Inhibition of the hydrosmotic response to antidiuretic hormone by 3,3'-diallyldiethylstilbestrol (DADES).

3,3'-diallyldiethylstilbestrol (DADES), a blocker of the facilitated diffusion of glucose, was found to interfere markedly with the hydrosmotic response to antidiuretic hormone and its related agonists. Frog urinary bladders were isolated and monitored for transmural net water flow. DADES was added either to the serosal or to the apical medium at concentrations ranging from 10(-4) M to 10(-6) M. Pretreatment for 30 min with apical 10(-4) M DADES drastically reduced the subsequent hydrosmotic response: (a) to oxytocin (4.4 x 10(-8) M) by 91.7 +/- 17.6% versus 6.2 +/- 7.8 in control; (b) to 8-bromo 3',5'-cyclic AMP by 93.5 +/- 19.4% versus 19.4 +/- 11.4%; (c) to serosal hyperosmolarity (mannitol 220 mOsm) by 99.3 +/- 0.5% versus 12.3 +/- 18.2%. This effect was dose-dependent. Inhibitory action of DADES was more effective on the apical side than on the serosal side (97.0 +/- 1.5 versus 45.8 +/- 10.8). Freeze-fracture studies revealed a modified distribution of the particles and unusual endocytotic pits and vesicles in the apical membrane of both granular and mitochondria-rich epithelial cells. These observations point to multiple and complex effects of the drug. Thus, it seems that DADES has numerous effects on urinary epithelium, which makes it a nonspecific inhibitor of water permeation. Conclusions on its use should therefore be drawn with suitable caution.

Effect of calcium and magnesium ions on the intestinal absorption of oleic acid in vitro.

The effect of Ca++ and Mg++ upon intestinal absorption of oleic acid was investigated using two in vitro models: rat isolated jejunal loops at 30 degrees C and 37 degrees C and mouse jejunal explants at 37 degrees C. At 30 degrees C or at 37 degrees C, Ca++ significantly increased 14C oleic acid uptake by rat isolated jejunal loops or mouse jejunal explants; at 37 degrees C, Ca++ significantly enhanced lipid exocytosis in rat intestinal loops but not in mouse jejunal explants; in both models, in the presence of Ca++ and at 37 degrees C, Mg++ significantly improved the esterification of oleic acid phospholipids and triacylglycerols, as shown by the increase in triacyglycerol synthesis in rat isolated intestinal loops or by the increase in triacylglycerols recovered from the incubation media of mouse jejunal explants; experiments carried out with rat isolated jejunal loops highlighted the determinant role of temperature in oleic acid absorption processes. The present work shows that the simultaneous presence of Ca++ and Mg++ did not impede oleic acid absorption processes but, on the contrary, enhanced them.

Microtubules and actin microfilaments in the amphibian bladder granular cells.

Microtubules and microfilaments were localized by an immunocytochemical method in the granular cells of the frog bladder after fixation and isolation. An extensive array of microtubules was observed in the granular cells with an orientation towards the luminal plasma membrane in the supranuclear zone. Actin filaments formed a continuous bundle that underlined the cellular membrane. After incubation in the presence of colchicine, nocodazole, or tubulozole, the microtubular network appeared fragmented but did not disappear completely. These observations are related to the role of the cytoskeleton in the permeability response of the frog bladder epithelium to vasopressin.

Isolation of large sheets of apical material from frog urinary bladder epithelial cells by freeze-fracture.

In the amphibian urinary bladder, water permeability is correlated with the insertion of intramembrane particle aggregates (IMPAs) into the apical plasma membrane (AM) of the granular cells. These aggregates are believed to contain water channels. Characterization of the IMPAs by comparing AM fractions of antidiuretic hormone (ADH)-treated and resting epithelia requires isolation and purification of AM-rich material, free of other cytoplasmic aggregate-containing organelles, in both cases. A technique derived from freeze-fracture was chosen to isolate large sheets of apical membrane material from frog (Rana esculenta) urinary bladder epithelium. The apical side was plated on a polylysine-coated glass slide, frozen with liquid nitrogen, and fractured. A nylon mesh was inserted between the glass slide and the bladder, in order to bring the fracture plane back to the AM periodically. Fluorescent markers were used to characterize the material having fractured with the glass slide. Samples were observed by fluorescence and phase contrast microscopy. We obtained evidence that numerous patches of fractured AM remained on the glass surface without nuclei. A phase contrast picture was obtained only at a high magnification, indicating a low thickness of the recovered material. Further characterization was made with SDS-PAGE. Protein contents of samples were extracted under various experimental conditions and the patterns of ADH-treated, resting AM samples, or whole epithelial cell crude homogenates, were compared. Staining of some bands increased under certain conditions, whereas many others disappeared. Both morphological and biochemical approaches demonstrate that the recovered material was apical in origin.

To what extent is microtubular network involved in antidiuretic response?

Antimitotic drugs markedly interfere with antidiuretic response, strongly implying that cytoskeleton integrity is essential to this function. This role of the cytoskeleton in controlling the epithelial transport has been seen as a necessary step in the translocation of the water channel containing particle aggregates and in their delivery to the apical membrane. We have now reexamined the exact role of the microtubular network by appropriate time course determinations, by the use of microtubule disruptive agents that lack of the side effects of colchicine, and by trying to visualize the apparent modifications of the microtubular network that accompany water permeability alterations using immunocytochemical techniques. Our results fully confirm that after microtubular network disruption, antidiuretic hormone-induced water permeability variations undergo typical alterations consisting in both a reduction in peak net water flow and a slowing down of its onset. At the same time, the microtubular network disappears in all the epithelial cells. We also show that colchicine-induced inhibition can still be observed in the presence of a prostaglandin synthetase inhibitor and that this inhibition is most likely to occur at a post-adenosine 3',5'-cyclic monophosphate level. These data, as well as results from other series with nocodazole, indicate that the reduction of the net water flow directly results from microtubular network disruption and not from side effects of the disrupting drugs. They also show that the hydrosmotic response is only partially dependent on the microtubular network, which probably has only a guidance role in the translocation of particle aggregates and their exocytotic fusion to the apical membrane.

ADH-induced water permeability: what role for the microtubular network?

1. ADH-induced intramembrane particle aggregates in the apical membrane of the epithelial cells are specifically related to water permeability in the epithelium. 2. Colchicine and nocadozole (both of which bind to tubulin) inhibit ADH-induced osmotic water flow in the amphibian bladder. 3. Microtubules may be involved in the translocation of the aggrephores prior to their insertion into the plasma membrane.

Effect of monensin on cell ultrastructure and glycoprotein migration in adult mouse jejunal epithelium in organ culture.

Explants from adult mouse jejunum were cultured for 3 h in a medium which contained both 3H-fucose (10 or 25 microCi/ml) and monensin (100 microM) or 3H-fucose only (control). Radiochemical analysis of cell fractions showed that 3H-fucose labelling of the brush border fraction decreased 42% in monensin-treated explants, suggesting that in absorptive cells the intracellular transport of newly synthesized glycoproteins to the apical plasma membrane had been inhibited. Electron-microscopic examination of treated explants revealed a variation in response to the drug from region to region. In some areas, both absorptive and goblet cells exhibited little alteration. In others, the Golgi cisternae of both absorptive and goblet cells were entirely replaced by large vacuoles, and in the latter cell type, the cisternae of the rough endoplasmic reticulum were greatly distended. Electron-microscopic radioautographic analysis showed that in absorptive and goblet cells exhibiting little morphological change, intracellular transport of newly synthesized glycoproteins was similar to that in controls. In regions where absorptive cells exhibited extensive Golgi modifications, intracellular transport remained normal in some cases; more often-however, there was a marked inhibition (over 70%) of transport of labelled glycoproteins to the apical surface. Transport to the basolateral membrane was never affected. In goblet cells exhibiting modifications of the Golgi apparatus and rough endoplasmic reticulum, no incorporation of 3H-fucose label in the Golgi apparatus occurred, suggesting a block of intracellular transport proximal to the site at which 3H-fucose is added. In absorptive cells, this does not appear to be the case, since the level of 3H-fucose incorporation in all treated cells remained similar to that in controls.

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

Explants from mouse jejunum were cultured for 3-7 h in the absence (control) or presence of colchicine (100 micrograms/ml) or nocodazole (10 micrograms/ml). In recovery experiments, explants 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.

Ontogeny of EGF receptors during postnatal development of mouse small intestine.

This study was aimed at characterizing epidermal growth factor (EGF) receptors during postnatal development of the maturing mouse small intestine at a time when this tissue is in the constant presence of high levels of intraluminal EGF. Binding studies with [125I]EGF on isolated epithelial cells demonstrate the continued presence of EGF receptors along the entire length of the small intestine with minimal binding at birth (3.67 +/- 0.30%/mg protein) and increasing steadily throughout postnatal life to reach adult values (10.39 +/- 0.90%/mg protein) by the 26th day, with maximum binding on the 21st day (13.4 +/- 2.2%/mg protein). This EGF binding pattern is similar for all three segments studied but was more pronounced for duodenum and jejunum, especially from day 14 on when binding capacity is more elevated in the proximal segments than in the ileum. Competition-inhibition curves of [125I]EGF by EGF reveal the presence of two classes of binding sites for which the number of sites and affinity constants are lower at birth than in adults. The continued presence of EGF receptors in mouse small intestine throughout the postnatal period thus provides support for the possible role of EGF as a modulator in the functional development of the maturing gastrointestinal (GI) tract.

Immunopathology of guinea pig autoimmune enterocolitis induced by alloimmunisation with an intestinal protein.

An enterocolitis has been induced in guinea pigs by alloimmunisation with a mucosal protein. A single dose of immunogen fails to provoke a synthesis of precipitant antibodies, but a high percentage of animals, injected with two or more doses, develop these antibodies. A specific cell mediated immune response was already detectable 30 days after a single dose of immunogen. At later periods and after multiple doses of immunogen positive results were still found, although in a lesser percentage of the animals. The pathology was characterised by the appearance of multiple mucosal ulcerations, congestion, oedema and localised haemorrhage. The target organs were mainly ileum and descending colon. At later periods a combination of mononuclear cell infiltration, fibrocytic proliferation and granuloma formation appeared together with the other lesions. When an intraluminal challenge was made 48 hours before death, a heavy mononuclear cell infiltration was present in the contact area. The lesions appeared to extend to areas which usually remained unaffected. The characteristic immunopathology of this autoimmune enterocolitis in guinea pigs possesses some of the features of human inflammatory bowel diseases and makes it a useful model for further studies.

Effect of monensin and nocodazole on the intestinal lipid esterification in mouse jejunal organ culture.

The ability of mouse jejunal explants to esterify a lipid emulsion containing oleic acid, palmitic acid and monopalmitin has been studied in different in vitro experimental conditions. The incubating lipid solution must have a minimum volume for obtaining optimal triglyceride esterification by the cultured intestinal mucosa. In our incubating conditions the exchange of oleic for palmitic acid does not significantly modify the amount of lipids esterified by the explants in 15 min. Monensin or nocodazole, added to the culture medium of intestinal explants for 3 hr, significantly change the amount of lipids esterified and secreted. The inhibition observed after nocodazole treatment disappears, however, when the explants are rinsed and the culture is allowed to continue for an additional 3 hr in a drug-free medium. These results suggest that the regulation of lipid metabolism can be studied in organ culture.

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.

Epidermal growth factor receptors in isolated adult mouse intestinal cells: studies in vivo and in organ culture.

In isolated intestinal cells from adult fed mouse, the binding of [125I]epidermal growth factor (EGF) was time and temperature dependent. Maximum binding was obtained after 30 min of incubation at 20 C. For a concentration of 5 X 10(-11)M [125I]EGF (180 microCi/micrograms), specific binding for isolated cells from duodenum, jejunum, and ileum was closely similar, with means of 9.4 +/- 0.9, 13.3 +/- 0.8, and 9.3 +/- 2.0%/mg protein, respectively. The binding increases along the crypt-villus axis. Inhibition dose-response analysis indicated high affinity binding with 50% inhibition at 3 X 10(-10) M unlabeled EGF. The specific binding decreased by 19% after 48 h of fasting. In the P1 fraction (microsome and lateral membranes) from scrapings or isolated cells of the jejunal mucosa, specific binding was 2.4 +/- 0.8 and 6.5 +/- 0.7%/mg protein, respectively. In the P2 fraction (brush border), specific binding was 6.9 +/- 1.6% and 16.3 +/- 0.7%/mg protein. After 24 h of organ culture, specific binding is not modified in duodenal explants. Moreover, in the presence of EGF (500 ng/ml) in the culture medium, the binding is decreased by 72%. These results show that isolated intestinal cells from adult mice possess a high concentration of EGF receptors that exhibit kinetic properties identical to those of other EGF target cells. Unlike insulin receptors, EGF receptors are numerous on the intestinal brush border and in intestinal crypts and decrease by fasting.

Influence of explantation procedure on the electrical and morphological properties of cultured neonatal rat ventricle cells.

The ultrastructure and electrophysiological properties of ventricle cells from newborn rats were studied before and after explantation. The cultured cells were dissociated either with trypsin or with collagenase, the latter enzyme being used with and without stirring with a magnetic bar. The explanted cells were studied 10 hr and 48 hr or more after explantation. At 10 hr after explantation, the cells exhibited fast-rising action potentials, but their myofibrils were disorganized, except for stirred collagenase-dispersed cells, which were also depolarized and inexcitable. At 2 days and later after explantation, all preparations had well-defined sarcomeres and myofibrils oriented in parallel similar to the ventricle before explantation, but the cells showed slow-response action potentials together with spontaneous activity. These findings suggest that the disorganization of myofibrils does not reflect damage to the surface membrane. Moreover, collagenase seems more damaging to the cells than trypsin under similar conditions (comparable periods of mechanical stirring), especially 10 hr after explantation.

Insulin receptors in isolated adult mouse intestinal cells: studies in vivo and in organ culture.

Isolated intestinal cells from adult mice possess a high concentration of insulin receptors. The binding capacity and the number of binding sites are higher in duodenum than in jejunum or ileum and in the upper part of the villus than in the crypts. The specific binding is, respectively, 11.8 +/- 1.0%, 9.1 +/- 4.0%, and 5.5 +/- 0.3%/mg . protein for duodenum, jejunum, and ileum. The number of high affinity sites per cell is, respectively, 11.0 X 10(3), 3 X 10(3), and 2.5 X 10(3). The number of low affinity sites per cell is, respectively, 11.0 X 10(4), 4.1 X 10(3), and 3.9 X 10(3). This specific binding increases to 15.9 +/- 0.9% after 24 h of fasting and to 24.5 +/- 2.2% mg protein after 48 h of fasting. This increase is due not only to an increment in the number of sites but also to alterations in affinity constants (K1, control, 0.380, 48-h fasting, 0.044 X 10(9) M-1; K2, control, 1.20, 48-h fasting; 2.61 X 10(7) M-1). The receptors are mainly located on the basolateral and internal membranes (P1, 9.4 +/- 0.7%/mg protein), but are also present on brush border membranes (P2, 2.6 +/- 1.1%/mg protein, P less than 0.01). After 24 h of organ culture, the specific binding is not modified in duodenal explants. Moreover, in the presence of insulin in the culture medium, the binding is decreased by 59%.

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis.

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.

Effects of epidermal growth factor (EGF) on adult mouse small intestine in vivo and in organ culture.

1. Exogenous administration of epidermal growth factor (EGF) has not modified the protein and DNA content, nor several brush border enzymes activities of duodenum, jejunum and ileum of intact and fasted adult mice. 2. Exogenous administration of EGF has not stimulated the DNA synthesis in the three regions of the small intestine of intact adult mice. 3. EGF has a stimulatory effect on DNA synthesis of fasted mice intestine 12 hr after injection. 4. In organ culture, EGF has not altered at any concentration (10, 50, 100, 200, 800 ng/ml), the same parameters in duodenal and jejunal explants taken from animals fasted 24 hr before being killed. 5. These last results suggest that the increase of DNA synthesis observed in vivo was not a direct effect of EGF administration. 6. Finally, the EGF content of serum af adult male mice was measured in fed and fasted mice and in the organ culture media.

Effect of pentagastrin on mouse gastric and small intestinal mucosa in vivo and in vitro.

1. Single administration of various doses (62.5, 125, 250, 500 micrograms/kg bodyweight) or multiple administration of a single dose (250 micrograms/kg body wt) of pentagastrin to fasted mice do not alter the protein and DNA content, nor the DNA synthesis in fundus of stomach, distal duodenum, and proximal jejunum. 2. Pentagastrin has also been added at different doses into an organ culture of adult mouse duodenum or jejunum. 3. The explants were taken from animals fasted for 24 or 48 hr before sacrifice. 4. Protein and DNA content, protein and DNA synthesis, activities of several brush border enzymes and morphology have not been modified. 5. Therefore, the drug does not seem to have a direct effect on the mouse intestinal mucosa. 6. These observations are compared to results obtained from other species.

Development of peroxisomes in amphibians. II. Cytochemical and biochemical studies on the liver, kidney, and pancreas.

The ontogeny of catalase-containing organelles was studied by cytochemical and biochemical methods in the liver, kidney, and pancreas during the development of Rana catesbeiana. The biochemical differentiation of peroxisome in the liver and kidney was compared to that of Xenopus laevis. Catalase activity was localized after incubation in DAB medium and studied biochemically by a spectrophotometric method. In Rana Catesbeiana the number of catalase-positive organelles per cell section is low in all three organs during premetamorphosis; their number increases substantially in the liver and kidney of froglets, while it remains almost stable in the pancreas. No further increase is observed in the adult. Biochemically, the liver, kidney, and pancreas of tadpoles exhibit, respectively, 12,22 and 63% of the catalase activity found in the adult tissues. After metamorphosis an important increase of catalase activity is particularly noted in liver and kidney, the activity being, respectively, 43 and 77% of that of adult bullfrogs. On the other hand, no change in catalase activity in the liver and kidney is noted during the entire development of Xenopus laevis. The present study illustrates the very different developmental pattern of catalase activity observed during the development of two anuran amphibians. The different development pattern of the same enzyme within the small intestine, liver, kidney, and pancreas in Rana catesbeiana is also stressed.

Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns.

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 +/- 0.9) as compared with scrapings of intestinal mucosa (14.8 +/- 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase-glucoamylase-isomaltase (band 4), alkaline phosphatase (bands 9-10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.