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The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a significant role in a multifunctional array of important physiological processes in insects. PK/PBAN analogs incorporating beta-amino acids were synthesized and evaluated in a pheromonotropic assay in Heliothis peltigera, a melanotropic assay in Spodoptera littoralis, a pupariation assay in Neobellieria bullata, and a hindgut contractile assay in Leucophaea maderae. Two analogs (PK-betaA-1 and PK-betaA-4) demonstrate greatly enhanced resistance to the peptidases neprilysin and angiotensin converting enzyme that are shown to degrade the natural peptides. Despite the changes to the PK core, analog PK-betaA-4 represents a biostable, non-selective agonist in all four bioassays, essentially matching the potency of a natural PK in pupariation assay. Analog PK-betaA-2 is a potent agonist in the melanotropic assay, demonstrating full efficacy at 1pmol. In some cases, the structural changes imparted to the analogs modify the physiological responses. Analog PK-betaA-3 is a non-selective agonist in all four bioassays. The analog PK-betaA-1 shows greater selectivity than parent PK peptides; it is virtually inactive in the pupariation assay and represents a biostable antagonist in the pheromonotropic and melanotropic assays, without the significant agonism of the parent hexapeptide. These analogs provide new, and in some cases, biostable tools to endocrinologists studying similarities and differences in the mechanisms of the variety of PK/PBAN mediated physiological processes. They also may provide leads in the development of PK/PBAN-based, insect-specific pest management agents.

The present study was aimed at investigating the effect of protein kinase C (PKC) activation on CRF receptor function of proopiomelanocortin (POMC) cells in culture. Incubation of tissues with the phorbol ester PMA selectively potentiated corticotropin-releasing factor (CRF)-stimulated ACTH secretion and cyclic AMP formation of anterior pituitary (AP) cells, while, in sharp contrast, it failed to similarly affect intermediate pituitary (IP) cells and AtT-20 corticotrophs exposed to CRF. Unexpectedly, however, long-term treatment of cultures with PMA, which depletes cell stores of PKC, resulted in a similar dramatic attenuation of stimulated peptide release from both corticotrophs and melanotrophs, while being without significant effect on cyclic AMP production. Exposure of cells to PMA did not change either basal or CRF-enhanced levels of POMC mRNA. We conclude that activation of PKC fails to synergize with CRF-mediated signalling in IP and AtT-20 cells, although optimal CRF receptor expression requires the presence of a functional kinase C pathway, thus suggesting cross-talks between both messenger systems.

In frog melanotrope cells, γ-aminobutyric acid (GABA) induces a biphasic effect, i.e. a transient stimulation followed by a more sustained inhibition of α-MSH release, and both phases of the GABA effect are mediated by GABA A receptors. We have previously shown that the stimulatory phase evoked by GABA A receptor agonists can be accounted for by calcium entry. In the present study, we have investigated the involvement of the chloride flux on GABA-induced [Ca 2+ ] c increase and α-MSH release. We show that GABA evokes a concentration-dependent [Ca 2+ ] c rise through specific activation of the GABA A receptor. The GABA-induced [Ca 2+ ] c increase results from opening of voltage-activated L- and N-type calcium channels, and sodium channels. Variations of the extracellular Cl − concentration revealed that GABA-induced [Ca 2+ ] c rise and α-MSH release both depend on the Cl − flux direction and driving force. These observations suggest for the first time that GABA-gated Cl − efflux provokes an increase in [Ca 2+ ] c increase that is responsible for hormone secretion.

We have investigated the presence of ACTH, alpha-MSH and beta-endorphin, three peptides which derive from the multifunctional precursor protein proopiomelanocortin (POMC) in the brain of the rainbow trout Salmo gairdneri. Using both the indirect immunofluorescence and peroxidase-antiperoxidase techniques, a discrete group of positive cells was identified in the hypothalamus, within the anterior part of the nucleus lateralis tuberis. alpha-MSH-containing neurons represented the most abundant immunoreactive subpopulation. Coexistence of alpha-MSH, ACTH and beta-endorphin was observed in the lateral part of the nucleus. ACTH- and beta-endorphin-containing cells were mainly distributed in the rostral and caudal regions of the nucleus. In the medial portion of the nucleus lateralis tuberis, numerous cells were only stained for alpha-MSH. Moderate to dense plexuses of immunoreactive fibers were observed in the ventral thalamus and the floor of the hypothalamus. Some of these fibers projected towards the pituitary. The concentrations of ACTH, alpha-MSH and beta-endorphin-like immunoreactivities were measured in microdissected brain regions by means of specific radioimmunoassays. Diencephalon, mesencephalon and medulla oblongata extracts gave dilution curves which were parallel to standard curves. The highest concentrations of POMC-derived peptides were found in the diencephalon (alpha-MSH: 4.28 +/- 0.43 ng/mg prot.; ACTH: 1.08 +/- 0.09 ng/mg prot.; beta-endorphin: 1.02 +/- 0.1 ng/mg prot.), while lower concentrations were detected in the mesencephalon, medulla oblongata and telencephalon. The present results demonstrate that various peptides derived from POMC coexist within the same cell bodies of the fish hypothalamus. Taken together, these data suggest that expression and processing of POMC in the fish brain is similar to that occurring in pituitary melanotrophs.

The presence of thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) has been demonstrated in the neural lobe and in the intermediate lobe of the frog pituitary by immunocytochemistry on ultrathin sections of neurointermediate lobes obtained by cryoultramicrotomy. In the neural lobe, separate populations of TRH- or NPY-immunoreactive nerve fibers were observed. Both neuropeptides were contained in dense-core secretory vesicles about 200 nm in diameter. In intermediate lobe cells, TRH- and NPY-like immunoreactivities were observed in the cytoplasmic matrix and more sparsely in secretory granules. Occasionally, immunoreactive TRH could be visualized at the plasma membrane level. In the nucleus, both peptides were detected in the euchromatin, in the vicinity of the heterochromatin and in the nucleolus. Conversely, gonadotropin-releasing hormone-like immunoreactivity could not be detected. These results provide immunocytological evidence for the presence of endogenous TRH and NPY in frog melanotrophs indicating that these peptides may participate in the regulation of intermediate lobe secretion.

The histology, immunohistochemistry, peptide synthesis and secretion as well as proliferation rate of rat intermediate lobe (IL) were studied in primary cultures. The cultures contained two populations of cells: melanotrophs either organized in free floating lobules or in lobules which attached to the dishes and formed a monolayer. Both populations retained their in vivo morphology: polyhedral cells with smooth, ovoid nuclei and a large number of cytoplasmic secretory coated vesicles, a well developed Golgi apparatus, abundant mitochondria and extensive areas of rough endoplasmic reticulum. The melanotrophs stained with varying intensity for α-MSH and in situ hybridization showed the presence of pro-opiomelanocortin (POMC) mRNA. 35 S-methionine incorporation combined with 2-D gel electrophoresis demonstrated POMC peptide synthesis and radioimmunoassay confirmed its secretion into the medium. 3 H-thymidine uptake in the attached melanotrophs was considerably higher than that in the free-floating melanotrophs, demonstrating the dependency of proliferation rate on the cytoarchitecture of the explant. The retention of melanotroph morphology, biosynthetic and proliferative capacity in vitro affords a valid model system for studying POMC gene expression.