PAM: diverse roles in neuroendocrine cells, cardiomyocytes, and green algae.

Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin, and insulin, each of which is stored in electron-dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the ER lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by peptidylglycine α-amidating monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes, and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell types.

A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells.

The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H+ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

PGP 9.5 immunocytochemical staining for pancreatic endocrine tumors.

AIMS/HYPOTHESIS: Protein gene product 9.5 (PGP 9.5) is a marker for neuroendocrine cells but has not been used for pancreatic islet cells and pancreatic endocrine tumors (PETs). Antibodies for PGP 9.5 are now commercially available for immunocytochemical study, with which immunostaining may be able to differentiate between benign and malignant PETs. RESULTS: All 4 kinds of normal islet cells were positively immunostained for PGP 9.5-moderately positive for ß-cells and strongly positive for δ-cells, whereas ganglion cells were immunostained more strongly than islet cells. Nine of 12 insulinomas were moderately to strongly positive for PGP 9.5. Two glucagonomas, 3 of 6 pancreatic polypeptidomas (PPomas), 3 of 9 gastrinomas, and 2 of 4 non-functioning PETs were negative for PGP 9.5. MATERIALS AND METHODS: Thirty-four PETs were immunocytochemically stained for PGP 9.5 using a rabbit polyclonal antibody together with immunostaining for 4 pancreatic hormones, chromogranin A (CgA), and gastrin. PETs consisted of 12 insulinomas, 2 glucagonomas, 1 somatostatinoma (SRIFoma), 6 PPomas, 9 gastrinomas, and 4 non-functioning PETs. CONCLUSION/INTERPRETATION: PGP 9.5 immunostaining was universally positive for 4 kinds of islet cells and was moderately to strongly positive for 9 of 12 (75%) insulinomas. All 22 non-ß-cell PETs were negative or weakly positive for PGP 9.5, and thus negative or weakly positive PGP 9.5 immunostaining may be used as a marker for potential malignancy and poor prognosis for non-ß-cell PETs.

Phospholipid scramblase-1-induced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1⁻/⁻ mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis.

Upregulation of manganese superoxide dismutase (SOD2) is a common pathway for neuroendocrine differentiation in prostate cancer cells.

Despite improvements in diagnosis of advanced prostate cancer (PCa), treatment is not efficient and 5-year survival is still low. Initially, the less abundant of cell types, neuroendocrine cells (NE), are involved in regulatory process but their physiological role is not fully understood. Among others, an increase in NE cells along with tumor progression has been commonly reported but their role in tumorigenesis or the molecular mechanisms of transdifferentiation is still a matter of debate. We have used human PCa cells (LNCaP) induced to differentiate to NE cells with several stimuli: androgen withdrawal, cyclic AMP or treatment with the antioxidant pineal hormone melatonin. PCa patients' specimens were also analyzed by western blotting and by immunocytochemistry. NE-like LNCaP cells express high levels of mitochondrial superoxide dismutase (MnSOD/SOD2) in addition to NE markers. MnSOD upregulation is mediated by NFkappaB transcription factor, mainly through p65 translocation into the nuclei. More importantly, overexpression of MnSOD induces the rise of NE-markers in LNCaP cells, showing that MnSOD upregulation might be instrumental for NE differentiation in PCa cells. Furthermore, MnSOD is highly expressed in advanced tumors of patients' when compared with control, nonpathological samples or with low-grade tumors, along with the presence of synaptophysin, a common NE marker. Also, fluorescence immunohistochemical analysis revealed that MnSOD colocalizes with NE markers in most of NE cells observed in PCa specimens. The present findings indicate that MnSOD is essential for NE transdifferentiation and mediates in part the differentiation process, which appears also to be critical in vivo.

betaPIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells.

Rho GTPases are crucial regulators of actin cytoskeletal rearrangements and play important roles in many cell functions linked to membrane trafficking processes. In neuroendocrine cells, we have previously demonstrated that RhoA and Cdc42 mediate part of the actin remodelling and vesicular trafficking events that are required for the release of hormones by exocytosis. Here, we investigate the functional importance of Rac1 for the exocytotic reaction and dissect the downstream and upstream molecular events that might integrate it to the exocytotic machinery. Using PC12 cells, we found that Rac1 is associated with the plasma membrane and is activated during exocytosis. Silencing of Rac1 by siRNA inhibits hormone release, prevents secretagogue (high K(+))-evoked phospholipase D1 (PLD1) activation and blocks the formation of phosphatidic acid at the plasma membrane. We identify betaPix as the guanine nucleotide-exchange factor integrating Rac1 activation to PLD1 and the exocytotic process. Finally, we show that the presence of the scaffolding protein Scrib at the plasma membrane is essential for betaPix/Rac1-mediated PLD1 activation and exocytosis. As PLD1 has recently emerged as a promoter of membrane fusion in various exocytotic events, our results define a novel molecular pathway linking a Rho GTPase, Rac1, to the final stages of Ca(2+)-regulated exocytosis in neuroendocrine cells.

The Coffin-Lowry syndrome-associated protein RSK2 controls neuroendocrine secretion through the regulation of phospholipase D1 at the exocytotic sites.

Together with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, fusogenic cone-shaped lipids, such as phosphatidic acid (PA), have been recently shown to be important actors in membrane fusion during exocytosis. Phospholipase D (PLD) appears to be the main provider of PA at the exocytotic site in neuroendocrine cells. We show here that ribosomal S6 kinase 2 (RSK2) stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino-terminal Phox-homology domain. In PC12 cells, depletion of RSK2 dramatically prevents PA synthesis at exocytotic sites and inhibits hormone release. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis.