Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat

Radioautography of tritiated diprenorphine in rat brain indicates anatomic distribution of receptors with a greater degree of precision than is possible using dissection techniques. The results of this study largely confirm those of others but indicate some differences in receptor distribution in the thalamus. Differential receptor binding in the periaquaductal gray matter with the highest counts lying laterally is an original observation.     

Tissue sources of sex steroids in rat ovaries during the preovulatory period

The method of quantitative histoenzymological analysis was used to determine the extent of participation of various structures of the ovary in the provision of the preovulatory synthesis of sex hormones. The activity of steroid dehydrogenases (3beta, 17beta, and 20alpha-OH), glucoso-6phosphric dehydrogenases, NAD and NADP-diaphorases was investigated. The synthesis of sex hormones proved to be realized by the mobilization of all the ovarian structures. At the early proestrus enhanced estrogen synthesis was provided by mature follicles, interstitial glands and the old corpora lutea. In the mid proestrus the active sources of progesterone and 20alpha-hydroxypregh-4en-3on synthesis are young corpora lutea and follicles; at this time the interstitial glands and old corpora lutea synthesized mainly the progesterone derivative.     

Dual effect of female sex steroids on drug-induced gastroduodenal ulcers in the rat.

Since the sexual dimorphism of gastroduodenal ulcers is well known and might possibly relate to the actions of sex hormones, we studied the role of the female sex steroids, progesterone and 17beta-estradiol in cysteamine-induced mucosal ulcers in female Wistar rats (200-220 g). Administration of cysteamine (400 mg/kg, s.c.) provoked macroscopic gastroduodenal mucosa injury as assessed planimetrically, an increase in microvascular permeability in the stomach and the duodenum as assessed by extravasation of radiolabelled albumin, and decreased gastroduodenal mucus levels as assessed by the Alcian blue technique. Ovariectomy (2 weeks before cysteamine) decreased plasma 17beta-estradiol level as assessed by radioimmunoassay, gastroduodenal macroscopic injury and albumin extravasation, and increased mucus levels following cysteamine challenge. Administration of progesterone (10-50 mg/kg/week, s.c.) attenuated in a dose-dependent manner cysteamine-induced gastroduodenal mucosa injury and microvascular leakage, while it increased mucus levels in the stomach and the duodenum. In contrast, administration of 17beta-estradiol (1-5 mg/kg/week, s.c.) dose-dependently augmented gastric and duodenal macroscopic mucosa lesions and microvascular injury provoked by cysteamine, and caused a further reduction in gastroduodenal mucus levels observed after cysteamine administration. In different experiments, ovariectomy decreased indomethacin-induced gastroduodenal injury. The injection of 17beta-estradiol (1-5 mg/kg/week) did not affect gastroduodenal damage, while treatment with progesterone (10-50 mg/kg/week) protected against indomethacin-provoked mucosa ulcers. It is concluded that female sex steroids play a role in drug-induced gastroduodenal ulcers by modulating microvascular permeability and mucus secretion.     

The influence of sex steroids on calcium- and magnesium-induced mitogenesis in isolated rat thymic lymphocytes

Elevated calcium and magnesium concentrations promoted mitotic activity in rat thymic lymphocyte cultures. Oestradiol inhibited calcium- but not magnesium-induced mitogenesis. One prerequisite for the mitogenic action of calcium is a raised intracellular concentration of cyclic adenosine 3′5′ monophosphate (cyclic AMP) but cyclic AMP-induced mitogenesis was insensitive to oestradiol. This suggests that the steroid blocks the mitogenic process at a stage preceding the endogenous cyclic AMP elevation. Furthermore the mitogenic actions of adrenaline, which stimulates adenylate cyclase (the enzyme responsible for cyclic AMP biosynthesis), and caffeine, which inhibits phosphodiesterase (the enzyme which degrades cyclic AMP) were also insensitive to oestradiol inhibition. This precludes a direct effect of the steroid on these enzymes. However, oestradiol did inhibit the mitogenic action of parathyroid hormone (PTH). Since the mitogenic action of PTH probably involves increased calcium entry to the cell, oestradiol may block this ion influx. The inhibition of calcium- and PTH-induced mitogenesis must be attributable to some structurally specific action of oestradiol. The steroids cholesterol, progesterone and testosterone all failed to reduce calcium-induced mitogenesis, whereas both α and β oestradiol were effective. In addition to its insensitivity to oestradiol inhibition, magnesium-stimulated mitosis was unaffected by both imidazole and calcitonin at concentrations which significantly reduced calcium-stimulated proliferation. These findings are compatible with the thesis that magnesium-induced mitogenesis does not involve the elevation of cyclic AMP concentrations.     

Role of sex steroids and the pituitary in regulating the level of a specific estrogen-binding protein in rat liver

The effects of androgens (A), estrogens (E) and hypophysectomy on the content of an unusual rat liver estrogen-binding protein (UEBP) were studied by the differential quantitative method of the UEBP content measurement. The UEBP content was shown to increase during maturation of male rats. After A injections the UEBP content was high only in the liver of prepubertal but not of mature or immature males. Castration or hypophysectomy of mature males equally caused a decrease in the UEBP content in mature males whereas subsequent administration of A made it completely return to normal. Hypophysectomy of castrated males did not alter the UEBP content. A single injection of E provoked an appreciable reduction in the UEBP level after several days. Administration of A interfered with the inhibitory action of E after simultaneous injection of A and E and recovered the E-induced lowering of the UEBP content upon administration of A following E. Hypophysectomy of castrated males did not affect significantly the UEBP level. The UEBP content was insensitive to the direct action of pituitary factors. The pituitary is necessary for the realization of the effects of E alone but not A. It is suggested that the regulatory role of A consists in the maintenance of the constant optimal UEBP level in rat liver.     

Metabolism of tritiated D-glucose anomers in rat erythrocytes

It was recently proposed that alpha-D-glucose 6-phosphate may undergo enzyme-to-enzyme channelling between glucokinase and phosphoglucoisomerase in rat pancreatic islets. The present study aims at exploring whether a different situation prevails in cells deprived of glucokinase, namely in erythrocytes. At anomeric equilibrium, the ratio between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH was lower in rat erythrocytes incubated for 60 min at 4 degrees C in the presence of 2.8 mM, rather than 8.3 mM, D-glucose. This coincided with both a greater relative increase in beta-D-[5-3H]glucose, as compared to alpha-D-[5-3H]glucose, conversion to 3HOH and an increase in the beta/alpha ratio for 3HOH generation from D-[5-3H]glucose in response to an increase in the anomeric concentration from 2.8 to 8.3 mM, the suppression of the difference between the beta/alpha ratios for 3HOH generation from D-[2-3H]glucose and D-[5-3H]glucose in the erythrocytes incubated at 8.3 mM, as distinct from 2.8 mM, alpha- and beta-D-glucose, and a [2-3H]/[5-3H] ratio for 3HOH generation lower than unity in erythrocytes exposed to alpha-D-glucose but not significantly different from unity in the presence of beta-D-glucose. These findings emphasize the relevance of alpha-D-glucose 6-phosphate channelling between hexokinase and phosphoglucoisomerase as a determinant of the difference between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH, and reveal that the regulation of such a tunnelling process by the concentration of the D-glucose represents, in rat erythrocytes, a mirror image of that observed in rat pancreatic islets. The regulation of this process thus tightly depends on the identity of the hexokinase enzyme mainly responsible for the phosphorylation of D-glucose in distinct cell types.     

Species,sex and inter-individual differences in DNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes

Sex steroids, due to the generally negative responses observed in routinely employed standard genotoxicity assays, are considered epigenetic carcinogens. Some doubts on this conviction are raised by the results of recent studies providing evidence that cyproterone acetate and two structural analogues, chlormadinone acetate and megestrol acetate, are genotoxic in female rats but only for the liver, and in primary human hepatocytes from donors of both genders. The experimental evidence suggests that the metabolic activation of these molecules to reactive species and the consequent formation of DNA adducts occur only in the intact hepatocyte. Since the possibility that other sex steroids cause a liver-specific genotoxic effect cannot be ruled out a priori, we investigated nine drugs of this family for their ability to induce DNA repair synthesis in primary cultures of rat and human hepatocytes. Each steroid was tested in cultures from at least two male and two female donors of each species. Hepatocytes were exposed for 20h to sub-toxic concentrations ranging from 1 to 50 micro M, and DNA repair induction was measured by quantitative autoradiography. In primary rat hepatocytes, induction of DNA repair indicative of a frankly positive response was detected in cultures from: 2/2 males and 3/3 females with drospirenone, 2/2 males and 1/2 females with ethinylestradiol, 1/2 males and 1/2 females with oxymetholone, 1/2 males with norethisterone, 1/4 females with progesterone, and 1/4 males with methyltestosterone. Consistent negative responses were obtained with testosterone and stanozolol. A few inconclusive responses were observed in rat hepatocytes exposed to progesterone, medroxyprogesterone, norethisterone, methyltestosterone and oxymetholone. In contrast, under the same experimental conditions the nine sex steroids provided frankly negative responses in the large majority of cultures of primary hepatocytes from both male and female human donors; the only exceptions being the inconclusive responses obtained in cultures from two of the donors exposed to norethisterone and to ethinylestradiol, and from one of the donors exposed to testosterone, methyltestosterone, and stanozolol. These results and previous findings concerning cyproterone and its structural analogues suggest that sex steroids differ for their ability to induce DNA repair, and that their genotoxicity may be: (i) different in rat and human hepatocytes, (ii) dependent on the sex of the donor, and (iii) affected by inter-individual variability.     

Role of endocytosis in cellular uptake of sex steroids

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.     

Axoplasmic flow of tritiated proline in the corticospinal tract of the rat

Summary The rates of axoplasmic transport were studied in the corticospinal tract of the rat by injecting tritiated proline into the sensory-motor cortex and subsequently analyzing the distribution of incorporated label in the spinal cord at intervals after injection. A mathematical model of the anatomy of the corticospinal tract was developed and used in analysis of the data. The rate of a fast component was calculated to be 240–420 mm per day, which is comparable with rates of fast components in the peripheral nervous system (PNS), but considerably greater than rates in other tracts in the central nervous system. A slow component was calculated to have a transport rate of 3–8 mm per day which is greater than rates found either in the CNS or PNS. This higher rate may be related to the greater length of the corticospinal tract as compared to other CNS tracts studied.This research was financed by the Veterans Administration research support awarded to Dr. Feringa by the Development Funds of the Department of Pathology, University of Michigan, and by the University of Michigan Medical Center Fund for Computing. The authors wish to express their appreciation to Ms. Linda Lee Austin for technical assistance, Ms. Diane Trakas and Ms. Barbara Reader for secretarial aid, and Mr. Richard Fritzler for assistance with graphics     

The content of five sex steroids in human testis

In order to assess whether intratesticular hormone content may be helpful for prediction of successful conception in men with fertility problems, five sex steroids, testosterone, dihydrotestosterone, androstenedione, estradiol and, for the first time epitestosterone, were measured in testicular tissue obtained by surgical retrieval from total 84 men. The group consisted of non-obstructive azoospermic men, aged 21-67 years who attended the centre for in vitro fertilization. Steroids after ether extraction and solvent partition were separated by high performance liquid chromatography and then measured by specific radioimmunoassays. The values varied considerably with means ± S.D. 2.43±2.47, 0.27±0.24, 0.080±0.13, 0.071±0.089 and 0.31±0.27 for testosterone, dihydrotestosterone, androstenedione, estradiol and epitestosterone, respectively.