Frank Ungar

Circadian Rhythm in the in vitro Response of Mouse Adrenal to Adrenocorticotropic Hormone

Adrenal corticosterone production resulting from adrenocorticotropic hormone (ACTH) stimulation in vitro depends upon the time of gland removal. This rhythm in adrenal reactivity to ACTH is out of phase with the corticosterone rhythm in serum of the mice used as donors of adrenals. The responsiveness of the gland to exogenous ACTH is highest when serum corticosterone levels are lowest.

Thin-layer chromatographic systems for adrenal corticosteroids

Abstract Six thin-layer chromatography solvent systems for the separation of corticosteroids are presented. Their application for the isolation of aldosterone and the 18-hydroxysteroids from bovine adrenal extracts is discussed.

The role of a carrier protein in cholesterol and steroid hormone synthesis by adrenal enzymes

Squalene and sterol carrier protein (SCP) of liver, known to be required for cholesterol synthesis by liver microsomal enzymes, functions with adrenal microsomal enzymes (Δ7-sterol Δ5-dehydrogenase and Δ5,7-sterol Δ7-reductase) catalyzing cholesterol synthesis from sterol precursors. Adrenal preparations contain a heat-stable protein which functions similarly to liver-SCP with adrenal and liver enzymes. Liver-SCP also can participate with an adrenal enzyme (solubilized from an acetone powder of mitochondria) in the conversion of cholesterol to pregnenolone. These findings indicate that an SCP-like protein not only participates in cholesterol synthesis and transport in the adrenal, but also in the initial steps of cholesterol metabolism to steroid hormones.

In vitro exploration of a circadian Rhythm in adrenocorticotropic activity of C mouse hypophysis

Ein circadianer Rhythmus in der adrenocorticotropen Wirkung der Hypophyse bei Inzucht-C-Mäusen wird durch einin vitro-Verfahren nachgewiesen. Es werden Angaben der Phasenunterschiede zwischen dem hypophysären Rhythmus und den gleichfalls circadian-periodischen Schwankungen gemacht: (1) im Serum-Corticosteron und (2) in der Reaktion der Nebenniere auf ACTH,in vitro.

Corticosteroid identification and corticosterone concentration in serum of Rana pipiens during dehydration in winter and summer

Abstract 1. 1. Inter-renal tissue from the frog Rana pipiens was incubated in vitro with 4-14C-progesterone, the radioactive products extracted, and separated in simple forms or as acetylated derivatives in six solvent systems using thin layer chromatography. 2. 2. Corticosterone and aldosterone (produced in a ratio of 3 : 1) were observed to be the major products. 3. 3. The concentration of corticosterone (B) in serum from R. pipiens acclimated at 25°C was measured in May (summer) and November (winter) under normal and dehydrating conditions. 4. 4. The concentration of B was constant in winter frogs irrespective of treatment and was ca. 2·2 μg/100 cm3 serum. 5. 5. In the summer, tap-water control frogs had a high concentration of B, namely 1·8μg/100 cm3, while dehydrated frogs had significantly decreased concentrations of B; 0·7 and 1·1 μg/100 cm3 in 0·125 M and 0·150 M NaCl, respectively. 6. 6. The distribution of glucocorticoids observed in the Amphibia and a possible cause of corticosterone response to dehydration was discussed.

Formation of 3α-hydroxy-5β-pregnan-20-one in the ovaries of catfish, Heteropneustes fossilis (bloch)

Abstract Labeled pregnenolone was incubated with minced and homogenized tissue obtained from the ovaries and kidney or head kidney of two varieties of catfish, Heteropneustes fossilis and Ictalurus punctatus. No 21-hydroxylated steroids such as DOC were found as conversion products with ovarian tissue. The major ovarian steroid produced by H. fossilis was 3α-hydroxy-5β-pregnan-20-one. After extensive chromatography it was identified by recrystallization to constant specific activity. A second steroid was isolated and tentatively assigned the structure 5β-pregnane-3α-20α-diol based on chromatographic mobilities. In vivo pretreatment with salmon gonadotropin but not ACTH enhanced the conversion rate to 3α-hydroxy-5β-pregnan-20-one in ovarian tissue in H. fossilis. No conversion products were identified in the ovarian tissue of the channel catfish, Ictalurus punctatus. Extensive conversion of precursor to corticosteroid products was observed with interranal tissue in both species. In vivo pretreatment with ACTH enhanced the formation of adrenocorticosteroid products by interrenal tissue.

Pineal modulation of ACTH 1–17 effect upon murine corticosterone production

In tests of corticosterone production in vitro, aqueous pineal homogenate (APH) modulates the effect of a short-chain ACTH analogue, ACTH 1-17, added to adrenals from different circadian stages. Adrenal and pineal glands from female B6D2F1 mice, standardized on staggered LD 12:12 regimens, were obtained at the same clock-hour from each room, in order to cover 6 different circadian stages. Adrenals from each circadian stage were bisected and incubated with APH from the same circadian stage (isophasic incubation) or from one of the other 5 circadian stages (heterophasic incubation). ACTH 1-17 (0.05 IU) was added to each incubation medium. After 4 hours of incubation at 37 degrees C with 95% O2 and 5% CO2, the media were stored at -20 degrees C until corticosterone RIA were done. APH was found to have a statistically significant modulatory effect upon the stimulation by ACTH 1-17 of adrenal corticosterone production in vitro. This APH effect changed rhythmically as a function of circadian stage from amplification over no effect to attenuation, as a so-called feed-sideward.

Circadian rhythmic pituitary adrenocorticotropic activity, rectal temperature and pinnal mitosis of starving, dehydrated c mice.

Summary Circadian rhythms in pituitary adrenocorticotropic activity, pinnal mitosis and rectal temperature, as well as in the corticosterone content of serum and adrenal, persist in inbred Bagg albino (C) mice deprived of food and water. A lead in phase of the rhythm in pituitary ACTH content over the circadian rhythm in serum corticosterone—an aspect of internal timing in a mammal—is apparent in starved and dehydrated mice as well as in mice with food and water available ad libitum. The rhythms in pinnal mitosis and rectal temperature, compared with each other or with the rhythms in adrenal and serum corticosterone, also show statistically significant differences in phase that characterize circadian systems in the absence, as well as presence, of food and water.

Descending and ascending chromatography of steroids using thin-layer chromatography sheets

Abstract The utility of newly available thin-layer chromatography (TLC) sheets was investigated using both descending and ascending chromatography. This was done for pure steroids and biological extracts. The advantages and disadvantages of these sheets relative to conventional TLC plates are discussed.

Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains

Cholesterol and α-tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4-month-old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37°C) and oxidized by adding (i) ferrous iron (Fe2+) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe2+ in the presence or absence of ascorbate produced primarily 7-keto- and 7-hydroxy-cholesterols and small amounts of 5α,6α-epoxycholesterol. Since brain contains high levels of ascorbate, any release of iron could result in oxysterol formation. Peroxynitrite oxidized α-tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 μM Fe2+ and ascorbate, α-tocopherol was oxidized while cholesterol remained unchanged. Thus, α-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA dit not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe2+ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7-oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may play an important role in brain tissue damage during oxidative stress.