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Biomedical subjects

S J Quirk

Publications and source records attributed to S J Quirk.

At least 19 recordsLinked to original sources
PubMed1990

Epithelial and adipose cells isolated from mammary glands of pregnant and lactating rats differ in 11 beta-hydroxysteroid dehydrogenase activity.

Both adipose and epithelial cells isolated from the mammary glands of pregnant and lactating rats show 11 beta-hydroxysteroid dehydrogenase (11-HSD) activity, as measured by conversion of corticosterone to 11-dehydrocorticosterone. Activity in adipose cells from pregnant rats is 3-fold higher than in lactating rats. Epithelial cells from pregnant rats show one-twentieth of the activity of adipose cells, and activity is lower still in epithelial cells from lactating rats. Explants incubated for 48 h extensively metabolized corticosterone to 11-dehydrocorticosterone, and to a much lesser extent to a second unknown metabolite which is found in tissue extracts but not conditioned medium. Mammary gland 11-HSD may thus constitute one of the physiological mechanisms preventing premature milk production in response to glucocorticoids.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1990

11 beta-hydroxysteroid dehydrogenase activity in the mammary gland.

Levels of 11 beta-hydroxysteroid dehydrogenase activity in mammary gland homogenates from pregnant and lactating Sprague-Dawley rats were determined by incubation with [3H]corticosterone under standard conditions, followed by thin-layer chromatography of incubated media. Enzyme activity was high in virgin and pregnant rats, but fell soon after parturition, suggesting a possible role for this enzyme in the co-ordinate regulation of glucocorticoid effects on milk protein synthesis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1989

Mammary gland whey acidic protein: ontogeny and changing patterns of steroid sensitivity.

Whey acidic protein (WAP) has been measured by radioimmunoassay in the mammary gland of rats over pregnancy and lactation, and in mammary gland explants incubated with glucocorticoid or progestin in vitro. The radioimmunoassay used a novel fusion protein, glutathione transferase-WAP (GT-WAP), which can be iodinated with ease, unlike the native protein. Mammary gland WAP levels were low (less than 100 ng/mg tissue) until 2-3 days before parturition, but rose to 3 micrograms/mg tissue at day 21 of pregnancy. Immediately after parturition WAP content decreased to 1 micrograms/mg tissue, and then increased to greater than 5 micrograms/mg tissue during mid-lactation. Similarly, alpha-lactalbumin content was low throughout pregnancy (less than 10 ng/mg tissue) until day 20. Thereafter, values rose on the last day of pregnancy and the first day of lactation, fell briefly in early lactation like WAP, and rose to plateau levels by day 11 of lactation. In vitro explants prepared from mid-pregnant rats (day 14) synthesized WAP in the presence of insulin and prolactin. The synthetic glucocorticoid RU26988 (11 beta,17 beta-hydroxy-17 alpha-(1-propynyl)-androsta-1,4,6-trien-3-one) progressively increased WAP production to a maximum of greater than 10-fold basal (in the presence of insulin and prolactin) at 300 nM, in contrast with alpha-lactalbumin which showed a biphasic dose-response curve in explants from mid-pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Steroid receptors, and the generation of closely coupled/biphasic dose-response curves.

In most steroid-modulated systems, responses are closely coupled to receptor occupancy, with half-maximal responses at Kd, and near-maximal at greater than or equal to 10 Kd. Though glucocorticoids have been reported to increase, and progesterone to lower, levels of various milk proteins, clear differences in glucocorticoid dose-response profiles have been previously described. In mouse mammary gland explants in vitro, Ono and Oka showed that casein synthesis was stimulated by cortisol to plateau levels with progressively higher doses; in contrast, the dose-response curve for alpha-lactalbumin (alpha-LA) was biphasic, with stimulation at low doses and a return to baseline at higher cortisol levels. Since cortisol is both non-physiologic in the mouse, and has higher affinity for Type I (mineralocorticoid-like) than Type II (glucocorticoid) receptors, this might reflect an induction of alpha-LA synthesis via Type I receptor occupancy, and a suppression via Type II receptor occupancy. To examine this possibility we have used highly selective Type II receptor ligands, and in addition have defined the role of progestins as candidate inhibitors of alpha-LA synthesis. We have thus incubated mid-pregnant rat mammary gland explants in medium including insulin and prolactin, with increasing concentrations of highly specific Type II ligand (RU26988 or RU28362) and/or the selective progestin ORG 2058, and determined tissue and medium content of alpha-LA 48 h later. RU26988/RU28362 increased alpha-LA to a maximum of 3-6-fold basal at approximately 3 nM, well below the Kd (37 degrees C) for Type II receptors (8-28 nM); levels fell progressively with increasing concentration of ligand, returning to baseline by 30-300 nM. ORG 2058 lowered both basal and RU26988-stimulated levels, with the half-maximally effective dose being approximately 10(-10) M, again well below Kd for receptor binding. To accommodate the observed biphasic dose-response curve for highly specific glucocorticoids, and the shift-to-the-left for both classes of effector, we have proposed a model of a single "turn-on" nuclear acceptor site for glucocorticoid receptors (glucocorticoid regulatory element: GRE) and multiple, pre-emptive "turn-off" GREs, occupancy of any one of which will abrogate transcription; for progestins, we propose that all the GRE are alternate "turn-off" sites. Consistent with such a hypothesis, Qasba and Safaya have shown that the alpha-LA genome contains six core consensus sequences (TGT TCCT) which are putative GRE, and von der Ahe et al. that both progesterone receptors and glucocorticoid receptors may bind to GRE sites.

Androstanols↗
PubMed1988

Alpha-lactalbumin production by rat mammary gland: prepartum emergence of resistance to glucocorticoids and progestins.

Explants of mammary glands from pregnant or lactating rats were shown to differ markedly in their response to glucocorticoid and progestin in terms of alpha-lactalbumin production. Tissue from day 14 pregnant rats had low initial tissue levels and basal production of alpha-lactalbumin, and showed a dose-biphasic response to glucocorticoids, with significantly higher production at 3 nM than 300 nM RU26988; the progestin ORG 2058 inhibited production. In explants from lactating rats (day 1 and day 5) tissue content and basal production of alpha-lactalbumin were markedly increased compared with day 14 pregnant, but were unaltered by glucocorticoid or progestin. Animals near parturition (day 19, day 20) showed a transitional pattern, with considerable variation between animals. Basal production and tissue content correlated closely; as levels rose, the tissues became progressively less responsive to the effects of both classes of steroid. We interpret these data as evidence for a switch, in terms of glucocorticoid control of alpha-lactalbumin gene expression pre-partum; the mechanism(s) underlying this switch remain to be explored.

Androstanols↗
PubMed1986

Mechanisms of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants.

The highly selective Type II glucocorticoid ligand RU28362 showed a clear biphasic effect on alpha-lactalbumin (alpha-LA) production in rat mammary gland explants, with a peak at 1 nM and a return to basal levels at 30-300 nM; dexamethasone showed a similar profile. Corticosterone, which has a higher affinity for Type I than Type II receptors, produced a variable response. In six out of eleven studies this was biphasic, with a maximum at 300 nM; in five no increase above baseline was seen. Classical Type I receptor ligands--aldosterone and deoxycorticosterone--showed responses parallel to their Type II agonist activity. We interpret these data as follows occupancy of Type I receptors does not increase alpha-LA production the response to selective Type II receptor ligands is truly biphasic and one explanation of this pattern may be the existence of both "turn-on" and "turn-off" acceptor sites in the nucleus.

Aldosterone↗
PubMed1986

Adrenocorticoid-dependent alpha-lactalbumin synthesis in rat mammary gland explants: antagonist studies.

Though adrenal steroids are required for the production of alpha-lactalbumin by mammary gland explants, the physiological class of steroid activity (mineralocorticoid, glucocorticoid) remains to be established. alpha-Lactalbumin production by mammary gland explants from mid-pregnant rats has been shown in previous studies to be increased by high doses of aldosterone, but not by deoxycorticosterone; in six of 11 experiments corticosterone, the physiological glucocorticoid in the rat, elevated alpha-lactalbumin; in five other studies corticosterone had no effect. In the present studies the mineralocorticoid antagonist, spirolactone, at very high doses (3-10 mumol/l) blocked the stimulatory effect on alpha-lactalbumin levels of both 30 nmol/l corticosterone and 3 nmol/l RU 26988, a pure synthetic glucocorticoid (Type II) receptor agonist. Receptor studies, however, indicated that this antagonism is consistent with Type II, glucocorticoid receptor occupancy by spirolactone. Since deoxycorticosterone is without agonist effect, and only very high doses of spirolactone affect alpha-lactalbumin synthesis, we conclude that the effect of adrenal steroids on alpha-lactalbumin production is a manifestation of glucocorticoid and not mineralocorticoid activity.

Aldosterone↗
PubMed1986

Specificity and mechanism of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants.

RU26988 [11 beta, 17 beta-dihydroxy-17 alpha-(1-propynyl)androsta-1,4,6-trien-3-one], a specific type II (glucocorticoid) ligand with negligible affinity for type I (mineralocorticoid) receptors, increased alpha-lactalbumin production in mammary gland explants from midpregnant rats when cultured in vitro in the presence of insulin (5 micrograms/ml) and rat PRL (1 microgram/ml). The dose-response curve was biphasic, over a 3-300 nM dose range, with maximum at 3-10 nM, followed by a progressive decline toward or to control levels at 300 nM. A similar dose-response curve was obtained with another type II-specific ligand, RU28362 [11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl)androsta-1,4,6-trien-3-one], and with dexamethasone. This biphasic response had been previously reported for mouse mammary gland explants using cortisol, which binds to both type I and type II receptors. Our experiments, therefore, show that occupancy of type II receptors alone can be responsible for the stimulation at low steroid concentrations, followed by the decrease from peak levels observed at higher steroid concentrations. On the basis of these data, we propose a model to account for these findings, based on ligand binding to a single class of receptors and the putative existence of both turn-on and turn-off glucocorticoid regulatory elements in the nucleus.

Androstanols↗
PubMed1985

Progestins specifically suppress alpha-lactalbumin synthesis and secretion.

Mammary gland explants from pregnant (day 12-15) rats were cultured with insulin and prolactin, and their content and secretion of alpha-lactalbumin determined after exposure to a wide range of doses (0.01-300 nM) of the specific synthetic progestin (ORG2058), alone or with a maximally stimulatory dose of the highly specific glucocorticoid RU26988. ORG2058 alone suppressed alpha-lactalbumin synthesis below baseline, with a half-maximal effect at a concentration of less than 0.1 nM; RU26988-stimulated secretion was similarly abrogated by ORG2058, similarly with a half maximally effective dose of less than 0.1 nM. We interpret these data as suggesting that (i) given the specificity and doses of the steroids used the effect of progestins on alpha lactalbumin synthesis is directly via progesterone receptor occupancy, and not by competing with glucocorticoids for glucocorticoid receptors and (ii) given the shift to the left in the alpha-lactalbumin response (half maximal less than 0.1 nM ORG2058) compared with receptor binding (Kd (37 degrees C) greater than 1 nM), one possible model for such sensitivity is that of multiple, independent regulatory elements on the chromatin controlling alpha-lactalbumin gene expression, occupancy of any one of which by an activated progesterone receptor is sufficient to abrogate transcription.

Androstanols↗
PubMed1984

Oestrogen administration induces progesterone receptors in lactating rat mammary gland.

Displaceable binding of the synthetic progestin [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20 dione) was found in ammonium sulphate treated cytosols from uterus and mammary gland from lactating rats. Specificity studies, however, showed that in mammary gland but not uterus this binding was exclusively to glucocorticoid receptors, and that specific progesterone receptors were undetectable. In contrast, after in vivo administration of oestradiol benzoate in oil (4 micrograms at -48, -24h), [3H]ORG 2058 bound predominantly to specific, high affinity progesterone receptors in lactating mammary gland. Thus though in lactation progesterone receptors are normally absent from the mammary gland, they can be induced by oestrogen administration.

Animals↗
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↗
PubMed1983

Ultrastructural evidence of variation in the number of secretory granules within the granulosa cells of the sheep corpus luteum.

The granulosa luteal cells of the sheep corpus luteum secrete their hormonal products by exocytosis of granules. Electron micrographs of randomly selected granulosa cells from nine corpora lutea at day 10 of the oestrous cycle were examined to obtain the cellular density of these granules. From the area of the cell, calculated using an x-y digitiser, and the number of granules observed, the number of intracellular secretory granules per microns 3 of luteal cell cytoplasm was calculated. There was a large variation in the number of granules per cell within the same corpus luteum and between corpora lutea taken at the same stage of the cycle. The number of intracellular granules in nine corpora lutea varied from 2.12 +/- 1.05 granules per microns 3 (mean, SD, n = 30) cells to 0.36 +/- 0.18 granules per microns 3 (mean, SD, n = 26). These morphological data suggest that the variation in granule synthesis in individual cells may contribute to the variation in hormone content of corpora lutea at the same stage of the cycle, and the episodic release of hormone into the plasma.

Animals↗
PubMed1983

Aldosterone-binding sites in pregnant and lactating rat mammary glands.

We have identified and characterized aldosterone-binding sites in the mammary gland of pregnant and lactating rats by using the highly specific glucocorticoid RU26988 (11 beta, 17 beta-dihydroxy-17 alpha-propynyl-androsta-1,4,6-triene-3-one) to eliminate aldosterone binding to classical glucocorticoid (type II)-binding sites. The hierarchy of affinity of steroids competing for aldosterone in the presence of RU26988 was similar in mammary glands of pregnant and lactating rats and kidneys of lactating rats: deoxycorticosterone greater than aldosterone greater than or equal to corticosterone greater than dexamethasone; this pattern is similar but not identical to that for the mineralocorticoid (type I) receptor of a classical mineralocorticoid target organ, the kidney of the virgin female rat, where aldosterone has a higher affinity than corticosterone. This pattern (aldosterone greater than corticosterone) was also found in renal cytosols of pregnant rats. To define these aldosterone-binding sites in terms of dissociation constant and number of sites, it was necessary to pretreat cytosols with charcoal. Without this treatment, curvilinear Scatchard plots were obtained. After pretreatment with charcoal, subsequent Scatchard analysis indicated a single class of site with low capacity (3-33 fmol/mg protein) and dissociation constants of about 1-3 nM in kidneys and mammary glands of pregnant and lactating rats. The occupancy of this site in vivo may depend on the relative tissue concentrations of aldosterone or corticosterone, which, in turn, are modulated by the levels of extravascular transcortin.

Aldosterone↗
PubMed1982

Glucocorticoid receptors in epithelial cells isolated from the mammary glands of pregnant and lactating rats.

We have isolated epithelial cell clusters from mammary glands of pregnant and lactating rats by collagenase-hyaluronidase-deoxyribonuclease digestion, followed by Ficoll density-gradient centrifugation. Clusters of greater than 90% viable cells were identified by light microscopy as essentially devoid of other cell types; the integrity of their subcellular organelles verified by electron microscopy. Binding characteristics of the synthetic glucocorticoid [3H]dexamethasone were studied in cytosols prepared from isolated cell clusters. Cytosols from both pregnant and lactating rats bound [3H]dexamethasone with high affinity to a single class of low capacity binding sites. In both types of cytosol the dissociation constant (Kd 4 degrees C approximately/nM) of the binding was similar; the number of sites per cell in lactating rats was approximately double that in pregnant rats. The specificity of binding was typical of a classical glucocorticoid receptor, with a hierarchy of affinity by competition studies dexamethasone greater than progesterone greater than aldosterone much much greater than testosterone = estradiol. In particular, no difference in progesterone affinity for these glucocorticoid receptors was seen between pregnancy and lactation. This suggests that reported differences in inhibitory action of progesterone, pregnancy versus post-partum, are not glucocorticoid-receptor mediated.

Aldosterone↗
PubMed1982

Progesterone receptors in mammary gland of the pregnant rat.

The characteristics of pregnant rat mammary gland progesteronee receptors have been studied by the use of two synthetic probes, [3H]R5020 (3H-labeled 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) amd [3H]ORG 2058 (3H-labeled 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione). [3H] ORG 2058 bound with high affinity (Kd at 4 C, approximately 0.5 nM) to an apparent single class of displaceable sites, with a hierarchy of competition as follows: ORG 2058 greater than R5020 greater than or equal to progesterone greater than dexamethasone. In contrast, [3H]R5020 showed binding in mammary gland cytosols which was nonlinear with protein concentration, of intermediate affinity (Kd at 4 C, approximately 5 nM), and poorly displaced by nonradioactive R5020; the hierarchy of competitors was R5020 greater than progesterone greater than dexamethasone = ORG 2058. Parallel studies in rat uterine cytosols showed both [3H]ORG 2058 and [3H]R5020 to bind with high affinity to an apparently identical class of sites; the hierarchy of competition for both was ORG 2058 greater than or equal to R5020 greater than progesteron greater than dexamethasone. We conclude that in pregnant rat mammary gland, as opposed to rat uterus and other tissues, R5020 is unsuitable as a progesterone receptor probe; however, the affinity, capacity, ad specificity characteristics of progesterone receptors in this tissue can be described by the use of ORG 2058.

Animals↗
PubMed1978

Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol.

1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca(2+) used, at least 80% of the cells fused after 30min at 37 degrees C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in ;ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca(2+), although some cells fused when no exogenous Ca(2+) was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of ;ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1-2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca(2+) into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed.

Calcium↗