Insulin-like Growth Factor I Regulation of Swarm Rat Chondrosarcoma Chondrocytes in Culture

Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [³H]thymidine and [³⁵S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10^-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [³H]thymidine incorporation and was comparable to that of serum for [³⁵S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [³H]thymidine incorporation and [³⁵S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [³H]thymidine and [³⁵S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [³H]thymidine and [³⁵S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action.     

Suppression of Anti-Candida Activity of Murine Neutrophils by Progesterone In Vitro: A Possible Mechanism in Pregnant Women's Vulnerability to Vaginal Candidiasis

Sex steroid hormones were examined for their effect on mycelial growth of Candida albicans, and the inhibitory activity of casein-induced murine peritoneal neutrophils against mycelial growth of C. albicans was examined in vitro using a crystal violet staining method or a [³H]glucose incorporation method. Four steroid hormones, danazol, estradiol, estriol and testosterone had no effect on mycelial growth of C. albicans, but progesterone appeared to convert the growth form of C. albicans from hyphal to yeast. Danazol (10^–6 m ) and progesterone (10^–5 m ) suppressed anti-Candida activity of neutrophils of non-treated mice, while testosterone, estradiol, and estriol did not. The anti-Candida activity of neutrophils of estradiol-pretreated mice was clearly suppressed by progesterone even at 10^–6 m which corresponded to its plasma concentration in pregnant women in the third trimester. The physiological significance of this suppressive effect of progesterone was discussed in relation to the vulnerability of pregnant women to vaginal candidiasis.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Regulation of cultured rat vaginal epithelial cells by 17 beta-estradiol and progesterone

We have previously described a technique to obtain short-term cultures of epithelial cells from Wistar rat vaginae. In order to improve the efficiency and life span of these cultures, in the present study we have cultured the vaginal cells with lethally irradiated 3T3 cell feeder layers. Under this condition, cells can grow for several weeks while retaining epithelial characteristics and can eventually be subcultured. The proliferative effect of the ovarian hormones in these cultures was studied using two different approaches, [Methyl-3H]Thymidine (3HTdr) incorporation and increase in cell number. Both assays indicated a proliferative effect of 17 beta-estradiol and progesterone at physiological concentrations. This proliferative effect was also shown in feeder layer-free cultures, ruling out an indirect effect through the mesodermal cells. The capacity of the hormones to modify terminal differentiation in the culture was also studied, using colony stratification as an indicator of differentiation. Progesterone and fetal calf serum had an inhibitory effect on terminal differentiation, whereas 17 beta-estradiol induced a stimulatory action. This culture model allowed us to show a direct effect of the ovarian hormones on vaginal cells in vitro and seems to be a useful model to study hormone-cell interactions in vitro.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Two sex steroid receptors in SC-115 mammary tumor cells

The growth of the SC-115 mammary carcinoma in mice is androgen dependent. Estrogens antagonize the androgen effect. The high affinity binding of androgens and estrogens has been studied in soluble extracts of the tumor, of primary culture cells and clone MI₁ cells.Results indicate that two distinct specific steroid hormone-binding sites (termed ‘receptors’) are found in all cytosol fractions. The androgen-receptor (A) binds testosterone, androstanolone, cyproterone (an anti-androgen), progesterone and estradiol, but only very weakly non-steroidal diethylstilbestrol. The estrogen-receptor (E) binds estrogenic substances such as estradiol and diethylstilbestrol, but no androgen. The apparent K_{D, eq} for A and E receptors of [³H]androstanolone and [³H]estradiol respectively, is identical (-0.5-1 nM at 4 °C). The affinity of estradiol for the A-receptor, when measured against [³H]androstanolone binding, indicates a K_i = 17.5 nM. The concentration of binding sites is of the order of 0.1 pmole/mg protein (somewhat higher for A than for E receptor) in MI₁ cell cytosol. Studies by ultracentrifugation through glycerol-Tris gradients (low salt medium) reveal the macromolecular nature of the cytosol A and E receptors (7–7.5 S). Evidence is presented of the transfer of the A and the E receptors to nuclei after incubation of tumor slices as well as of clone MI₁ cells with the corresponding hormones.Experiments suggest that the two different binding sites are present on two separated macromolecular moieties. After incubation at 37 °C of tumor slices with 10–20 nM [³H]testosterone, or with 10 nM [³H]estradiol, the corresponding radioactive hormone-receptor complexes are, as expected, found in the nuclear KCl extracts. In parallel experiments, where slices are incubated with non-radioactive hormones at the same concentration and the nuclear KCl extracts subsequently treated by radioactive steroids, no available androgen-binding sites are found in the nuclei after exposure to estradiol, nor estrogen-binding sites after exposure to testosterone.Therefore, in the same cell, two receptors are present which bind androgens and estrogens with high affinity, and one given hormone (estradiol) can be specifically bound (with different affinities) by two different receptors which, however, discriminate a synthetic analog (diethylstilbestrol). The data may give some molecular background for interpreting responses to the same hormone which may differ at various concentrations, for studying effects of analogs, and for analysing the control of tumor growth by antagonistic steroids.     

Effects of peptides of pituitary origin on the formation of C21 steroid by fetal calf adrenal cells in culture.

Corticotrophic activity of opiate-like peptides was assessed by their ability to stimulate the formation of C₂₁ steroids from [³H] progesterone by three-day old cultures of fetal calf adrenal cells. ACTH_1–39, ACTH_α1–24 and a purified preparation of pituitary ovine β-endorphin caused a marked increase in 17α and 21-hydroxylation while a preparation of pure synthetic porcine β-endorphin gave a minimal stimulation. The activity of the purified ovine β-endorphin preparation could not be accounted for by contamination by ACTH or by a synergistic action between the two peptides. The novel pituitary factor described here may be due to a contaminant of the β-endorphin peak which is different from ACTH_1–39.     

Rapid modulation by progesterone and tamoxifen of estradiol effects on nuclear histone acetylation in the uterus of the fetal guinea pig

The effect of estradiol, progesterone, tamoxifen, estradiol + progesterone or estradiol + tamoxifen on the [³H]acetylation of histones in the fetal uterus of guinea pig was studied. The fetuses were injected subcutaneously ‘in situ’ with the hormones or $\text{tamoxifen}\text{+ [}{}^3\text{H}\text{]}\text{acetate}$ alone. In 10 min, estradiol stimulated the acetylation of histone 10–12-times with respect to the control animals. Progesterone and tamoxifen blocked this effect. It is suggested that histone acetylation is an early step induced by estrogen action during intrauterine life and that progesterone and tamoxifen suppress this mechanism very effectively.     

Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr^db/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

Insulin binding in cultured Chinese hamster kidney epithelial cells: The effect of serum in the medium

Summary [¹²⁵I]Insulin (porcine) binding to an epithelial cell line established from a Chinese hamster kidney, CHK-AC_E−100, showed an optimum at pH 8.0 and reached a maximum after 2.5 h incubation at 25°C. Dissociation of bound [¹²⁵I]insulin was facilitated by the addition of unlabeled insulin in the dilution buffer. Porcine insulin effectively competed for [¹²⁵I]insulin binding to the cultured cells and was 30 and 90 times as potent as guinea pig insulin and porcine proinsulin in causing 50% inhibition of [¹²⁵I]insulin binding; glucagon was completely ineffective. Scatchard analysis of the binding data yielded a curvilinear plot and a capacity of 0.6 ng/10⁶ cells; the average affinity of the empty receptor, , was calculated to be 1.78×10⁸ M ⁻¹ and that of the filled receptor, , 0.57×10⁸ M ⁻¹. Substitution of fetal bovine serum (FBS) in the culture medium with bovine calf, bovine newborn, or bobby calf serum altered insulin binding characteristics in the cells and reduced cell growth. Insulin binding characteristics of cells grown in hormone-supplemented medium containing 0 to 0.1% FBS were similar to those of cells grown in minimum essential medium (MEM) containing 2 to 5% FBS. The data indicated that the established Chinese hamster kidney epithelial cell line CHK-AC_E−100 possessed specific insulin receptors and the characteristics of the receptors could be manipulated by changing the serum in culture medium.     

Vasopressin stimulates sulfate incorporation into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture

The effects of lysine vasopressin (1–100 ng/ml) on the 24 h incorporation of [³⁵SO₄⁻] into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture has been investigated. The hormone enhances sulfate incorporation into proteoglycans released in the medium and those associated with the cell layer. This enhancement was independent of cell density or stimulation of cell division by the hormone or calf serum. These observations provide evidence that the hormone stimulation of sulfate incorporation is not directly linked to hormone stimulation of cell division.     

Electron microscope analysis of amplifying ribosomal DNA from Xenopus laevis.

The inhibition of human prostatic epithelial cell (MA-160) replication by cAMP and certain analogs was explored in tissue cultures. When untreated fetal bovine serum was used to supplement the culture medium, cyclic AMP (cAMP) markedly inhibited cell growth. The inhibition was reversed by equimolar concentrations of uridine. Inhibition by 8-methyl-thio-cAMP (MES) was somewhat less effective and was not reversed by uridine. After heat treatment of the fetal bovine serum, which inactivated the cAMP phosphodiesterases, cAMP became less effective in cell growth inhibition, whereas the activity of MES remained unaltered. Dibutyryl cAMP (db-cAMP) had no effect on cell growth, however, when combined with the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), significant retardation of cell replication was observed. Cells treated for 24 h with 0.5 mM MES took up and incorporated significantly less [³H]TdR and [³H]uridine than control cells. Treatment of cells with 0.5 mM cAMP for 24 h, on the other hand, resulted in both substantially increased [³H]TdR uptake and increased [³H]uridine incorporation into RNA. The effects of similar treatment with db-cAMP plus MIX closely paralleled those of MES with marked inhibition of the uptake and incorporation of both thymidine and uridine.     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.     

Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf.

The synthesis and appearance of carbohydrate-rich macromolecules by epithelial cells of the developing secondary palate was examined with concanavalin A (CON A) binding and [³H]glucosamine labeling. The amount of [¹²⁵I]CON A bound to the epithelial surface of the rat palatal shelf in vitro increased from day 15 of gestation to day 16 when initial adhesion to the opposite shelf occurs in vivo. Visualization of CON A binding by electron microscopy using the peroxidase method revealed a dramatic increase in binding between days 15 and 16 of gestation, most apparent on the medial-edge epithelial surface. The incorporation in vivo of [³H]glucosamine during this period into the medial-edge epithelial cells was detected with autoradiography. These results show that a glycoprotein-rich surface material appears on the superficial cells of the medial-edge epithelium prior to adhesion of the apposing shelves.     

Demonstration and maintenance of mucus secretion in cultured human gallbladder epithelial cells

Summary The method of human gallbladder epithelial cell culture has been developed successfully with active mucus secretory function. Human gallbladder epithelial cells were dissociated by Dispase digestion from the specimens obtained by cholecystectomy for uncomplicated gallbladder stone cases. The dissociated cells formed a monolayer in Eagle’fs minimum essential medium supplemented with 10% fetal bovine serum within 24 h after the inoculation. These cells were maintained for at least 2 wk without fibroblastic overgrowth. Cultured cells contained periodic acid Schiff-positive material in cellular cytoplasm for 3 d. On transmission electron microscopy these materials were identified as mucous secretory granules. Mucous secretory function was determined by [³H]glucosamine incorporation. Sixty percent of the secreted glycoproteins labeled with [³H]glucosamine was eluted in excluded fractions of Sepharose 4B gel filtration, which were considered to be mucous glycoprotein, because they were found to be resistant to proteoglycan-specific enzymes such as hyaluronidase, chondroitinase ABC, heparitinase, and heparinase. The mucous glycoprotein secretion was maintained for 3 d and found to be inhibited in a dose-dependent manner by monensin (10⁻⁷ to 10⁻⁵ M) which is a known blocker of secretory function.