Biomedical subjects
Publications and source records attributed to J H Clark.
This is a report of a 16-day-old, breast-fed infant who presented with metabolic acidosis and no history of drug ingestion. Salicylate intoxication was demonstrated as the probable cause of the acidosis.
Nafoxidine (1-[2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]ethyl]-pyrrolidine hydrochloride); a triphenylethylene derivative] and estradiol cause translocation of the estrogen receptor to the nucleus, elevate the levels of cytoplasmic progesterone receptors, and do not effect levels of nuclear progesterone receptor in hormone-dependent and -independent MXT-3590 mammary tumor lines. Cytoplasmic and nuclear receptor levels were measured by the dextran-coated charcoal assay and a modified protamine sulfate nuclear exchange assay, respectively. Both estradiol (2.5 microgram) and nafoxidine (40 microgram) stimulate partial growth in the independent line. These data are the first evidence of the estrogenic effects of a so-called antiestrogen (nafoxidine) on the quantity of progesterone receptors in a hormone-independent experimental mammary tumor. The growth effects of antiestrogens demonstrated here in ovariectomized mice should alert clinicians to be watchful of these effects when using such drugs in the treatment of human breast cancer. These results also confirm recent suggestions that growth and progesterone receptor synthesis are controlled separately by estrogenic compounds. (Endocrinology 108: 668, 1981)
Estrogen administration to mature-ovariectomized rats causes the activation or stimulation of secondary nuclear estrogen-binding sites (type II) in the uterus which can interfere with estrogen receptor (type I) measurement. Earlier reports from our laboratory have shown that quantitation of type I sites in the presence of the type II site is very difficult and can only be achieved by graphic analysis of saturation curves which employ a wide range (0.4-40 NM) of [3H]estradiol concentrations in nuclear exchange assay. The studies presented in this manuscript describe simple methods which can be used to separately quantitate both nuclear estrogen-binding sites using a single concentration of [3H]estradiol. Since the nuclear type II site does not bind [3H]estradiol in the presence of reducing agent, type I sites can be easily quantitated by incubating nuclei (37 C for 30 min) in Tris-EDTA buffer containing 0.1-1.00 mM dithiothreitol using a single saturating concentration of [3H]estradiol. Conversely, a single concentration of [3H]estradiol (40-80 nM) can be used to quantitate the nuclear type II site by incubating nuclei in Tris-EDTA buffer under conditions (4 C for 60 min) which do not measure occupied nuclear estrogen receptor. Therefore, by using the appropriate buffer system, type I and type II sites can be easily separated in mixed binding systems. In addition, we also demonstrate that Nafoxidine does not bind to the nuclear type II site. Therefore, it can be used as a competitive inhibitor of [3H]estradiol binding to type I sites and permit the measurement of type II sites without interference from type I sites. These techniques should be applicable to autoradiographic or fluorescence studies which cannot discriminate between steroid binding to these two classes of nuclear estrogen-binding sites.
The question of the presence of specific glucocorticoid receptors in the rat uterus was reassessed. A high speed supernatant obtained from uteri of ovariectomized or intact 45-day old rats was incubated 18 h at 4 c in the presence of [3H]triamcinolone acetonide ([3H]TA) with or without excess dexamethasone, followed by a 10 min exposure to dextran-coated charcoal. A specific uterine glucocorticoid receptor was detected with a Kd=5.1 nM and the number of binding sites equal to 1.1 pmol/uterus. [3H]dexamethasone was observed to underestimate the number of specific receptor sites, probably due to the instability of the dexamethasone-receptor complex during treatment with charcoal. In addition, unlabeled TA was unsatisfactory for determining nonspecific binding because of the cross-reactivity with the progesterone receptor also present in uterine cytosol. The uterine glucocorticoid receptor exhibited the appropriate steroid specificity. Sucrose density gradient analysis of uterine cytosol revealed peaks of dexamethasone-competable [3H]TA binding at 7.2S and 5.75 in low salt and at 4S in 0.4M KCL. Similar components were observed in cytosol and nuclear fractions, respectively, 20 min after in vivo injection of 25 mu Cl [3H]TA. The physical characteristics of the specific glucocorticoid receptor were consistent with those determined for other receptor proteins. The documentation of the presence of glucocorticoid receptors in the rat uterus is important physiologically because of numerous reports of glucocorticoid effects on this reproductive tissue. If receptors could not be demonstrated, extensive studies of the mechanism of these effects would be necessary for a full understanding of uterine endocrinology.
Glutathione (gamma-glutamyl-cysteinyl-glycine) concentrations of plasma and whole blood were measured for lactating Holstein cows. Samples were taken from both the internal iliac artery and mammary vein to calculate arteriovenous differences across the mammary gland. Glutathione in plasma was extremely low, the there was no significant arteriovenous difference of this tripeptide across the mammary gland. In contrast, glutathione in whole blood was 200-fold greater than in plasma and showed a significant arteriovenous difference across the mammary gland. In vivo studies have shown that when arteriovenous differences of free amino acids in plasma are quantitated, there appears to be a shortage of cysteine for synthesis of milk protein. Using estimated blood flow and arteriovenous differences, we calculated that mammary gland uptake of glutathione from whole blood is more than enough to account for the amount of cysteine secreted in milk. Implications regarding availability to the mammary gland of glutathione from whole blood are discussed.
Investigations were to determine if bovine mammary tissue could synthesize orotate and to assess the influence of amino acids on production of orotate. Mammary tissue from 6 Holstein cows was obtained immediately after slaughter. Tissue slices were incubated in either a modified Krebs-Ringer bicarbonate buffer, the same medium containing a physiological concentration of amino acids, or a third medium containing a fourfold concentration of the amino acid mixture. In all experiments, tissue slices were preincubated in the physiological amino acid medium for 5, 10, and 15 min (30 total min) with fresh medium changes after each incubation with one of the three treatment media for three consecutive 20-min periods. Tissue slices incubated in the physiological amino acid medium for a total of 90 min synthesized 28.5 microgram orotate/g wet tissue after correction for the initial tissue contribution. Orotate secreted by the tissue slices increased as concentration of amino acids increased. Lactose released into the medium was not affected by amino acid concentration. Bovine mammary tissue can synthesize substantial amounts of orotate, and tissue synthesis of orotate but not lactose increased as amino acid concentration increased.
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The release of luteinizing hormone-releasing hormone (LHRH) from mitochondrial-synaptosomal fractions (P2) of basomedial hypothalamus was examined under various conditions. Less than 3% of the LHRH in P2 suspensions was released under control conditions while the addition of 60 mM KCl or NaCl effected an 8-fold increase in LHRH as measured by radioimmunoassay. Equiosmolar sucrose effected only a 1.8-fold increase in LHRH release. The stimulatory effects of both Na+ and K+ were significantly inhibited by Mn2+ or La3+. Two forms of released LHRH were observed, one soluble and the other particulate. Soluble LHRH release was effected by hypertonic sucrose or 60 mM KCl and was not inhibited by Ca2+ antagonists. The release of particulate LHRH was unaffected by hpertonic sucrose, was stimulated 10-fold by 60 mM KCl, and was abolished with Ca2+ antagonists. These results suggest that the released soluble LHRH results from nonspecific leakage while the release of particulate LHRH reflects a Ca2+-dependent secretory process.
Mitochondrial-synaptosomal fractions (P2) from the basomedial hypothalamus of adult ovariectomized rats were employed to study the effects of estradiol benzoate (EB) and progesterone (P) on the release of luteinizing hormone-releasing hormone (LHRH). Treatment of ovariectomized rats with 5 or 50 micrograms of EB significantly reduced the total LHRH released from P2 under both control and K+-stimulated conditions. Furthermore, rats given 50 microgram EB demonstrated cyclic variations in the magnitude of inhibition of LHRH release. Comparison of LHRH release from P2 of rats sacrificed at 0900 hr with that from those sacrificed at 1500 hr revealed a small but persistent facilitation of LHRH release each afternoon. This facilitation, associated with an increase in the soluble component of LHRH release, was absent when rats also received 5 mg of P. No effects on LHRH release were observed when 17 beta-estradiol alone or when P was applied to P2 in vitro. The data show that the regulatory effects of estrogen and progesterone given in vivo on LHRH secretion can be observed in a subcellular fraction of the hypothalamus containing neurosecretory cell terminals.
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Rate sedimentation of the 900 X G supernatants (S1) of hypothalamic homogenates from untreated male rats or ovariectomized rats with or without 5 micrograms estradiol benzoate (EB) revealed two populations of LHRH particles: a minor, slowly sedimenting one (peak 1) and a major, more rapidly sedimenting one (peak 2). Some LHRH-containing material also sedimented to the bottom of the gradient. The ovariectomized rats displayed more heterogeneity of particulate LHRH than did the male rats. Furthermore, the administration of EB to ovariectomized rats altered the relative sedimentation pattern of LHRH. In ovariectomized rats, hypotonic shock of S1 prior to rate sedimentation eliminated peak 2 and post-peak 2 LHRH and increased free LHRH at the top of the gradient. Peak 1 LHRH was still present and was elevated after EB treatment. Also, EB treatment lowered the free LHRH at the top of the gradient. These data demonstrate that the administration of EB to an ovariectomized rat alters the subcellular distribution of LHRH.
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Crude nuclear preparations from the rat uterus and the MXT mouse mammary tumor contain endogenous nucleases whose activity results in the loss of estrogen-binding sites from these fractions due to DNA breakdown. Although this is not a significant problem for studies in the uterus, the majority of the receptors present in nuclei from the tumor were lost from the particulate fraction under the standard conditions for the [3H]estradiol exchange assay. The addition of 5 mM Ca++ to either of these nuclear preparations resulted in the aggregation of the released estrogen receptors with particulate components and, hence, a total inhibition of receptor release into the soluble (wash) fractions. Ca++ had no effect on the affinity of the receptors for [3H]estradiol, although in some cases it slowed the overall rate of binding. The addition of Ca++ to the nuclear estrogen exchange assay may therefore prove to be a simple yet powerful aide in studies on the potential hormonal responsiveness of human and experimental mammary tumors.
A hormone-independent mouse mammary tumor line was derived by transplantation of a hormone-dependent line (MXT-3590) into ovariectomized-adrenalectomized animals and subsequent transplantation of surviving tumors. Although the tumor line is hormone independent, it contains a full complement of estrogen and progesterone receptors which are similar to those found in hormone-dependent tumors. This similarity includes the criteria of nuclear translocation of the estrogen receptor and subsequent stimulation of progesterone receptor synthesis. In addition to the estrogen receptor, a secondary estrogen-binding site (type II) is also present; this binding site was previously described in the hormone-dependent companion tumor line. Thus, while this variant tumor line contains the biosynthetic pathways that control estrogen and progesterone receptor synthesis, its growth does not depend on these steroids. Therefore, it may provide a good model for the study of this class of hormone-independent tumors.
Previous investigations have shown that hypothalamic luteinizing hormone releasing hormone (LHRH) is localized within a subcellular fraction eqivalent or similar to the synaptosome or nerve ending particle. In the present study, the ovariectomized adult rat was employed to investigate the effects of estrogen on the distribution of LHRH among various subcellular fractions of the hypothalamus. In addition, the liberation of LHRH from a synaptosomal fraction by repeated freeze-thaw cycles was examined before and after in vivo estrogen treatment. Following a single injection of 5 micrograms of estradiol benzoate (EB), the LHRH concentration of the synaptosomal fraction rose as much as 100% in 27 h. Increasing the dose of EB to 50 micrograms shortened the latency of the rise and prolonged its duration, but did not alter its magnitude compared to the lower dose. Furthermore, the LHRH in that synaptosomal fraction was significantly more resistant to solubilization by repetitive freezing and thawing than the LHRH in a synaptosomal fraction from rats which had not received EB. These observations led us to conclude that estrogens can alter the physical properties of the hypothalamic LHRH-containing particles or the storage form of the peptide within those particles so that it is less easily released. This phenomenon may represent one aspect of the regulatory effects of estrogens on hypothalamic LHRH secretion.