In vitro growth rate of placental fibroblasts is developmentally regulated.

Placental cells of mesenchymal origin were used to study the regulation of fetal growth at the cellular level. A significant difference in the in vitro growth rates of placental fibroblasts was observed as a function of gestational age. Cells derived from 10-19-wk placentae exhibited proliferative rates two to three times greater than cells derived from 7-9-wk placentae (16-30 h vs. 30-60 h, P less than 0.001). The proliferation rate remained stable throughout multiple passages in culture. Additionally, these two groups of cell strains exhibited marked differences in their responsiveness to mitogenic stimuli. Using maximal effective concentrations, insulin-like growth factor I interacted synergistically with epidermal growth factor and fibroblast growth factor to stimulate DNA synthesis in cells derived from 10-19-wk placentae. By contrast, the interaction of insulin-like growth factor 1 with epidermal growth factor and fibroblast growth factor exhibited significantly less synergy in 7-9-wk cells. These findings argue that the accelerated growth rate of human fetal cells results primarily from developmental events intrinsic to the cells and is associated with enhanced responsiveness to the mitogenic action of peptide growth factors.

IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): evidence for MAP kinase-dependent and MAP kinase-independent mechanisms.

OBJECTIVE: IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model. DESIGN: The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo(b30) choriocarcinoma cells using the non-metabolizing amino acid analog, [(3)H]-alpha-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation. RESULTS: Maximal stimulating concentrations of IGF-I (100 ng/ml) stimulated AIB transport by 30-40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-I-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 miccroM), an inhibitor of MAP kinase activation, inhibited 20-30% of basal AIB transport but did not inhibit IGF-I-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms. CONCLUSIONS: These data demonstrate that IGF-I stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo(b30) cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs.

Evidence for carrier-mediated transport of glucocorticoids by human placental membrane vesicles.

Glucocorticoid uptake by isolated placental membrane vesicles has been studied in an attempt to identify a membrane-mediated carrier mechanism. A preliminary communication from this laboratory has reported that uptake of the glucocorticoid corticosterone by these vesicles was a time-dependent, saturable, osmotically sensitive process (Fant, M.E., Harbison, R.D. and Harrison, R.W. (1979) J. Biol. Chem. 254, 6218-6221), but did not conclusively demonstrate a carrier mechanism. Further studies of labeled corticosterone uptake by placental vesicles are described herein which indicate that steroid uptake by these vesicles is a carrier-mediated process. We found that corticosterone uptake was temperature-sensitive, and an apparent phase-transition effect on the rate of uptake was seen to occur at approximately 16 degrees C. Treatment of the vesicles with phospholipase A2 and the sulfhydryl group attacker, p-chloromercuriphenylsulfonate, inhibited corticosterone uptake. In contrast to our previous findings in intact cells, neuraminidase treatment of membranes did not inhibit steroid uptake, perhaps indicating a species variation. Lastly, it was possible to show that corticosterone movement across the membrane exhibited countertransport, a phenomenon common only to carrier-mediated transport mechanisms. These studies show that placental vesicles accumulate corticosterone by a carrier-mediated mechanism.

A potential role for endothelin-1 in human placental growth: interactions with the insulin-like growth factor family of peptides.

Endothelin-1 (Et-1) stimulated DNA synthesis in placental fibroblasts in a dose-dependent manner, as measured by [3H]thymidine incorporation (ED50, 0.2-0.3 ng/mL). Insulin-like growth factor-I (IGF-I) interacted synergistically with Et-1 to potentiate the stimulation of DNA synthesis. Additionally, Et-1 stimulated the turnover of phosphoinositides in a time- and concentration-dependent manner (ED50, 1 ng/mL), as measured by a 2- to 3-fold increase in the total accumulation of [3H]inositol phosphates. This was accompanied by a 2- to 3-fold rise in intracellular calcium, as measured by fura-2 fluorescence. IGF-I, however, had no potentiating effect on Et-1-stimulated phosphoinositide turnover or increase in cytosolic Ca2+. The ability of Et-1 to stimulate the production of IGF-II and IGFBPs by placental fibroblasts was then studied. Western ligand blot analysis using an [125I]IGF-II probe revealed the presence of six major binding proteins corresponding to 42, 38, 35, 32, 31, and 24 kilodaltons. Et-1 (50 ng/mL) stimulated all binding fractions concordantly. This was accompanied by a similar increase in immunoreactive IGF-II secretion, as assessed by a specific RIA. No increase in immunoreactive IGF-I was observed. The ability of the placenta to produce Et-1 was examined by Northern blot analysis. Placentae at 14 and 17 weeks gestation expressed small amounts Et-1 mRNA, whereas significantly higher levels of mRNA were expressed at term. These data suggest that the human placenta produces Et-1 in a developmentally regulated manner that may act via paracrine and/or autocrine mechanisms to regulate placental growth.

An autocrine/paracrine role for insulin-like growth factors in the regulation of human placental growth.

Tissue derived from preterm (9-19 weeks gestation) and term (38-41 weeks gestation) human placentae were examined for their ability to synthesize and secrete insulin-like growth factors (IGFs) in organ culture. IGF-I was measured by a specific RIA, and IGF-II by a rat placental membrane radioreceptor assay. First, explants of placental tissue were maintained in organ culture. These explants secreted immunoreactive IGF-I (IR-IGF-I). There were no differences in the IR-IGF-I content of media conditioned by term and preterm placentae under these conditions. The similarity of this material to authentic human IGF-I was supported by parallel displacement in a specific RIA and coelution during Sephadex G-50 gel filtration. Second, monolayer cultures of fibroblasts from normal human preterm placentae (15-19 weeks gestation) were established. Confluent monolayers of these fibroblasts secreted IR-IGF-I (3-10 pg/10(5) cells X 40 h). IR-IGF-I secretion was reversibly inhibited by 5.3 microM cycloheximide, suggesting that the IR-IGF-I was the result of de novo protein synthesis. IR-IGF-I secretion was stimulated 5-fold by platelet-derived growth factor (0.6 U/ml). The response of monolayers of placental fibroblasts to IGF-I also was tested. IGF-I stimulated alpha-[3H]aminoisobutyric acid transport in these fibroblasts, with half-maximal stimulation occurring at 2-3 ng/ml. Stimulation of alpha-[3H]aminoisobutyric acid uptake by IGF-I correlated with specific binding of [125I]iodo-IGF-I. Half-maximal inhibition of [125I]iodo-IGF-I binding occurred at 2-3 ng/ml IGF-I. Placental tissue also secreted IGF-II-like activity, as measured by radioreceptor assay. Media conditioned by placental explants contained 15-20 ng/mg protein X 48 h, and media conditioned by placental fibroblasts contained 3-7 ng/10(5) cells X 40 h IGF-II determined by radioreceptor assay. These data support the hypothesis that the human placenta produces IGFs (IGF-II and/or IGF-I) that act locally to regulate placental growth.

Soluble HLA-G in human placentas: synthesis in trophoblasts and interferon-gamma-activated macrophages but not placental fibroblasts.

The HLA class Ib antigen, HLA-G, is highly expressed in early gestation placentas where it is believed to modulate maternal-fetal immunological interactions. In this study, soluble isoforms (sHLA-G) encoded by intron 4-retaining transcripts were identified in first trimester placentas by immunohistochemistry using a mAb specific for the C-terminus of sHLA-G. Immunoreactive sHLA-G protein was localized to trophoblast cells and to villous mesenchymal cells with the morphological features of macrophages. Reverse transcriptase polymerase chain reaction analysis which used primers specific for intron 4 and the 3' untranslated region of the HLA-G gene showed that transcripts encoding sHLA-G were present in the trophoblast-derived Jeg-3 cells as well as interferon-gamma-activated myelomonocytic U937 cells but were absent and uninducible in placental fibroblasts. These results indicate that placental sHLA-G is synthesized in trophoblast cells and activated placental macrophages and support the postulate that placenta-derived sHLA-G modulates maternal and fetal immune cell functions during pregnancy.

Insulin-like growth factor binding proteins (BP) from human placenta are immunologically related to the growth hormone dependent binding protein in adult human serum (BP-53).

Preterm human placentae produce IGF-specific binding proteins. Placental binding proteins are immunologically similar to the growth hormone dependent binding protein in human serum (BP-53) and not the growth hormone independent binding protein (BP-28) in human serum and amniotic fluid. Placental production occurs in mesenchymal cells located in the villous core.

Production of insulin-like growth factor binding protein(s) (IGF-BPs) by human placenta: variation with gestational age.

Preterm human placentae produce insulin-like growth factor- (IGF-)specific binding protein(s). Competitive binding experiments revealed at least two distinct, high-affinity binding sites for IGF-I and IGF-II. Gel permeation chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the binding protein(s) suggested that they possess a molecular weight in the 45 to 50 kD range. In contrast, term placental tissue produced almost no detectable binding protein activity.

Type I insulin-like growth factor receptors in the BeWo choriocarcinoma cell (b30 clone) during cell differentiation.

The expression of insulin-like growth factor (IGF) receptors in the differentiating human trophoblast was studied using the b30 clone of the BeWo choriocarcinoma cell line (BeWob30) as a model system. This clonally derived cell line differentiates over 48-72 h, in culture, to form syncytiotrophoblasts when intracellular cAMP levels are elevated by exposure to 100 microM forskolin (FSK). IGF receptors were studied at various times during the differentiation process by measuring the specific binding of [125I]-IGF-I and [125I]-IGF-II to attached cells. First, [125I]-IGF-I bound to a single class of binding sites in the untreated cells (KD approximately 1-2 x 10-10 M) that exhibited binding specificity characteristic of the type I IGF receptor (IGF-I > or = IGF-II > > Insulin). FSK treatment resulted in a two- to threefold increase in the number of these binding sites. Increased receptor expression was observed as early as 24 h after FSK treatment and remained elevated for at least 72 h. Next, [125I]-IGF-II bound to two classes of binding sites in the untreated cells, a high-affinity (KD approximately 2.5 x 10(-10) M), low-capacity site and a low-affinity (KD approximately 6 x 10(-9) M), high-capacity site. The Bmax and KD of the high-affinity site suggested that it represented the type I IGF receptor. Competition studies revealed that 15-20 per cent of total [125I]-IGF-II binding only was sensitive to IGF-I competition in the untreated cells. After FSK treatment, however, unlabelled IGF-I inhibited 60-70 per cent of specific [125I]-IGF-II binding. Scatchard analysis revealed a two- to fourfold increase in the number of both binding sites with no change in their respective binding affinities. Cross-linking analysis demonstrated that [125I]-IGF-II bound to two structurally distinct binding sites in the untreated BeWob30 cell consistent with both the type I and II IGF receptors. After FSK treatment, however, there was an increase in the relative amount of [125I]-IGF-II associated with the higher affinity type I IGF receptor. The BeWob30 cells expressed no insulin receptors at any stage of differentiation. These data demonstrate that the BeWob30 choriocarcinoma cell line expresses both type I and II IGF receptors. Induction of cell differentiation is associated with an increase in type I IGF receptors expressed at the cell surface. These receptors bind IGF-II with high-affinity, providing additional binding capacity for locally available IGF-II. These data are consistent with specific roles for the type I IGF receptor in regulating differentiated trophoblast cell function. Furthermore, the early rise in type I IGF receptor number suggests they may play a regulatory role in the differentiation process itself.

Human placental endothelin: expression of endothelin-1 mRNA by human placental fibroblasts in culture.

Endothelin-1 (ET-1) mRNA is expressed by the human placenta in a developmentally regulated manner and has been shown to stimulate the growth of placental mesenchymal cells. The ability of placental fibroblasts to express preproET-1 mRNA was studied to determine if ET-1 could potentially participate via autocrine mechanisms in the proliferation of placental fibroblasts. Fibroblasts were isolated from normal placentae at various gestational ages (7-19 weeks and term) and their abilities to express preproET-1 mRNA in culture evaluated by Northern analysis. Sparse, rapidly growing cultures of placental fibroblasts expressed preproET-1 mRNA at each gestational age in the presence of 10% FBS. The regulation of preproET-1 expression in placental fibroblasts was studied by exposing cells to known mitogenic stimuli. Quiescent, confluent monolayers of placental fibroblasts expressed no detectable levels of preproET-1 mRNA under basal conditions. Epidermal growth factor (EGF, 10 mg/ml), transforming growth factor-beta 1 (TGF-beta 1, 5 ng/ml), or interleukin 1 beta (IL-1 beta) alone, had no significant effect on steady state preproET-1 mRNA levels. Cycloheximide, an inhibitor of protein synthesis, increased the steady state levels of preproET-1 mRNA at a concentration of 10 micrograms/ml. In the presence cycloheximide, IL-1 beta markedly stimulated preproET-1 mRNA expression, whereas EGF was less effective. TGF-beta 1 had no effect in the presence or absence of cycloheximide. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA, 20 nM) exerted a small stimulatory effect on preproET-1 mRNA expression which was not influenced by cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Glypican-3 (GPC3) expression in human placenta: localization to the differentiated syncytiotrophoblast.

The expression of glypican-3 (GPC3), a heparan-sulfate proteoglycan associated with the Simpson-Golabi-Behmel fetal overgrowth syndrome, was studied in normal human placental tissue and cell lines derived from human placentae. Cytotrophoblasts derived from term placentae expressed GPC3 mRNA at low levels in culture. GPC3 mRNA expression increased markedly during trophoblast differentiation. By contrast, fibroblast cell lines derived from normal placentae did not express GPC3 in culture. Similarly, choriocarcinoma cell lines derived from human placentae (BeWo, JAR, and JEG) failed to express GPC3 mRNA. In situ hybridization confirmed the localization of GPC3 mRNA to the syncytiotrophoblast. Furthermore, immunohistochemical staining of paraffin imbedded placental tissue demonstrated intense staining of the syncytiotrophoblast cell layer and less intense staining of cytotrophoblasts. No staining of mesenchymal elements was noted. These data confirm the presence of GPC3 in human placenta and suggest it is expressed by the differentiated syncytiotrophoblast at term.

Developmentally regulated expression of IGF binding protein-3 (IGFBP-3) in human placental fibroblasts: effect of exogenous IGFBP-3 on IGF-1 action.

Preterm, human, placental fibroblasts exhibit growth rates, in vitro, that vary with gestational age. The observed increase in proliferation rate is associated with enhanced mitogenic responsiveness to IGF-I. IGFBPs can either potentiate or inhibit IGF action at the cellular level. The production of IGFBPs by placental fibroblasts was studied as potential modulators of their responsiveness to IGFs. Human placental fibroblasts were obtained at various gestational ages and maintained in culture. IGFBP-3 protein and mRNA expression were assessed by Northern and ligand blot analyses. First, media conditioned by fibroblasts, in culture, were subjected to ligand blot analysis. Multiple species of IGFBPs were present in each cell line tested. IGFBP-3, migrating as a doublet at approx. 38/42 kDa, was the predominant IGFBP species present. Other IGFBPs of 22-35 kDa were also present. The secretion of IGFBP-3 exhibited a marked decrease at 10-15 weeks gestation relative to 8-9 week fibroblasts but began to increase again by 19 weeks. We next studied the expression of IGFBP-3 mRNA. Total cellular RNA was obtained from rapidly growing cells and subjected to Northern analysis. Placental fibroblasts exhibited decreased steady state levels of IGFBP-3 mRNA at 10-15 weeks gestation consistent with its decreased protein expression. The ability of IGFBP-3 to influence IGF-1 stimulated DNA synthesis was studied in 10 week placental fibroblasts as measured by [3H]thymidine incorporation. IGFBP-3 inhibited IGF-1 (3.3 nM) stimulated DNA synthesis in a dose-dependent manner when added simultaneously with IGF-1 or preincubated with the cells for 48 h prior to the addition of IGF-1. By contrast, maximum effective concentrations of IGFBP-3 (52 nM) potentiated the effect of IGF-1 50-200% when preincubated with bovine fibroblasts for 48 h prior to the addition of IGF-1. These data suggest that IGFBP-3 production is developmentally regulated in human placental fibroblasts and inhibits their mitogenic response to IGF-1. The regulated expression of IGFBP-3 may contribute to the altered growth rate and IGF responsiveness exhibited by placental fibroblasts, in vitro.

Circulating levels of IGFs and IGF binding proteins in human cord serum: relationships to intrauterine growth.

Cord sera were obtained from 44 term, human infants exhibiting various patterns of intrauterine growth and were assayed for IGF-1, IGF-2, and IGFBP-1, 2, and 3 by specific RIAs. Serum levels were correlated with birth weight (BW), ponderal index (PI), and placental weight (PW). Total IGF-1 levels correlated significantly with BW (r = 0.392), PW (r = 0.351), and PI (r = 0.481). By contrast, the correlation of IGF-2 with birth weight was not statistically significant (r = 0.264, P = 0.091). The association of IGF-2 with PI, however, was significant (r = 3.348, P = 0.024). IGFBP-3 exhibited significant correlations with BW, PI, and PW, similar to those seen with IGF-1. IGFBP-1 and IGFBP-2, however, were not significantly related to growth parameters. IGF-1 levels correlated strongly with IGFBP-3 levels (r = 0.646, P = 0.001). By contrast, IGF-1 correlated with the reciprocal of both IGFBP-1 and IGFBP-2. Based upon in vitro affinity constants, theoretical concentrations for each [IGF/IGFBP] complex, free IGFs, and free IGFBPs were calculated for each infant. Multiple regression analysis was performed including all 11 calculated variables and correlated with each growth parameter. This analysis revealed that an integrated expression of IGF activity exhibited stronger correlations with growth than each individual peptide species (BW, r = 0.681; PI, r = 0.660; PW, r = 0.658). These data further support roles for IGF related peptides (IGFRPs) in human fetal and placental growth and suggest regulatory/counterregulatory roles for the IGFBPs. It also supports the hypothesis that individual IGFRPs interact in a complex manner to define 'net IGF activity' in relation to fetal growth and/or metabolic status.

IGF binding protein-1 (IGFBP-1) is preferentially associated with the fetal-facing basal surface of the syncytiotrophoblast in the human placenta.

IGF receptors are expressed in a spatially polarized manner on the syncytiotrophoblast cell membrane. We therefore examined the hypothesis that IGFBPs expressed at the maternal-fetal interface interact with distinct surfaces of the syncytiotrophoblast membrane to modulate IGF function. Membrane vesicles were prepared specifically from the maternal-facing, microvillous membrane (MVM) and the fetal-facing, basal membrane (BM) surfaces of the syncytiotrophoblast. The association of IGFBPs with each membrane preparation was determined by ligand blot analysis. A doublet migrating at 38/42 kD was detected in both MVM and BM preparations. Selective immunoprecipitation followed by ligand blot analysis identified this IGF binding species as IGFBP-3. Additionally, a protein migrating at approximately 29 kD was associated primarily with the BM. This protein was identified as IGFBP-1 by both immunoprecipitation and ligandblotting techniques. Non-denaturing PAGE revealed five distinct bands corresponding to different degrees of phosphorylation. The phosphorylation pattern of BM-associated IGFBP-1 was identical to that of native IGFBP-1 in amniotic fluid. Immunohistological analysis of term placenta revealed IGFBP-1-specific staining of the syncytiotrophoblast and the fetal capillary/pericapillary bed. The localization of IGFBP-1 to a distinct compartment within the fetal placenta, not in proximity to the syncytiotrophoblast type I IGF receptor, suggests it may play a role in regulating/targeting IGF activity within the stromal compartment or by exerting IGF-independent effects on the basal surface of the syncytiotrophoblast. The nature of its binding to the BM has not been determined.

Heavy drug abuse in Sweden 1979 - a national case-finding study.

An investigation was carried out as a case-finding study to estimate the scale of heavy drug abuse in Sweden. Just over 8200 persons were reported as heavy drug abusers, 80% of these as injecting. For the majority of those reported more than one type of drug was indicated. There was concurrent abuse of alcohol by a majority of the heavy drug abusers. After correction of non-response with a modified capture-recapture technique and for erroneous classification, the scale of heavy drug abuse was estimated at 10000 - 14000 persons.

Insulin and insulin-like growth factors in human development: implications for the perinatal period.

The physiologic and cellular mechanisms regulating fetal growth cannot be adequately described by regulatory mechanisms important postnatally. This review summarizes recent advances in clinical medicine, cell and molecular biology, and physiology showing the central and essential roles of insulin and the insulin-like growth factor family of peptides in regulating fetal growth. Moreover, the importance of insulin-like growth factors in tissue-specific growth regulation during critical periods of development suggest that these mechanisms may also be relevant to the pathogenesis of tissue injury in the preterm infant, and may offer therapeutic strategies aimed at reducing morbidity associated with prematurity. Illustrations of how the insulin-like growth factor axis may represent potential therapeutic targets for specific clinical problems facing the newborn are briefly discussed.

Insulin-like growth factors (IGFs) and IGF binding proteins-1, -2, and -3 in newborn serum: relationships to fetoplacental growth at term.

Cord sera were obtained from term, Chilean newborns exhibiting various patterns of intrauterine growth and assayed for IGF-1, IGF-2, IGFBP-1, IGFBP-2, and IGFBP-3 by specific radioimmunoassays (RIA). Serum levels of each peptide were correlated with birth weight (BW), ponderal index (PI), and placental weight (PW). Total IGF-1 levels correlated with BW (r = 0.665, P = 0.0001). PI (r = 0.527, P = 0.004), and PW (r = 0.596, P = 0.0017). In contrast, IGF-2 failed to correlate with any growth parameter. Of the three binding proteins, IGFBP-3 exhibited the strongest relationship to each growth parameter. IGFBP-3 correlated significantly with BW (r = 0.71, P < 0.0001), PI (r = 0.782, P < 0.0001), and PW (r = 0.57, P = 0.0029). In addition IGFBP-3 levels positively correlated to IGF-1 levels (r = 0.614, P = 0.0005). By contrast, circulating IGFBP-1 and IGFBP-2 were inversely related to IGF-1 levels. All five peptides were subjected to multiple regression analysis and related to BW. Significant relationships between the predicted BW and the actual BW were observed in these infants (r = 0.802, P = 0.0006). The BWs of a cohort of unrelated North American infants were also predicted using the Chilean-derived equation and found to be significantly related to their actual BWs (r = 0.453, P = 0.0033). These relationships were strengthened by the inclusion of estimated gestational age (EGA) as an independent variable. These data point to particularly important roles for IGF-1 and IGFBP-3 in regulating fetal growth at term, and suggest that they are regulated in a coordinated manner during the latter stage of gestation. Furthermore, they suggest that IGFBPs play multiple, and potentially opposing, regulatory roles in modulating IGF action. Lastly, an integrated expression of IGF activity derived from one population significantly correlated with newborn BW in a geographically and culturally distinct population.

Mercury, cadmium, lead and selenium in ringed seals (Phoca hispida) from the Baltic Sea and from Svalbard.

The concentrations of mercury (Hg), cadmium (Cd), lead (Pb) and selenium (Se) were determined in liver, kidney and muscle samples from 20 Baltic ringed seals (Phoca hispida botnica) (3-32 years), and from 17 ringed seals (Phoca hispida) (0-20 years) from Svalbard, in the Arctic. The concentrations of Hg and Se were considerably higher in the Baltic ringed seals, but the Cd concentrations lower than in the Svalbard ringed seals. There was no big geographical difference with respect to Pb concentrations. Se and Hg concentrations showed a significant positive correlation in both regions. By comparison with earlier studies on Baltic seals, the metal concentrations have remained at the same level since the 1980s. Of the metals we studied, only the level of Hg in Baltic ringed seals can be considered high (mean 53 mg/kg, range 6.5-124 mg/kg wet wt. for liver), but probably not high enough to cause metal intoxication. No pathological changes associated with metal intoxication were observed in the seals.

Heterogeneity of AtT-20 cell glucocorticoid binding sites: evidence for a membrane receptor.

In previous studies we have found that intact AtT-20 cells contained two glucocorticoid binding sites with distinctly different affinities and specificity. In this paper, the nature of these sites was investigated by studying glucocorticoid binding to cytosol and to plasma membranes isolated from AtT-20 mouse pituitary tumor cells. Plasma membrane vesicles were isolated from AtT-20 cells and found to take up alpha-aminoisobutyric acid, indicating that they were properly oriented and functionally intact. Corticosterone bound to these vesicle in a time- and temperature-dependent manner. The binding exhibited a glucocorticoid preference since non-glucocorticoids such as progesterone, testosterone or estradiol were unable to inhibit binding. In addition, binding specificity differed from that of the cytoplasmic receptor since the synthetic glucocorticoids were also ineffective competitors. The major inhibitors of binding were corticosterone greater than 11-dehydrocorticosterone greater than 11-ketoprogesterone greater than cortisol. In other complementary studies, AtT-20 cell cytosol was tested to determine whether heterogenous soluble sites exhibiting a preference for the natural vs the synthetic steroid could also be identified. We found that binding sites for both steroid classes were approximately similar in number, specificity and behavior on ion-exchange chromatography. We conclude that, in addition to a classical soluble cytoplasmic glucocorticoid receptor, AtT-20 cells contain plasma membrane glucocorticoid binding sites. The affinity and specificity of these sites for the natural ligand, corticosterone, suggest that they play an important role in the subcellular mechanism of glucocorticoid action.