Neuropeptides mediate cell-cell signaling in the nervous and endocrine systems. The neuropeptidome is the spectrum of peptides generated from precursors by proteolysis within dense core secretory vesicles (DCSV). DCSV neuropeptides and contents are released to the extracellular environment where further processing for neuropeptide formation may occur. To assess the DCSV proteolytic capacity for production of neuropeptidomes at intravesicular pH 5.5 and extracellular pH 7.2, neuropeptidomics, proteomics, and protease assays were conducted using chromaffin granules (CG) purified from adrenal medulla. CG are an established model of DCSV. The CG neuropeptidome consisted of 1239 unique peptides derived from 15 proneuropeptides that were colocalized with 64 proteases. Distinct CG neuropeptidomes were generated at the internal DCSV pH of 5.5 compared to the extracellular pH of 7.2. Class-specific protease inhibitors differentially regulated neuropeptidome production involving aspartic, cysteine, serine, and metallo proteases. The substrate cleavage properties of CG proteases were assessed by multiplex substrate profiling by mass spectrometry (MSP-MS) that uses a synthetic peptide library containing diverse cleavage sites for endopeptidases and exopeptidases. Parallel inhibitor-sensitive cleavages for neuropeptidome production and peptide library proteolysis led to elucidation of six CG proteases involved in neuropeptidome production, represented by cathepsins A, B, C, D, and L and carboxypeptidase E (CPE). The MSP-MS profiles of these six enzymes represented the majority of CG proteolytic cleavages utilized for neuropeptidome production. These findings provide new insight into the DCSV proteolytic system for production of distinct neuropeptidomes at the internal CG pH of 5.5 and at the extracellular pH of 7.2.

The classical small molecule neurotransmitters are essential for cell-cell signaling in the nervous system for regulation of behaviors and physiological functions. Metabolomics approaches are ideal for quantitative analyses of neurotransmitter profiles but have not yet been achieved for the repertoire of 14 classical neurotransmitters. Therefore, this study developed targeted metabolomics analyses by full scan gas chromatography/time-of-flight mass spectrometry (GC-TOF) and hydrophilic interaction chromatography-QTRAP mass spectrometry (HILIC-MS/MS) operated in positive ionization mode for identification and quantitation of 14 neurotransmitters consisting of acetylcholine, adenosine, anandamide, aspartate, dopamine, epinephrine, GABA, glutamate, glycine, histamine, melatonin, norepinephrine, serine, and serotonin. GC-TOF represents a new metabolomics method for neurotransmitter analyses. Sensitive measurements of 11 neurotransmitters were achieved by GC-TOF, and three neurotransmitters were analyzed by LC-MS/MS (acetylcholine, anandamide, and melatonin). The limits of detection (LOD) and limits of quantitation (LOQ) were assessed for linearity for GC-TOF and LC-MS/MS protocols. In neurotransmitter-containing dense core secretory vesicles of adrenal medulla, known as chromaffin granules (CG), metabolomics measured the concentrations of 9 neurotransmitters consisting of the catecholamines dopamine, norepinephrine, and epinephrine, combined with glutamate, serotonin, adenosine, aspartate, glycine, and serine. The CG neurotransmitters were constitutively secreted from sympathoadrenal chromaffin cells in culture. Nicotine- and KCl-stimulated release of the catecholamines and adenosine. Lithium, a drug used for the treatment of bipolar disorder, decreased the constitutive secretion of dopamine and norepinephrine and decreased nicotine-stimulated secretion of epinephrine. Lithium had no effect on other secreted neurotransmitters. Overall, the newly developed GC-TOF with LC-MS/MS metabolomics methods for analyses of 14 neurotransmitters will benefit investigations of neurotransmitter regulation in biological systems and in human disease conditions related to drug treatments.

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. Graphical Abstract ᅟ.

The goal of this study was to define profiles of secreted neuropeptide and catecholamine neurotransmitters that undergo co-release from sympathoadrenal chromaffin cells upon stimulation by distinct secretagogues. Chromaffin cells of the adrenal medulla participate in the dynamic responses to stress, especially that of ‘fight and flight’, and, thus, analyses of the co-release of multiple neurotransmitters is necessary to gain knowledge of how the stress response regulates cell–cell communication among physiological systems. Results of this study demonstrated that six different secretagogues stimulated the co-release of the neuropeptides Met-enkephalin, galanin, NPY, and VIP with the catecholamines dopamine, norepinephrine, and epinephrine. Importantly, the quantitative profiles of the secreted neurotransmitters showed similarities and differences upon stimulation by the different secretagogues evaluated, composed of KCl depolarization, nicotine, carbachol, PACAP, bradykinin, and histamine. The rank-orders of the secreted profiles of the neurotransmitters were generally similar among these secretagogues, but differences in the secreted amounts of each neurotransmitter occurred with different secretagogues. Epinephrine among the catecholamines showed the highest level of secretion. (Met)enkephalin showed the largest levels of secretion compared to the other neuropeptides examined. Levels of secreted catecholamines were greater than that of the neuropeptides. These data support the hypothesis that profiles of secreted neuropeptide and catecholamine neurotransmitters show similarities and differences upon stimulation by distinct secretagogues. These results illustrate the co-release of concerted neurotransmitter profiles that participate in the stress response of the sympathoadrenal nervous system.

Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.

Background and Aims: N-truncated pyroglutamate (pGlu)-amyloid-β [Aβ(3-40/42)] peptides are key components that promote Aβ peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because Aβ deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aβ(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length Aβ(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and QC are colocalized with Aβ in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters. Methods: Purified DCSV were assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitter secretion. Neuron-like chromaffin cells were analyzed for cosecretion of pGlu-Aβ, QC, Aβ, and neuropeptides. The cells were treated with a QC inhibitor, and pGlu-Aβ production was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC secretion. Results: Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ and QC undergo activity-dependent cosecretion with Aβ and enkephalin and galanin neurotransmitters. The QC inhibitor decreased the level of secreted pGlu-Aβ. The human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that was colocalized with QC. Conclusions: pGlu-Aβ and QC are present with Aβ in DCSV and undergo activity-dependent, regulated cosecretion with neurotransmitters.

This study investigated human-induced pluripotent stem cell (hiPSC) -derived neurons for their ability to secrete neurotransmitters in an activity-dependent manner, the fundamental property required for chemical neurotransmission. Cultured hiPSC neurons showed KCl stimulation of activity-dependent secretion of catecholamines--dopamine (DA), norepinephrine (NE), and epinephrine (Epi)--and the peptide neurotransmitters dynorphin and enkephlain. hiPSC neurons express the biosynthetic enzymes for catecholamines and neuropeptides. Because altered neurotransmission contributes to schizophrenia (SZ), we compared SZ to control cultures of hiPSC neurons and found that SZ cases showed elevated levels of secreted DA, NE, and Epi. Consistent with increased catecholamines, the SZ neuronal cultures showed a higher percentage of tyrosine hydroxylase (TH)-positive neurons, the first enzymatic step for catecholamine biosynthesis. These findings show that hiPSC neurons possess the fundamental property of activity-dependent neurotransmitter secretion and can be advantageously utilized to examine regulation of neurotransmitter release related to brain disorders.

Pyroglutamate amyloid-beta peptides (pGlu-Abeta) are particularly neurotoxic forms of Abeta, and they accumulate with full-length Abeta peptides in Alzheimer's disease (AD) brains. pGlu-Abeta peptides are N-terminally truncated forms of full-length Abeta peptides (flAbeta(1-40/42)) with modification of the N-terminal glutamate to form pGlu-Abeta(3-40/42). The significant presence of pGlu-Abeta in AD brains may be important in the development of AD because pGlu-Abeta initiates formation of oligomeric neurotoxic Abeta forms. Beta-secretase processing of amyloid precursor protein (APP) produces flAbeta(1-40/42), but it is not yet known whether the beta-secretase BACE1 or the alternative beta-secretase cathepsin B (CatB) participate in the production of pGlu-Abeta. Therefore, experiments examined the effects of gene knockout of these proteases on pGlu-Abeta and flAbeta brain levels, amyloid plaque load, and memory deficits in APPLon AD mice, which express APP-695 and have the wild-type (wt) β-secretase activity found in most AD patients. APPLon mice with knockout of the CatB or BACE1 gene were generated, and were assessed for memory deficits by the Morris water maze test. Brain extracts were measured for levels of pGlu-Abeta and flAbeta peptides by ELISAs, and amyloid plaque load was assessed by quantitative immunohistochemistry. Knockout of the CatB gene reduced brain levels of pGlu-Abeta(3-40/42), flAbeta(1-40/42), and pGlu-Abeta/Abeta plaque load in APPLon mice. Expression of the CatB gene increased levels of pGlu-Abeta and Abeta peptides, as well as amyloid plaque load. Substantial improvements in memory deficits resulted from knockout of CatB. But knockout of the BACE1 gene had no effect on levels of these Abeta forms and had no effect on memory in the APPLon mouse model of AD. CatB participates in the production of pGlu-Abeta and flAbeta, and the absence of the CatB gene improves memory deficits in the APPLon AD mouse model. These findings suggest that inhibitors targeting CatB will be useful in therapeutic treatment of AD patients.

Pyroglutamate amyloid-β peptides (pGlu-Aβ) are particularly pernicious forms of amyloid-β peptides (Aβ) present in Alzheimer's disease (AD) brains. pGlu-Aβ peptides are N-terminally truncated forms of full-length Aβ peptides (flAβ(1-40/42)) in which the N-terminal glutamate is cyclized to pyroglutamate to generate pGlu-Aβ(3-40/42). β-secretase cleavage of amyloid-β precursor protein (AβPP) produces flAβ(1-40/42), but it is not yet known whether the β-secretase BACE1 or the alternative β-secretase cathepsin B (CatB) participate in the production of pGlu-Aβ. Therefore, this study examined the effects of gene knockout of these proteases on brain pGlu-Aβ levels in transgenic AβPPLon mice, which express AβPP isoform 695 and have the wild-type (wt) β-secretase activity found in most AD patients. Knockout or overexpression of the CatB gene reduced or increased, respectively, pGlu-Aβ(3-40/42), flAβ(1-40/42), and pGlu-Aβ plaque load, but knockout of the BACE1 gene had no effect on those parameters in the transgenic mice. Treatment of AβPPLon mice with E64d, a cysteine protease inhibitor of CatB, also reduced brain pGlu-Aβ(3-42), flAβ(1-40/42), and pGlu-Aβ plaque load. Treatment of neuronal-like chromaffin cells with CA074Me, an inhibitor of CatB, resulted in reduced levels of pGlu-Aβ(3-40) released from the activity-dependent, regulated secretory pathway. Moreover, CatB knockout and E64d treatment has been previously shown to improve memory deficits in the AβPPLon mice. These data illustrate the role of CatB in producing pGlu-Aβ and flAβ that participate as key factors in the development of AD. The advantages of CatB inhibitors, especially E64d and its derivatives, as alternatives to BACE1 inhibitors in treating AD patients are discussed.

Beta-amyloid (A beta) peptides are secreted from neurons, resulting in extracellular accumulation of A beta and neurodegeneration of Alzheimer's disease. Because neuronal secretion is fundamental for the release of neurotransmitters, this study assessed the hypothesis that A beta undergoes co-release with neurotransmitters. Model neuronal-like chromaffin cells were investigated, and results illustrate regulated, co-secretion of A beta(1-40) and A beta(1-42) with peptide neurotransmitters (galanin, enkephalin, and NPY) and catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). Regulated secretion from chromaffin cells was stimulated by KCl depolarization and nicotine. Forskolin, stimulating cAMP, also induced co-secretion of A beta peptides with peptide and catecholamine neurotransmitters. These data suggested the co-localization of A beta with neurotransmitters in dense core secretory vesicles (DCSV) that store and secrete such chemical messengers. Indeed, A beta was demonstrated to be present in DCSV with neuropeptide and catecholamine transmitters. Furthermore, the DCSV organelle contains APP and its processing proteases, beta- and gamma-secretases, that are necessary for production of A beta Thus, A beta can be generated in neurotransmitter-containing DCSV. Human IMR32 neuroblastoma cells also displayed regulated secretion of A beta(1-40) and A beta(1-42) with the galanin neurotransmitter. These findings illustrate that A beta peptides are present in neurotransmitter-containing DCSV, and undergo co-secretion with neuropeptide and catecholamine neurotransmitters that regulate brain functions. (C) 2013 Elsevier Inc. All rights reserved.

J. Neurochem. (2012) 122, 512–522.AbstractPeptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N‐ and/or C‐terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin‐like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N‐terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR‐ME and KK‐ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and immunofluorescence deconvolution microscopy. Purified human cathepsin H sequentially removes N‐terminal basic residues to generate ME, with peptide products characterized by nano‐LC‐MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N‐terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production.

Proteases are required for processing precursors into active neuropeptides that function as neurotransmitters for cell-cell communication. This study demonstrates the novel function of human cathepsin V protease for producing the neuropeptides enkephalin and neuropeptide Y (NPY). Cathepsin V is a human-specific cysteine protease gene. Findings here show that expression of cathepsin V in neuroendocrine PC12 cells and human neuronal SK-N-MC cells results in production of (Met)enkephalin from proenkephalin. Gene silencing of cathepsin V by siRNA in human SK-N-MC cells results in reduction of (Met)enkephalin by more than 80%, illustrating the prominent role of cathepsin V for neuropeptide production. In vitro processing of proenkephalin by cathepsin V occurs at dibasic residue sites to generate enkephalin-containing peptides and an ∼24-kDa intermediate present in human brain. Cathepsin V is present in human brain cortex and hippocampus where enkephalin and NPY are produced and is present in purified human neuropeptide secretory vesicles. Colocalization of cathepsin V with enkephalin and NPY in secretory vesicles of human neuroblastoma cells was illustrated by confocal microscopy. Furthermore, expression of cathepsin V with proNPY results in NPY production. These findings indicate the unique function of human cathepsin V for producing enkephalin and NPY neuropeptides required for neurotransmission in health and neurological diseases.

Secretion and extracellular accumulation of neurotoxic beta-amyloid peptides (Abeta) represent a major factor in the development of Alzheimer's disease that results in memory deficit. Knowledge of the secretory pathway(s) utilized by Abeta peptides can enhance understanding of the neurobiology of Abeta. Because neurons undergo activity-dependent secretion of Abeta and neurotransmitters, this study sought to investigate the hypothesis of that Abeta undergoes co-secretion with neurotransmitters via the regulated secretory pathway that provides activity-dependent secretion of neurotansmitters. Neuronal-like chromaffin cells in primary culture were subjected to activity-dependent secretion, induced by KCl depolarization (50 mM, 30 min), and stimulated secretion of Abeta was measured by ELISA. Selected neuropeptides and catecholamines (dopamine, norepinephrie, and epinephrine) were measured in the secretion media. In addition, dense core secretory vesicles were purified from chromaffin cells and their content of Abeta peptides with neurotransmitters was measured. APP processing components (beta- and gamma-secretases) were assessed by western blots. Neuronal immunofluorescence confocal microscopy assessed localization of Abeta peptides with neurotransmitters. The data show that Abeta peptides undergo activity-dependent secretion from neuronal chromaffin cells (induced by KCl depolarization). Furthermore, Abeta undergos cosecretion with the peptide neurotransmitters (neuropeptides) and catecholamines. Investigation of the secretory vesicles that provide regulated secretion of signaling molecules from these cells showed that isolated dense core secretory vesicles (DCSV) of neuronal-like chromaffin cells contain Abeta peptides with multiple peptide neurotransmitters of enkephalin, NPY, galanin, VIP, and somatostatin. Furthermore, these secretory vesicles contain the catecholamine neurotransmitters dopamine, norepipnephrine, and epinephrine that are co-secreted with Abeta and neuropeptides. This organelle contains full-length APP, Abeta peptides, and components for beta- and gamma-secretases that produce Abeta. These APP processing components co-reside with processing proteases for neuropeptide biosynthesis, as well as with catecholamine biosynthetic enzymes. The colocalization of Abeta with neuropeptides in secretory vesicles was further illustrated by cellular immunofluorescence confocal microscopy. These findings indicate the biosynthesis and secretion of Abeta peptides in dense core secretory vesicles with neuropeptide and catecholamine neurotransmitters, suggesting that Abeta peptide forms function with multiple neurotransmitter systems.

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction.

Dynorphin opioid neuropeptides mediate neurotransmission for analgesia and behavioral functions. Dynorphin A, dynorphin B, and alpha-neoendorphin are generated from prodynorphin by proteolytic processing. This study demonstrates the significant role of the cysteine protease cathepsin L for producing dynorphins. Cathepsin L knockout mouse brains showed extensive decreases in dynorphin A, dynorphin B, and alpha-neoendorphin that were reduced by 75%, 83%, and 90%, respectively, compared to controls. Moreover, cathepsin L in brain cortical neurons was colocalized with dynorphins in secretory vesicles, the primary site of neuropeptide production. Cellular coexpression of cathepsin L with prodynorphin in PC12 cells resulted in increased production of dynorphins A and B. Comparative studies of PC1/3 and PC2 convertases showed that PC1/3 knockout mouse brains had a modest decrease in dynorphin A, and PC2 knockout mice showed a minor decrease in alpha-neoendorphin. Overall, these results demonstrate a prominent role for cathepsin L, jointly with PC1/3 and PC2, for production of dynorphins in brain.

Dynorphins, as well as other opioid neuropeptides, have an established role in analgesia and pain processing among other central nervous system functions. Dynorphin neuropeptides, as well as numerous peptide neurotransmitters, are generated by proteolytic processing of respective proneuropeptide precursors. Processing of proneuropeptides has recently been found to be mediated by the recently discovered cathepsin L cysteine protease in secretory vesicles. To assess the role of secretory vesicle cathepsin L in dynorphin production, studies in cathepsin L gene knockout mice and with cathepsin L protease expression were conducted. In brains of cathepsin L gene knockout mice, substantial reduction of prodynorphin‐derived neuropeptides was observed. Brain levels of dynorphin A, dynorphin B, and a‐neoendorphin were decreased by more than 70% in cathepsin L knockout mice compared to wild‐type. Expression of cathepsin L with prodynorphin in neuroendocrine PC12 cells resulted in increased production of dynorphin A, indicating cathepsin L mediated conversion of prodynorphin to the dynorphin peptide product. Moreover, colocalization of dynorphins with cathepsin L in secretory vesicles of neuronal cells was demonstrated by immunofluorescence confocal microscopy. These results demonstrate a significant role for secretory vesicle cathepsin L in the produciton of dynorphin opioid neuropeptides.

The production of the peptide hormones ACTH, α-MSH, and β-endorphin requires proteolytic processing of POMC which is hypothesized to utilize dual cysteine- and subtilisin-like protease pathways, consisting of the secretory vesicle cathepsin L pathway and the well-known subtilisin-like prohormone convertase (PC) pathway. To gain knowledge of these protease components in human pituitary where POMC-derived peptide hormones are produced, this study investigated the presence of these protease pathway components in human pituitary. With respect to the cathepsin L pathway, human pituitary contained cathepsin L of 27–29 kDa and aminopeptidase B of ~64 kDa, similar to those in secretory vesicles of related neuroendocrine tissues. The serpin inhibitor endopin 2, a selective inhibitor of cathepsin L, was also present. With respect to the PC pathway, human pituitary expresses PC1/3 and PC2 of ~60–65 kDa, which represent active PC1/3 and PC2; peptide hormone production then utilizes carboxypeptidase E (CPE) which is present as a protein of ~55 kDa. Analyses of POMC products in human pituitary showed that they resemble those in mouse pituitary which utilizes cathepsin L and PC2 for POMC processing. These findings suggest that human pituitary may utilize the cathepsin L and prohormone convertase pathways for producing POMC-derived peptide hormones.

BACKGROUND/AIMS:The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues. METHODS:This study utilized methods consisting of Western blots, immunoelectron microscopy, and immunofluorescence microscopy for evaluation of the localization of endopin protease inhibitors in neuroendocrine tissues. RESULTS:Immunoelectron microscopy with these selective antisera demonstrated the localization of endopins 1 and 2 within secretory vesicles of adrenal medulla (bovine). Cellular immunofluorescence confocal microscopy illustrated the high level of colocalization of endopins 1 and 2 with enkephalin and NPY neuropeptides that are present in secretory vesicles of adrenal medullary chromaffin cells in primary culture. Tissue distribution studies (by Western blots) showed the expression of endopins 1 and 2 in bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues. CONCLUSIONS:These results implicate endopins 1 and 2 as endogenous protease inhibitors in neuropeptide-containing secretory vesicles and neuroendocrine tissues.

Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.