melitten and mezerein

Follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin, a novel gonadal glycoprotein hormone, has been shown to have chronic inhibitory effects on the secretion of both FSH and luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) in vitro. The present study was designed to investigate the acute effects of bovine FSP on GnRH-stimulated gonadotropin secretion and to examine the potential subcellular sites of this action of FSP using cultured pituitary cells. Anterior pituitaries from adult male Sprague-Dawley rats were enzymatically dispersed and cultured for 48 h, after which the cells were treated with bovine FSP for 6 h, followed by a 4 h stimulation with secretagogues in the continued presence of FSP. Results showed that the 35 kDa form of bovine FSP (0.1-3 nM) dose-dependently suppressed GnRH-stimulated FSH and LH secretion, with inhibition of 38 and 25%, respectively, at 3 nM. In addition, FSP suppressed gonadotropin secretion in response to activators of protein kinase C (phorbol 12-myristate 13-acetate (PMA) and mezerein) and a calcium ionophore (A23187). However, FSP had no effect on gonadotropin secretion evoked by melittin, an activator of phospholipase A2. Furthermore, 35 kDa bovine FSP did not compete with GnRH for GnRH binding sites in a direct competition study and treatment of cultured pituitary cells with FSP (0.1-3 nM) for 10 h did not alter the number of GnRH binding sites on the cell membranes. Finally, similar inhibitory effects on gonadotropin secretion in response to GnRH, PMA and mezerein were obtained with the 31 and 39 kDa forms of bovine FSP, each at a concentration of 1 nM. We conclude from the present study that FSP acutely inhibits GnRH-stimulated gonadotropin secretion in cultured pituitary cells, and that FSP exerts its action beyond the GnRH receptor, possibly by affecting the protein kinase C and/or the calcium-calmodulin systems.

Topics: Animals; Calcimycin; Cells, Cultured; Diterpenes; Enzyme Activation; Follicle Stimulating Hormone; Follistatin; Glycoproteins; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Male; Melitten; Phospholipases A; Phospholipases A2; Pituitary Gland, Anterior; Protein Kinase C; Rats; Rats, Inbred Strains; Terpenes; Tetradecanoylphorbol Acetate

To further characterize the subcellular mechanisms by which inhibin suppresses GnRH-stimulated gonadotropin release, anterior pituitary cells from adult male Sprague-Dawley rats were treated on day 2 of culture with or without purified 31-kDa bovine inhibin (1-300 pM) for a further 3 days. On day 5, the pretreated cells were washed and incubated in the absence or presence of various secretagogues for 4 h. At the end of the stimulation, the media were saved, and cells were lysed for measurement of both extracellular and intracellular FSH and LH by specific RIAs. Released hormone was expressed as the proportion of total (released plus intracellular) hormone that was available for release in each case. This manipulation of the data corrects for the differential effect of the inhibin pretreatments to suppress intracellular FSH before the stimulation period. Pretreatment for 3 days with inhibin suppressed the proportions of FSH and LH released during 4 h in response to 1) phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase-C, by maxima of 48% and 53% with inhibin median inhibitory concentrations (IC50) of 17 and 18 pM, respectively; 2) mezerein (100 nM), another type of activator of protein kinase-C, by maxima of 49% and 50% with inhibin IC50 of 19 and 20 pM, respectively; 3) high extracellular K+ (60 mM) by 42% (P less than 0.01) and 38% (P less than 0.01), respectively, with 130 pM inhibin; 4) the calcium ionophore, A23187 (100 microM) by maxima of 54% and 56% with IC50 of 18 and 17 pM, respectively; and 5) GnRH (10 nM) by maxima of 52% and 53% with IC50 of 18 and 19 pM, respectively. However, inhibin had no effect on the proportional release of gonadotropin induced by melittin, an activator of phospholipase-A2. Finally, inhibin had no effect on ACTH release either under basal conditions or in response to CRF (10 nM), phorbol 12-myristate 13-acetate (100 nM), or A23187 (100 microM). We conclude that inhibin suppresses the stimulated release of hormones from gonadotrophs in part by a mechanism common to both gonadotropins that is independent of the previously described inhibitory effect of inhibin on the GnRH receptor. The results are consistent with an action at a site(s) beyond the GnRH receptor, such as protein kinase-C and calmodulin.

Topics: Adrenocorticotropic Hormone; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cells, Cultured; Diterpenes; Enzyme Activation; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Inhibins; Luteinizing Hormone; Male; Melitten; Pituitary Gland, Anterior; Potassium; Protein Kinase C; Rats; Rats, Inbred Strains; Receptors, LHRH; Terpenes; Tetradecanoylphorbol Acetate

The induction of aryl hydrocarbon hydroxylases in lymphocytes is dependent on their activation. The tumor-promoting phorbol esters which induce blast formation and DNA synthesis in lymphocytes enable polycyclic aromatic hydrocarbons to induce aryl hydrocarbon hydroxylases. Melittin, the major constituent of bee venom, acts synergistically with these phorbol esters in enhancing both lymphocyte activation and hydroxylase synthesis. Since aryl hydrocarbon hydroxylases convert procarcinogens to carcinogens these results suggest that tumor promotion by phorbol esters may be associated with their ability to affect the induction of these enzymes. This hypothesis is supported by the finding that phorbol and phorbol esters which lack tumor-promoting activity fail to enhance induction of aryl hydrocarbon hydroxylases in lymphocytes. However mezerein, although rather ineffective in promoting tumors, activates lymphocytes and permits polycyclic aromatic hydrocarbons to induce their hydroxylases effectively.

Topics: Aryl Hydrocarbon Hydroxylases; Benz(a)Anthracenes; Diterpenes; DNA; Drug Synergism; Humans; Lymphocyte Activation; Lymphocytes; Melitten; Phorbol Esters; Phorbols; Pokeweed Mitogens; Terpenes; Thymidine