Catecholamine Storage Vesicles Research Articles

Plasma catestatin importance and correlation in patients with dipper and non-dipper hypertension patients

Catestatin (CST) is a metabolite of chromogranin A, a soluble protein in catecholamine storage vesicles acting as a feedback inhibitor of catecholamine secretion. This study aims to investigate the correlation of catestatin with dipper and non-dipper status in hypertensive patients. Hypertensive (n=355) and healthy subjects (n=129) were randomly selected at an outpatient clinic. Blood pressure and heart rate were measured according to relevant guidelines. Plasma catestatin level, plasma glucose, lipid levels were measured. An ambulatory blood pressure monitor was performed to determine the dipping patterns in hypertensive subjects. Univariate and multivariate analyses were conducted to assess factors affecting catestatin levels. Left ventricular mass was higher among hypertensives compared with healthy controls (178±47 g versus 213±52 g in healthy controls and hypertensives, p<0.05), but there was no significant difference between the two hypertensive groups 205±43 g versus 228±77 g in dippers versus non-dippers, p=0.54). Average systolic blood pressure (SBP) and the percentage of systolic blood pressure dipping were statistically significantly negative correlated with CST (r=-0.316, p<0.05 for systolic dipping%, r=-0.283, p<0.05 for ASBP). In multivariate analysis for the prediction of catestatin levels, systolic dipping % and mean SBP were found to be independent predictors of plasma catestatin level. Plasma catestatin levels were lower among hypertensives compared to healthy controls and modest inverse correlations between catestatin and dipping percentage of systolic blood pressure and also between catestatin and average systolic blood pressure were noted. In hypertensive subjects, lower plasma catestatin levels may alert clinicians to the non-dipping status of the blood pressure.

Chromogranin B: intra‐ and extra‐cellular mechanisms to regulate catecholamine storage and release, in catecholaminergic cells and organisms

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under-expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic-stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over-expression of CHGB was achieved by transduction of a CHGB-expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic-triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic-stimulated catecholamine release by ~ 72%. One conserved-region CHGB peptide inhibited nicotinic-triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi-directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra-cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

Iodine-123-metaiodobenzylguanidine scintigraphy in risk stratification of sudden death in heart failure

Metaiodobenzylguanidine (MIBG) is a false neurotransmitter noradrenaline analogue that is taken up by the ‘uptake 1’ transporter mechanism in the cell membrane of presynaptic adrenergic neurons and accumulates in catecholamine storage vesicles. Since it is practically unmetabolized, it can be labeled with a radioisotope (iodine-123) in scintigraphic exams to noninvasively assess the functional status of the sympathetic innervation of organs with a significant adrenergic component, including the heart. Studies of its application in nuclear cardiology appear to confirm its value in the assessment of conditions such as coronary artery disease, heart failure, arrhythmias and sudden death.Heart failure is a global problem, with an estimated prevalence of 2% in developed countries. Sudden cardiac death is the main cause of its high mortality. The autonomic nervous system dysfunction, including sympathetic hyperactivity, that accompanies chronic heart failure is associated with progressive myocardial remodeling, declining left ventricular function and worsening symptoms, and contributes to the development of ventricular arrhythmias and sudden death.Since 123I-MIBG cardiac scintigraphy can detect changes in the cardiac adrenergic system, there is considerable interest in its role in obtaining diagnostic and prognostic information in patients with heart failure.In this article we present a literature review on the use of 123I-MIBG scintigraphy for risk stratification of sudden death in patients with heart failure.

Utilização da cintigrafia com iodo-123-metaiodobenzilguanidina na estratificação do risco de morte súbita na insuficiência cardíaca

Metaiodobenzylguanidine (MIBG) is a false neurotransmitter noradrenaline analogue that is taken up by the ‘uptake 1’ transporter mechanism in the cell membrane of presynaptic adrenergic neurons and accumulates in catecholamine storage vesicles. Since it is practically unmetabolized, it can be labeled with a radioisotope (iodine-123) in scintigraphic exams to noninvasively assess the functional status of the sympathetic innervation of organs with a significant adrenergic component, including the heart. Studies of its application in nuclear cardiology appear to confirm its value in the assessment of conditions such as coronary artery disease, heart failure, arrhythmias and sudden death.Heart failure is a global problem, with an estimated prevalence of 2% in developed countries. Sudden cardiac death is the main cause of its high mortality. The autonomic nervous system dysfunction, including sympathetic hyperactivity, that accompanies chronic heart failure is associated with progressive myocardial remodeling, declining left ventricular function and worsening symptoms, and contributes to the development of ventricular arrhythmias and sudden death.Since 123I-MIBG cardiac scintigraphy can detect changes in the cardiac adrenergic system, there is considerable interest in its role in obtaining diagnostic and prognostic information in patients with heart failure.In this article we present a literature review on the use of 123I-MIBG scintigraphy for risk stratification of sudden death in patients with heart failure.

The anti-fibrinolytic SERPIN, plasminogen activator inhibitor 1 (PAI-1), is targeted to and released from catecholamine storage vesicles

Recent studies suggest a crucial role for plasminogen activator inhibitor-1 (PAI-1) in mediating stress-induced hypercoagulability and thrombosis. However, the mechanisms by which PAI-1 is released by stress are not well-delineated. Here, we examined catecholaminergic neurosecretory cells for expression, trafficking, and release of PAI-1. PAI-1 was prominently expressed in PC12 pheochromocytoma cells and bovine adrenomedullary chromaffin cells as detected by Northern blotting, Western blotting, and specific PAI-1 ELISA. Sucrose gradient fractionation studies and immunoelectron microscopy demonstrated localization of PAI-1 to catecholamine storage vesicles. Secretogogue stimulation resulted in corelease of PAI-1 with catecholamines. Parallel increases in plasma PAI-1 and catecholamines were observed in response to acute sympathoadrenal activation by restraint stress in mice in vivo. Reverse fibrin zymography demonstrated free PAI-1 in cellular releasates. Detection of high molecular weight complexes by Western blotting, consistent with PAI-1 complexed with t-PA, as well as bands consistent with cleaved PAI-1, suggested that active PAI-1 was present. Modulation of PAI-1 levels by incubating PC12 cells with anti-PAI-1 IgG caused a marked decrease in nicotine-mediated catecholamine release. In summary, PAI-1 is expressed in chromaffin cells, sorted into the regulated pathway of secretion (into catecholamine storage vesicles), and coreleased, by exocytosis, with catecholamines in response to secretogogues.

Proteomic Analysis Yields an Unexpected Trans -Acting Point in Control of the Human Sympathochromaffin Phenotype

The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and gives rise to a catecholamine release-inhibitory fragment. Because genetic variation in the proximal human CHGA promoter predicts autonomic function and blood pressure, we explored how a common genetic variant alters transcription of the gene. Bioinformatic analysis suggested that the common G-462A promoter variant (rs9658634) may disrupt as many as 3 transcriptional control motifs: LEF1, COUP-TF, and PPARγ-RXRα. During electrophoretic mobility shifts, chromaffin cell nuclear proteins bound specifically to the A (though not G) allele of CHGA promoter G-462A. On oligonucleotide affinity chromatography followed by electrospray ionization followed by 2-dimensional (tandem) mass spectrometry analysis of A allele eluates, the transcription factor LEF1 (lymphoid enhancer-binding factor-1) was identified. Interaction of LEF1 with the A allele at G-462A was confirmed by supershift. On cotransfection, LEF1 discriminated between the allelic variants, especially in chromaffin cells. Allele specificity of trans-activation by LEF1 was transferable to an isolated G-462A element fused to a heterologous (SV40) promoter. Because β-catenin (CTNNB1) can heterodimerize with LEF1, we tested the effect of cotransfection of this factor and again found A allele-specific perturbation of CHGA transcription. Common genetic variation within the human CHGA promoter alters the interaction of specific factors in trans with the promoter, with LEF1 identified by proteomic analysis and confirmed by supershift. Coexpression experiments show functional effects of LEF1 and CTNNB1 on CHGA promoter. The findings document a novel role for components of the immune and WNT pathways in control of human sympathochromaffin phenotypes.

Catecholamine Storage Vesicles: Role of Core Protein Genetic Polymorphisms in Hypertension

Hypertension is a complex trait with deranged autonomic control of the circulation. The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Catecholamine storage vesicles (or chromaffin granules) of the adrenal medulla contain remarkably high concentrations of chromogranins/secretogranins (or “granins”), catecholamines, neuropeptide Y, adenosine triphosphate (ATP), and Ca2+. Within secretory granules, granins are co-stored with catecholamine neurotransmitters and co-released upon stimulation of the regulated secretory pathway. The principal granin family members, chromogranin A (CHGA), chromogranin B (CHGB), and secretogranin II (SCG2), may have evolved from shared ancestral exons by gene duplication. This article reviews human genetic variation at loci encoding the major granins and probes the effects of such polymorphisms on blood pressure, using twin pairs to probe heritability and individuals with the most extreme blood pressure values in the population to study hypertension.

Catecholamine-producing cells in the synovial tissue during arthritis: modulation of sympathetic neurotransmitters as new therapeutic target

BackgroundThe proinflammatory and anti-inflammatory role of the sympathetic nervous system in early and late inflammation is an unresolved paradox. A drastic loss of sympathetic nerve fibres in the synovial tissue...

Common Functional Genetic Variants in Catecholamine Storage Vesicle Protein Promoter Motifs Interact to Trigger Systemic Hypertension

The purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease. CHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5' region. Here we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits. The A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p=3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT<AA<<CA<AT on both systolic blood pressure (SBP) and diastolic blood pressure (DBP), accounting for approximately 2.3% to approximately 3.4% of SBP/DBP variance; the haplotype effects on BP in vivo paralleled those on promoter activity in cella. Site-by-site interactions at A-296C and A-261T yielded highly nonadditive effects on SBP/DBP. The CHGB haplotype effects on BP were also noted in an independent (African ancestry) sample. In normotensive twins, parallel effects were noted for a pre-hypertensive phenotype, BP response to environmental stress. The common CHGB promoter variants A-296C and A-261T, and their consequent haplotypes, alter binding of specific transcription factors to influence gene expression in cella as well as BP in vivo. Such variation contributes substantially to risk for human hypertension. Involvement of the sex-specific factor SRY suggests a novel mechanism for development of sexual dimorphism in BP.

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [ 3H]NE uptake capacity, an effect characterized by a robust decrease in the V max of the transport of [ 3H]NE. As expected, reserpine did not displace the binding of [ 3H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [ 3H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [ 3H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca 2+/Ca 2+–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [ 3H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl- p-tyrosine, increased [ 3H]NE uptake and eliminated the inhibitory effects of reserpine on [ 3H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca 2+-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

Long human CHGA flanking chromosome 14 sequence required for optimal BAC transgenic “rescue” of disease phenotypes in the mouse Chga knockout

Chromogranin A (CHGA) plays a catalytic role in formation of catecholamine storage vesicles and also serves as precursor to the peptide fragment catestatin, a catecholamine secretory inhibitor whose expression is diminished in the hypertensive individuals. We previously reported the hypertensive, hyperadrenergic phenotype of Chga-/- knockout (KO) mice and rescue by the human ortholog. In the present study, we compare two humanized CHGA mouse models. Into the Chga null background, by bacterial artificial chromosome transgenesis human CHGA transgene has been introduced. Both lines have the complete approximately 12 kbp CHGA gene integrated stably in the genome but have substantial differences in CHGA expression, as well as consequent sympathochromaffin biochemistry and physiology. A mouse model with longer-insert HumCHGA31 displays integration encompassing not only CHGA but also long human flanking sequences. This is in contrast to mouse model HumCHGA19 with limited flanking human sequence co-integrated. As a consequence, HumCHGA19 mice have normal though diminished pattern of spatial expression of CHGA, and 14-fold lower circulating CHGA, with failure to rescue KO phenotypes to normalcy. In the longer-insert HumCHGA31 mice, catecholamine secretion, exaggerated responses to environmental stress, and hypertension were all alleviated. Promoter regions of the transgenes in both HumCHGA19 and HumCHGA31 display minimal CpG methylation, weighing against differential "position effects" of integration, and thus suggesting that lack of cis elements required for optimal CHGA expression occurs in HumCHGA19 mice. Such "humanized" CHGA mouse models may be useful in probing the physiological consequences of variation in CHGA expression found in humans, with consequences for susceptibility to hypertension and cardiovascular disease.

Complex Renal Traits

Many if not most of the common disease states encountered in internal medicine, nephrology in particular, are complex traits1: Typically poorly understood and likely to be multifactorial, with etiologic contributions from both heredity and environment. Family or twin studies can establish the relative roles of heredity or environment trait determination, and indeed, familial aggregation or heritability is documented for such traits as hypertension2 and chronic kidney disease,3 whereas less common traits, including adult and juvenile polycystic kidney disease, display simpler, Mendelian hereditary patterns. The contribution of polymorphisms at specific loci are now documented for hypertensive ESRD, including the nonmuscle myosin variant, MYH9 ,4 and the catecholamine storage vesicle protein chromogranin A, CHGA .5 Systematic family studies have not been conducted in acute kidney injury (AKI), perhaps because of the transient nature of the illness coupled with unavailability of appropriate first-degree relatives for study during hospitalization. Despite a recent explosion of studies and investigations of complex diseases, results have been mixed, incomplete, and inconsistent.6 Such studies are compelling insofar as they aim to tackle important medical questions regarding traits that are likely influenced by genes, the environment, or interaction between the genes and the environment, highlighting the difficulty and intricacy of such traits. Since 2007, developments in nanotechnology have enabled genome-wide association (GWA) studies, which typically study very large numbers (many hundreds to several thousands) of disease cases and controls at up to approximately 5 to 10 × 105 single-nucleotide polymorphisms (SNPs) at once.7 In such hypothesis-free approaches, many new susceptibility traits have been revealed, unveiling …

One-electron oxidation of catecholamines generates free radicals with an in vitro toxicity correlating with their lifetime

One-electron oxidation of dopamine by ferricyanide generates a highly reactive free radical intermediate that inactivates the V-type H +-ATPase proton pump in catecholamine storage vesicles, i.e., the driving force in both the vesicular uptake and the storage of catecholamines, in a cell-free in vitro model system at pH 7.0. Electron paramagnetic resonance spectroscopy revealed that a radical with g = 2.0045, formed by this oxidation, was relatively long-lived ( t 1/2 obs = 79 s at pH 6.5 and 25°C). Experimental evidence is presented that the observed radical most likely represents dopamine semiquinone free radical, although an o-quinone free radical cannot be ruled out. Oxidation of noradrenaline and adrenaline by ferricyanide generated similar isotropic radicals, but of shorter half-lives (i.e., 43 and 5.3 s, respectively), and the efficacy of inactivation of the H +-ATPase correlated with the half-life of the respective catecholamine free radical (i.e., dopamine > noradrenaline ≫ adrenaline). Thus, the generation of relatively long-lived semiquinone free radicals, although at low concentrations, in dopaminergic and noradrenergic neurons may represent a common mechanism of cytotoxicity linked to neurodegeneration of the respective neurons related to Parkinson disease.

Catecholamine storage vesicles and the metabolic syndrome: The role of the chromogranin A fragment pancreastatin.

Chromogranins or secretogranins (granins), present in secretory granules of virtually all neuroendocrine cells and neurones, are structurally related proteins encoded by different genetic loci: chromogranins A and B, and secretogranins II through VI. Compelling evidence supports both intracellular and extracellular functions for this protein family. Within the cells of origin, a granulogenic or sorting role in the regulated pathway of hormone or neurotransmitter secretion has been documented, especially for chromogranin A (CHGA). Granins also function as pro-hormones, giving rise by proteolytic processing to an array of peptide fragments for which diverse autocrine, paracrine, and endocrine activities have been demonstrated. CHGA measurements yield insight into the pathogenesis of such human diseases as essential hypertension, in which deficiency of the catecholamine release-inhibitory CHGA fragment catestatin may trigger sympathoadrenal overactivity as an aetiologic culprit in the syndrome. The CHGA dysglycaemic fragment pancreastatin is functional in humans in vivo, affecting both carbohydrate (glucose) and lipid (fatty acid) metabolism. Pancreastatin is cleaved from CHGA in hormone storage granules in vivo, and its plasma concentration varies in human disease. The pancreastatin region of CHGA gives rise to three naturally occurring human variants, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptide's potency to inhibit cellular glucose uptake. These observations establish a role for pancreastatin in human intermediary metabolism and disease, and suggest that qualitative hereditary alterations in pancreastatin's primary structure may give rise to interindividual differences in glucose disposition.

The chromogranin A fragment catestatin: specificity, potency and mechanism to inhibit exocytotic secretion of multiple catecholamine storage vesicle co-transmitters

Secretory granules of chromaffin cells and neurons co-store and release, by exocytosis, the acidic soluble protein chromogranin A (human, CHGA; rodent, Chga) along with catecholamines, neuropeptides and adenosine triphosphate (ATP). CHGA serves as a pro-protein and upon proteolytic cleavage it generates active peptides, including catestatin (human CHGA352-372), first discovered in adrenal medullary chromaffin granules. Studies in our laboratory demonstrated that catestatin acts at the nicotinic acetylcholine receptor to inhibit catecholamine secretion. However, the specificity of catestatin to exert nicotinic-cholinergic antagonism among its co-transmitters is not clearly known, nor is the potential effect of catestatin on multiple vesicle co-transmitters understood. Here we probed the specificity of catestatin's actions among its co-transmitters: catecholamines, ATP, and neuropeptide Y (NPY). We studied the effects of each transmitter on exocytotic secretion of its co-transmitters from PC12 chromaffin cells, stimulating secretion by triggering physiological pathways at multiple sites. We observed that, among chromaffin granule co-transmitters, only catestatin and NPY inhibited catecholamine release induced by nicotinic-cholinergic stimulation; catestatin was more than tenfold more potent than NPY in this setting. We also stimulated norepinephrine secretion by other chromaffin cell agonists: catestatin blocked norepinephrine release induced by nicotine, but not by other agents (such as membrane depolarization) acting at later stages in the secretory pathway, nor by agents acting on other receptor classes. By contrast, NPY acted less specifically, blocking norepinephrine release triggered by either nicotine or membrane depolarization. Catestatin inhibited nicotinic-cholinergic co-release of all classes of chromaffin granule co-transmitters: catecholamines, chromogranins, neuropeptides, and ATP. Naturally occurring variants of human catestatin (Gly364Ser and Pro370Leu) exhibited parallel changes in potency to inhibit secretion of catecholamines and ATP. We conclude that, among the chromaffin granule co-transmitters, catestatin acts as the most specific and potent inhibitor of physiological pathway (nicotinic-cholinergic) stimulated secretion. Furthermore, catestatin generally inhibits nicotinically triggered exocytotic release of multiple co-transmitters from chromaffin granules. The results have physiological and pharmacological implications for co-transmission in the sympathochromaffin system.

Vitamin C Is an Important Cofactor for Both Adrenal Cortex and Adrenal Medulla

The adrenal gland is among the organs with the highest concentration of vitamin C in the body. Interestingly, both the adrenal cortex and the medulla accumulate such high levels of ascorbate. Ascorbic acid is a cofactor required both in catecholamine biosynthesis and in adrenal steroidogenesis. Here we provide an overview on the role of vitamin C in the adrenal cortex and medulla derived from in vitro and in vivo studies. In addition, recent insights gained from an animal model with a deletion in the gene for the ascorbic acid transporter will be summarized. Mutant mice lacking the plasma membrane ascorbic acid transporter (SVCT2) have severely reduced tissue levels of ascorbic acid and die soon after birth. There is a significant decrease of tissue catecholamine levels in the adrenals. On the ultrastructural level, adrenal chromaffin cells in SVCT2 null mice show depletion of catecholamine storage vesicles, signs of apoptosis, and increased glycogen storage. Decreased plasma levels of corticosterone and altered morphology of mitochondrial membranes indicate additional effects of the deficiency on adrenal cortical function. The data derived from these animal models and various cell culture studies confirm a crucial role for vitamin C for both the adrenal cortex as well as the adrenal medulla further underlining the interdependence of the two endocrine systems united in one gland.

Intracellular Protein Trafficking into Catecholamine Storage Vesicles

Intracellular Protein Trafficking into Catecholamine Storage Vesicles

Proteolytic Cleavage of Chromogranin A (CgA) by Plasmin: SELECTIVE LIBERATION OF A SPECIFIC BIOACTIVE CgA FRAGMENT THAT REGULATES CATECHOLAMINE RELEASE

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.

Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion.

Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.

Energy-dependent accumulation of calcium antagonists in catecholamine storage vesicles

The calcium antagonists verapamil, nitrendipine, mibefradil, and amlodipine accumulate in chromaffin granule ghosts with apparent equilibrium partition coefficients [(mol/mg membrane lipid)/(mol/mg solvent water)] of 246 ± 105 (N = 8), 2700 ± 600 (N = 4), 7400 ± 2200 (N = 4), and 8100 ± 1100 (N = 5), respectively. In the presence of 1.2 mM MgATP, the partition coefficients were 854 ± 206 (N = 10), 2300 ± 600 (N = 4), 32,700 ± 8,900 (N = 7), and 20,300 ± 5,000 (N = 11) for verapamil, nitrendipine, mibefradil, and amlodipine, respectively. Except for nitrendipine, the apparent partition coefficients in the presence of MgATP were significantly different from the control ( P < 0.001). For amlodipine and verapamil, the vacuolar H +-ATPase inhibitors bafilomycin A 1 (30 nM) and N-ethylmaleimide (2 mM) and the protonophore (uncoupler) carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μM) completely blocked the increase in partition coefficients in response to MgATP. The extra amlodipine, mibefradil, and verapamil that accumulated in response to MgATP were released into the medium by CCCP (10 μM) by 18% (N = 5), 30% (N = 5), and 88% (N = 5) for amlodipine, mibefradil, and verapamil, respectively. Thus, amlodipine, mibefradil, and verapamil, but not nitrendipine, accumulate in catecholamine storage vesicles in response to Δμ H+ generated by the endogenous V-type H +-ATPase, and are partially released by de-energetisation. Hence, these calcium antagonists can reach unexpectedly high concentrations in certain target cells, and give pharmacodynamic properties not shared by nitrendipine.