Roger Busche

Role of short‐chain fatty acids in the hind gut

Short-chain fatty acids (SCFA) are produced by microbial fermentation in the hindgut in considerable amounts. Most of the anions in hindgut contents are SCFA, mainly acetate, propionate and butyrate. SCFA are rapidly absorbed. Mechanisms involved in the transepithelial transport are discussed. Besides the contribution to the overall energy metabolism of animals or men, SCFA have a number of further important effects on the colonic mucosa. Factors affecting the pH of compartments in the mucosa, cell swelling, stimulation of mucin release and of mucosal blood flow are mentioned. Controversial reports are known on the role of SCFA in the metabolism of colonocytes. In spite of the conflicting opinions on the interaction between SCFA metabolism and the development of colitis ulcerosa, diverticulosis and colorectal cancer seems to exist. The obscure differences between the effects of SCFA on cell proliferation, differentiation and apoptosis of colonocytes in vivo and in vitro indicate that besides direct effects of SCFA systemic effects such as neural and humoral factors are of crucial importance. The opposing effects of SCFA on proliferation and apoptosis in normal colonocytes and in colonic cancer cells may open possibilities for prevention and/or therapy of patients with colonic diseases.

Maintenance and regulation of the pH microclimate at the luminal surface of the distal colon of guinea‐pig

1 The fluorescent dye 5‐N‐hexadecanoyl‐aminofluorescein (HAF) was used to study the mechanisms involved in maintaining a relatively constant luminal surface pH (pHs) in the distal colon of the guinea‐pig. The fatty acyl chain of the HAF molecule inserts into the apical membrane of epithelial cells. This allows a continuous measurement of the surface pH for several hours. 2 The localization of HAF was confirmed by confocal laser‐scanning microscopy and by using monoclonal antibodies against fluorescein. The insertion of HAF into the apical membrane of the colonocytes did not change the transepithelial conductance or the short‐circuit current of the epithelium. 3 With the HAF method a pH microclimate was confirmed at the colonic surface. Although the pH of the bulk luminal solution was decreased in bicarbonate‐containing solution from 7.4 to 6.4 the pHs changed only in the range 7.54‐6.98. 4 In the absence of bicarbonate pHs almost followed changes of bulk luminal pH. In the presence of bicarbonate there was a decrease in pHs after removal of chloride from the luminal side and an increase in pHs after addition of butyrate to the luminal solution. This suggests the involvement of a bicarbonate‐anion exchange in bicarbonate secretion: a Cl−‐HCO3− as well as a short‐chain fatty acid−‐HCO3− exchange. 5 The apical K+‐H+‐ATPase in the distal colon of guinea‐pig has little influence on pHs in the presence of physiological buffer concentrations. 6 Our findings indicate that bicarbonate plays a major role in maintaining the pH microclimate at the colonic surface.

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.

Withdrawal of butyrate from the colonic mucosa triggers "mass apoptosis" primarily in the G0/G1 phase of the cell cycle.

Abstract.Butyrate exerts a trophic effect on the colonocytes and plays a protective role in ulcerative colitis. In the present study, we investigated the effect of butyrate withdrawal on the colonic mucosa of the guinea-pig. The samples were mounted in Ussing chambers and bathed for 45, 60, 90 and 150 min with standard Ringer solution with or without sodium butyrate. Light and electron microscopy for morphology, electrophysiological methods for testing tissue function, histochemistry using the TUNEL method for localization of apoptotic cells and flow cytometry for cell cycle analysis were applied. Morphological observations revealed that butyrate deprivation caused a time-dependent hypoplasia and a rapid triggering of massive apoptosis as substantiated by the TUNEL assay. The epithelium, however, did not show discontinuities at any time. Electrophysiological data confirmed that no leakage of the epithelium had occurred. In the control specimens, the mucosa underwent a moderate reduction in height; apoptotic epithelial cells were infrequently observed. Cell cycle analysis of co-lonocytes isolated from the mucosa deprived of butyrate revealed a decrease in the percentage of cells occupying each phase of the cycle, especially the G0/G1 phase. Thus, in the absence of butyrate, apoptosis was enhanced and cell renewal reduced. The trophic protective action exerted by butyrate in both physiological and pathological conditions could derive from its capacity to modulate survival and death of colonocytes.

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A

The intracellular pH (pHi) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na+/H+ exchange and a stilbene-insensitive Cl− /HCO3− exchange mechanism have been identified in the basolateral membrane. Removal of Na+ from the basolateral solution caused a decrease of pHi by 0.50±0.09 unit (n=4). Amiloride or Na+-free solution at the apical side had no effect on pHi. Cl− removal at the basolateral side led to an increase of pHi by 0.20±0.03 unit (n=4) whereas apical removal had no influence on pHi. This effect was independent of Na+ and was insensitive to 0.2 mM 4,4′-diisothiocyanatodihydrostilbene-2, 2′-disulphonic acid. A basolateral Cl−/ HCO3− exchanger is the most likely explanation for this observation. The Na+/H+ exchange mechanism in the basolateral membrane is an acid extruder, whereas the C1−/HCO3− exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.

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

Abstract. 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.

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

Abstract.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 (pHi) is therefore essential in these cells. The aim of the present study was to examine the effects of SCFA and of ammonia on pHi, on pHi regulation and to characterize the mechanisms involved in pHi 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. pHi 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 pHi markedly, whereby ammonia caused larger changes in pHi than SCFA. In contrast, addition of SCFA to the mucosal solution had no effect on pHi and ammonia increased pHi only slightly. Basolaterally located pHi regulation mechanisms, Na+-H+ exchange and Cl–-HCO3– exchange, are involved mainly in returning pHi to normal values. It is concluded that, due to apparently lower permeability of the apical membranes, the caecal and colonic epithelium is protected against pHi disturbances caused by the naturally high luminal SCFA and NH3 concentrations. The major regulation mechanisms of pHi are located in the basolateral membrane of the enterocytes.

Surface pH at the Basolateral Membrane of the Caecal Mucosa of Guinea Pig

Abstract. Since the major mechanisms responsible for regulation of intracellular pH of enterocytes are located in the basolateral membrane, respective effects may be expected on pH in the compartment near the basolateral membrane. A method was established to estimate the pH at the basolateral membrane (pHb) of isolated caecal epithelia of guinea pig using pH-sensitive fluorescein attached to lectin (lens culinaris). In the presence of bicarbonate and a perfusion solution-pH of 7.4, pHb was 7.70 ± 0.15. In the absence of bicarbonate or chloride as well as by inhibition of the basolateral Cl−-HCO−3 exchange with H2-DIDS, pHb was reduced near to solution-pH. Inhibition of the basolateral Na+-H+ exchanger by adding a sodium- and bicarbonate-free, low-buffered solution increased pHb. Decrease of pH of serosal perfusion solution to 6.4 provoked a similar decrease of pHb to solution pH. Short-chain fatty acids (SCFA) added to the mucosal solution caused a slight decrease of pHb. SCFA added to the serosal side alkalized pHb. However, in the presence of bicarbonate pHb returned quickly to the initial pHb, and after removal of SCFA a transient acidification of pHb was seen. These responses could not be inhibited by MIA or H2-DIDS. We conclude that no constant pH-microclimate exists at the basolateral side. The regulation of the intracellular pH of enterocytes reflects pHb. The slightly alkaline pHb is due to the bicarbonate efflux. Data support the presence of an SCFA−-HCO−3 exchange.

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.

Effect of SCFA on intracellular pH and intracellular pH regulation of guinea-pig caecal and colonic enterocytes and of HT29-19a monolayers.

The response of the intracellular pH (pHi, measured with BCECF) of the caecal and distal colonic epithelium of guinea pig and of monolayers of HT29 clone 19a cells on the addition of short-chain fatty acids (SCFA) was assessed. Addition of SCFA to the luminal side of these cells had no major effect on pHi, independent of whether the apical Na+/H+ exchange or the apical K+/H+ ATPase was inhibited or not. Addition of SCFA to the serosal side, on the other hand, caused a marked decrease of pHi, followed by an effective regulation back to basal values, and after removal of the acid, the cells became alkalinized. Intracellular pH is mainly regulated by mechanisms in the basolateral membrane. The basolateral Na+/H+ exchanger and the Cl-/HCO3- exchanger were mainly responsible for pHi regulation. Inhibition studies are consistent with a NHE-1 type Na+/H+ exchanger in the basolateral membranes. The apical Na+/H+ exchanger of caecal enterocytes and in HT29 cells, and the apical K+/H+ ATPase in the apical membrane of the distal colon have no or little influence on pHi regulation. The comparison shows that the HT29-19a cell line is an adequate model for studying pHi phenomena of hind gut epithelial cells.