Parathyroid hormone increases epidermal growth factor receptors in cultured human trophoblastic cells from early and term placenta.

The effect of PTH on the epidermal growth factor (EGF) receptor was analyzed during the in vitro differentiation of human cytotrophoblasts. The cytotrophoblasts were isolated by a trypsin-DNase method from first trimester and term placentas and purified on a Percoll gradient. In culture, these cells aggregated and fused together to form a syncytium. This in vitro differentiation was associated with a 2-fold increase in 125I-EGF binding after 48 h of culture. The addition of 0.1 microM PTH (PTH-treated cells) to the culture medium induced a significant 2- to 3-fold increase (P less than 0.005) in EGF binding. The effect was dose related with a maximum obtained at a 1 nM concentration. Scatchard analyses revealed that PTH-treated cells possess a 2-fold higher number of high affinity sites as compared to control cells from early placenta (0.71 +/- 0.06 pmol/mg protein and 0.34 +/- 0.04 pmol/mg protein, respectively) and from term placenta (1.24 +/- 0.10 pmol/mg protein and 0.61 +/- 0.07 pmol/mg protein, respectively). The apparent Kd values for high affinity sites (0.15 nM) and for low affinity sites (4 nM) were not altered either by the gestational age of the cells or by PTH treatment. With respect to the EGF-dependent phosphorylation in membranes of trophoblast cells in culture, it was found that the phosphorylation of two major proteins of 175 kilodaltons and 35 kilodaltons, is greatly increased in PTH-treated cell membranes in the presence of EGF. This PTH-induced effect on EGF receptors was associated with an augmented functional response of trophoblastic cells to EGF. PTH increased the EGF-stimulated secretion of hCG. These results demonstrate that PTH increases the number of biologically active EGF receptors during the in vitro differentiation of human trophoblast cells. This PTH-induced effect suggests a role for this hormone in the regulation of the growth and the endocrine functions of these cells.

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.

Regulation of growth hormone secretion in human trophoblastic cells in culture.

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10(5) cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10(-6) M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.

Characterization and differentiation of human first trimester placenta trophoblastic cells in culture.

A preparation of highly enriched isolated cytotrophoblasts was obtained from first trimester placenta using dispase incubation of villous tissue at 4 degrees C, followed by a spontaneous cell release at 37 degrees C. After 24 h of culture, 90-95% of the cells were immunostained by anticytokeratin antibody, showing their epithelial characteristic. After 48 h of culture, these cells differentiated into syncytiotrophoblast, as shown by optic and electron microscopic study. The secretion of hCG, and of its free alpha and beta subunits, and the secretion of hPL were studied as a function of cell culture time. While the level of secreted hCG and its free subunits was stable during 72 h of culture, the hPL level was undetectable during the first 48 h of culture, increasing continuously afterwards. Addition of dibutyryl cAMP from the start or after 96 h of cell culture induced an increase of hCG production and of its free subunits and also stimulated the secretion of hPL. This suggests that these cells maintained the capacity to respond to stimuli which increased intracellular cAMP level. Such a cell culture is of interest in further determining the mechanisms of early gestation involved in the differentiation and growth of placental cytotrophoblasts, and in the regulation of their endocrine functions.

Comparative determination of the asialoglycoprotein receptor by ligand and antibody binding in hepatocytes from normal and diabetic rats.

The number of cell surface and total asialoglycoprotein receptors was investigated in normal and diabetic rat hepatocytes using 2 methods: ligand and polyclonal antibody binding. An identical number of immunoreactive receptors was found in both types of cells, while the ligand binding activity of cell surface receptors was reduced by 58% in diabetic rats compared with normal ones, or by 33% for total cell receptors.

[In vitro secretion of somatostatin (SRIH) by human adenomatous somatotropic cells. Relation with somatotropic hormone (GH) release and modulation by thyroliberin (TRH)]. / Sécrétion in vitro de somatostatine (SRIH) par les cellules somatotropes adénomateuses humaines. Relation avec la libération d'hormone somatotrope (GH) et modulation par la thyrolibérine (TRH).

SRIH and GH secretions by GH-secreting adenomatous human pituitary cells were analyzed in vitro in a perifusion system. Of the 13 adenomas studied, 7 secreted SRIH, in variable amounts (50 to 700 pg/ml/2 min., corresponding to 600 10,700 pg for the total experiment. SRIH secretion increased during the perifusion, the highest levels being observed at the end of the perifusion. GH secretion also varied from one adenoma to the other (6 to 500 ng/ml). In most cases, the secretion profiles were negatively correlated, GH secretion decreasing while SRIH secretion was increasing. In the presence of 10(-7) M TRH, GH secretion increased while that of SRIH decreased. The hypothesis of a paracrine and/or an autocrine role for SRIH as well as its possible in situ synthesis are discussed.

Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats.

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.

Study of hepatic binding protein activity in jejuno-ileal by-passed rat hepatocytes.

The kinetic constants of internalization of asialoorosomucoid were determined for normal and jejuno-ileal by-passed rat hepatocytes. In by-passed rats the maximum velocity of asialoorosomucoid internalization is decreased 3-fold, without any modification of apparant constant of internalization. Moreover, the rate constant of internalization was the same in the two groups of rats. These data suggest that the process of asialoorosomucoid internalization is not altered in by-passed hepatocytes and that the decrease of maximal velocity is only due to a decrease of total uptake receptor number.

Endocytosis and binding of asialoorosomucoid by hepatocytes from rats with jejunoileal bypass.

Rats with jejunoileal bypass were used to study the biological activity of the hepatic binding protein. Hepatocytes were prepared 11 weeks after surgical procedure, and presence of asialoorosomucoid in serum has been determined. Compared to control rat hepatocytes, endocytosis of [3H]asialoorosomucoid by bypassed rat hepatocytes was decreased by about 60%. This was due to a decreased number of total and surface receptors. In most sera, an accumulation of asialoorosomucoid was found. A reduction of protein synthesis or turnover could be considered.

Asialoorosomucoid degradation by normal and diabetic rat hepatocytes.

Using high concentrations of extracellular [3H]asialoorosomucoid we obtained the steady-state level of [3H]asialoorosomucoid endocytosis by isolated hepatocytes from normal and streptozotocin diabetic rats. At the steady-state of the overall reaction, the intracellular amount of [3H]asialoorosomucoid did not change with time, the apparent overall rate of [3H]asialoorosomucoid degradation was close to that of [3H]asialoorosomucoid internalization, in both normal and diabetic rat hepatocytes. Although in diabetic cells the intracellular amount of [3H]asialoorosomucoid was about three-times lower than in normal cells, the same fraction of intracellular asialoorosomucoid was degraded per time interval by both normal and diabetic cells. The apparent first-order rate constant of steady-state degradation was about 0.011 min-1 for both normal and diabetic cells. In diabetic rat hepatocytes, the decrease of the clearance of serum asialoglycoproteins was directly correlated to the variation of cell surface receptor number, without any modification of internalization and degradation steps.

Diabetes-induced variation in hepatic binding protein.

The total capacity of hepatocytes to bind asialoorosomucoid was measured on normal and streptozotocin diabetic rats. 4 days after the streptozotocin injection, a slight decrease of total receptor concentration was observed while a more marked reduction of cell surface receptor occurred. In animals sacrificed 11 days after the streptozotocin injection, the total capacity of hepatocytes to bind asialoorosomucoid was about 70% of the normal level.

Membrane lipids of hepatocytes, Kupffer cells and endothelial cells.

The membrane lipid composition of isolated hepatocytes, Kupffer cells and endothelial cells was determined. The hepatocytes are characterized by a lower quantity of gangliosides, cholesterol, sphingomyelin and a reduced cholesterol/phospholipid molar ratio when compared to the two other liver cell types. The main gangliosides of Kupffer and endothelial cells are the GM3 species, and those of hepatocytes are of the polysialogangliosides species.

Interactions of insolubilized lectins with membrane glycoproteins in presence of detergents.

The effects of several detergents commonly used to solubilize membrane glycoproteins have been investigated on the binding of hepatoma cell surface [3H]-galactoglycoproteins to, and their elution from, concanavalin A or Ricinus communis lectins conjugated to Sepharose 4B. The optimum conditions (pH, ionic strength) in the presence of ionic [sodium deoxycholate (DOC) and sodium dodecyl sulphate (SDS)] and non-ionic detergents (Triton X-100) at a constant concentration were determined in order to ascertain which would yield the better efficiency. The effects of different detergent concentrations on binding and elution were then studied. The range of concentrations for each detergent to be used without modifying efficiency was determined. Triton X-100 and DOC (0.1-1%) did not change the efficiency on Ricinus lectin-Sepharose, whereas SDS, at a concentration greater than 0.05%, caused a dramatic decrease in efficiency. On concanavalin A-Sepharose, by contrast, the non-ionic detergent had no effect on the efficiency at all the concentrations tested (0.1-1%), while concentrations of more than 0.5% DOC and 0.1% SDS significantly decreased both binding and elution.

Toxic effect of Ricinus lectin on hepatoma cells in relation to enzyme modification of the cell surface.

With regard to the toxic effects of Ricinus lectin, neuraminidase-treated hepatoma cells have been found to be the most sensitive, and untreated hepatoma cells the least. Cells treated with neuraminidase and galactose oxidase exhibited an intermediate sensitivity. At 37 degrees C, the number of Ricinus lectin molecules bound to untreated, neuraminidase-treated and neuraminidase and galactose oxidase-treated cells required to being about 30% toxicity within 2 h was 15 . 10(5), 7.5 . 10(5) and 11.5 . 10(5) molecules/cell, respectively. This difference was rather small and suggests that the additional binding sites exposed following enzyme treatment were as efficient in mediating lectin toxicity as those present before enzyme treatment. Positive cooperativity was observed during Ricinus lectin binding to enzyme-treated cells at 37 degrees C and the apparent association constant increased with the increase of binding site occupancy. The binding sites on enzyme-treated cells appeared to be homogeneous since under different physical conditions (4 degrees C) the shape of the Scatchard plot could be altered in such a way as to produce a single line of slope. In contrast to enzyme-treated cells, untreated cells did not exhibit a positive cooperative process either at 37 degrees C or at 4 degrees C. We found that the toxicity of Ricinus lectin paralleled the irreversible specific binding of lectin, suggesting that only this was able to mediate the toxic effect. Our results are discussed in terms of the possible entry into the cells of Ricinus lectin and this occurs more rapidly in enzyme-treated than in untreated cells. This difference agrees with the sequence of events proposed: (i) Binding of Ricinus lectin; (ii) Clustering of lectin binding sites; and (iii) Endocytosis.

Specific modifications of hepatoma cell-surface glycoproteins with enzymes. Effects on in vitro growth as investigated by the use of lectins.

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.