Profiles of secreted neuropeptides and catecholamines illustrate similarities and differences in response to stimulation by distinct secretagogues

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

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles

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

Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

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

Metabolomics Analyses of 14 Classical Neurotransmitters by GC-TOF with LC-MS Illustrates Secretion of 9 Cell–Cell Signaling Molecules from Sympathoadrenal Chromaffin Cells in the Presence of Lithium

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

Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.

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