Journal of steroid biochemistry最新文献

The gastrointestinal tract is a well characterized target tissue for aldosterone, where it regulates electrolyte transport, particularly in the descending colon. Previous studies have demonstrated the presence of aldosterone receptors in gastrointestinal tissues. We have used specific cRNA probes for the rat mineralocorticoid receptor to explore both the distribution and ontogeny of mineralocorticoid receptor gene expression in the gastrointestinal tract.

Mineralocorticoid receptor gene expression is found throughout the small and large intestine, but is absent from the stomach. The highest levels are observed in the distal colon, and significant expression is found in the duodenum; in both tissues levels of expression are higher than those in kidney. In both the developing duodenum and colon, mineralocorticoid receptor gene expression precedes the development of the full physiological response to aldosterone. These findings emphasise the colon as an important target tissue for aldosterone, and raise the question of potential roles for aldosterone in the duodenum.

Estrogen metabolism was evaluated in freshly isolated kidney and liver microsomes and in primary kidney cell cultures from Syrian hamsters, a potential experimental model for examining the possible role(s) of estrogens in tumor initiation and development. Initial velocity studies of the conversion of estradiol to 2-hydroxyestradiol, as determined by the ³H₂O release assay with the substrate [2-³H]estradiol, resulted in similar apparent K_ms of estrogen 2-hydroxylase of 2.85 and 6.25 μM for liver and renal microsomes, respectively. The apparent V_max for freshly prepared liver microsomes was 0.13 nmol·mg⁻¹-min⁻¹, while that for renal microsomes was 0.040 nmol · mg⁻¹ · min⁻¹. Evaluation of estrogen metabolism was also performed in primary cell cultures of hamster kidney cells, consisting of 75% epithelial cells. [6,7-³H]Estradiol (10 μM) was incubated for 0, 24 and 48 h in primary kidney cell cultures, and the organic soluble metabolites analyzed by reverse-phase HPLC. The cultures from untreated, castrated hamsters metabolize [³H]estradiol to yield small quantities of estrone and significant amounts of polar metabolites, while no catechol estrogens were isolated. Estrogen metabolism by diethylstilbestrol-treated (DES-treated) hamster kidney cell cultures also provided small quantities of estrone and no evidence of catechol estrogens. Additionally, larger amounts of additional polar metabolites were isolated in the cultures from DES-treated hamsters. Finally, levels of estrogen 2-hydroxylase were detected in these cultures using the ³H₂O release assay. Thus, the short-term primary kidney cell cultures from the Syrian hamster are capable of metabolizing estrogens. Furthermore, the enzymatic processes appear to be available for the conversion of any catechol estrogens formed into more polar metabolites. These investigations in intact cells, capable of performing all biochemical processes, complement both in vivo and subcellular biochemical studies and may aid in elucidating the roles of estrogens and estrogen metabolism in the initiation and development of estrogen-induced, estrogen-dependent kidney tumors in the Syrian hamster.

A method for analysing 26-hydroxycholesterol (260HC) in serum and tissue samples using solid phase extraction and high-performance liquid chromatography is reported. This procedure was used to measure the levels of 260HC in the sera of apparently healthy subjects and of 18 patients with angiographically proven atherosclerosis. Sixteen of the patients had levels within or below the range detected in the apparently healthy subjects (125–294 ng/ml), indicating that high 260HC levels cannot be a major factor in the development of atherosclerosis. However, when the patients and the normal subjects were combined in a group, there was a significant positive correlation (r = 0.5, P < 0.01) between serum cholesterol and serum 260HC, and that correlation approached significance for each of the individual groups (P = 0.06 for each group). These results suggest that there is an association between cholesterol and 260HC levels in human serum.

A sensitive assay of aromatization of 16α-hydroxylated androgens, 16α-hydroxyandrostenedione (16α-OHA), 16α,19-dihydroxyandrostenedione [16α,19-(OH)₂A], and 16α hydroxy-19-oxo androstenedione (16α-OH-19-oxo A), was developed using reversed phase high-performance liquid chromatography with a coulometric detector. The estrogens, estriol and 16α-hydroxyestrone, were simultaneously detected in quantities as low as 300 pg of the estrogens formed in an assay by an internal standard method. Apparent K_m and V_max of the microsomal aromatase for 16α-OHA, 16α,19-(OH)₂A or 16α-OH-19-oxo A were 1.06, 4.00 or 571 μM and 0.014, 0.087 or 1.67 pmol/min/μg protein, respectively. The results show that the 19-oxo steroid has extremely low affinity for aromatase relative to the other substrates.

The purpose of this study was to determine if the similarity of effect of progesterone and androgens on antagonism of estrogen-induced prolactin release also applied to the regulation of LH and FSH release. An additional objective was to examine the effect of the antiandrogen, flutamide, upon the ability of progesterone to induce gonadotropin secretion. Using the ovariectomized estrogen-primed immature rat, testosterone propionate suppressed LH and FSH secretion, whereas dihydrotestosterone only suppressed serum LH levels. In contrast, progesterone significantly elevated both serum LH and FSH levels. Thus, with respect to regulation of gonadotropin secretion, the effects of androgens and progesterone were dissimilar. In the estrogen-primed ovariectomized immature rat, flutamide was found to suppress LH, FSH and PRL secretion. Progesterone (0.8 mg/kg body wt) was incapable of overcoming this suppressive effect of flutamide. The effect of flutamide on gonadotropin secretion required estrogen priming. The effect of flutamide in suppressing LH, FSH and PRL release was not through suppression of an adrenal steroid as shown by adrenalectomy or the use of RU486. In the PMSG primed immature rat, flutamide had no effect on basal gonadotropin levels or ovulation. However, flutamide antagonized progesterone and triamcinolone acetonide-induced gonadotropin surges and blocked their ability to facilitate ovulation. These studies demonstrate that in the ovariectomized estrogen-primed immature rat flutamide has potent neuroendocrine regulatory ability leading to suppression of LH, FSH and PRL release. Flutamide also blocked progesterone and triamcinolone acetonide induced gonadotropin surges and ovulation in PMSG-primed immature female rats.

The protective effect of androgen against the cell death of mouse uterine epithelium was evaluated by examining the retention of 5'-[¹²⁵I]iodo-2'-deoxyuridine ([¹²⁵I]IdUrd) incorporated into the whole uterus and the apoptotic index (percentage of the apoptotic cells to the total cells) which is a good index of physiological cell death. Castrated adult female mice were daily injected with oestradiol-17β for 3 days, followed by the injection of [¹²⁵I]IdUrd. Thereafter, these mice were daily injected with only the vehicle or 5α-dihydrotestosterone (DHT), and the ¹²⁵I-radioactivity retained in the whole uterus was determined. When only the vehicle was injected, the ¹²⁵I-radioactivity retained in the whole uterus rapidly decreased but injections of DHT reduced the loss of ¹²⁵I-radioactivity. The effect of DHT on the retention of ¹²⁵I-radioactivity depended on doses of DHT and was abolished by the pure antiandrogen, flutamide. The apoptotic index of uterine cells was examined by a similar experimental protocol, but without an injection of [¹²⁵I]IdUrd. Injections of only the vehicle caused marked increases in the apoptotic indices of both luminal and glandular epithelia, but injections of DHT decreased them significantly. The apoptotic index of stroma was not affected by the injection of DHT. The present results indicate that androgen reduces the cell death of mouse uterine epithelium through the androgen receptor.

The activity of purified bovine adrenocortical P-450_11β on the C₁₈-steroid, 4-estrene-3,17-dione (19-norandrostenedione), is described. The major steroid products were separated by HPLC and identified by GC-MS, and ¹H- and ¹³C-NMR as 11β-, 18- and 6β-hydroxylated derivatives of 19-norandrostenedione. The turnover numbers of the 11β-, 18-and 6β-hydroxylase reactions were 45, 7.5 and 1.9 (mol/min/mol of P-450_11β), respectively, with a common K_m of 44 μM. All of these activities required the presence of the electron donating system consisting of NADPH, adrenal ferredoxin (adrenodoxin) and its reductase. These findings provide additional insights into the versatile catalytic roles of P-450_11β in the adrenal cortex, in which it may act on C₁₈-19-nor-steroids in addition to its known activities on C₂₁ and C₁₉-steroids.