Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60 degrees C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.     

Allosteric properties of phosphofructokinase from the epithelial cells of thermally injured rat small intestine

1. The allosteric properties of phosphofructokinase from the epithelial cells of thermally injured rat small intestine were studied and compared with those properties of the normal rats. 2. The fructose 6-phosphate saturation curve of mucosal phosphofructokinase from thermally injured rats (3 days post injury, 33% of body surface area) displayed cooperatively; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8.0, v 0.5/V, was 0.42 +/- 0.02 in the normal rats and 0.22 +/- 0.03 in the injured rats. 3. The enzyme from thermally injured rats was very sensitive to inhibition by ATP as compared to that from normal rats. 4. The enzyme from thermally injured rats was inhibited by citrate and phosphocreatine in a synergistic manner with ATP. 5. Activation under nearly cellular conditions was produced by ADP, AMP and glucose-1,6-biphosphate. 6. In general, the mucosal enzyme of thermally injured rats was more susceptible to inhibition or activation by various metabolites than the enzyme of the normal rats. 7. These results may suggest that mucosal phosphofructokinase of thermally injured rats may not be subject to the same control mechanism as the normal rats in vivo due to changes in the concentrations of fructose-2,6-biphosphate.     

α-Adrenergic inhibition of cyclic AMP accumulation in epithelial cells isolated from rat small intestine

The present work shows that α-adrenergic agonists induce the suppression of basal and hormone-stimulated cyclic AMP levels in rat intestinal epithelial cells. Epinephrine (100 μM) suppresses by 35% the cyclic AMP levels evoked by the vasoactive intestinal peptide (VIP). The adrenergic agent induces a similar percentage of inhibition at 15, 30 and 37°C. Addition of epinephrine 20 min prior to, on 5 or 20 min after VIP yields the same magnitude of inhibition as when performed together with the stimulus. The α-adrenergic agent does not alter the K_0.5 of VIP in stimulating cyclic AMP production but reduces its efficacy. Epinephrine also suppresses prostaglandin E₁- and E₂-stimulated cyclic AMP levels by about 35%. The lowest effective concentration of epinephrine required to suppress VIP-stimulated cyclic AMP levels is 0.1 μM, half-maximal (K_0.5) and maximal effects being observed at 5 and 100 μM, respectively. Norepinephrine has the same efficacy but a slightly lower potency (K_0.5 = 18 μM) than epinephrine. Phenylephrine acts as a partial agonist of very low potency; clonidine has very little intrinsic activity and antagonizes the inhibition by epinephrine. The inhibition of VIP-stimulated cyclic AMP levels is observed in the absence of any blocking agents. It is not affected by the β blocker propranolol, but is completely reversed with α blockers with the following order of potency: dihydroergotamine>yohimbine>phentolamine. Yohimbine is much more potent than prazosin, which only partially reverses the inhibition induced by epinephrine. It is concluded that α-adrenoreceptors of the α₂ subtype mediate the suppression of VIP-stimulated cyclic AMP levels in intestinal epithelial cells. This effect is likely to be due to the inhibition of adenylate cyclase within intact cells as epinephrine is able to reduce adenylate cyclase activity of intestinal epithelial cell plasma membranes.     

L-MBP is expressed in epithelial cells of mouse small intestine

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.     

Methodical aspects of obtaining isolated small intestine epithelial cells

Based on the data from literature and of the author's results, the methods for isolation of small intestine epithelial cells have been analyzed by the following criterion: object of investigation, procedures for cell isolation--mechanical, chemical, enzymatic, and biochemical properties.     

Quantitative changes in alkaline phosphatase in epithelial cells of rat small intestine from birth to weaning

Synopsis Alkaline phosphatase activity has been measured in mucosal scrapings of rat small intestine from birth to weaning. The levels of activity were then compared with those found in brush border fractions and cell residues over the same period. The amount of activity found in the residues was too high and variable to be accounted for in terms of diffusion, and it was concluded that during the developmental period considerable quantities of enzyme were present in the cytoplasm.The duodenal changes were similar, in form, in both mucosal scrapings and brush border, but the changes in the ileal brush borders were markedly different from those found in the ileal mucosal scrapings. A peak in the activity measured in the brush border fraction was observed during the weaning period. A partial explanation for the observed changes is suggested but a detailed analysis was not possible at the present stage in the work.     

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

Close relationships between the cells of the immune system and the epithelial cells in the rat small intestine

Summary A possible contribution of the intestinal epithelium to the immune defence system was studied by electron microscopy in the rat small intestine. The cells of the immune system (CIS) such as lymphocytes, eosinophils and macrophages penetrate the basal lamina into the epithelium and make close relationships with the absorptive cells. At the points of close apposition, the two cell membranes run parallel at a regular distance of about 20 nm. On the other hand, about 5% of the intestinal absorptive cells also penetrate the basal lamina into the lamina propria via their basal protrusions and show a similar close association with CIS. The basal protrusions contain many microfilaments; this indicates that they are structures with a definite function rather than a simple hernia. These findings are discussed with respect to the transport of antigenic molecules and of intercellular communication between CIS and the intestinal epithelium.     

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.     

Catalase particles in the epithelial cells of the guinea-pig small intestine

Synopsis Purified preparations of epithelial cells have been made from the guinea-pig small intestine. Homogenates of these preparations have been analysed by centrifugation in a zonal rotor. The results confirm the presence of lysosomes in these cells and indicate the existence of catalase particles which equilibrate in a sucrose gradient at a density of between 1.21 and 1.23 and which have a different distribution from other subcellular particles except lysosomes. Injection of Triton WR-1339 into fasting animals enables the separation of lysosomes and catalase particles.     

Slow-waves in rat small intestine

Rat small intestine generates rhythmic slow-wave activity. The slow-waves are not eliminated by ouabain application or incubation in potassium free solution. Exposure to low sodium or calcium free solution decreases slow-wave activity. Incubation in sodium and calcium free solution eliminates activity. It is concluded that rat small intestinal slow-waves may not result from the same mechanism as in the cat.