Rolf Ekman

Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis.

The immune and the nervous systems are anatomically closely related and interact with each other by molecules common to both systems, such as cytokines and neurotransmitters. The purpose of this study was to investigate the participation of catecholamines in the neuroimmunological network. The ability of immune cells to produce catecholamines was examined by a highly sensitive capillary electrophoresis assay, which permits detection of easily oxidized catecholamines in the zeptomole (10−21) range. In addition, the effects of catecholamines on in vitro proliferation, differentiation and apoptosis of lymphocytes were assessed. Mouse spleen cells and macrophages contained on average 7 × 10−17 and 2 × 10−17 mole dopamine per cell, respectively. In the former cell population also norepinephrine was found. Several mouse B‐ and T‐cell hybridomas were also shown to contain endogenously produced dopamine in levels ranging from 7 × 10−20 to 2 × 10−18 mole dopamine per cell. In addition, one of the T‐cell hybridomas proved to synthesize norepinephrine. The dopamine production of lymphocytes was blocked by the tyrosine hydroxylase inhibitor α‐methyl‐p‐tyrosine, whereas incubation with the precursor L‐DOPA increased the dopamine content. Incubation with L‐DOPA, dopamine and norepinephrine dose‐dependently suppressed mitogen induced proliferation and differentiation of mouse lymphocytes. Even short‐time pretreatment of lymphocytes with L‐DOPA and dopamine strongly suppressed lymphocyte proliferation and cytokine production. Incubation of lymphoid cells with L‐DOPA, dopamine and norepinephrine dose‐dependently induced apoptosis which, at least partly, explains the suppressive effects of catecholamines on lymphocyte function. Our results demonstrate that catecholamines: (i) are actively produced by lymphocytes and (ii) have the capacity to act as auto‐ and/or paracrine regulators of lymphocyte activity through induction of apoptosis.

Mu- and delta-opioid receptor antagonists decrease proliferation and increase neurogenesis in cultures of rat adult hippocampal progenitors

Opioids have previously been shown to affect proliferation and differentiation in various neural cell types. In the present study, cultured rat adult hippocampal progenitors (AHPs) were shown to release β‐endorphin. Membrane preparations of AHPs were found to bind [125I]β‐endorphin, and immunoreactivity for mu‐ and delta‐opioid receptors (MORs and DORs), but not for kappa‐opioid receptors (KORs), was found on cells in culture. Both DNA content and [3H]thymidine incorporation were reduced after a 48‐h incubation with 100 µm naloxone, 10 µm naltrindole or 10 µmβ‐funaltrexamine, but not nor‐binaltorphimine, suggesting proliferative actions of endogenous opioids against MORs and DORs on AHPs. Furthermore, analysis of gene and protein expression after incubation with MOR and DOR antagonists for 48 h using RT‐PCR and Western blotting suggested decreased signalling through the mitogen‐activated protein kinase (MAPK) pathway and lowered levels of genes and proteins that are important in cell cycling. Cultures were incubated with naloxone (10 or 100 µm) for 10 days to study the effects on differentiation. This resulted in an approximately threefold increase in neurogenesis, a threefold decrease in astrogliogenesis and a 50% decrease in oligodendrogenesis. In conclusion, this study suggests that reduced signalling through MORs and DORs decreases proliferation in rat AHPs, increases the number of in vitro‐generated neurons and reduces the number of astrocytes and oligodendrocytes in culture.

Catecholaminergic suppression of immunocompetent cells

Abstract The synthesis of catecholamines by immunocompetent cells has now been demonstrated. In addition, catecholamines have been discovered inside the nuclear membrane and may therefore interact in the transcription process. Here, Jonas Bergquist and colleagues describe the powerful impact that catecholamines exert on the immune system by downregulation of proliferation and differentiation, and induction of apoptosis.

The synaptic-vesicle-specific proteins rab3a and synaptophysin are reduced in thalamus and related cortical brain regions in schizophrenic brains

Two synaptic-vesicle proteins, rab3a and synaptophysin, have been studied on post-mortem brain tissues of schizophrenics and healthy controls. We found significantly reduced levels of rab3a in thalamus (p<0.001); for both proteins in gyrus cinguli and hippocampus (p<0.0001); for rab3a in frontal and parietal cortex (p<0.05); and no differences in temporal cortex or cerebellum in schizophrenics compared with controls. Reduced synaptic density may be a prominent feature of the molecular neuropathology of schizophrenia.

Processing of neuropeptide Y, galanin, and somatostatin in the cerebrospinal fluid of patients with Alzheimer's disease and frontotemporal dementia

Alzheimers disease (AD) and frontotemporal dementia (FTD) are two prevalent neurodegenerative disorders for which the causes are unknown, except in rare familial cases. Several changes in neuropeptide levels as measured by radioimmunoassay (RIA) have been observed in these illnesses. Somatostatin (SOM) levels in cerebrospinal fluid (CSF) are consistently decreased in AD and FTD. Neuropeptide Y (NPY) levels are decreased in AD, but normal in FTD. Galanin (GAL) levels increase with the duration of illness in AD patients. The majority of studies of neuropeptides in CSF have not been verified by HPLC. The observed decrease in a neuropeptide level as measured by RIA may therefore reflect an altered synthesis or extracellular processing, resulting in neuropeptide fragments that may or may not be detected by RIA. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) has been shown to be a powerful technique in the analysis of biological materials without any pre-treatment, by detecting peptides and proteins at a specific mass-to-charge (m/z) ratio. We studied the processing of the neuropeptides NPY, NPY, SOM and GAL in the cerebrospinal fluid of patients with AD (n = 3), FTD (n = 3) and controls (n = 2) using MALDI-MS. We found that considerable inter-individual variability exists in the rate of neuropeptide metabolism in CSF, as well as the number of peptide fragments formed. Certain patients showed differences in the processing of specific neuropeptides, relative to other patients and controls. This analysis of the metabolic processing of neuropeptides in CSF yielded a large amount of data for each individual studied. Further studies are required to determine the changes in neuropeptide processing that can be associated with AD and FTD. With further investigations using MALDI-MS analysis, it may be possible to identify a neuropeptide fragment or processing enzyme that can be correlated to these disease states.

MASS SPECTROMETRY OF PEPTIDES IN NEUROSCIENCE

This review focuses on the contributions of modern mass spectrometry to neuropeptide research. An introduction to newer mass spectrometric techniques is provided. Also, the use of mass spectrometry in combination with high-resolution separation techniques for neuropeptide identification in biological samples is illustrated. The amino acid sequence information that is important for the identification and analysis of known, novel, or chemically modified neuropeptides may be obtained using mass spectrometric techniques. Because mass spectrometry techniques can be used to reflect the dynamic properties associated with neuropeptide processing in biological systems, they may be used in the future to monitor peptide profiles within organisms in response to environmental challenges such as disease and stress.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of proteins in human cerebrospinal fluid.

Matrix-assisted laser desorption/ionization time-of-flight mass spectra of proteins in cerebrospinal fluid analyzed without prior purification are presented. Less than 100 fmol amounts of proteins in the 10,000 to 20,000 u mass range and linked to human disease (multiple sclerosis, Alzheimers disease, and stroke) were detected in a complex mixture of proteins and peptides, in the presence of high concentrations of salts, lipids and free amino acids. The mass resolution was sufficient to distinguish between the non-hydroxylated and hydroxylated forms of a 13,400 u protein. Simple fractionation of the cerebrospinal fluid using microbore-reversed phase high performance liquid chromatography improved signal-to-noise ratios in the mass spectra. High-accuracy peptide mass mapping and database searching were utilized to confirm the identity of several proteins. The presented results show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry could be used as a tool to perform rapid screening of chemically altered proteins in small volumes of biological fluids.

Identification of nuclei associated proteins by 2D-gel electrophoresis and mass spectrometry

In clinical neuroscience as well as in many other clinical disciplines, the completion of the human genome project offers a new possibility to identify and localize the products of the genes, the proteins. Nuclear proteins are synthesized in the cytoplasm and imported into the nucleus by multiple pathways. The mechanisms by which nuclear accumulation of different molecular species occur are unclear but it is apparent that changes in the cellular and molecular events associated with the accumulation of nuclear proteins sometimes precedes transformation of cells into diseased states. The significance of the accumulation and the operation of nuclear proteins remain to be elucidated in detail. Such knowledge will play a key role in the understanding of the regulation of transcription and its disturbances in several of our most devastating diseases. In this paper we present a strategy to identify nuclear associated proteins in small samples by using two-dimensional electrophoresis and mass spectrometry. We have used human blood lymphocytes as a model, but the method should be rather general for any kind of tissue. Twenty two proteins were randomly chosen, and of these 18 proteins were identified by database searching of mass spectrometric data, obtained from in-gel tryptic digests of the spots. Thirteen proteins recently described with nuclear localization and function were identified, and five proteins; calgranulin B, glyceraldehyde-3-phosphate dehydrogenase (G3P2), a TATA-binding protein (ATBP), tubulin beta chain and moesin were also identified as being nuclear associated. The presented data clearly shows of the great role of two-dimensional gel electrophoresis and modern mass spectrometry in the excavation of the protein patterns on the subcellular level, and the ability to use small samples well suited for clinical screening.

Covariation of plasma ghrelin and motilin in irritable bowel syndrome

We have previously shown that ghrelin is mainly localized to the stomach but also occurs, together with the prokinetic hormone motilin, in endocrine cells in the proximal small intestine. This study explored ghrelin and motilin concentrations in plasma in relation to gastrointestinal motility and whether plasma ghrelin is changed in patients with irritable bowel syndrome (IBS). Nine patients with severe IBS and 10 healthy subjects underwent stationary antro-duodeni-jejunal manometry; blood was sampled during similar motility phases in the two groups. The motility phases were monitored and blood samples were collected during fasting and after food intake. Plasma was analyzed for two forms of ghrelin (octanylated and desoctanylated) as well as for motilin. In IBS patients circulating motilin levels covaried with total ghrelin levels (r=0.90; p<0.004), octanylated ghrelin (r=0.77; p<0.02) and desoctanylated ghrelin (r=0.69; p<0.04). No such correlations were seen in the control group. Octanylated ghrelin comprised 35.3+/-3.9% (mean+/-SEM) of the total circulating ghrelin in the IBS patients compared to 40.4+/-4.5% (mean+/-SEM) in the control group (NS). Ghrelin covaried with motilin in plasma in IBS but not in plasma from healthy subjects. This suggests the two peptides act together in IBS.

Processing of neuropeptide Y and somatostatin in human cerebrospinal fluid as monitored by radioimmunoassay and mass spectrometry

The processing of four neuropeptides, neuropeptide Y (NPY) 1-36, NPY (18-36), somatostatin (SOM) 1-28, and SOM (15-28) was studied in human cerebrospinal fluid (CSF) by using a novel combination of methods that included radioimmunoassay (RIA) and mass spectrometry. Untreated CSF samples were chromatographed using reversed-phase high pressure liquid chromatography (RP-HPLC) followed by NPY-RIA or SOM-RIA. These results were compared with those obtained by incubating CSF with exogenous synthetic peptides and directly detecting peptide fragments by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS). Using this combination of methods, we were able to determine the probable identities of peptides/peptide fragments recognized in radioimmunoassays. The most important NPY-immunoreactive components in CSF were found to be NPY (1-36) and NPY (3-36). Metabolic products of SOM (15-28) were found to contribute to SOM-like immunoreactivity (SOM-LI) in CSF, but SOM (1-28) only to a lesser degree. Differences in the rate of neuropeptide processing were observed. These differences depended more on the length of the peptide than its sequence. NPY (18-36) and SOM (15-28) were rapidly and extensively processed, whereas NPV (1-36) and SOM (1-28) were processed much more slowly in CSF. The production of SOM (15-28) from SOM (1-28) by enzymes in CSF was not observed. Also, the presence of a disulfide bond in the somatostatins appeared to stabilize them against enzymatic digestion of the ring structure. The results detailed in this report confirm MALDI-MS important role in studies of neuropeptide processing in CSF.