Epidermal growth factor: structure, location, phosphorylation and regulation of its receptor

Epidermal growth factor (EGF) is a Mr 6045 polypeptide first characterized for its ability to stimulate mitogenesis in epidermal and epithelial cells. The first step in the action of the growth factor is its binding to specific, high affinity membrane receptors. These receptors have been studied in a number of tissues and cell culture lines. The level of EGF receptors is modulated by many agents. EGF down-regulates its receptor. In addition, the number of EGF receptors is decreased by other growth factors (platelet-derived growth factor; transforming growth factor), by many tumor promoters and by viral transformation. Several hormones also can regulate EGF binding in its target tissues.     

Extremely rapid degradation of [3H] methionine-enkephalin by various rat tissues in vivo and in vitro

Thirty sec after the intrajugular injection of [³H] methionine-enkephalin (met-enkephalin) in the rat, the radioactivity was already distributed in an apparent volume of 53 ml and the metabolic clearance rate calculated from the characteristics of the plasma disappearance curve was 10 ml/min. As shown by partition chromatography plasma extracts obtained 15 sec after injection of [³H] met-enkephalin, only 5% of the total radioactivity migrated as the intact pentapeptide, while no detectable intact pentapeptide remained 2 min after injection, thus indicating a half-life of [³H] met-enkephalin of the order of 2 to 4 sec. Incubation of rat cerebral tissue with [³H] met-enkephalin indicates that the first step in the breakdown of met-enkephalin in both plasma and brain tissue is cleavage of the Tyr-Gly amide bond. These data offer an explanation for the low activity of met-enkephalin after intraventricular or intravenous administration.     

Corticotropin-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Ovine corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in rat anterior pituitary homogenate at an ED₅₀ value of 70 nM. GTP increases the stimulatory effect of CRF on [³²P] cyclic AMP formation in a rat adenohypophysial particulate fraction and in bovine anterior pituitary plasma membranes. The present data show that CRF stimulates adenylate cyclase activity in the anterior pituitary gland at least partly through a guanyl nucleotide-dependent mechanism.     

Immunohistochemical localization of beta-LPH in the human hypothalamus.

In order to establish the presence of β-LPH and to clearly identify the nervous structures containing β-LPH in the human hypothalamus, an immunohistochemical localization of β-LPH was performed in this tissue. The immunohistochemical technique involved use of a specific antiserum to human β-LPH and the peroxidase-antiperoxidase complex. Immunostained neuronal cell bodies were observed in the arcuate nucleus whereas β-LPH-positive nervous fibers could be detected in a large area extending rostro-caudally from the anterior part of the paraventricular nucleus up to the mammillary bodies. Staining was completely abolished by previous immunoabsorption with β-LPH while β-endorphin and ovine γ-LPH_1–47 only partially prevented immunostaining. Although it cannot be excluded that the precursor 31K molecule, β-LPH_1–58 and/or β-endorphin are detected by the immunostaining, it is likely that β-LPH is at least partly responsible for the positive reaction.     

Localization of 17 beta-hydroxysteroid dehydrogenase throughout gestation in human placenta

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) is the enzyme responsible for the formation of all sex steroids in gonadal as well as extragonadal tissues. To obtain more information about the age-specific expression of 17 beta-HSD in the human placenta, we have localized this enzyme by immunocytochemistry at the light microscopic level at different periods of gestation. In the 7- and 9-week-old placenta, immunostaining was detected exclusively in the cytoplasm of the syncytiotrophoblast. Between the tenth and thirteenth weeks of gestation, immunolabeling was also observed in the cytoplasm of the cytotrophoblastic cells, suggesting that these cells could be transiently involved in the biosynthesis of sex steroids. Interestingly, between the fourteenth and twenty-fifth weeks of gestation, 17 beta-HSD was observed in both the cytoplasm and nucleus of the syncytiotrophoblast. The reaction product was much more intense in nuclei than in cytoplasm. During the last trimester of gestation, strong immunocytochemical staining was observed in all the nuclei of the syncytiotrophoblast, the cytoplasm being unstained. The meaning of this nuclear staining for 17 beta-HSD is still unclear and remains to be extensively investigated.     

Steroid glucuronides: Human circulatory levels and formation by LNCaP cells

We studied the relationship between circulating androsterone glucuronide, androstane-3,17β-diol glucuronide and androstane-3β,17β-diol glucuronide concentrations and adrenal as well as testicular C-19 steroids in men. Among the three 5-reduced steroid glucuronides, androsterone glucuronide is the predominant C-19 steroid measured in plasma and its levels are markedly elevated compared to those of the non-conjugated steroid. The marked rise in testosterone during puberty was strongly correlated with the increase in both androsterone glucuronide and androstane-3,17β-diol glucuronide, thus suggesting that testicular C-19 steroids are the main precursors of the steroid glucuronides. We also found that the presence of testicular androgen in plasma contributes to approx. 70% of plasma androsterone glucuronide and androstane-3,17β-diol glucuronide. Our data suggest that the adrenal C-19 steroids remaining in circulation after castration in men are converted into potent androgen which are then glucuronidated by UDP-glucuronyltransferase. We also demonstrated that the human prostate cell line LNCaP is capable of converting to a large extent androstenedione into androsterone glucuronide. Our data further confirm that glucuronidation is a major pathway of steroid metabolism in steroid target tissues.     

Differential inhibition of dehydrogenase and 5-ene→4-ene isomerase activities of purified 3β-hydroxysteroid dehydrogenase. Evidence for two distinct sites

The success in synthesis of [³H]5-androstene-3,17-dione, the intermediate product in the transformation of DHEA to 4-androstenedione by 3β-hydroxysteroid dehydrogenase/ 5-ene→4-ene isomerase (3β-HSD) offers the opportunity to determine whether or not the two activities reside in one active site or in two closely related active sites. The finding that N,N-dimethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxamide (4-MA) inhibits competitively and specifically the dehydrogenase activity whereas a non-competitive inhibition type with a K_i value 1000 fold higher was observed for the isomerase activity, indicated that dehydrogenase and isomerase activities belong to separate sites. Using 5-dihydro-testosterone and 5-androstane-3β,17β-diol, exclusive substrates for dehydrogenase activity, it was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.     

Age-related changes in central nervous system enkephalins and substance P

Concentrations of substance P and met- and leu-enkephalins were measured by radioimmunoassay in discrete rat brain nuclei of young (4–5 months) and old (24–26 months) rats. The substance P content of n. anterior (hypothalami), n. ventromedialis, n. premamillaris ventralis, n. interstitialis striae terminalis, n. entopeduncularis and n. dorsalis raphes is reduced in old rats. The met-enkephalin content is decreased in n. suprachiasmaticus, n. arcuatus and n. premamillaris ventralis while the leu-enkephalin content of n. preopticus medialis, n. suprachiasmaticus, n. paraventricularis, n. ventromedialis and n. premamillaris ventralis is decreased in old rats.     

Specific progesterone receptors in dimethylbenzanthracene (DMBA)-induced mammary tumors.

Properties of a progesterone receptor present in the cytosol (105,000 xg supernatant) of dimethylbenzanthracene (DMBA)-induced mammary tumors were studied using the highly potent progestin [3H]R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of [3H] R 5020 is associated with components migrating at 7-8S and 4S. Low affinity binding of the synthetic progestin is eliminated by treatment with dextran-coated charcoal. [3H] R 5020 binding is highly progestin-specific since it is easily displaced by unlabelled norgestrel, R 5020 and progesterone while estradiol-17beta, dihydrotestosterone, testosterone, testosterone and diethylstilbestrol have much lower activity. Dexamethasone and cortisol have little, if any, effect on [3H] R 5020 binding.