J. S. Hugon

Membrane associated particles: Distribution in frog urinary bladder epithelium at rest and after oxytocin treatment

SummaryThe fine structure of the plasmic membrane of the frog urinary bladder epithelium has been studied with a freeze-etching technique. The results reveal that the external face of the cytoplasmic leaflet (A face) of the apical membrane of epithelial cells is characterized by: 1) A markedly reduced number of membrane associated particles compared to the value observed on the A face of the lateral membranes of these cells. 2) The existence after oxytocin treatment of numerous spots of clustered particles.These characteristics are present in unfixed glycerinated cells but are more clearly observed after prefixation of glycerol treated cells with glutaraldehyde. The oxytocin induced clustering is completely reversible.It is suggested that these ultrastructural features are related to the permeability properties of the apical barrier and to their control by antidiuretic hormone.

Loss of microtubules and alteration of glycoprotein migration in organ cultures of mouse intestine exposed to nocodazole or colchicine.

SummaryExplants from mouse jejunum were cultured for 3–7 h in the absence (control) or presence of colchicine (100 gm/ml) or nocodazole (10 μg/ml). In recovery experiments, expiants were cultured in fresh medium for an additional period. To label glycoproteins, 3H-fucose was added during the last 3 or 6 h of the initial culture or recovery period. Subcellular fractionation studies revealed that colchicine and nocodazole inhibited migration of labelled glycoproteins to the brush border (P2) by 40–45%. Radioautographic studies of absorptive cells showed that colchicine and nocodazole inhibited labelling of the microvillous border by 67% and 87%, while labelling of the basolateral plasma membrane increased by 114% and 275%. Immunocytochemical studies revealed that both colchicine and nocodazole caused the virtual disappearance of the microtubular network in the absorptive cells. It is possible that some glycoproteins normally destined for the microvillous border are rerouted to the basolateral membrane. The observed loss of microtubules after drug treatment suggests that microtubules may play a role in the intracellular migration of membrane glycoproteins. Additional support for this concept is provided by the fact that in recovery experiments the distribution of label returned to control values after the microtubular network became re-established.

Intestinal brush border enzyme activities in developing amphibian rana catesbeiana

Abstract 1. 1. Several intestinal enzyme activities have been measured on homogenates in pre- and post-metamorphosed frogs and in adults. 2. 2. Disaccharidase, glucoamylase, alkaline phosphatase and γ-GTP activities are present along the entire small intestine in larvae and adults. 3. 3. A weak and sometimes significant gradient for all brush border enzyme activities can be shown along the digestive tract of the pre-metamorphosis animals. 4. 4. In the newly metamorphosed animals, all the activities increase specially in the first part of the intestines. 5. 5. These observations are compared to the modifications of the same enzymes in the post-natal period of the mammals.

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

SummarySuspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.

Organ culture of adult mouse intestine: II. Mitotic activity, DNA synthesis and cellular migration after 24 and 48 hours of culture

SummaryDNA synthesis, mitotic activity and cell migration have been measured in organ culture of adult mouse jejunum during the first 48 hr. Explants cultured in DMEM-HEPES-NCTC-135 medium present a sharp decrease of mitotic activity in the first hours of culture, but mitoses are restored to 80% of the controls between 24 and 48 hr. DNA synthesis follows the same pattern. Cell migration continues during culture.

Organ culture of adult mouse intestine

SummaryExplants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase is measured during the first 24h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.

Ultrastructural localization of alkaline phosphatase activity in the intestine of developingRana Catesbeiana

SummaryAlkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog,Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.

Effect of glutaraldehyde and lead on the activity of hepatic glucose-6-phosphatase

SummaryThe sensitivity of mouse liver glucose-6-phosphatase activity towards glutaraldehyde fixation has been analysed by biochemical and cytochemical means. The degree of enzymatic inhibition and various enzymatic properties have been studied. Several differences have been observed in the Km determination, the sensitivity to pH 5 and the activity related to pH between fixed and unfixed enzymes. The role of Pb++ ions in the cytochemical media has also been estimated.It is concluded that several enzymatic differences appear between fixed and unfixed enzymes and that the inhibition by Pb ions is dependent on the buffer and on the amount of substrate used.

Immediate and localized response of intestinal mucosal enzyme activities in streptozotocin-diabetic mice

Abstract 1. 1. Short-term effects of induced-diabetes on intestinal digestive function were investigated in mice. 2. 2. Enhanced brush border enzyme activities show different time patterns in their immediate response to Streptozotocin diabetes. 3. 3. These increases are not only localized at the brush border membrane level, but also in microsomal and eytosol fractions. 4. 4. Accumulation of brush border-destined enzymes in the cytoplasm may be due to defective insulinsensitive mechanisms involved in normal cell processes.

Lipid esterification and secretion by the mouse intestine in organ culture

Explants of adult mouse jejunum were cultured for different time periods and incubated in presence of a lipid diet emulsified by sodium taurocholate or complexed with albumin. The esterification of fatty acids and the secretion of triglycerides and phospholipids were measured and compared to the lipid absorption observed in vivo after the perfusion of the same diet. The results show that, in vitro, the enterocytes esterify the fatty acids present in the medium and secrete them with a yield improving during the culture and even better than the absorption observed in the in vivo experiments.