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The neuroendocrine marker genes Ptprn and Ptprn2 encode protein tyrosine phosphatase receptors N and N2, 2 members of protein tyrosine phosphatase receptors void of enzymatic activity, and whose function and mechanism of action have not been elucidated. To explore the role(s) of Ptprn and Ptprn2 on the hypothalamic-pituitary-adrenal axis, we used mice in which both genes were knocked out (DKO). The focus in this study was on corticotrophs and melanotrophs from the anterior and intermediate lobes of the pituitary gland, respectively. In both sexes, DKO caused an increase in the expression of the corticotroph/melanotroph genes Pomc and Tbx19 and the melanotroph-specific gene Pax7. We also found in vivo and in vitro increased synthesis and release of beta-endorphin, alpha-melanocyte-stimulating hormone, and ACTH in DKO mice, which was associated with increased serum corticosterone levels and adrenal mass. DKO also increased the expression of other melanotroph-specific genes, but not corticotroph-specific genes. The dopaminergic pathway in the hypothalamus and dopaminergic receptors in melanotrophs were not affected in DKO mice. However, hyperplasia of the intermediate lobe was observed in DKO females and males, accompanied by increased proopiomelanocortin immunoreactivity per cell. These results indicate that protein tyrosine phosphatase receptor type N contributes to hypothalamic-pituitary-adrenal function by being involved in processes governing postnatal melanotroph development and Pomc expression. [ABSTRACT FROM AUTHOR]

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through Gs and Gq/11 protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the Gq/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP3-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the Gs signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and β-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner. [ABSTRACT FROM AUTHOR]

Using electron microscopy, we studied the morphology of secretory granules in rat pars intermedia cells. We found figures of apparent intergranule fusion, characterized by a tight association of two granules. The fusion was detected in around 2% of all measured granules, indicating a low occurrence of intergranule fusion. To study whether intergranule fusion affects the distribution of granule diameters a simple probabilistic model was developed. It is based on the theory that larger granules are formed by fusion of two or more spherical granules of fixed size, and that the surface of a newly formed granule is equal to the sum of fused granule membranes. The model accounts for the bias on granule diameter measurements due to sectioning of granules. Although the electron microscopy data strongly indicates the existence of intergranule fusion in rat melanotrophs, this process as modelled in the present work does not contribute to the granule diameter distribution significantly. It is likely that in addition to the fusion of larger granules, other processes, such as fusion of microvesicles, may affect the distribution of granule diameters.

Abstract: In this work we have compared the ultrastructural characteristics of major pancreatic endocrine cells, pituitary melanotrophs and adrenal chromaffin cells in the normal mouse strain (wild type, WT) and mice with a known secretory deficit, the Rab3a knockout strain (Rab3a KO). For this purpose, pancreata, pituitary glands and adrenal glands from the Rab3a KO and from the WT mice were analysed, using conventional transmission electron microscopy (TEM). In order to assess the significance of the presence of Rab3a proteins in the relevant cells, we focused primarily on their secretory vesicle morphology and distribution. Our results showed a comparable general morphology in Rab3a KO and WT in all assessed endocrine cell types. In all studied cell types, the distribution of secretory granules along the plasma membrane (number of docked and almost-docked vesicles) was comparable between Rab3a KO and WT mice. Specific differences were found in the diameters of their secretory vesicles, diameters of their electron-dense cores and the presence of autophagic structures in the cells of Rab3A KO mice only. Occasionally, individual electron-dense round vesicles were present inside autophagosome-like structures; these were possibly secretory vesicles or their remnants. The differences found in the diameters of the secretory vesicles confirm the key role of Rab3a proteins in controlling the balance between secretory vesicle biogenesis and degradation, and suggest that the ablation of this protein probably changes the nature of the reservoir of secretory vesicles available for regulated exocytosis. [Copyright &y& Elsevier]

Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca2+ concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca2+ sensitivity in endocrine cells, accurate and fast determination of Ca2+ dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a robust exocytotic activity. Slow increases in the [Ca2+]i revealed exocytotic phases with different Ca2+ sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca2+ sensitivity 'classes' of secretory vesicles have been observed: a highly Ca2+-sensitive, a medium Ca2+-sensitive and a low Ca2+-sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca2+ sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca2+ sensor protein. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol-4, 5-bisphosphate [PI(4,5)P2] has been implicated in the priming of large dense-core vesicles in many secretory cells; however, its role in the Ca2+-dependent secretory activity in pituitary cells remains elusive. We assessed the effect of elevated intracellular PI(4,5)P2 on the kinetics of Ca2+-dependent exocytosis, using a whole-cell patch-clamp technique in wild-type mouse melanotrophs from fresh pituitary tissue slices. We found that 1 μmol/L PI(4,5)P2 significantly increased Ca2+-dependent exocytosis of vesicles that need to go through ATP-dependent reactions; however, the exocytosis of release-ready vesicles (ATP-independent release) and voltage-activated Ca2+ currents remained unaffected. We suggest that PI(4,5)P2 increases the size of the readily releasable vesicle pool by regulating the effectiveness of vesicular mobilization and fusion in an ATP-dependent manner. [ABSTRACT FROM AUTHOR]

α-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three α-neurexin genes show a severe reduction of synaptic release. Although analyses of α-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+channels, it remained unclear whether α-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult α-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that α-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for α-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells. [ABSTRACT FROM AUTHOR]

Abstract: In melanotrophs, neuroendocrine cells from the intermediate lobe of the rat pituitary gland, glutamate causes a rise in intracellular [Ca2+] suggesting the presence of ionotropic NMDA and non-NMDA AMPA/K receptors. However, the Ca2+-dependent release of the major peptide hormone, α-melanocyte stimulating hormone (α-MSH), in response to glutamate stimulation has not been studied yet in this cell model. Significant spontaneous secretion of the peptide, which results in hormone deposits on the perimeter of the cells, has been confirmed by using confocal microscopy. Co-staining with a membrane area marker FM 1-43, which co-localized with the immunocytochemically marked hormone deposits, showed that fusion-competent sites on the plasma membrane coincided with secretion-competent sites. Stimulation of the cells with glutamate and high K+ saline induced a significant increase in the plasma membrane area covered with α-MSH deposits compared to control cells incubated with glutamate and CNQX, a glutamate channel blocker. The optical approach to monitor the secretory activity of a single neuroendocrine cell revealed that glutamate stimulates the release of α-MSH at distinct exocytotic membrane domains only. [Copyright &y& Elsevier]

Eukaryotic cells internalize extracellular macromolecules by endocytosis and it was shown that Rab5 protein is required for this process. While it is clear that endocytosis consists of vesicle fission from the plasma membrane, the role of Rab5 protein in the plasma membrane surface area changes is still unclear. Here we studied whether Rab5 is required for membrane surface area changes in rat melanotrophs—cells deriving from the pituitary pars intermedia. The presence of this protein in melanotrophs was probed by immunocytochemistry and its putative role in membrane area dynamics was monitored electrophysiologically with membrane capacitance measurements as this parameter directly reflects changes in membrane surface area. We found that Rab5 protein exists in melanotrophs. At [Ca2+]i < 3 μM, endocytosis-dominated membrane capacitance decrease was found to be blocked by microinjection of specific Rab5 antibody. At high [Ca2+]i, Rab5 antibody did not affect the steady-state increase in membrane capacitance, while it elevated the rate of membrane capacitance increase, which is consistent with an inhibition of endocytosis. [ABSTRACT FROM AUTHOR]

Abstract We have previously reported that voltage-dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2 ). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR), Ca2+ -imaging and whole-cell, patch-clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1–4 ). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+ ]i ). The maximum reduction in [Ca2+ ]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT-PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+ ]i were mimicked by the selective EP3 agonist, ONO-AE-248, whereas three other EP agonists, ONO-DI-004 (EP1 ), ONO-AE1-259 (EP2 ) and ONO-AE1-329 (EP4 ), had little or no effect on [Ca2+ ]i . All four G-protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT-PCR. Dopamine concentration-dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2 . [ABSTRACT FROM AUTHOR]

Corticotropin and melanotropin producing cells were localized in the adenohypophysis of normal Lerots by using antibodies against synthetic corticotropins (anti β1-24 ACTH, anti α17-39 ACTH, anti α25-39 ACTH), and melanotropins (anti αMSH, anti βMSH). All the anticorticotropin sera stained the same cells both in the anterior lobe and in the intermediate lobe. The anti αMSH serum only stained a few cells, exclusively located in the intermediate lobe. These αMSH cells were not stained with anticorticotropin antibodies. The anti βMSH serum revealed all the cells stained with anticorticotropin and anti αMSH sera. Absorption tests showed that the 4-10 heptapeptide common to ACTH and MSH, is not responsible for the immunohistochemical staining. The staining of only some corticotrophs with the anti 4-10 ACTH serum might indicate the presence in these cells of a peptide with an accessible 4-10 site. These results are discussed [ABSTRACT FROM AUTHOR]

The adenohypophysial primordium of Xenopus laevis tadpoles at stages 33/34 to 46 (Nieuwkoop and Faber, 1956) were examined immuno-histologically for α-MSH, β-MSH and ACTH. β-MSH was demonstrated from stage 37/38 onwards, and α-MSH from stage 39. No signs of ACTH production were detected. α-MSH and β-MSH occurred in the same cells. No differences were found in the intensity of immunofluorescence between tadpoles which were kept on a black and a white background. The present study lends no support to the hypothesis concerning the derivation of α-MSH from ACTH. The observations made suggest that the morphological formation of the pars intermedia is accomplished during stages 37/38 to 39. [ABSTRACT FROM AUTHOR]

The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against γ-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against α-MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against α-MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against β-endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against γ-endorphin. These results correlate with biochemical data in favour of mainly - if not exclusively - intragranular processing of POMC. Except for γ-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of γ-endorphin appears to be a later step in POMC processing, occurring in some mature SV. [ABSTRACT FROM AUTHOR]

Melanotrophic cells of the pars intermedia of the rabbit were studied at the electron-microscopic level by means of the immuno-gold technique with the use of antisera against γ-MSH and α-MSH. Both antibodies labelled all secretory vesicles stored in the peripheral cytoplasm, but secretory vesicles and intrasaccular condensations in the Golgi area reacting for γ-MSH were not labelled with the antibody against α-MSH. Since this antibody appears to recognise only the acetylated form of α-MSH, the present observations suggest that acetylation occurs at a stage subsequent to the Golgi packaging, during maturation of the secretory vesicles. Thus, the morphological evidence supports biochemical data in favour of intragranular processing of opiomelanocortin in melanotrophic cells. [ABSTRACT FROM AUTHOR]

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (Cm). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1μM [Ca2+]i. Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis. [ABSTRACT FROM AUTHOR]

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (C m ). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1μM [Ca 2+ ] i . Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.