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Biomedical subjects

J W Funder

Publications and source records attributed to J W Funder.

At least 235 records · Page 13Linked to original sources
PubMed1984

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. III. Hormone receptor status and responsiveness.

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.

Breast Neoplasms↗
PubMed1984

Expression of the gene for arginine vasopressin in Brattleboro rats.

There is now overwhelming evidence--radio-immunoassay, immunofluorescence, Northern blot analysis with highly specific cDNA probes--for the production of vasopressin and its congeners in the rat ovary as well as the hypothalamus. Ontogeny studies and studies over the oestrous cycle suggest that such production is controlled by different mechanisms in the two tissues. Studies in Brattleboro rats suggest that the production of vasopressin and its congeners involves different post-transcriptional mechanisms in these tissues. In Brattleboro rats hypothalamic mRNA is either not translated, or yields a very short-lived product, whereas ovarian levels of immunoreactive vasopressin are indistinguishable from those in control animals. The mechanism of this tissue specificity, and its implications in terms of potential paracrine roles for vasopressin and, or its congeners awaits explanation.

Animals↗
PubMed1983

Cells in neonatal rat hypothalamus primary culture--an immunofluorescence study.

Hypothalami from 1 day neonatal rats were dissociated and cultured for 4-16 days. Using immunofluorescence and antisera against neurofilament (NF) peptides, glial fibrillary acidic protein (GFAP), galactocerebroside and fibronectin we have distinguished neurons, astrocytes, oligodendrocytes and fibroblast-like cells in culture. Astrocytes initially grew as islets of 15-30 cells which dispersed as the culture aged. These cells, together with fibronectin-reactive flat cells, formed a monolayer upon which ovoid and process-bearing cells grew after 4 days in culture. Neurofilament-positive neurons constituted 5-10% of the total cell population. In maturing cultures the number of neurons decreased and fibroblasts increased. Oligodendrocytes represented less than 1% of total cell population. These studies emphasize the necessity of using the complementary techniques of morphology and immunocytochemistry for the characterization of hypothalamic neural cells in vitro.

Animals↗
PubMed1983

Coffee contains potent opiate receptor binding activity.

Opiate receptor-active peptide fragments (exorphins) have been identified recently in casein and gluten hydrolysates, and morphine has been found in bovine and human milk. To determine whether similar peptides or alkaloids occur in other foodstuffs, we have screened potential sources using a rat brain homogenate assay to detect opiate receptor activity. We report here that instant coffee powders from a variety of manufacturers compete with tritiated naloxone for binding to opiate receptors in the rat brain membrane preparations, with no significant difference between normal and decaffeinated coffee. The receptor binding activity resembles that seen with opiate antagonists, in that there was no change in the half-maximal effective dose (ED50) in the presence of 100 mM Na+; on bioassay, the activity was similarly shown to be antagonistic and specific for opiate-induced inhibition of twitch. Preliminary characterization of the activity reveals that it has a molecular weight (MW) in the range 1,000-3,500, is heat-stable, ether-extractable, not modified by enzymatic digestion with papain, and clearly separable from caffeine and morphine on TLC. As its concentration in an average cup of coffee is five times the ED50, these data suggest that drinking coffee may be followed by effects mediated via opiate receptors, as well as effects of caffeine.

Animals↗
PubMed1983

Effects of steroids on fibroblasts: identification of glucocorticoid-regulated proteins by two-dimensional gel electrophoresis.

Fibroblasts cultured from normal human forearm or genital skin were exposed to dexamethasone (DM) 10(-9)-10(-7) M, deoxycorticosterone (DOC) 10(-6)M, or vehicle for 4-16 h and [35S]methionine-labelled proteins compared by two-dimensional gel electrophoresis. No effects on cell number or [35S]methionine incorporation were seen with either steroid, nor any effects of DOC on patterns of protein synthesis. In contrast, after 4 h incubation DM consistently (24/24 gels) caused increased abundance of a protein spot designated k (kappa; Mr approximately 41 000, pKi approximately 6.5). After 16 h steroid exposure, levels of protein kappa remained elevated; abundance of a second protein spot omega (omega; Mr approximately 42 000, pKi approximately 5.4) was consistently (8/8 gels) lower than in control fibroblasts. We interpret these data as evidence for glucocorticoid-specific effects on human skin fibroblasts, and that (within the pKi and molecular weight range studied) the observed changes in abundance of proteins kappa and omega constitute the human fibroblast glucocorticoid domain.

Child↗
PubMed1983

Glucocorticoid-induced proteins in bovine endothelial cells.

High circulating levels of corticosteroids are associated with elevated blood pressure and an increased incidence of vascular damage. We have used high resolution two-dimensional gel electrophoresis and autoradiography to analyse direct steroid effects on the newly synthesized proteins in bovine aortic endothelial cells. At medium concentrations of 10(-7) M, corticosteroids but not sex steroids increased the synthesis of two endothelial proteins: el (M.W. approximately 43K, pI approximately 6.3) and e2 (M.W. approximately 28K, pI approximately 5.9). The responses were consistent for different endothelial cell cultures, different gel runs, and for natural and synthetic glucocorticoids, including the 'pure' synthetic glucocorticoid, RU 26988. Mineralocorticoids (aldosterone, deoxycorticosterone) at 10(-7) M were less potent in their effect on the synthesis of e2, and had no discernible effect on the synthesis of e1. These specific effects on cellular protein synthesis demonstrate that glucocorticoids can act directly on endothelial cells. These direct actions may thus mediate altered endothelial cell function in clinical and experimental cardiovascular conditions characterized by corticosteroid excess.

Aldosterone↗
PubMed1983

Adrenalectomy and glucocorticoid administration in vivo do not affect production of 6-oxoprostaglandin F1 alpha by rat aortae.

1. Rats were bilaterally adrenalectomized and treated for periods of up to 32 days with doses of dexamethasone equivalent to physiological and supraphysiological endogenous glucocorticoid secretion. Aortic production of 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha) was measured by radioimmunoassay of the supernatant fluid from rings of aorta incubated in vitro. 2. Up to 32 days after adrenalectomy, neither adrenalectomy nor dexamethasone (2 micrograms/day and 20 micrograms/day) influenced aortic 6-oxo-PGF1 alpha production. 3. High doses of dexamethasone (60 micrograms/day for 6 days) in the presence or absence of mineralocorticoid (11-deoxycorticosterone, 600 micrograms/day) had no effect on aortic 6-oxo-PGF1 alpha production. 4. In contrast to the lack of effect of dexamethasone administered in vivo, incubation of aortic discs for 5 h with dexamethasone (1 mumol/l) in vitro caused inhibition of aortic 6-oxo-PGF1 alpha production to 23% of control levels. 5. Alteration in endogenous glucocorticoid secretion is unlikely to be involved in the physiological regulation of aortic 6-oxo-PGF1 alpha production.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity.

There is current evidence for two classes of hippocampal glucocorticoid receptors (GR)--one classical, [3H]dexamethasone [( 3H]Dex)-binding sites in glial cells, and the other [3H]corticosterone-preferring sites in neuronal cells. In the presence of 1 microM of the synthetic glucocorticoid RU26988 (11 beta, 17 beta-dihydroxy-17 alpha-propynylandrost-1,4,6,-trien-3-one) to exclude tracer from [3H]Dex sites, hippocampal cytosol from adrenalectomized/ovariectomized Sprague-Dawley rats binds [3H]Dex to sites (Kd at 4 degrees C, 0.77 X 10(-9) M; 65 fmol/mg of protein) with the following order of specificity: aldosterone (Aldo) = 9 alpha-fluorocortisol (9 alpha F-cortisol) = deoxycorticosterone (DOC) = corticosterone greater than cortisol much greater than Dex; [3H]Aldo, [3H]DOC, and [3H]corticosterone binding show identical specificity in the presence of RU26988. Addition of 1% adrenalectomized/ovariectomized rat plasma (but not plasma heated at 56 degrees C for 30 min) alters the specificity to: 9 alpha F-cortisol greater than or equal to Aldo greater than or equal to DOC much greater than Dex greater than or equal to corticosterone greater than or equal to cortisol, consistent with sequestration of DOC, corticosterone, and cortisol by transcortin and similar to classical mineralocorticoid receptor (MR) binding of [3H]Aldo in renal cytosol (9 alpha F-cortisol greater than or equal to Aldo greater than or equal to DOC much greater than corticosterone greater than or equal to cortisol greater than or equal to Dex). Separation of other renal binders from transcortin by hydroxylapatite adsorption established the intrinsic specificity of [3H]Aldo binding to MR as: DOC greater than or equal to Aldo greater than or equal to 9 alpha F-cortisol greater than or equal to corticosterone greater than cortisol much greater than Dex, parallel to that of the [3H]corticosterone-binding sites in hippocampus. These studies suggest (i) that hippocampal [3H]corticosterone-binding sites and renal MR may have identical intrinsic specificity for steroids, with apparent specificity differences the result of tissue-specific sequestration of naturally occurring steroids other than Aldo and (ii) that an identical steroid-binding species may thus be occupied under physiological conditions by a mineralocorticoid in one tissue (kidney) and a glucocorticoid in another (hippocampus).

Aldosterone↗
PubMed1983

Insulin hypoglycaemia and cholinergic blockade: response of plasma immunoreactive beta-endorphin.

Six normal subjects were studied to investigate the mechanisms involved in the release of immunoreactive beta-endorphin (ir-beta EP) in response to insulin hypoglycaemia. Insulin alone (0.2 U/kg body weight) was followed after a 30 min lag period by a congruent to 2.5 fold elevation of plasma ir-beta EP, with a return to basal levels by 90 min. Concurrent infusion of 10% dextrose at 250 ml/h for 2 h prevented the increase in plasma ir-beta EP levels, suggesting an effect of hypoglycaemia rather than a direct effect of insulin. Premedication with atropine 0.6 mg at -30 min was followed by hypoglycaemia equivalent to that seen with insulin alone, but no increase in plasma ir-beta EP, suggesting the involvement of cholinergic mechanisms in the neural mediation of ir-beta EP release in response to hypoglycaemia.

Adult↗
PubMed1983

Affinity of liquorice derivatives for mineralocorticoid and glucocorticoid receptors.

Liquorice abuse causes a syndrome of pseudohyperaldosteronism. Much less commonly, glucocorticoid-like effects have been reported. The electrolyte-active principle of liquorice is glycyrrhizic acid (GI), which can be hydrolyzed to glycyrrhetinic acid (GE). Previous studies have reported that GE, but not GI, may occupy mineralocorticoid and glucocorticoid receptors. We here report that both GE and GI can bind to both mineralocorticoid and glucocorticoid receptors. The affinity of GI for mineralocorticoid receptors is four orders of magnitude lower than aldosterone and for glucocorticoid receptors five orders of magnitude lower than dexamethasone. The affinity, though low, is sufficient to explain the mineralocorticoid-like side effects, given the large amount of liquorice required to produce such a syndrome.

Aldosterone↗
PubMed1983

Cystic fibrosis and normal fibroblasts have identical glucocorticoid receptor profiles and induced protein responses.

The possibility of differences in glucocorticoid sensitivity between normal and cystic fibrosis fibroblasts was studied in cultured cells derived from forearm skin biopsies. Both normal and cystic fibrosis cells bound tritiated dexamethasone with high affinity (Kd 37 degrees C approximately equal to 10 nmol/1) to a limited class of sites (concentration approximately equal to 50,000/cell); no difference between cystic fibrosis and normal cells was seen in the nuclear transfer of receptor-bound tritiated dexamethasone. After 4 h exposure to dexamethasone and analysis of 35S-methionine labelled proteins by 2-dimensional gel electrophoresis, both normal and cystic fibrosis cells showed increased abundance of protein kappa (molecular weight approximately equal to 41,000; pKi approximately equal to 6.5) over non-steroid exposed gels. After 16 h exposure, in both cystic fibrosis and normal cells exposed to dexamethasone a decrease in protein omega (mol. wt approximately equal to 42,000; pKi approximately equal to 5.4) was seen compared with control. We interpret these data as evidence against a difference in glucocorticoid receptor of effector mechanisms between cystic fibrosis and normal fibroblasts.

Adolescent↗
PubMed1983

Correlations between fibroblast androgen receptor levels and clinical features in abnormal male sexual differentiation and infertility.

Androgen receptor binding of radiolabelled methyltrienolone (3H-R1881) was determined in cultured genital skin fibroblasts from 17 normal male controls and from 65 males with genital abnormalities. Analysis of the results was performed with patients grouped according to the predominant clinical features. Of 16 patients with complete male pseudohermaphroditism, 12 had androgen receptor (AR) deficiency, one had "receptor-positive" Testicular Feminization, and in three, alternative diagnoses were established (17 alpha hydroxylase deficiency; 20,22-desmolase deficiency; mixed gonadal dysgenesis). In contrast, only four of 16 patients investigated for ambiguous genitalia (phallus intermediate between male and female, perineal urethra) showed AR deficiency. Borderline or slightly low AR levels were found in each of four boys in whom the sole abnormality was micropenis. In 26 boys with penile hypospadias, however, only one had low AR. Very low levels of AR (levels comparable to those seen in complete Testicular Feminization) were found in two of three men with infertility associated with abnormally high serum testosterone levels.

Adult↗
PubMed1983

Stress-induced changes in plasma, pituitary and hypothalamic immunoreactive beta-endorphin: effects of diurnal variation, adrenalectomy, corticosteroids, and opiate agonists and antagonists.

Immunoreactive beta-endorphin (ir-beta EP) levels were determined in plasma, anterior pituitary (AP), neuro-intermediate lobe (N-IL) and hypothalamus (HT) of intact rats and adrenalectomized rats subjected to swimming stress. In intact rats, plasma ir-beta EP was elevated fourfold, and ir-beta EP levels in AP and N-IL significantly reduced after 30 min swimming; in contrast, HT ir-beta EP in these animals was not affected. In adrenalectomized animals, similar but even more marked changes were found in plasma and tissue ir-beta EP following stress. The effect of diurnal variation, morphine and naloxone on stress-induced changes in tissue ir-beta EP was also examined; in addition, the influence of acute and chronic treatment of adrenalectomized animals with dexamethasone or deoxycorticosterone was explored. Plasma ir-beta EP of rats swum in the afternoon was significantly higher than of those swum in the morning; no differences were detected in AP, N-IL or HT ir-beta EP. Acute morphine administration (10 mg/kg) did not affect levels of ir-beta EP in any tissue, baseline or post-stress; however, naloxone (10 mg/kg) consistently and significantly prevented the reduction in N-IL ir-beta EP otherwise found following prolonged stress. Both acute and chronic dexamethasone (2 and 20 micrograms) attenuated the stress-induced changes found in AP and plasma ir-beta EP in a dose-related manner; equivalent effects, however, were not seen in deoxycorticosterone (20, 200 micrograms) treated animals. In contrast, the stress-induced reduction found in N-IL ir-beta EP was influenced neither by acute nor chronic treatment with dexamethasone or deoxycorticosterone. These results suggest that changes found in AP and plasma ir-beta EP after swimming stress are glucocorticoid-sensitive, but do not involve pathways including opiate receptors; in contrast, though baseline levels of N-IL ir-beta EP are mineralocorticoid-sensitive, the changes after swimming stress are steroid-independent and involve the action of endogenous opioids.

Adrenal Cortex Hormones↗
PubMed1983

Prolonged foot-shock induced analgesia: glucocorticoids and non-pituitary opioids are involved.

In rats, the analgesic response induced by prolonged intermittent foot-shock is abolished by naloxone or adrenalectomy, and is restored in adrenalectomized animals by the administration of dexamethasone or corticosterone, but not deoxycorticosterone. Levels of immunoreactive beta-endorphin in the anterior pituitary are significantly elevated by adrenalectomy alone, and further by dexamethasone and corticosterone; in contrast, immunoreactive beta-endorphin levels in the neuro-intermediate lobe are raised by deoxycorticosterone. These findings suggest that analgesic responses produced by prolonged intermittent foot-shock involve both the pituitary-adrenal axis and endogenous, non-pituitary opioids.

Adrenalectomy↗
PubMed1983

Neonatal rat hypothalamus cell culture: neuron subpopulations secrete immunoreactive beta-endorphin but not immunoreactive ACTH.

Primary cultures of dissociated hypothalamic cells were prepared from 1-day-old rat neonates. Studies with cell-specific antisera revealed the presence of neurons, glial cells, oligodendrocytes and fibroblast-like cells. By immunohistochemistry, two morphologically distinct cells in culture were positive for immunoreactive beta-endorphin (IR-beta-EP). The medium derived from these cultures contained radioimmunoassayable IR-beta-EP, but not IR-ACTH. These data suggest that, in rat neonatal hypothalamic cultures, two subpopulations of cells exist which store and secrete IR-beta-EP, but not IR-ACTH.

Adrenocorticotropic Hormone↗
PubMed1983

Defective nuclear accumulation of androgen receptors in disorders of sexual differentiation.

Nuclear transfer of androgen receptors (AR) and glucocorticoid receptors (GR) was determined in cultured genital skin fibroblasts from 10 normal controls and eight patients with abnormalities of the external genitalia. In whole cell studies, cultures were incubated for 20 min at 37 degrees C with [3H]methyltrienolone (3H-R1881) or tritiated dexamethasone, and specific binding was determined in whole cell, cytoplasmic, and crude nuclear fractions. Between normal and affected fibroblasts no difference was seen in cellular levels of GR, or in cytoplasmic and nuclear distribution of GR. In normal fibroblasts, cytoplasmic binding of 3H-R1881 represented 56%, and crude nuclear binding 44%, of total binding; in fibroblasts from five of the eight patients similar values (cytoplasmic 55% and nuclear 44%) were seen for 3H-R1881 binding. In fibroblasts from the other three patients no decrease in total cellular levels of AR were seen; nuclear compartmentalization, however, was much lower (approximately 20%) than in other cultures. In vitro reconstitution studies, combining 3H-R1881-loaded cytosol with naive nuclei, lead us to suggest that the defect in nuclear compartmentalization lies at the level of the nuclear acceptor site rather than the cytoplasmic binder in affected cells. We interpret the data to suggest that defective nuclear binding of AR complexes may be involved in a proportion of cases of abnormal development of the external genitalia.

Adolescent↗
PubMed1983

Steroid effects on protein synthesis in cultured smooth muscle cells from rat aorta.

To examine the direct effects of steroids on vascular smooth muscle, we have incubated rat aortic vascular smooth muscle cells in culture either steroid-free, or with natural and synthetic corticosteroids (RU26988, dexamethasone, corticosterone, 9 alpha-fluorocortisol, aldosterone, deoxycorticosterone) or sex steroids (estradiol, 5 alpha-dihydrotestosterone). At the end of 24 h, cultures were pulsed with [35S]methionine for 2 h, the cells lysed, and patterns of incorporation of isotope into protein determined by two-dimensional gel electrophoresis and autoradiography. Neither estradiol nor 5 alpha-dihydrotestosterone altered protein synthetic profiles compared with control (steroid-free) incubations. In contrast, cultures exposed to the six corticosteroids at 10(-7) M showed an identical pattern of response (6 proteins increased, 6 proteins decreased). This response appears to be glucocorticoid specific, since the mineralocorticoids (9 alpha-fluorocortisol, aldosterone, and deoxycorticosterone) did not have any effects over and above those seen with the pure glucocorticoid RU26988. We interpret these data as evidence for a putative glucocorticoid domain of at least 12 proteins in rat vascular smooth muscle cells. In contrast, there appear to be no comparable estrogen-, androgen-, or mineralocorticoid-specific changes in these cells.

Animals↗