Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Rapid Internalization and Intracellular Metabolic Processing of Exogenous Ganglioside by Cerebellar Granule Cells Differentiated in Culture

Ganglioside GM1, tritiated at the level of the long chain base (sphingosine) [( Sph-3H]GM1), sialic acid (N-acetylneuraminic acid) [( NeuAc-3H]GM1), or terminal galactose [( Gal-3H]GM1) was supplied to cerebellar granule cells differentiated in vitro, and its metabolic processing was followed with pulse time. Using [Sph-3H]GM1 and [NeuAc-3H]GM1 the formation of radioactive compounds of catabolic origin (GM2, GM3, lactosylceramide, glucosylceramide, and ceramide) started being detectable at 10-15 min of pulse, whereas compounds of biosynthetic origin (GD1a, GD1b, GT1b, O-acetylated GT1b, spingomyelin, and sialoglycoprotein) appeared after 15-30 min of pulse. Using [Gal-3H]GM1 two radioactive substances were formed, GD1a and GT1b, with the former (produced by direct sialosylation of GM1) appearing after 30 min of pulse and the latter (formed by biosynthetic recycling of released galactose) appearing after 2 h. The radioactivity linked to all metabolites increased with increasing pulse time until 4 h. The percentage of GM1 taken up and subjected to metabolic processing was found to increase from 1.8% after 10 min of pulse to 12.5% after 4 h. Cerebellar granule cells were able to release enzymes of lysosomal origin, beta-D-N-acetylhexosaminidase and beta-D-galactosidase, into the culture medium, with the release being markedly decreased by the absence in the medium of fetal calf serum, a condition that was used for studying exogenous GM1 uptake and metabolization. However, these enzymes exerted no activity at the pH of the culture medium, and no radioactive gangliosides, besides GM1, were detected in the culture medium during pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

New approaches to the study of sphingolipid enriched membrane domains: the use of electron microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture. To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3 x 10(-7) M [1-(3)H]sphingosine. [1-(3)H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-(3)H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy. Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.     

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture

We studied the effect of fetal calf serum and serum protein fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine β-globulins reduced this transfer, although to a lesser extent than whole serum. α-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A γ-globulin fraction failed to exhibit any effect on the uptake of [¹⁴C]phosphatidylcholine by the cells.All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles preincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine to parenchymal cells.These observations were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of the liposomal surface membrane as a result of the binding of certain serum proteins. On the other     

New approaches to the study of sphingolipid enriched membrane domains: The use of microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture.To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3×10^–7 M [1-³H]sphingosine. [1-³H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-³H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy.Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture.

We studied the effect of fetal calf serum and serum proteins fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296--307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine beta-globulins reduced this transfer, although to a lesser extent than whole serum. alpha-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A gamma-globulin fraction failed to exhibit any effect on the uptake of [14C]phosphatidylcholine by the cells. All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles reincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine ot parenchymal cells. These observation were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of tm proteins. On the other hand, we cannot rule out that plasma membrane modifications are involved in the mechanism of inhibition as well.     

Growth of Myeloma MPC-11 Cells in Serum-free Growth Factor Supplemented Medium

A myeloma MPC-11 cell line, which originated in a mouse plasma cell tumor, was able to proliferate in the absence of serum in a synthetic medium supplemented with transferrin, ethanolamine and selenium. The cells showed almost the same growth rate in this medium as in medium with 10% fetal calf serum added. Immunoglobulins secreted into the serum-free medium by the cells were easily separated from medium components by polyacrylamide gel electrophoresis.     

Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

Hyaluronic acid associated with the surfaces of cultured fibroblasts is linked to a serum-derived 85-kDa protein.

Hyaluronic acid (HA) was extracted from the cell layer of cultured mouse dermal fibroblasts with 6 M guanidine HCl in the presence of 8% (w/v) Zwittergent. HA could be separated from the bulk of extracted proteins by consecutive isopycnic centrifugation and gel and ion-exchange chromatography under dissociative conditions. The final preparation was the complex of HA (viscosity average molecular weight approximately 2 x 10(6)) and a protein of Mr approximately 85,000 in a molar ratio of 1:1. Since the extraction procedure employed has been shown to break most noncovalent bonds between HA and proteins, they would appear to be covalently linked. However, the HA-binding protein remained unlabeled even after long incubation of the cells in the presence of a highly radioactive amino acid mixture, suggesting that it is an exogenous protein derived from the fetal calf serum added to culture medium. The presence of a HA-binding 85-kDa protein could in fact be demonstrated in fetal calf serum as well as sera from various other sources. This protein cross-reacted with antibodies raised against the HA-protein complex purified from cultured mouse dermal cells and was retained on octyl-Sepharose. Like the cell-derived 85-kDa protein, the serum 85-kDa protein, once bound to HA, could not be released from the complex by various dissociative procedures. These results, taken together, suggest that the hydrophobic serum protein can be intercalated into cell surface membranes, thereby mediating the binding of HA to the cell surface.     

Incorporation In Vivo and In Vitro of Radiolabeled Sphingolipid Precursors into Paramecium tetraurelia Lipids

ABSTRACT Paramecium tetraurelia contains high concentrations of ethanolamine sphingolipids, especially in its ciliary membrane. Three ethanolamine sphingophospholipids with different long chain bases (dihydrosphingosine, sphingosine and phytosphingosine), and their phosphonyl analogs, were previously identified and characterized. In the present study, radiolabeling experiments on lag- and log-phase cells were performed to investigate the extent of sphingolipid biosynthetic capacities of the ciliate. Long chain bases of sphingolipids are formed by an initial condensation reaction of serine with a fatty-coenzyme A. Thus, radiolabeled palmitic acid, stearic acid and serine were used as precursor compounds in these experiments. The results indicated that (1) sphingolipid precursors were incorporated into every major lipid fraction. (2) ethanolamine sphingophosphonolipids accumulated faster than the ethanolamine sphingophospholipids, (3) in contrast to these sphingolipids, the glycerolipid, phosphatidyethanolamine. accumulated faster than its phosphono analog, and (4) palmitic acid, but not stearic acid, was incorporated into the long chain bases of ethanolamine sphingophospho- and sphingophosphonolipids. consistent with an earlier report demonstrating that these lipids contain only C,g long chain bases. Since P. tetraurelia takes up serine and other water-soluble substrates very slowly, and catabolizes fatty acids rapidly, label is randomized in intact cells. Thus, cell-free protocols provide useful experimental systems for studies of sphingolipid biosynthesis than do intact organisms, when the uptake of precursor substrates are slow.     

Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: correlation with prostaglandin E2-induced growth inhibition

When L-1210 murine leukemia cells were incubated with 60 microM PGE2 in culture medium containing fetal calf serum for various time, cell proliferation was inhibited dependent on incubation time. However, when the medium containing PGE2 was changed every 6 h during 24 h exposure to cells, growth inhibition became much weaker. Moreover, when the medium containing PGE2 was aged by preincubating without cells at 37 degrees C, growth inhibitory effect of the medium was enhanced with preincubation time, suggesting that active growth inhibitory compound(s) accumulated during preincubation. In culture medium containing fetal calf serum, PGE2 degraded time-dependently and the major product was identified as PGA2 by HPLC. Furthermore, when cells were incubated with the medium containing 60 microM[3H]PGE2 or the same medium aged by preincubation, we observed that the radioactivity was taken up by the cells time-dependently, and identified the incorporated radioactivity as PGA2. This uptake was closely correlated with decrease in viable cell number during incubation. These results suggested that growth inhibitory effect of PGE2 was due to the metabolic dehydration of PGE2 to PGA2, and PGA2, after taken up by cells, exerted cell growth inhibition.     

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.     

Transforming growth factor-beta1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-beta1 (TGF-beta1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-beta1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-beta1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-beta1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.     

Deacylation of ceramide, triacylglycerol and phospholipids in guinea pig epidermal cells

The epidermal cells obtained by mild trypsinization of guinea pig skin were first labeled with [3H]palmitate. Interestingly, free ceramide was one of the major radioactive products. The radioactivity of ceramide was located exclusively in the N-acyl moiety, and palmitate was incorporated as such, without detectable chain modification. Sphingomyelin and glucosylceramide were not significantly labeled in spite of the active synthesis of ceramide. In order to investigate the action of lipolytic enzymes, the labeled cells were chased in a tracer-free medium. Bovine serum albumin (or fetal calf serum) and ionophore A23187 markedly stimulated the deacylation of lipids. The effects of the two stimulators were, however, distinctly different from each other. Bovine serum albumin (or fetal calf serum) caused principally the deacylation of triacylglycerol and ceramide, with little effect on that of phospholipids. In contrast, the effect of the ionophore was confined to phospholipid deacylation. In the presence of the ionophore, phosphatidylcholine and phosphatidylethanolamine were degraded to a similar extent. Palmitate was cleaved from both C-1 and C-2 positions of phosphatidylcholine. The results suggest that the lipolytic systems of the epidermal cells can respond to extracellular stimuli, and that the action of triacylglycerol lipase and ceramidase is controlled differently from that of phospholipases.     

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.     

IL 1 requirement for B cell activation revealed by use of adult serum

Fetal calf serum is an essential component of the culture medium developed by Mishell and Dutton for the immunization of murine spleen cells in vitro. Serum from adult donors (mouse, human, rabbit) does not support antibody synthesis in this system. This "deficiency" of adult serum can be overcome with IL 1. Adult serum in the presence of IL 1 is as effective in stimulating a B cell response against xenogeneic red cells as fetal calf serum. We attribute the capacity of fetal calf serum to support an immune response in the absence of exogenous IL 1 to serum factors that cause macrophages to release IL 1 endogenously. Our findings strengthen the notion that IL 1 plays an essential role in the process of B cell activation and suggests that the use of fetal calf serum should be avoided in studies concerned with the function of interleukin 1.     

Androstenedione-mediated inhibition of 11 beta-hydroxylation in monolayer cell cultures of fetal calf adrenals

The possibility that the formation of androstenedione by fetal calf adrenal cells in culture is linked to their decreased ability to form cortisol and corticosterone was investigated. Fetal calf adrenal cells metabolise radioactive adrostenedione to two major products which coelute on thin layer chromatography with 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone. When the cells are incubated with 11-deoxycortisol or 11-deoxycorticosterone in the presence of androstenedione there is a dose dependant inhibition of cortisol and corticosterone formation. Further studies with progesterone showed an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in cells incubated simultaneously with androstenedione. The results demonstrate that exogenous androstenedione can have dramatic effects on steroidogenesis in the fetal calf adrenal and suggest that the accumulation of androstenedione in the medium of cultured andrenocortical cells is responsible, at least in part, for the decreased formation of cortisol and corticosterone.