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Biomedical subjects

E R Simpson

Publications and source records attributed to E R Simpson.

At least 271 records · Page 15Linked to original sources
PubMed1982

The binding of high and low density lipoproteins to human placental membrane fractions.

It was shown previously that human placental trophoblastic cells use principally lipoprotein cholesterol for progesterone biosynthesis and that the rate of de novo synthesis of cholesterol is low. In addition, it was demonstrated that cholesterol derived from maternal plasma low density lipoprotein (LDL) rather than high density lipoprotein (HDL), is the principal source of placental cholesterol. In the present investigation, membrane fractions derived from human placenta were used to identify and characterize specific binding sites for both HDL and LDL. Pretreatment of membrane fractions with heparin resulted in an increase in the specific binding capacity for [125I]iodo-LDL 1.5 times that in membrane fractions not pretreated with heparin. Heparin pretreatment did not affect significantly the specific binding capacity of placental membranes for [125I]iodo-HDL. The specific binding capacity for [125I]iodo-LDL was 107 ng LDL protein mg-1 membrane protein, with an approximate Kd of 77 microgram LDL protein ml-1 in membranes pretreated with heparin. The specific binding capacity for [125I]iodo-HDL was much greater, equal to 323 ng HDL protein mg-1 membrane protein, with an approximate Kd of 152 microgram HDL protein ml-1. Each [125I]iodolipoprotein was specifically displaced by the corresponding respective nonradiolabeled lipoprotein. Preincubation of membranes with trypsin and pronase caused reductions in the specific binding capacity for [125I]iodo-LDL of 88% and 100%, respectively. Incubation of membranes with heparin caused displacement of [125I]iodo-LDL. However none of these treatments affected [125I]iodo-LDL. However none of these treatments affected [125I]iodo-HDL binding capacity. Similar binding sites for "125I]iodo-LDL and [125I]iodo-HDL were demonstrated in cells prepared from human placenta by trypsin digestion and maintained in monolayer culture.

Binding Sites↗
PubMed1982

Human chorionic gonadotropin binding to human fetal testes as a function of gestational age.

The characteristics of binding of hCG to testicular tissue obtained from human abortuses of 10--24 weeks gestational age were studied. Specific, saturable binding of [125I]hCG was demonstrated using homogenates of human fetal testicular tissue. The equilibrium dissociation constant ranged from 0.4 x 10(-10) M to 5.5 x 10(-10) M, a finding that is indicative of a high affinity receptor. The capacity to bind hCG was low, but varied strikingly with gestational age. The binding capacity for hCG of tissues from abortuses of gestational age less than 15 weeks and greater than 22 weeks was consistently less than 10.0 pg x mg-1 tissue (2.2 fmol x mg-1 tissue). The binding capacity for hCG of tissues from abortuses of gestational age between 15--20 weeks ranged from 2.4--29.8 pg x mg-1 tissue (0.5--6.5 fmol x mg-1 tissue) with the majority of values being greater than 10 pg x mg-1 tissue (2.2 fmol x mg-1 tissue). On the other hand, receptors for hCG in human fetal ovarian tissue were undetectable, irrespective of gestational age. It is concluded that specific high affinity binding sites for hCG are present in human fetal testes and that the binding capacity is maximum between gestational ages of 15--20 weeks. This increase in binding capacity parallels the surge in testosterone production known to occur during the same period of development. These results suggest that the increase in fetal plasma levels of testosterone during this time in gestation is the result of an increase in the sensitivity of the fetal testis to hCG caused by an increase in the number of hCG receptors, and that hCG most likely is responsible for stimulation of fetal testicular steroidogenesis in utero at this time of gestation.

Chorionic Gonadotropin↗
PubMed1981

Cholesterol metabolism in cancer cells in monolayer culture. III. Low-density lipoprotein metabolism.

The metabolism of low-density lipoprotein (LDL) was studied in neoplastic and non-neoplastic cells of human gynecological origin, in monolayer cultures. The neoplastic cells were derived from epidermoid vaginal carcinoma, epidermoid cervical carcinoma and endometrial adenocarcinoma, in various degrees of differentiation. The non-neoplastic cells were cervical fibroblasts and epithelial cells from proliferative endometrial glands. Both neoplastic and non-neoplastic cells assimilated and degraded LDL in a similar fashion to other human cells (e.g. skin fibroblasts). However, the neoplastic cells metabolized LDL at a higher rate than the non-neoplastic cell (e.g. epidermoid cervical cancer cells metabolized LDL at a 20 times higher rate than did cervical fibroblasts). Such a high rate of LDL metabolism probably enables continuously replicating cancer cells to obtain the large amounts of cholesterol required for cell membrane synthesis. If a high rate of LDL metabolism proves to be a general property of cancer cells, such a property could prove useful for tumor chemotherapy, providing cytotoxic chemicals could be incorporated within the LDL molecule.

Adenocarcinoma↗
PubMed1981

Low-density lipoprotein as a potential vehicle for chemotherapeutic agents and radionucleotides in the management of gynecologic neoplasms.

Cholesterol metabolism was studied in cells from two established gynecologic cancer cell lines which were maintained in monolayer cultures. The cell lines were derived and established from poorly differentiated epidermoid cervical carcinoma (EC-50) and endometrial adenocarcinoma (AC-258). The specific activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme of cholesterol de novo synthesis, in AC-258 cells (1,700 pmoles x mg-1 microsomal protein x min-1) was three times higher than that found in EC-50 cells (550 pmoles x mg-1 microsomal protein x min-1). However, epidermoid cervical cancer cells (EC-50) metabolized low-density lipoprotein (LDL), the major transport vehicle for cholesterol in plasma, at a very high rate (14,000 ng x mg-1 cell protein x 6 hours). This rate is fifteen times greater than the rate observed in fetal adrenal tissue and fifty times greater than the rate observed in nonneoplastic gynecologic tissue, each in organ culture. Both cancer cells (EC-50 and AC-258) in monolayer culture were shown to have specific receptors for LDL. These cancer cells demonstrate no defect in LDL metabolism, and lysosomal degradation of LDL was blocked by chloroquine. From the results of studies of specific binding of LDL in tissues obtained from nude mice it was demonstrated that membrane fractions prepared from EC-50 cells, after propagation in the mice, contained fifteen to thirty times more specific binding capacity for [125I]iodo-LDL than vital organs of the mouse, such as the liver, heart, lung, kidney, or brain. The results of these studies are suggestive that certain tumor cells might have a higher affinity for LDL than normal tissues and cytotoxic drugs or radionucleotides ligated to the LDL macromolecule may be utilized for the specific delivery of these agents.

Adenocarcinoma↗
PubMed1981

Failure of contraceptive steroids to modify human chorionic gonadotrophin secretion by hydatidiform mole tissue and choriocarcinoma cells in culture.

The effect of steroids contained in oral contraceptives, namely ethinylestradiol:17 alpha-ethinyl-1,3,5,(10)-estratriene-3, 17-diol (E) and norethindrone acetate:17 beta-acetoxy-17-ethinyl-4-estren-3-one (N), on cell replication and human chorionic gonadotropin (hCG) secretion by choriocarcinoma cells in monolayer culture and by hydatidiform mole tissue maintained in organ culture were studied. The steroids were added to the culture medium individually or in combination to achieve a range of concentrations (10-10 to 10-4), within and beyond the presumed concentration of these substances in the blood of women taking oral contraceptives. The effect of luteinizing hormone releasing hormone (LHRH) on hCG secretion by choriocarcinoma cells in monolayer culture also was investigated. The rate of hCG production by either choriocarcinoma cells in monolayer culture or by hydatidiform mole tissue maintained in organ culture was not affected by the hormones used in this study; indeed hCG secretion remained reasonably unchanged even with high concentrations of steroids (up to 10-4 M) or LHRH (up to 10-4 mg x ml-1). Cell replication, as measured by increase in amount of cellular protein and DNA, was not stimulated by either of these compounds.

Cell Division↗
PubMed1981

Evidence for a higher molecular weight precursor of cholesterol side-chain-cleavage cytochrome P-450 and induction of mitochondrial and cytosolic proteins by corticotropin in adult bovine adrenal cells.

Adult bovine adrenal cortical cells in monolayer culture were used to study the induction of cholesterol side-chain-cleavage cytochrome P-450 by corticotropin (ACTH). In the presence of 1 microM ACTH, there was a 4-fold increase in cortisol production by these cells over a 72-hr period and a corresponding increase in total cytochrome P-450 content. The incorporation of [35S]methionine into a number of cellular proteins was stimulated by the presence of ACTH in the culture medium, whereas the incorporation into other proteins was decreased. The temporal profile of these changes varied from one protein to another. Examination of the incorporation of [35S]methionine into mitochondrial protein showed an increased production of a radiolabeled protein that comigrated with the form of cytochrome P-450 known as side-chain-cleavage cytochrome upon incubation with ACTH. Thus, it appears that the cytochrome P-450scc content is increased in bovine adrenal cortical cells exposed to ACTH. Cytochrome P-450scc, synthesized in a cell-free translation system directed by RNA isolated from bovine adrenal cortical tissue or from cells, had a molecular weight of 54,500. Cytochrome P-450scc isolated from bovine adrenal mitochondria had a molecular weight of 49,000. Thus, cytochrome P-450scc is synthesized as a larger precursor that must be processed by proteolytic cleavage before or upon insertion into the mitochondrion.

Adrenal Cortex↗
PubMed1981

Estrogen formation in stromal cells of adipose tissue of women: induction by glucocorticosteroids.

Stromal cells prepared from adipose tissue of women were maintained in monolayer culture to study the regulation of aromatase activity by hormones. Aromatase activity was stimulated 20- to 100-fold by dexamethasone at a concentration of 250 nM. Half-maximal stimulation of aromatase activity was attained at a dexamethasone concentration of 2.7 nM. The stimulatory effect of dexamethasone was apparent after a preincubation time of 4 hr, and stimulation was maximal after 24 hr of preincubation. The stimulatory effect of dexamethasone was observed only when fetal calf serum also was present in the culture medium. Of the various steroids tested, dexamethasone was the most potent in stimulating aromatase activity. Cortisol was less effective than dexamethasone, whereas corticosterone, at a concentration of 250 nM, caused only a small stimulation of aromatase activity. Progesterone and deoxycorticosterone (250 nM) did not affect aromatase activity. Cytosolic fractions prepared from stromal cells that had been maintained in monolayer culture were found to contain a homogenous population of sites that specifically bound [3H]dexamethasone with relatively high affinity (Kd = 2.9 nM) and low capacity (38 fmol per mg of protein). The stimulatory effect of dexamethasone on aromatase activity was prevented by simultaneous incubation with cortisol 21-mesylate (0.1-10 microM), a compound known to block the binding of glucocorticosteroids to cytoplasmic receptors. The stimulatory effect of dexamethasone also was prevented by incubation of the cells with cycloheximide or actinomycin D. These findings are suggestive that glucocorticosteroids act to increase aromatase activity in stromal cells by inducing the synthesis of new enzyme protein.

Adipose Tissue↗
PubMed1981

Lipoprotein utilization and cholesterol synthesis by the human fetal adrenal gland.

A model proposed for regulation of steroidogenesis, lipoprotein utilization and cholesterol metabolism in HFA tissue is presented in Fig 17. We envision that the role of ACTH and cAMP in steroidogenesis and cholesterol metabolism is as follows. ACTH binds to specific receptors on the surface of the cells of the HFA gland and as a consequence, adenylate cyclase is activated, leading to increased formation of cAMP. cAMP causes activation of protein kinase that leads, presumably, to phosphorylation of specific proteins. This leads to the initiation of reactions that give rise to increased activity of key enzymes and levels of proteins involved in adrenal cholesterol metabolism. Presumably, the action of ACTH causes an increase in the activity of cholesterol side chain cleavage, the rate-limiting step in the conversion of cholesterol to steroid hormones. We suggest that once the mitochondrial cholesterol side-chain cleavage system is fully activated by ACTH, the supply of cholesterol to the mitochondria becomes rate-limiting for steroidogenesis. To meet this demand for cholesterol, a further action of ACTH results in an increase in the number of LDL receptors. LDL binds to specific receptors on the cell surface that are localized in coated pits. LDL is internalized by a process of adsorptive endocytosis and the internalized vesicles fuse with lysosomes and the protein component of LDL is hydrolyzed by lysosomal proteolytic enzymes to amino acids. The cholesteryl esters of LDL also are hydrolyzed to give rise to fatty acids and cholesterol. The liberated cholesterol is available for utilization in the biosynthesis of steroid hormones and other cellular processes. In addition, ACTH stimulates the activity of HMG CoA reductase and, thus, the rate of de novo cholesterol biosynthesis. In this way sufficient cholesterol is obtained to provide for precursor cholesterol to maintain the high rate of steroid synthesis by the HFA. HDL is not utilized as a source of cholesterol by the HFA. Because of the rapid rate of utilization of LDL by the HFA, fetal plasma levels of LDL are low and the activity of the HFA is a primary determinant of these levels. Thus, in the case of anencephaly, in which the activity of the adrenal is very low, plasma levels of LDL are 2--3 times higher than in normal fetuses, whereas plasma HDL levels are similar. In addition, in the normal neonate plasma LDL levels rise rapidly after birth, and this event is coincident with the involution of the fetal zone of the adrenal. The fetal liver is likely to be the major source ultimately of the LDL-cholesterol utilized by the HFA. Consequently, factors that regulate cholesterol and lipoprotein synthesis in the fetal liver may, in turn, affect the steroidogenic activity of the HFA through regulation of the supply of cholesterol precursor. Thus, if trophic factors for the HFA other than ACTH exist, an important site of their action might be the fetal liver, rather than a direct action to influence the rate of synthesis of steroids by the fetal adrenal.

Adrenal Glands↗
PubMed1981

Effects of adrenocorticotropic hormone on low density lipoprotein receptors of human fetal adrenal tissue.

In the present investigation, the mechanism(s) whereby ACTH stimulates low density lipoprotein (LDL) metabolism in human fetal adrenal was evaluated. Plasma membrane fractions were prepared from fetal adrenal tissue fragments incubated in lipoprotein-poor serum with or without ACTH, and the binding of [125I]iodo-LDL to such membrane fractions was examined. The mean specific binding capacity for [125I]iodo-LDL by membrane fractions prepared from four separate fresh human fetal adrenal glands was 1370 +/- 168 ng mg-1 protein (mean +/- SE), and the concentration of [125I]iodo-LDL producing half-maximal binding was 20.8 +/- 1.2 ng ml-1. Thus, the presence of high affinity, low capacity binding sites for LDL in human fetal adrenal tissue was confirmed. When human fetal adrenal tissue was maintained in organ culture for 2 days in medium containing lipoprotein-poor serum in the absence of ACTH, and plasma membrane fractions were subsequently prepared, the binding capacity for LDL in such membrane fractions was the same as or slightly greater than that in membrane fractions prepared from fresh tissue. When ACTH was present in the culture medium, the binding capacity for LDL was doubled compared to that in membrane fractions prepared from tissues incubated in the absence of ACTH. The rate of [125I]iodo-LDL degradation by human fetal adrenal tissue maintained in medium containing ACTH was also twice that of tissue maintained in the absence of ACTH. These results demonstrate that ACTH causes an increase in the number of LDL-binding sites in human fetal adrenal tissue in vitro. This is one mechanism whereby ACTH stimulates LDL metabolism in this tissue.

Adrenal Glands↗
PubMed1981

Plasma lipoprotein regulation of progesterone biosynthesis by human corpus luteum tissue in organ culture.

The role of plasma lipoproteins in supplying cholesterol for progesterone biosynthesis by human corpus luteum tissue in culture was investigated. Progesterone secretion by tissue fragments maintained in organ culture reached a maximum rate by the third day and subsequently declined. Maximal secretion of progesterone was dependent on the presence of both low density lipoprotein (LDL) and hCG in the culture medium, whereas high density lipoprotein (HDL) was ineffective in supporting progesterone biosynthesis. Human corpus luteum tissue degraded [125I]iodo-LDL by a mechanism which was saturable, and degradation of [125I]iodo-LDL was stimulated by hCG. Although 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was present in microsomes prepared from fresh human corpus luteum tissue, the activity of this enzyme in microsomes prepared from tissue maintained in culture for 3 days was virtually undetectable. Fresh human corpus luteum tissue contained 3 times more unesterified cholesterol than esterified cholesterol. It is concluded that LDL, but not HDL, is the major source of cholesterol used by the human corpus luteum for progesterone biosynthesis.

Adult↗
PubMed1981

The role of cyclic adenosine 3',5'-monophosphate in cholesterol metabolism and steroidogenesis by the human fetal adrenal gland.

In the present investigation we studied the role of cAMP as a mediator of ACTH action in human fetal adrenal (HFA) tissue. We have characterized the response to ACTH, dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and cholera toxin (CT) with respect to steroidogenesis, low density lipoprotein (LDL) binding, degradation of LDL, and the rate of de novo synthesis of cholesterol. The rate of dehydroisoandrosterone sulfate secretion was similar in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT. In contrast, cortisol secretion by HFA tissue was more sensitive to dbcAMP and CT than to ACTH. In membrane preparations obtained from HFA tissue maintained in the presence of ACTH, dbcAMP, or CT, there was a 2 to 3-fold increase of specific binding of [125I]iodo-LDL. In HFA tissue maintained in the presence of ACTH or CT, the rate of degradation of LDL was significantly increased compared to tissue maintained in the lipoprotein-poor serum alone. Finally, in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT there was a 6- to 10-fold stimulation of the rate of incorporation of [14C]acetate into cholesterol. We conclude that steroidogenesis, LDL binding, and degradation, as well as de novo synthesis of cholesterol, are probably stimulated in HFA tissue via a cAMP-mediated pathway.

Adrenal Glands↗