strongly positive reaction staining of the sebaceous glands. In the hairless species, a distinct intracellular staining was restricted to cells of the thick vital epidermis in the hippopotamus, whereby in the Str. corneum positive intercellular reactions appeared. In the dolphin, on the contrary, positive intercellular reactions became visible in the vital epidermis, whereas in the Str. corneum the lipids concentrated in large longitudinal intracellular vesicles.

pH gradients and a micro-pore filter at the luminal surface affect fluxes of propionic acid across guinea pig large intestine.

A neutral pH microclimate had been shown at the luminal surface of the large intestine. The aim was to estimate to what extent fluxes of propionic acid/propionate are affected by changes of the luminal pH when this microclimate is present, largely reduced or absent. Fluxes of propionic acid/propionate (J(Pr)) across epithelia from the caecum, the proximal and the distal colon of guinea pigs were measured in Ussing chambers with and without a filter at the luminal surface. With bicarbonate and with a neutral or an acid pH of mucosal solutions (pH 7.4 or 6.4), mucosal-to-serosal fluxes (J(ms)(Pr) ) were 1.5 to 1.9-fold higher at the lower pH, in bicarbonate-free solutions and carbonic anhydrase (CA) inhibition 2.1 to 2.6-fold. With a filter at the mucosal surface and with bicarbonate containing solutions, J (ms) (Pr) was not or only little elevated at the lower pH. Without bicarbonate J(ms)(Pr) was clearly higher. We conclude that the higher J(ms)(Pr) after luminal acidification is due to vigorous mixing in Ussing chambers resulting in a markedly reduced unstirred layer. Therefore, an effective pH microclimate at the epithelial surface is missing. J(ms)(Pr) is not or is little affected by lowering of pH because in the presence of bicarbonate the filter maintains the pH microclimate. However, in bicarbonate-free solutions J(ms)(Pr) was higher at pH 6.4 because a pH microclimate does not develop. Findings confirm that 30-60% of J(ms)(Pr) results from non-ionic diffusion.

A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells.

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.

Excessive apoptosis of guinea pig colonocytes may lead to an imbalance between phagocytosis and degradation in vivo.

The success or failure of the clearance of apoptotic cell remains depends on the ability of phagocytic cells to recognize, phagocytoze, and digest these remains prior to their lysis, which would cause tissue inflammation. We have recently shown that, after mass-induced apoptosis of guinea pig colonocytes in vivo, phagocytosis by resident macrophages, although efficient, does not prevent a pre-inflammatory response of the mucosa. The present study has investigated the cause(s) of this clearance failure. Immunohistochemistry and transmission electron microscopy were applied. Antibodies directed against the epithelial plasma membrane protein E-cadherin, the lysosomal membrane protein LAMP-1, and the lysosomal matrix protease cathepsin-D were used. The results revealed that: (1) anti-E-cadherin labeled the membrane of epithelial apoptotic bodies internalized in macrophages, (2) double and triple labeling demonstrated that the anti-LAMP-1 and anti-cathepsin-D antibodies recognized and were co-localized in lysosomes and/or phagolysosomes in macrophages but left E-cadherin-positive structures unlabeled, (3) the more numerous were the E-cadherin-positive inclusions in macrophages, the smaller was the number of those that stained positive for lysosomal markers. In parallel with electron microscopy, these findings showed that not all apoptotic bodies phagocytozed by macrophages were subsequently digested, suggesting that the phagocytotic ability of these cells was not matched by their digestive capability.

Intracellular pH regulation in guinea-pig caecal and colonic enterocytes during and after loading with short-chain fatty acids and ammonia.

Absorption of short-chain fatty acids (SCFA) and ammonia implies considerable fluxes of protons across the epithelium of the large intestine. Efficient regulation of intracellular pH (pH(i)) is therefore essential in these cells. The aim of the present study was to examine the effects of SCFA and of ammonia on pH(i), on pH(i) regulation and to characterize the mechanisms involved in pH(i) regulation in surface enterocytes of the guinea-pig caecal and colonic mucosa. Intact epithelia from the caecum and the distal colon were mounted in a microperfusion chamber. pH(i) was measured by fluorescence microscopy using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Addition of SCFA or ammonia to the serosal side changed the enterocyte pH(i) markedly, whereby ammonia caused larger changes in pH(i) than SCFA. In contrast, addition of SCFA to the mucosal solution had no effect on pH(i) and ammonia increased pH(i) only slightly. Basolaterally located pH(i) regulation mechanisms, Na(+)-H(+) exchange and Cl(-)-HCO(3)(-) exchange, are involved mainly in returning pH(i) to normal values. It is concluded that, due to apparently lower permeability of the apical membranes, the caecal and colonic epithelium is protected against pH(i) disturbances caused by the naturally high luminal SCFA and NH(3) concentrations. The major regulation mechanisms of pH(i) are located in the basolateral membrane of the enterocytes.

Permeability properties of apical and basolateral membranes of the guinea pig caecal and colonic epithelia for short-chain fatty acids.

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes.

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.