Production of albumin and α-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone

Summary When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, α-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.     

Differential effects of concanavalin A, serum and cytokines on proteoglycan elaboration and DNA synthesis by mononuclear cells from human peripheral blood.

Human blood derived mononuclear cell (MC) cultures required concanavalin A (Con A) stimulation to synthesize and secrete into the medium high levels of a protease-resistant proteoglycan (PG) containing predominantly chondroitin sulfate (CS), which was elaborated largely by T-cells in culture. PG and DNA synthesis were studied in MC cultures in the absence and presence of Con A as well as serum and some biologically active polypeptide factors. In the presence of Con A, stimulation of PG synthesis was substantially greater in T-cell enriched cultures than in B-cell enriched cultures. DNA synthesis was also stimulated in the presence of Con A. This stimulation was concentration-dependent, but required the presence of serum for additional responses. DNA and cell proliferation were stimulated by interleukin-2 (IL-2), but PG production was not stimulated by conditioned media, IL-1, IL-2, IL-3, or transforming growth factor-beta (TGF-beta). Our results indicate that the elaboration of PG from T-cells of human MC is independent of the effects of regulatory peptides on cell proliferation and DNA synthesis.     

Cyclooxygenase products formed by primary cultures of cells from human chorion laeve: influence of steroids

Cells were isolated from human chorion laeve obtained at term (38-40 weeks gestation) by elective caesarean section and were maintained in primary culture for 1 week in defined media supplemented with 10% fetal calf serum. The production of various cyclooxygenase products by the cultures was examined. Little or no prostaglandin (PG) F2 alpha, 6-keto-PGF1 alpha, thromboxane B2, or 13,14-dihydro-15-keto-PGF2 alpha was found. In contrast, the cells produced PGE2 which was low on day 0, increased during culture to a maximum on day 1 or 2, then declined to low levels. When cells were grown in the presence of media containing cortisol, dexamethasone, progesterone, and estradiol (at 10(-7) or 10(-9) M), the glucocorticoids (at 10(-7) and 10(-9) M), but not estrogen or progesterone, markedly inhibited the increase in PGE2 output. There was no difference in the protein content and thymidine incorporation of cells grown in the presence of glucocorticoids when compared with controls. This inhibitory effect was not sensitive to cycloheximide (1 microgram/mL) indicating protein synthesis may not be involved in the process. These studies indicate that PGE2 is the major prostaglandin formed by primary cultures of chorion laeve and that prostaglandin metabolism in the chorion is sensitive to glucocorticoid inhibition.     

Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [¹⁴C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.     

Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone

Osteoprogenitor cells present in single-cell suspensions prepared from fetal rat calvaria (RC) form discrete mineralized three-dimensional bone nodules when cultured long-term in the presence of ascorbic acid and beta-glycerophosphate. These cells (CFU-O) constitute less than 1% of the total cell population under standard culture conditions and their number is increased in the presence of dexamethasone. Using the formation of the bone nodule as a marker for CFU-O, we have now analyzed the proliferation and differentiation capacity of these CFU-O by redistribution and continuous subculture experiments in the presence and absence of dexamethasone. Cell redistribution experiments showed no increase in nodule number after one population doubling with either treatment. After 5.4 population doublings of the entire RC population, nodule number increased up to 2.0-fold in control cultures and 4.5-fold in cultures containing 10 nM dexamethasone. Continuous subculture experiments in which cultures were split 1:3 every 3 day for up to seven subcultures showed that nodule number decreased in parallel with the split ratio in the absence of dexamethasone, while with dexamethasone nodule number was elevated above the number present in primary cultures for 1 or 2 subcultures after which nodule number decreased with the split ratio. Bone nodules were present for up to 18 population doublings. Measurements of nodule area by automated image analysis showed that dexamethasone increased nodule size and that nodule size decreased from primary to 1st to 2nd subculture with or without dexamethasone. The data suggest that dexamethasone selectively stimulates the proliferation of osteoprogenitor cells and that these progenitor cells have a limited capacity for generating daughter cells capable of expressing the bone phenotype.     

The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid induction of growth hormone synthesis in a strain of rat pituitary cells

Conditions were determined for measuring growth hormone synthesis by a clonal strain of rat pituitary cells grown in suspension culture. Incubation of the cells with [3H]leucine in either continuous labeling or pulse-chase experiments showed that secretion of newly synthesized growth hormone commences only after a lag of about 15 min. The pulse-chase experiments also demonstrated that there is no detectable degradation by the cells of growth hormone. Thus growth hormone synthesis could be measured, in the absence of complications arising either from secretion or degradation of growth hormone, by incubating the cells with [3H]leucine for 10 min. Exposure of cells grown under the usual culture conditions to dexamethasone (a synthetic glucocorticoid) led to an average stimulation of specific growth hormone synthesis (growth hormone synthesis/total cytoplasmic protein synthesis) of only 2.6-fold. However, two other growth conditions were found in which dexamethasone routinely yielded a 5- to 15-fold stimulation of specific growth hormone synthesis. One of these conditions, involving substitution of 10% fetal calf serum for the normal serum supplement, was employed in subsequent experiments. A stimulation of specific growth hormone synthesis could be observed at 10(-9) M dexamethasone, and the maximum stimulation was observed at dexamethasone concentrations of about 10(-8) to 10(-7) M. There was a lag of about 6 h before a stimulation by dexamethasone of specific growth hormone synthesis was detected. Thereafter, the stimulation increased in a nearly linear fashion until maximum stimulation was reached at about 48 h.     

Mouse embryo fibroblast proliferation and prostaglandin production in medium supplemented with fetal bovine serum or serum substitutes (Ultroser SF and G): role of glucocorticoids

The growth of DBA/2 mouse embryo fibroblasts, as well as their prostaglandin (PG) production, was compared under 3 different culture conditions: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2% Ultroser SF (steroid-free) or with 2% Ultroser G (containing steroids). The effect of the absence or presence of glucocorticoids on both parameters was more precisely investigated. In FBS-supplemented cultures, dexamethasone had a stimulatory effect on cells characterized by a slow growth rate, whereas it markedly inhibited proliferation in rapidly growing fibroblasts. The experiments carried out with serum substitutes (Ultroser SF and G) strongly corroborated the role of the absence or presence of glucocorticoids on fibroblast proliferation. Manipulations of glucocorticoid concentrations in Ultroser SF by adding 5 x 10(-8) M dexamethasone or in Ultroser G by adding 10(-6) M RU 486 reversed the effect of the absence of glucocorticoid in the first case, or in the latter case the effect of the presence of glucocorticoid on both cell growth and PG production. Progesterone had no effect by itself. Our results emphasize the importance of performing complete kinetic studies to investigate the effect of a given factor on cell proliferation in vitro, since glucocorticoids may have opposite effects on fibroblast proliferation according to their cell growth pattern in vitro.     

Dexamethasone stimulates insulin receptor synthesis in cultured rat hepatocytes

The ability of the glucocorticoid dexamethasone to modulate the insulin receptor was examined directly in primary cultures of hepatocytes prepared from adult male rats. Hepatocytes were cultured in a defined medium in the presence and absence of dexamethasone, 0.1 microM. The exposure of hepatocytes to dexamethasone resulted in a time-dependent (steady state by 32 h) increase in insulin binding in both intact hepatocytes and Triton X-100-soluble extracts (total insulin receptor content). The enhanced insulin binding found in soluble extracts of dexamethasone-treated hepatocytes was the result of an increase in insulin receptor number without a change in receptor affinity. In order to assess the mechanism by which dexamethasone "up-regulates" the insulin receptor, the heavy isotope density-shift technique was used to analyze insulin receptor turnover in control and dexamethasone-treated hepatocytes. Hepatocytes were initially cultured for 32 h in standard culture media containing only "light" (14C, 12C, 1H) amino acids. In hepatocytes exposed to dexamethasone, a 417% increase in insulin binding in Triton X-100-soluble extracts was observed. After 32 h, when steady state binding is achieved in dexamethasone-treated cultures, parallel cultures of hepatocytes incubated in the absence and presence of dexamethasone were washed and subsequently cultured in media containing "heavy" amino acids (15N, 13C, 2H). The time-dependent disappearance of light insulin receptor (receptor degradation) and appearance of heavy insulin receptor (receptor synthesis) were monitored using CsCl gradients to resolve the two density species of receptor. At steady state, the rate of receptor synthesis (k8) was 2.94 and 0.62 fmol of insulin bound h-1 in dexamethasone-treated and control hepatocytes, respectively. In contrast to this large increase in the rate of receptor synthesis observed in dexamethasone-treated cells, the first order rate constant for decay (k d) was the same in dexamethasone-treated (0.074 h-1) and in control (0.077 h-1) hepatocytes. We therefore conclude that glucocorticoid-induced up-regulation of the insulin receptor in the liver is due to stimulation of insulin receptor synthesis.     

Biosynthesis of alpha-fetoprotein in cultured hepatoma cells

Pulse and pulse-chase experiments demonstrated that a heterogeneous polypeptide with an apparent Mr = 68,000 was the first intracellular anti-alpha-fetoprotein (AFP)-precipitable polypeptide synthesized by rat Mc-A-RH-7777 hepatoma cells. The 68,000-dalton polypeptide may consist of polypeptides with apparent molecular weights ranging from 68,000 to 70,000. It was the precursor of two intracellular anti-AFP-precipitable polypeptides of 69,000 and 73,000 apparent molecular weight. The latter were secreted into the medium without further processing. The anti-AFP-precipitable polypeptides in both cells and medium incorporated [3H]glucosamine, indicating that these polypeptides are at least partially glycosylated. The 68,000-dalton polypeptide in cells was bound mostly to concanavalin A-Sepharose, whereas the 69,000-dalton polypeptide was entirely unbound. The 73,000-dalton polypeptide consisted of concanavalin A-bound and -unbound variants. Tunicamycin completely abolished the uptake of [3H]glucosamine into anti-AFT-precipitable polypeptides in both cells and medium, and the resulting polypeptide of apparent Mr = 66,000 did not bind to concanavalin A-Sepharose. Tunicamycin did not affect the synthesis or secretion of AFP by hepatoma cells.     

Processing of atriopeptin prohormone by nonmyocytic atrial cells.

Atriopeptin (AP) is synthesized and stored in the mammalian atria as a 126 amino acid prohormone (AP126). Upon secretion, the prohormone undergoes site specific proteolysis within the atria to yield the carboxyl terminal 28 amino acid hormone (AP28). The atrial cell responsible for AP126 bioactivation has not yet been determined. Primary neonatal rat atrial cell cultures were generated with and without depletion of nonmyocytic cells. The molecular form of AP detected in the conditioned media of mixed cultures was determined to be AP126. Addition of dexamethasone to these cultures resulted in the appearance of a peptide that co-migrated with AP28. In contrast, no AP126 processing was detected in the conditioned media of myocyte enriched cultures when grown in the presence of dexamethasone. Readdition of nonmyocytic atrial cells to myocyte enriched cultures successfully reconstituted the steroid induced AP126 processing. Incubation of recombinant AP126argarg with nonmyocytic atrial cell cultures resulted in the generation of AP28argarg. We conclude that a nonmyocytic atrial cell is responsible for AP126 processing in vitro.     

Lack of inhibitory effect of alpha-human atrial natriuretic polypeptide on cortisol secretion in cultured adrenocortical adenoma cells from the patients with Cushing's syndrome

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on cortisol secretion by adrenocortical adenoma cells from patients with Cushing's syndrome (CS cells) in primary monolayer cultures, compared to cultured normal adrenal cells, were studied. alpha-hANP significantly inhibited cortisol secretion by human normal adrenal cells in culture, but had no direct effect on cortisol secretion from CS cells, in the presence or absence of 10(-8) M ACTH. alpha-hANP enhanced the accumulation of intracellular cyclic GMP in normal adrenal cells in culture, but not in CS cells. Visualization of [125I] iodo-alpha-hANP-specific binding sites by an in vitro receptor autoradiographic technique showed that these sites were lacking in adrenocortical adenoma tissues. These results suggest that the loss of alpha-hANP inhibitory effect on cortisol secretion in CS cells may be due to the absence of alpha-hANP receptor sites.     

Glucocorticoid enhancement of glucocorticoid production by cultured ovine adrenocortical cells

The present study examines the effect of chronic treatment with glucocorticoids on the steroidogenic activity of ovine adrenocortical cells in vitro. Cells cultured in the presence of 10(-9) to 10(-5) M dexamethasone produced more glucocorticosteroids in response to ACTH1-24, forskolin or 8 BrcAMP than did control cells. Such an enhancing effect required more than 5 h of treatment and was maximal at 30 h; it was both concentration-dependent and steroid-specific. The maximal secretion of corticosteroids was observed when cells were exposed to 10(-7) M dexamethasone; with higher concentrations the response to ACTH1-24 decreased steadily; the ED50 was 2.8 +/- 0.8 nM. Cortisol and corticosterone enhanced ACTH1-24-induced steroidogenesis to the same extent as dexamethasone, but at concentrations roughly 100-fold higher than for dexamethasone. Testosterone and 17 beta-oestradiol had no enhancing effect. Dexamethasone not only enhanced the maximal steroidogenic response to ACTH1-24 but also decreased its ED50 3-fold. Treatment of cultures with the antiglucocorticoid RU 38486 resulted in a dose-dependent, time-dependent, decrease in ACTH1-24-induced corticosteroid output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. The production of corticosteroids by dexamethasone-treated cells incubated in the presence of 22(R)-hydroxycholesterol or of exogenous pregnenolone was similar to that of control cells. The enhancing effect of dexamethasone was also observed when cultures were performed in the absence of insulin and/or in serum-free media. These data suggest that chronic exposure to glucocorticoids is necessary for the full steroidogenic activity of ovine adrenocortical cells. Moreover, they indicate that glucocorticoids exert their effect at least at two different levels in the cell: (i) on the adenylate cyclase system and (ii) at step(s) beyond cAMP but before pregnenolone formation.     

EFFECTS OF DEXAMETHASONE AND NERVE GROWTH FACTOR ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE AND ADRENALINE IN ORGAN CULTURES OF NEWBORN RAT SUPERIOR CERVICAL GANGLION

Abstract— Phenylethanolamine N -methyltransferase (PNMT) and adrenaline (A) have been studied in organ cultures of neonatal rat sympathetic ganglia. Organ culture for 2 days without added nerve growth factor (NGF) caused a fall in noradrenaline (NA) and PNMT contents but there was no change in dopamine (DA) or A contents compared to controls. However, in the presence of dexamethasone, there was a marked increase in both PNMT activity and A content, but no change in NA or DA content. Addition of NGF to cultures stimulated with dexamethasone caused no further significant change in PNMT activity or A content, whereas both NA and DA were increased. Prolonged culture without NGF, in the presence of dexamethasone resulted in reductions in both NA and DA content, but the high levels of PNMT activity and A content were sustained. The results are consistent with the hypothesis that both PNMT and A are not contained in the noradrenergic cell bodies but are located chiefly within the small intensely fluorescent cells in sympathetic ganglia.     

Mitogenic effect of fibroblast growth factor on early passage cultures of human and murine fibroblasts

Fibroblast growth factor (FGF), a polypeptide that has been shown to stimulate division in 3T3 cells, was tested for mitogenic effects on diploid, early-passage cells from human and murine sources. The quantitative assay of [3H]thymidine incorporation into acid-insoluble material showed that FGF at low concentrations (10 minus 9 M) was more effective than additional serum for provoking the initiation of DNA synthesis in human foreskin fibroblasts or mouse fibroblasts maintained in 5 or 10% serum, respectively. The growth of the human fibroblasts was twice as fast in the presence of FGF plus 10% calf serum as it was in the presence of 10% calf serum or 20% fetal calf serum alone. The addition of FGF to primary cultures of mouse fibroblasts in 0.4% serum resulted in a twofold increase in cell number compared to controls. In contrast to results obtained with 3T3 cells, neither insulin nor a glucocorticoid potentiated the effects of FGF on either human or mouse cells.