ABSTRACT
Chronic administration (6 days) of LH-RH to adenohypophyseal cells in monolayer culture leads to a 4-fold increase of the total content (cells plus medium) of radioimmunoassayable LH. A 75 % increase was observed at an initial concentration of 1 × 10−10 M LH-RH while a progressive increase to 300 % over control was measured at a concentration of 1 × 10−6 M of the synthetic neurohormone. Total FSH content was increased 3-fold under similar conditions, a plateau of stimulation being reached at an initial concentration of 1 × 10−9 M LH-RH. Chronic treatment with TRH leads to 2- to 2.5-fold stimulation of total TSH content. These data show clearly that synthetic TRH and LH-RH, besides stimulating the release of respectively TSH and LH and FSH, lead to a marked stimulation of the synthesis of their trophic hormones. At least in the case of LH and FSH, increased synthesis occurs during the first 24 h of incubation in presence of the neurohormones. Half-maximal (3- to 4-fold) stimulation of cyclic AMP accumulation in incubated rat adenohypophyseal tissue is observed at a concentration of 10−10 to 10−9 M of the LH-releasing hormone. A 2-fold increase of cyclic AMP accumulation is measured 2 to 6 min after addition of synthetic TRH while addition of a purified fraction of GH-RH leads to a rapid and marked (up to 10-fold) increased concentration of cyclic AMP. The rates of release of LH and FSH and of GH follow very closely the observed changes of the intracellular concentration of cyclic AMP, both as a function of dose of the neurohormone and time of incubation. Data obtained with theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, indicate that the changes of cyclic AMP concentration are secondary to increased adenylate cyclase activity in the specific adenohypophyseal cells. Chronic administration of N6-monobutyryl cyclic AMP to anterior pituitary cells in culture leads to a 2-fold stimulation of total LH content, thus indicating that the effects of synthetic LH-RH on both release and synthesis of LH can be mimicked by cyclic AMP. In short-term experiments, the incorporation of [3H] leucine into GH and PRL separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis is stimulated 2-fold by the presence of 2 mM N6-monobutyryl cyclic AMP during the preceding 2 h of incubation. The cyclic nucleotide, although stimulating both the rate of synthesis and release of GH and PRL, does not affect the over-all intracellular transit time of GH and PRL. Measurements of specific activity of the intracellular and released hormones show that newly-synthesized GH and PRL are preferentially released under stimulatory conditions. Since cyclic AMP stimulates the synthesis of at least LH, GH and PRL and that the short-term stimulatory effects of the cyclic nucleotide appear to operate at the translational level, phosphorylation of ribosomal proteins and some pertinent properties of the ribosome-associated protein kinase were studied. The ribosome-associated protein kinase is stimulated by cyclic AMP and by cyclic IMP at respective Km values of 1 × 10−6 and 1 × 10−5 M. Phosphorylation of ribosomal proteins was stimulated by cyclic AMP in the cell-free system and by N6-2′-O-dibutyryl cyclic AMP in intact cells. As shown by polyacrylamide gel electrophoresis, the radioactivity was mainly associated with one protein band. The data suggest a possible mechanism by which cyclic AMP could enhance protein synthesis at the translational level in the adenohypophysis. In order to investigate the metabolic requirements for the transport, packaging and storage of secretory products in somatotrophs and mammotrophs, quantitative electron microscope autoradiography was performed on fragments of rat anterior pituitary tissue pulse labelled for 3 min with [3H] leucine and incubated in chase medium for various lengths of time in the presence or absence of KCN, a respiratory inhibitor or of dinitrophenol, an inhibitor of respiratory phosphorylation. Radioactivity accumulates in the Golgi complex within 10 min and is almost exclusively found in the secretory granules after 60 min. The transport of proteins from the cisternae of the rough endoplasmic reticulum to the Golgi complex and from the Golgi apparatus to the secretory granules represent two energy-dependent steps.
Induction of mitochondrial proteins in MA-10 Leydig tumour cells with human choriogonadotropin