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Biomedical subjects

J Weisz

Publications and source records attributed to J Weisz.

At least 73 records · Page 4Linked to original sources
PubMed1982

A comparison of 2-hydroxyestradiol and U-0521 (3'4'-dihydroxy-2-methylpropiophenone, Upjohn) as in situ and in vitro inhibitors of tyrosine hydroxylase.

Feedback inhibition of tyrosine hydroxylase by catechols was evaluated using in situ and in vitro enzyme assays. The three catechol compounds used were norepinephrine, 2-hydroxyestradiol, and 3'4'-dihydroxy-2-methylpropiophenone (U-0521, Upjohn); representing endogenous catecholamines, catechol estrogens, and a synthetic catechol, respectively. The in situ experiments were performed with dissociated retinal cells from rats and with stationary phase adrenergic-like neuroblastoma cells (N1E-115). The catechol estrogen, 2-hydroxyestradiol, resembled the endogenous catecholamine in its potency to inhibit in vitro and in situ tyrosine hydroxylations with IC50 values of 10 microM in vitro and 100 microM in situ. The drug U-0521, which has been used as an inhibitor of catechol-O-methyltransferase (COMT), was also found to be an inhibitor of tyrosine hydroxylase. Further, it was shown to be more potent than the natural catechols, both in vitro and in situ, with IC50 values of 30--600 nM.

Adrenal Glands↗
PubMed1982

Effect of gonadotrophin-releasing hormone on circulating levels of immunoreactive luteinizing hormone in fetal rats.

Concentrations of luteinizing hormone (LH) were measured in plasma samples obtained from rat fetuses after injecting them with synthetic gonadotrophin-releasing hormone (GnRH) in utero. On days 17 1/2, 18 1/2 or 19 1/2 post-conception (p.c.), fetuses in one uterine horn were injected subcutaneously with 100 ng GnRH in 2 microliters of saline. Fetuses in the other uterine horn were injected with saline (four litters at each gestational age). Plasma samples were collected beginning 30 min after injection of GnRH. The concentration of LH in 20 microliters of fetal plasma was measured by a microvolume radioimmunoassay. On day 17 1/2 p.c., LH was detectable only in plasma samples of GnRH-injected fetuses. In 18 1/2- and 19 1/2-day-old fetuses LH reached detectable levels (greater than 6 ng/ml) in most saline-injected fetuses. In 18 1/2- and 19 1/2-day-old fetuses of both sexes, plasma LH titers of GnRH-injected fetuses were significantly higher than in saline-injected littermates. On day 19 1/2 GnRH-treated females had higher levels of plasma LH than similarly treated males. Thus fetal rat pituitaries were shown to have the capacity to release LH in vivo in response to circulating GnRH as early as 17 1/2 days p.c.

Analysis of Variance↗
PubMed1982

The catechol estrogen, 2-hydroxyestradiol-17 alpha, is formed from estradiol-17 alpha by hypothalamic tissue in vitro and inhibits tyrosine hydroxylase.

The ability of estradiol-17 alpha to serve as a substrate for estrogen-2/4-hydroxylase in rabbit hypothalamic tissue was determined and compared to that of estradiol-17 beta. Both 2- and 4-hydroxy metabolites of estradiol-17 alpha were formed by the hypothalamic tissue in vitro. The rates of formation of 2-hydroxyestradiol (2-OHE2)-17 alpha and -17 beta were similar as were their kinetic constants (Km and Vmax). In addition, 2-OHE2-17 alpha was shown to inhibit purified rat adrenal tyrosine hydroxylase with a potency comparable to that of 2-OHE2-17 beta, a finding similar to that reported by others with respect to catechol-o-methyltransferase. Since estradiol-17 alpha has a markedly reduced affinity for estrogen receptors compared with extradiol-17 beta, this steroid could be useful in studies designed to distinguish between receptor mediated effects of estrogens and effects that locally formed catechol estrogens may have through their direct interaction with catecholaminergic system in neural tissue.

Animals↗
PubMed1982

In vitro assays of aromatase and their role in studies of estrogen formation in target tissues.

In recent years, there has been increasing recognition of the importance of steroid aromatization in organs besides the steroidogenic ones. Estrogens formed from C-19 precursors in peripheral tissues can clearly contribute to the levels of estrogens in the circulation, while aromatization in target cells may be an important determinant of the concentration of estrogens to which a particular population of target cells is exposed. Assays sufficiently sensitive and simple enough to permit quantification and characterization of aromatase activity in peripheral and target tissues are now available. One of these, based on the quantification of 3H2O formed from [1-beta-3H]androstenedione has, in our hands, a sensitivity of 15 fmol, is highly reproducible, and is relatively simple. It has been validated for stoichiometry between amounts of 3H2O and [6,7-3H]estradiol formed from [1-3H]androstenedione and [6,7-3H]androstenedione, respectively. Using this assay, we have been able to quantify aromatase activity in discrete brain nuclear regions dissected from Vibratome sections of fetal rat brains and thereby to identify in discrete areas sex differences and temporal changes during development that are obscured when larger tissue specimens are used. However, aromatase activity in brain nuclei as well as other target organs, including breast tissue, is likely to be concentrated in specialized cell populations. This heterogeneity limits the interpretation that can be placed on data obtained from standard "test tube" assays on homogenized tissue. We have used quantitative cytochemical assays for enzymes and cytochrome P-450 to identify regional specialization in the membrana granulosa of preovulatory follicles and localize cells that may be involved in steroidogenesis, including aromatization. Our findings underscore the need for new quantitative cytochemical and immunocytochemical assays to localize and to measure the amount and activity of aromatase in situ within identified populations of target cells.

Animals↗
PubMed1981

Catechol estrogen formation by brain tissue: a comparison of the release of tritium from [2-3H]estradiol with [6,7-3H]2-hydroxyestradiol formation from [6,7-3H]estradiol by rabbit hypothalami in vitro.

In the course of establishing an assay for estrogen-2-hydroxylase activity, a detailed comparison was made between the formation of tritiated water (3H2O) and [6,7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6,7-3H-labeled estradiol, respectively. The amounts of both 3H2O and [6,7-3H]2-OHE2 formed were stimulated several-fold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S2, 17,500 X g supernatant, for tritium release; S3, 100,000 X g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100,000 X g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 mM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 microM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/2 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using 3H2O formation from [2-3H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE2 is inhibited, thereby making it practical to quantify enzyme activity on the basis of the amount of catechol estrogen formed. Evidence is also presented that the release of 3H2O from [2-3H]estradiol by hypothalamic tissue, unrelated to 2-OHE2 formation, may be enzymatically mediated.

Animals↗
PubMed1981

Catechol estrogen formation by brain tissue: characterization of a direct product isolation assay for estrogen-2- and 4-hydroxylase activity and its application to studies of 2- and 4-hydroxyestradiol formation by rabbit hypothalamus.

A direct product isolation assay for quantifying the formation of 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from [6,7-3H]estradiol by rabbit hypothalami in vitro was developed, and the assay was used to characterize some properties of estrogen-2- and 4-hydroxylase activity in this tissue. The reaction was carried out under conditions that minimized further metabolism of enzymatically formed catechol estrogens. A simple two-step separation procedure, involving the use of a neutral alumina column, followed by thin layer chromatography, was developed to isolate the enzymatically formed catechol estrogens in a radiochemically homogeneous form. The detergent, Tween-80, was found to activate the enzyme and was used routinely at a concentration of 0.1% in the assay. The formation of 2-OHE2 was linear up to 10 min and with increasing protein concentrations up to 150 micrograms/incubation. Similar values were obtained for 4-OHE2. Maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 190 and 270 pmol/mg protein . 10 min, respectively. The apparent Km values with respect to estradiol for 2-OHE2 and 4-OHE2 were 125 and 150 microM, respectively. The highest specific activity for the enzyme was present in the 100,000 X g supernatant (S3), while the activity in the microsomal fraction (P3) was less than that in the original homogenate. Enzyme activity depended on the presence of NADPH and oxygen and was inhibited by CO as well as by high concentrations of SKF-525A. Estrogen-2- and 4-hydroxylase activity in rabbit hypothalamus differed from that in rat liver in two respects. In the liver, enzyme activity was localized in the microsomal fraction and was virtually abolished by Tween-80. In contrast, enzyme activity in rabbit hypothalamus was maximal in the soluble fraction (100,000 X g supernatant)and was stimulated by the detergent.

Animals↗
PubMed1980

Maternal stress alters plasma testosterone in fetal males.

Titers of testosterone in plasma were determined by radioimmunoassay in male rat fetuses of stressed and control mothers on days 17, 18, 19, 21, and 23 (the day of birth) after conception. In fetuses of stressed mothers, testosterone concentrations were highest on day 17, declined on days 18 and 19, and then remained unchanged. In the control fetuses, testosterone increased from relatively low concentrations on day 17 to the highest amounts on days 18 and 19, and then declined. Thus, the persistence of feminine and impaired masculine sexual behavior in male offspring of stressed mothers could be due to the absence of a surge of circulating testosterone during days 18 and 19 after conception, a period postulated to be critical in the development of the central nervous system in the rat.

Animals↗
PubMed1980

Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring.

Testosterone and progesterone titers were determined by RIA in the plasma of pregnant rats and their male and female fetuses from day 17 of gestation through the day of birth and in male and female neonates on days 3 and 5 post partum. Males had significantly higher mean testosterone levels than females from day 18 of gestation through day 5 post partum. Sex differences in plasma testosterone concentrations were greatest in the fetuses on days 18 and 19 of gestation when testosterone levels peaked in the males. Instances in which female fetuses had testosterone titers equal to or greater than their male littermates were found on every day of gestation except day 18. Mean testosterone concentrations in plasma of female fetuses were high throughout gestation (greater than 1000 pcg/ml). Testosterone concentrations decreased in both sexes after birth. Differences between the sexes remained significant, and although there was an overlap in the values for males and females, testosterone concentrations in females exceeded those of their male littermates in only one out of nine pairs of samples on day 5 and in none of seven pairs on day 3 post partum. There were no significant differences in progesterone levels in plasma of males and females, either pre- or postnatally. Progesterone titers changed as a function of days post conception in both the fetuses and their mothers. In the fetuses, progesterone levels declined progressively from day 18 post conception through the day of birth, while in the mother they rose from days 18 to 19 then declined between days 20 and 21 of pregnancy. Fetuses had lower progesterone titers than their mothers. From these data, we conclude that day 18 and possibly day 19 post conception represent a critical period during which the central nervous system of the male is primed by high levels of testosterone. Thereafter, the process of masculinization is completed by exposure to testosterone levels that are relatively low and need not be consistently higher than those of female littermates.

Aging↗
PubMed1979

A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase activity in the membrana granulosa of the ovulable type of follicle of the rat.

During the last four days of follicular development prior to ovulation, the activities of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells. In all three regions of the MG of follicles of the ovulable type, 3 beta OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3 beta OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the eripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in prestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation of Type II H, in the peripheral region was constant throughout the estrous cycle. In contrast, in the antral region and cumulus, Type II H generation was greater in diestrus-1 and diestrus-2 than on either proestrus or estrus.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Steroid metabolism by the lung: conversion of dihydrotestosterone to 5 alpha-androstan-3 alpha, 17 beta-diol by rat lung tissue in vitro.

The potential of lung tissue of adult male rats metabolize dihydrotestosterone (DHT) in vitro was examined. Within 3 min, a homogenate of 100 mg lung tissue, cleared of blood by perfusion before homogenization, metabolized 90% of the [3H]DHT substrate. Appoximately 80% of the DHT was converted to 5 alpha-androstan- alpha, 17 beta-diol. The amount of 5 alpha-androstan-3 alpha, 17 beta-diol formed during a 5-min incubation increased linearly, with substrate concentrations ranging from 3.3 x 10(-8) to 3 x 10(-6) M. Thus, the capacity of rat lung tissue to metabolize DHT in vitro and the rate of 3 alpha-reduction of DHT are sufficiently great to consider the possibility that lung may be responsible for the rapid clearance of DHT from the circulation in this species.

Androstane-3,17-diol↗
PubMed1979

Inhibition of steroid 5 alpha-reductase in vivo: effect on suppression of luteinizing hormone and stimulation of accessory sex organs by testosterone in the orchidectomized rat.

An inhibitor of 5 alpha-reductase, the 17 beta-carboxylic acid derivative of testosterone (testosterone-17 beta CA), has been used to evaluate the importance of the 5 alpha-reduction of testosterone in its action on the suppression of LH secretion in male rats. The potential of testosterone-17 beta CA to inhibit the formation of 5 alpha-dihydrotestosterone (DHT) was first demonstrated in vitro. When homogenates of hypothalami or anterior pituitary glands were incubated with [3H]Testosterone in the presence of a 50-fold excess of testosterone-17 beta CA, the formation of labelled DHT was inhibited by more than 80%. Adult male rats that had been castrated for 1-2 months were fitted with chronic intravenous catheters and implanted with silicone elastomer sheets: one group received one sheet, 0.5-2.0 cm2 in size containing 1.6% testosterone, a second group received one 50 cm2 sheet containing 1.6% testosterone-17 beta CA and a third group received two sheets, one sheet 50 cm2 in size containing 1.6% testosterone-17 beta CA and the second ranging in size from 0.5 to 2.0 cm2 and containing 1.6% testosterone. Blood was withdrawn daily from each rat over a 4-5 day period after implantation of the steroids and the level of LH in the plasma was measured by radioimmunoassay. The seminal vesicles and the ventral prostate gland were removed at autopsy on day 4 or 5; the weights of these organs were shown to have increased progressively as the size of the implant of testosterone increased. In contrast, the level of LH in the plasma was suppressed to a comparable extent by implants of testosterone between 0.6 and 2 cm2, whereas a 0.5 cm2 implant of testosterone had no effect. Implants of testosterone-17 beta CA alone did not influence the weight of the accessory organs or the level of LH. When testosterone-17 beta CA and testosterone were implanted together, the growth-promoting effect of the latter on the accessory sex organs was significantly reduced. The effectiveness of testosterone in suppressing the level of LH in the plasma of these animals was not influenced by the presence of testosterone-17 beta CA and in certain instances the level was raised.

5-alpha Reductase Inhibitors↗