Rab3a is critical for trapping alpha-MSH granules in the high Ca²âº-affinity pool by preventing constitutive exocytosis.

Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca²âº-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca²âº-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca²âº] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and ß-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca²âº-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca²âº-sensitive release-ready vesicles as determined by slow photo-release of caged Ca²âº. Radioimmunoassay revealed that α-MSH, but not ß-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca²âº-dependent release of α-MSH.

Brain-derived neurotrophic factor stimulates growth of pituitary melanotrope cells in an autocrine way.

Brain-derived neurotrophic factor (BDNF) is expressed in the mammalian pituitary gland, in both the anterior and intermediate lobes, where its functional significance is unknown. Melanotrope cells in the intermediate pituitary lobe of the amphibian Xenopus laevis also produce BDNF, which co-exists in secretory granules with α-melanophore-stimulating hormone (α-MSH), a peptide that causes pigment dispersion in dermal melanophores during adaptation of the toad to a dark background. Xenopus melanotropes are highly plastic, undergoing very strong growth to support the high biosynthesis and release of α-MSH in black-adapted animals. In this study we have tested our hypothesis that this enhanced growth of the melanotrope is maintained by autocrine release of BDNF. Furthermore, since the extracellular-regulated kinase (ERK) pathway is a major component of BDNF signaling in neuronal plasticity, we investigated its involvement in melanotrope cell growth. For these purposes melanotropes were treated for 3 days in vitro, with either an anti-BDNF serum or a recombinant tropomyosin-receptor kinase B (TrkB) receptor fragment to eliminate released BDNF, or with the ERK inhibitor U0126. We also applied a novel inhibitor of the TrkB receptor, cyclotraxin-B, to test this receptor's involvement in melanotrope cell growth regulation. All treatments markedly reduced melanotrope cell growth. Therefore, we conclude that autocrine release of BDNF and subsequent TrkB-dependent ERK-mediated signaling is important for melanotrope cell growth during its physiologically induced activation.

BDNF stimulates Ca2+ oscillation frequency in melanotrope cells of Xenopus laevis: contribution of IP3-receptor-mediated release of intracellular Ca2+ to gene expression.

Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating the animal's skin color adaptation through secretion of α-melanophore-stimulating hormone (α-MSH). To fulfill this function optimally, the melanotrope cell undergoes plastic changes in structure and secretory activity in response to changed background light conditions. Xenopus melanotrope cells display Ca(2+) oscillations that are thought to drive α-MSH secretion and gene expression. They also produce brain-derived neurotrophic factor (BDNF), which stimulates in an autocrine way the biosynthesis of the α-MSH precursor, pro-opiomelanocortin (POMC). We have used this physiological adaptation mechanism as a model to investigate the role of BDNF in the regulation of Ca(2+) kinetics and Ca(2+)-dependent gene expression. By dynamic video imaging of isolated cultured melanotropes we demonstrated that BDNF caused a dose-dependent increase in Ca(2+) oscillation frequency up to 64.7±2.3% of control level. BDNF also induced a transient Ca(2+) peak in Ca(2+)-free medium, which was absent when calcium stores were blocked by thapsigargin and 2-aminoethoxydiphenyl borate, indicating that BDNF stimulates acute release of Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores. Moreover, we show that thapsigargin inhibits the expression of BDNF transcript IV (by 61.1±28.8%) but does not affect POMC transcript. We conclude that BDNF mobilizes Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores and propose the possibility that the resulting Ca(2+) oscillations selectively stimulate expression of the BDNF gene.

Id1, Id2 and Id3 are induced in rat melanotrophs of the pituitary gland by dopamine suppression under continuous stress.

In rats under continuous stress (CS) there is decreased hypothalamic dopaminergic innervation to the intermediate lobe (IL) of the pituitary gland, which causes hyperactivation and subsequent degeneration of melanotrophs in the IL. In this study, we investigated the molecular basis for the changes that occur in melanotrophs during CS. Using microarray analysis, we identified several genes differentially expressed in the IL under CS conditions. Among the genes up-regulated under CS conditions, we focused on the inhibitor of DNA binding/differentiation (Id) family of dominant negative basic helix-loop-helix (bHLH) transcription factors. RT-PCR, Western blotting and in situ hybridization confirmed the significant inductions of Id1, Id2 and Id3 in the IL of CS rats. Administration of the dopamine D2 receptor agonist bromocriptine prevented the inductions of Id1-3 in the IL of CS rats, whereas application of the dopamine D2 antagonist sulpiride induced significant expressions of Id1-3 in the IL of normal rats. Moreover, an in vitro study using primary cultured melanotrophs demonstrated a direct effect on Id1-3 inductions by dopamine suppression. These results suggest that the decreased dopamine levels in the IL during CS induce Id1-3 expressions in melanotrophs. Because Id family members inhibit various bHLH transcription factors, it is conceivable that the induced Id1-3 would cooperatively modulate gene expressions in melanotrophs under CS conditions to induce hormone secretion.

Exploring modulation of action potential firing by artificial graft of fast GABAergic autaptic afferences in hypophyseal neuroendocrine melanotrope cells.

Excitable cells are connected via electrical and chemical synapses to form a complex plastic network that transmit and modify action potential trains. In vivo and in vitro studies suggest that powerful type A GABAergic self-innervations, known as autapses, play a central role in the shaping of spike discharges. Herein, we have investigated the effects of artificial autaptic activity on action potential firing in cultured hypophyseal neuroendocrine melanotrope cells removed from any synaptic input. Type A autaptic conductances were introduced by using a dedicated dynamic-clamp device, based on a digital signal processor. The results indicate that cells grafted with autaptic dynamic-clamp are subjected to a modulation of both evoked firing and artificial GABA(A)-currents. The autaptic feedback reduced the action potentials amplitude, decreasing both the overshoot and the after-hyperpolarization potential (AHP). In return, the reduced voltage changes diminished the autaptic current amplitudes. The overall effect was a decrease of the cell firing rate. The introduction of a variable autaptic delay strongly altered the cell responses. Under certain conditions, the artificial autaptic current formed an additional component of the AHP which was markedly augmented. This action resulted in a stabilization of the action potential train leading to a more regular firing. In conclusion, it appeared that the autaptic feedback was very dependent on functional parameters such as the autaptic distance. Further investigations are in progress to complete our model, taking the autaptic plasticity into account.

Chronic stress elicits prolonged activation of alpha-MSH secretion and subsequent degeneration of melanotroph.

Prolonged stress affects homeostasis in various organs and induces stress-associated disorders. We examined the cellular changes of pituitary gland under the continuous stress condition using a rat model in which rats were kept in a cage filled with water to a height of 1.5 cm for up to 5 days. Among the pituitary hormone mRNAs, proopiomelanocortin mRNA was up-regulated specifically in the intermediate lobe (IL) of this rat model. Additionally, the peripheral blood levels of alpha-melanocyte stimulating hormone (alpha-MSH), a major product of proopiomelanocortin in IL were increased. The alpha-MSH secreting cells, melanotrophs, showed a markedly developed endoplasmic reticulum and Golgi apparatus in the early phase of the experiment. Subsequent continuous stress caused remarkable dilation of the endoplasmic reticulum, disruption of the Golgi structure, and the degeneration of some melanotrophs. In addition the dopaminergic nerve fibers from hypothalamus were markedly decreased in IL. A dopamine antagonist elicited the similar morphologic changes of melanotroph in normal rat. These findings suggest that prolonged stress suppressed hypothalamus-derived dopamine release in IL, which elicited over-secretion of alpha-MSH from the melanotrophs. The present study also suggests that prolonged hyperactivation of endocrine cells could lead to disorder of secretion mechanisms and eventual degeneration.

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect.

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.

Biostable beta-amino acid PK/PBAN analogs: agonist and antagonist properties.

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.

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis.

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.

Distinct role of Rab3A and Rab3B in secretory activity of rat melanotrophs.

Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.

The regulation of alpha-MSH release by GABA is mediated by a chloride-dependent [Ca2+]c increase in frog melanotrope cells.

In frog melanotrope cells, gamma-aminobutyric acid (GABA) induces a biphasic effect, i.e. a transient stimulation followed by a more sustained inhibition of alpha-MSH release, and both phases of the GABA effect are mediated by GABAA receptors. We have previously shown that the stimulatory phase evoked by GABAA 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 [Ca2+]c increase and alpha-MSH release. We show that GABA evokes a concentration-dependent [Ca2+]c rise through specific activation of the GABAA receptor. The GABA-induced [Ca2+]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 [Ca2+]c rise and alpha-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 [Ca2+]c increase that is responsible for hormone secretion.