Administration of human pancreatic growth hormone-releasing factor (GRF) analogs enhances responsiveness of cultured rat pituitary cells to GRF

The aim of this study was to investigate whether anterior pituitary responsiveness to human pancreatic growth hormone-releasing factor containing 29 amino acids (GRF-29) can be modulated by GRF-29 itself. Male rats were injected (sc) daily for 3 days with 50 ug of GRF-29, or were treated twice daily for 14 days with 5 ug of [D-Ala-2]-GRF-29 (a potent GRF agonist). Control animals were injected with saline. After the last injection, pituitaries were removed, dispersed, cultured for 96 h and then challenged with either GRF-29 or [D-Trp-6]-LHRH (a LHRH agonist). Cultured cells from analog-treated rats were more responsive to GRF-29 stimulation than were cells obtained from controls. In contrast, neither treatment altered the response to [D-Trp-6]-LHRH. These studies indicate that periodic administration of GRF analogs can increase hypophyseal GRF responsiveness. Such control may be an important component in the physiological regulation of GH secretion and has important implications for potential therapeutic uses of GRF analogs.     

Autoregulation of testicular luteinizing hormone receptors in hypogonadal (hpg/hpg) mice

The autoregulation of testicular luteinizing hormone (LH) receptors was studied in hypogonadal (hpg/hpg) and normal mice. The basal concentration of LH receptors was more than three-fold higher in hpg/hpg than in normal mice. After injection of hCG, hpg/hpg mice showed a decrease in LH receptor levels which was not observed in normal mice. Plasma testosterone was undetectable in hpg/hpg mice, even after treatment with a single dose of hCG. Plasma prolactin levels were higher in hpg/hpg than in normal mice. The increase in basal LH receptor levels is thought to be due to a compensatory mechanism in which elevated prolactin could play a role. The differences between hpg/hpg and normal mice in the autoregulation of LH receptors observed could be due to the hypersensitivity of the physiologically immature testis in hpg/hpg mice to the action of hCG, to gonadotropin deficiency, particularly during the earlier stages of development, or to a direct effect of the hpg locus on the metabolism of LH receptors.These studies were supported by NIH Grants HD 12642 and HD 12671 (AB) and Grant CA-24145 (WGB).     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Effects of different batches of iodine on properties of I-hFSH and characteristics of radioligand-receptor assays

Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCi carrier free Na ¹²⁵I (Amersham Corp., 15 mCi/μg I₂) in the presence of lactoperoxidase. Incorporation of ¹²⁵I into hFSH was determined by the method of [7.]Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20°C) using different calf testis membrane preparations having similar receptor characteristics. Each ¹²⁵I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of K_a and R_t. Incorporation of ¹²⁵I into FSH was relatively constant over the large number of experiments (62.4 ± 6.4 μCi/μg; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of ¹²⁵I-hFSH was related to the lot of ¹²⁵I utilized, and was significantly (P ≤ 0.01) lower and more variable (28.7 ± 10.5 μCi/μg). Maximum bindability of ¹²⁵I-hFSH was not correlated to specific activity (r = 0.06) but was negatively correlated to hFSH ¹²⁵I incorporation (r = −0.47; P ≤ 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na¹²⁵I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.     

牛生长激素结构不均一性的研究

本文报导用常规方法分离纯化的牛生长激素,在还原性SDS-11％聚丙烯酰胺凝胶电泳中呈分子量很接近的两条主带(22KD,21.5KD)。用单克隆抗体和多克隆抗体亲和层析技术分析了不同分子量形式的牛生长激素的转化,结果表明:牛生长激素可能在pH5.5条件下转化为21.5KD分子,在pH8.3条件下则转化18KD分子。这几种形式的牛生长激素均保留与抗体的结合力,但亲和力不尽相同,如在亲和层析的洗脱性质上存在差异。已检验分离并部分纯化了18KD分子以备作进一步的研究。     

双抗体免疫沉淀法纯化牛生长激素poly(A)~+RNA的研究

我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91％可以被牛生长激素抗体沉淀。用SDS-11％PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。     

Influence ofl-thyroxine,l-triiodothyronine,thyroid stimulating hormone,or estradiol on the cell kinetics of cultured mammary cancer cells

Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T₃),l-thyroxine (T₄), a thyroid-stimulating hormone (TSH), and/or estradiol (E₂: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G₂+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T₃, T₄, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S+G₂+M fraction as does E₂. Furthermore, our data indicate that E₂ and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics. This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium).