Glycoconjugate distribution and mobility on apical membranes of absorptive cells of suckling rat ileum in vivo

The luminal membrane of ileal absorptive cells in suckling rats includes two domains: microvillar membranes and deep invaginations between microvilli. We examined the fates of foreign macromolecules that bind to anionic or saccharide sites on these domains after infusion into ligated loops in vivo. Cationized ferritin (CF) and ferritin-RCAI (beta-galactosyl) binding sites were distributed over the entire apical membrane. Ligands bound to apical invaginations were rapidly endocytosed, but ligands on microvilli were not. After CF binding, anionic sites on microvilli were mobile in the plane of the membrane and formed CF clusters at the tip and base of each microvillus. RCAI binding sites did not cluster. Wheat germ agglutinin (WGA, sialic acid) labeling was restricted to microvillus tips of mature cells but was dispersed over the microvillar surfaces of lower villus cells. Ferritin conjugates of Concanavalia ensiformis (Con A), Ulex europaeus agglutinin (UEA), and Dolichos biflorus agglutinin (DBA) did not bind to cell surfaces in vivo. Aldehyde fixation dramatically altered lectin binding patterns, resulting in unmasking and labeling of Con A, WGA, and DBA binding sites that were unavailable in vivo.     

Maintenance of adult rat ventral prostate in organ culture

Androgens postpone the involution of prostatic tissue cultured in a chemically defined medium. However, after 1 week quantitative changes are accentuated and qualitative signs of involution appear. Other factors in addition to androgens are obviously needed for the adequate maintenance of the tissue. This study was aimed at the improvement of the culture conditions by adding insulin, corticosterone, epidermal growth factor, prolactin, and transferrin alone or in different combinations with testosterone and each other. A morphometric model developed for prostate culture was used, and maintenance and proliferation of the tissue were further followed by weighing the cultured explants and by measuring the incorporation of (3H) thymidine into DNA. The androgen dependency was seen in all the studied morphometric parameters at least for 2 weeks-i.e., in volume density and height of epithelium (VVEP and h), reflecting the epithelial maintenance, in volume density and width of interacinar tissue (VVIT and lambda AP), reflecting stromal tissue, and in volume density and diameter of lumen (VVLU and DLU), reflecting the secretory function. Testosterone (10(-7) M) also prevented partially the quantitative involution of the tissue within 2 weeks. Insulin, acting independently of androgen, was a potent mitogen, which, according to morphometric results, exerted its effect mainly on prostatic epithelium. Stratification of epithelium was frequently seen. Corticosterone had no effect on epithelian proliferation, but it probably stimulated secretion and inhibited the growth of stroma. By combining these three hormones it was possible to maintain not only the structure but also the amounts of tissue components practically unaltered at least for 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical staining of the rat adenohypophysis in organ culture

Rat anterior pituitaries were cytologically studied following cultivation in organ culture, with and without the addition of hypothalamic and cortical extracts. Although five distinct cell types could be identified with classical stains in the uncultivated glands, the peroxidase-labeled antibody technique (using antibodies against STH, LTH, FSH, LH and TSH) showed that not all of the immune-specific cell types were being identified with the classical stains. This discrepancy was magnified following culture as chromophilic cells seen with classic stains decreased in number with an increase in culture time. The peroxidase technique, however, revealed that all cells remained constant in type and number regardless of time in culture. While the addition of either hypothalamic or cortical extract to the culture medium produced cytological alterations demonstrated by the classical dyes, the antibody technique showed no such alterations. Such a comparison of staining techniques emphasizes the hazards of relying solely on histological procedures to reveal the hormonal activity of the pituitary gland.     

Immunochemical staining of the rat adenohypophysis in organ culture.

Rat anterior pituitaries were cytologically studied following cultivation in organ culture, with and without the addition of hypothalamic and cortical extracts. Although five distinct cell types could be identified with classical stains in the uncultivated glands, the peroxidase-labeled antibody technique (using antibodies against STH, LTH, FSH, LH and TSH) showed that not all of the immune-specific cell types were being identified with the classical stains. This discrepancy was magnified following culture as chromophilic cells seen with classic stains decreased in number with an increase in culture time. The peroxidase technique, however, revealed that all cells remained constant in type and number regardless of time in culture. While the addition of either hypothalamic or cortical extract to the culture medium produced cytological alterations demonstrated by the classical dyes, the antibody technique showed no such alterations. Such a comparison of staining techniques emphasizes the hazards of relying solely on histological procedures to reveal the hormonal activity of the pituitary gland.     

A morphometric analysis of rat ventral prostate in organ culture

A stereologic, morphometric method was used for determining the quantitative and qualitative changes in the rat ventral prostate during organ culture. The volume density of epithelium (VVEP), lumina (VVLU), and interstitium (VVIT) as well as the proportion of epithelium of the tissue, VVEP/(VVEP + VVIT), the length density of tubular structures (LV), mean diameter of the lumina (DLU), height of the epithelium (h), and mean width of the interacinar tissue (lambda AP) were evaluated. In the perfusion-fixed prostate these parameters gave the closest approximation for the in vivo situation: VVEP was 0.19 mm3/mm3; h 19.5 micron; VVEP/(VVEP + VVIT) 0.63 mm3/mm3; VVLU 0.70 mm3/mm3; DLU 230 micron; VVIT 0.11 mm3/mm3; lambda AP 84.1 micron; and LV 17.5 mm/mm3. Dissection of the prostate for culture caused leakage of prostatic secretions and a consequent diminution of VVLU (0.41 mm3/mm3) and DLU (151 micron), as well as folding of the epithelium and distortion of the interacinar tissue (VVIT 0.25 mm3/mm3). During 10 days culture in a defined medium, the prostate underwent involutive changes including loss of total weight, a decrease of VVEP, and an increase of VVIT. During the first day a stimulatory effect on the epithelium occurred, which might have been caused by a loss of androgenic control or by substances in leaking secretions. Testosterone postponed many of these changes and maintained the secretory function better. Thus, on day 4 the morphology of the prostate resembled better the situation in vivo than at the beginning of involutionary culture. However, later during culture changes, similar to those found without testosterone but weaker, were noted. Statistical analysis of data showed that it is more advisable to use many pieces from one prostate lobe rather than to use many animals. Analysis of one slice is also sufficient to give relevant data on that piece.     

Differentiation of alpha cells in the fetal rat pancreas grown in organ culture

Alpha cells can be demonstrated in embryonic rat pancreas by a modification of the Grimelius silver nitrate technique. At the start of culture at 18 days of gestation, alpha cells are closely associated with exocrine tubules and beta cells. After ten days of culture they are arranged peripherally around a mass of beta cells, in islets that are characteristic of adult rat pancreas.     

Degradation of fetal rat cartilage in organ culture: effect of Streptomyces hyaluronidase

Degradation of cartilage matrix was studied by characterizing the size and amount of products released from cultures of fetal rat long bones into the medium. Na32SO4 was administered to pregnant rats and the fetal radii and ulnae were explanted 24 hours later and cultured in chemically defined medium. Under these conditions, 35S-activity is released from the cartilagenous ends of the explants into the culture medium gradually over 72 hours. The 35S-labeled molecules in the medium were smaller than proteoglycan monomers and larger than papain-released products when chromatographed on Sepharose 2B. The amount of 35S-activity released into the medium decreased when heat-inactivated rat serum or plasma or heparin was added to the medium. Adding proteases or hyaluronidases or freeze-thawing the tissue enhanced release of 35S-activity. Streptomyces hyaluronidase was chromatographed on CM-cellulose and Ultrogel to remove protease activity. The protease-free enzyme released 35S-labeled molecules, similar in size to proteoglycan monomers, from cartilage which was heated before culturing to inactivate endogenous enzymes. These results indicate that hyaluronidase can degrade matrix by releasing proteoglycan monomers. Other treatments released smaller 35S-labeled molecules. These findings indicate that endogenous enzymes degrade matrix proteoglycans into smaller units.     

Appearance of contractile endothelin-B receptors in rat mesenteric arterial segments following organ culture

The aim of this study was to examine how different procedures for organ culture affect the expression of contractile endothelin(ET)-B receptors in a branch of the rat mesenteric artery. In fresh segments, ET-1 and ET-3 induced similar strong contractions, ET-1 being 20-fold more potent, whereas neither of the selective ET_B receptor agonists, sarafotoxin 6c (S6c) nor IRL 1620, induced significant contractions. In segments cultured for 1 day, ET-3 was only 3-fold less potent as ET-1, and S6c and IRL 1620 induced concentration-dependent contractions which were about 60% of the ET-1 induced contraction. The maximum contractile response to S6c was not altered in segments cultured with foetal calf serum or in buffer solution, but was reduced to about 20% of the control value when cultured in glucose-free buffer solution. The contraction to S6c was abolished in segments placed in cold (4 °C) buffer solution. Removal of the endothelium had no effect on the S6c-induced contractions. Arteries cultured at isometric tension (at 2 mN) for 1day achieved the same contractile response for ET_B agonists as resting segments. Pressurized arteries (60 mmHg) did not constrict to S6c when mounted as a fresh segment but demonstrated a strong contraction after 1 day at this transmural pressure. This study suggests that the appearance of ET_B receptor mediated contraction following organ culture is not dependent on specific nutrients, endothelial factors or absence of intrinsic tension, but is a metabolically active process.     

Long-term organ culture of embryonic rat pancreas in a chemically defined medium.

Embryonic rat pancreas anlagen have been grown in a chemically defined medium, subdivided biweekly and recultured for a total time of 10 weeks. The total mass increment during this time was in excess of 1000-fold. Samples were removed for light and electron microscope examination and for periodic measurement of enzymatic activity. Morphogenesis and cytodifferentiation occurred, peripheral outgrowth of epithelial buds was followed by the formation of interconnecting tubular structures and, eventually, by the appearance of distinctive acinar cells with zymogen granules. Mitotic figures became conspicuous at the periphery of explants within a day after each subdivision resulting in the formation of new tubular structures and acini. In general, the central area of the explants presented more mature acini with zymogen granules than was manifested at the periphery. The enzymatic activities of amylase, lipase, and chymotrypsin developed maximally during the first week of culture, reached a plateau level by the second week, and remained at a relatively constant level throughout the 10-week culture period.     

Comparative study of proliferation of suckling and adult rat hepatocytes in primary culture in response to growth-stimulating factors

The replicative responses of suckling and adult rat hepatocytes in primary culture to growth-stimulating factors were compared. By addition of L-proline alone, the [3H]-thymidine labeling of suckling rat hepatocytes was dramatically enhanced, but that of adult ones was not. Epidermal growth factor (EGF), insulin, triiodothyronine (T3) and glucagon also enhanced the labeling of suckling rat hepatocytes regardless of the presence or the absence of L-proline. On the other hand, in the absence of L-proline, only EGF enhanced the labeling of adult rat hepatocytes, and, in the presence of L-proline, insulin as well as EGF enhanced the labeling. In the presence of growth factors and L-proline, the number of suckling rat hepatocytes increased up to about 143%, whereas that of adult rat hepatocytes hardly increased. Thus, a remarkable difference in replicative responses to growth factors and L-proline was observed between suckling and adult rat hepatocytes in primary culture.     

Cell differentiation in the terminal tubule of fetal rat submandibular gland in organ culture

Submandibular glands from 17-day-old rat fetuses were maintained in organ culture for five days in a medium consisting of Eagle's MEM (87%), horse serum (10%), and chick embryo extract (3%). Each day of the culture period explants were incubated for the demonstration of peroxidase activity and processed for light and electron microscopic observations. In some experiments cultures were exposed to ³H-thymidine one hour prior to fixation and incubation for the demonstration of peroxidase activity. Labelling index was determined using radioautographs of 1 μ Epon-embedded sections. At the time of explantation the submandibular gland rudiment consisted of undifferentiated epithelial cells arranged in cords. On day 3 of culture two additional cell types could be distinguished: terminal tubule cells and proacinar cells. The proacinar cells were characterized by peroxidase activity in their granules and cytoplasm. By day 4 acinar cells begin to appear. On the fifth day of culture the four cell types of the terminal tubule were present in the following proportions: undifferentiated cells, 44%; terminal tubule cells, 19%; proacinar cells, 31%; acinar cells, 6%. These results indicate that the cytodifferentiation of the secretory unit of rat submandibular gland in vitro is comparable to the differentiation in vivo.     

Role of calcium in osmotic and nonosmotic release of vasopressin from rat organ culture

In the present study the role of calcium (Ca) in the stimulation of arginine vasopressin (AVP) release from the cultured rat hypothalamoneurohypophyseal complex (HNC) was examined in response to three different stimuli, 56 mM potassium chloride, an increase in medium osmolality from 290 to 310 mosmol/kg H2O, or 1 X 10(-6) M angiotensin II (ANG II). With all three stimuli AVP release from rat HNC explants was enhanced by increasing Ca concentration in the medium from 0 to 1.8 mM Ca. However, high concentrations of Ca (8 mM) inhibited the response of AVP release to either hyperosmolality or angiotensin II. Chemically dissimilar blockers of cellular Ca uptake, verapamil (5.2 X 10(-6) or 5.2 X 10(-5) M) or nifedipine (5.8 X 10(-6) or 5.8 X 10(-5) M), completely abolished AVP release from rat HNC explants in response to the three different stimuli in 1.8 mM Ca. In a normal concentration of medium Ca (1.8 mM) a Ca ionophore, A23187 (3.8 X 10(-5) M), significantly enhanced the osmotic and nonosmotic (ANG II-stimulated) release of AVP from rat HNC explants compared with controls without Ca ionophore. This effect of Ca ionophore to enhance AVP release was more evident in a lower Ca medium (0.9 mM Ca in the hyperosmolality study and 0.3 mM Ca in the ANG II study). These results therefore indicate that cellular Ca uptake is an important modulator of osmotic and nonosmotic AVP release from the intact rat hypothalamoneurohypophyseal system. The influence of extracellular Ca on the osmotic and nonosmotic release of AVP is also demonstrated.     

Maintenance of normal human breast organoids within rat mammary fat pads in organ culture

Summary Normal human breast organoids, derived by collagenase digestion of reduction mammaplasty tissue specimens, have been cultured in vitro for up to 28 days after injection into organ cultures of virgin rat mammary fat pads. The culture medium was serum-free Waymouth's MB 752/ 1 with hormonal additives. The rat mammary tissue responded well to growth-promoting and lactogenic stimuli in the culture medium, in agreement with previous investigations. Using immunohistochemistry casein was identified in rat epithelia exposed to lactogenic medium. Human organoids in culture remained viable but did not show hormone-responsiveness. Electron microscopy confirmed the presence of both luminal epithelial cells and myoepithelial cells.The serum-free culture of normal human breast organoids in a three-dimensional matrix provides a system in which to study factors controlling growth and differentiation.     

Antisecretory effects of berberine in rat ileum

The in vitro antisecretory effects of the alkaloid berberine (1.0 mM) on intestinal ion secretion and mucosal adenylate cyclase and Na-K-ATPase activities were studied in the rat ileum. Mucosal berberine did not alter the individual basal net ion fluxes and basal adenylate cyclase activity but decreased short-circuit current (Isc) and increased the net absorption of chloride plus bicarbonate. In the cholera toxin-treated tissue, mucosal berberine stimulated absorption of Na and Cl and inhibited the increased adenylate cyclase activity but did not change the specific Na-K-ATPase activity, whereas serosal berberine stimulated Na secretion and decreased Isc. Mucosal berberine also decreased Isc, increased Cl permeability, and reversed the ion secretion induced by dibutyryl cyclic AMP, the heat-stable enterotoxin of Escherichia coli, and methylprednisolone administration. The antisecretory effects of mucosal berberine may be explained by stimulation of a Na-Cl-coupled absorptive transport process. The mechanism of action of serosal berberine remains to be elucidated. However, it is clear that mucosal berberine affects intestinal ion transport by mechanisms different from stimulation of the Na pump and probably at a step distal to the production or degradation of cyclic AMP or cyclic GMP.     

Intestinal absorption of ferritin in the suckling rat

Extensive absorption of ferritin was demonstrated in the proximal and distal ileum of 8–12 day-old rats. In some regions, ferritin droplets were distributed in the basal cytoplasm of the columnar epithelium and in the lamina propria one hour after feeding. Subsequently, iron-positive material was localized adjacent to vessels of the lamina propria and in macrophages. Absorption of lesser quantities was demonstrated in the duodenum.