Gene networks specific for innate immunity define post-traumatic stress disorder.

The molecular factors involved in the development of Post-Traumatic Stress Disorder (PTSD) remain poorly understood. Previous transcriptomic studies investigating the mechanisms of PTSD apply targeted approaches to identify individual genes under a cross-sectional framework lack a holistic view of the behaviours and properties of these genes at the system-level. Here we sought to apply an unsupervised gene-network based approach to a prospective experimental design using whole-transcriptome RNA-Seq gene expression from peripheral blood leukocytes of U.S. Marines (N=188), obtained both pre- and post-deployment to conflict zones. We identified discrete groups of co-regulated genes (i.e., co-expression modules) and tested them for association to PTSD. We identified one module at both pre- and post-deployment containing putative causal signatures for PTSD development displaying an over-expression of genes enriched for functions of innate-immune response and interferon signalling (Type-I and Type-II). Importantly, these results were replicated in a second non-overlapping independent dataset of U.S. Marines (N=96), further outlining the role of innate immune and interferon signalling genes within co-expression modules to explain at least part of the causal pathophysiology for PTSD development. A second module, consequential of trauma exposure, contained PTSD resiliency signatures and an over-expression of genes involved in hemostasis and wound responsiveness suggesting that chronic levels of stress impair proper wound healing during/after exposure to the battlefield while highlighting the role of the hemostatic system as a clinical indicator of chronic-based stress. These findings provide novel insights for early preventative measures and advanced PTSD detection, which may lead to interventions that delay or perhaps abrogate the development of PTSD.

Genetic regulation of catecholamine synthesis, storage and secretion in the spontaneously hypertensive rat.

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

Effects of intensive insulin therapy alone and with added pioglitazone on renal salt/water balance and fluid compartment shifts in type 2 diabetes.

OBJECTIVE: To evaluate the effects of intensive insulin therapy alone or with added pioglitazone on renal salt/water balance and body fluid compartment shifts in type 2 diabetes. METHODS: A total of 25 insulin-treated, obese patients with type 2 diabetes were randomized to pioglitazone 45 mg (n = 12) or placebo (n = 13) and treated intensively for 12-16 weeks to achieve equivalent glycaemic control. We measured total body water (TBW) and extracellular/intracellular fluid by bioimpedance analysis; plasma/RBC volume with I(131)albumin; sodium handling by fractional excretion of sodium/lithium (FeNa/FeLi) and other renal/hormonal parameters. RESULTS: Intensification of insulin therapy and the addition of pioglitazone significantly improved glycaemia (HbA1C 7.8-7.2% and 7.6-7.1%) and increased body weight (1.7 and 4.9 kg) respectively. TBW increased 1.7 l with insulin alone (65% intracellular) and 1.6 l with added pioglitazone (75% extracellular) (p = 0.06 and 0.09 respectively). Plasma volume increased 0.2 +/- 0.1 l with insulin alone (p = 0.05) and 0.4 +/- 0.1 l with added pioglitazone (p < 0.05). Extravascular, extracellular (interstitial) fluid increased significantly and more with added pioglitazone (0.8 +/- 0.2 l, p < 0.01) than with insulin alone (0.4 +/- 0.2 l, p = ns). At steady-state, FeLi (marker of proximal-tubular sodium delivery to the distal nephron) increased significantly with added pioglitazone (12.4 +/- 1.3 to 18.0 +/- 3.2%) vs. no significant change with insulin alone (15.4 +/- 1.2 to 14.5 +/- 2.3%). There were no significant changes in the other parameters. CONCLUSION: In intensively insulin-treated obese type 2 diabetic patients, at equivalent glycaemic control, the addition of pioglitazone causes greater weight gain, but a similar increase in body water that is mainly extracellular and interstitial compared with intracellular increase with insulin therapy alone. Pioglitazone also increases the filtered load of sodium reabsorbed at the distal nephron with no net change in FeNa.

The Presence of a High Peak Feature Within Low-Average Shear Stimuli Induces Quiescence in Venous Endothelial Cells.

Wall shear stress (WSS) is an important stimulus in vascular remodelling and vascular lesion development. The current methods to assess and predict the risk associated with specific unsteady WSS consider the WSS mean values or the presence of reverse phases described by the oscillatory shear index. Recent evidence has shown that the accuracy of these methods is limited, especially with respect to the venous environment. Unsteady WSS are characterised by several features that may individually affect endothelial cells. Consequently, we assessed the effects of averaged WSS (TAWSS), temporal WSS gradient (TWSSG), maximum WSS (WSS peak) and reverse phase (OSI) by applying different WSS profiles to venous EC in-vitro, using a real-time controlled cone-and-plate cell-shearing device for 24 h. We found that TWSSG and WSS peak affect cell elongation and alignment respectively. We also found that the WSS waveforms with a peak of 1.5 Pa or higher significantly correlate with the induction of a protective phenotype. Cell phenotype induced by these high peak waveforms does not correlate to what is predicted by the hemodynamic indices currently used. The definition of reliable hemodynamic indices can be used to inform the computational models aimed at estimating the hemodynamic effects on vascular remodelling.

Common genetic variants in the chromogranin A promoter alter autonomic activity and blood pressure.

Chromogranin A (CHGA) is stored and released from the same secretory vesicles that contain catecholamines in chromaffin cells and noradrenergic neurons. We had previously identified common genetic variants at the CHGA locus in several human populations. Here we focus on whether inter-individual variants in the promoter region are of physiological significance. A common haplotype, CGATA (Hap-B), blunted the blood pressure response to cold stress and the effect exhibited molecular heterosis with the greatest blood pressure change found in Hap-A/Hap-B heterozygotes. Homozygosity for three minor alleles with peak effects within the haplotype predicted lower stress-induced blood pressure changes. The G-462A variant predicted resting blood pressure in the population with higher pressures occurring in heterozygotes (heterosis). Using cells transfected with CHGA promoter-luciferase reporter constructs, the Hap-B haplotype had decreased luciferase expression compared to the TTGTC (Hap-A) haplotype under both basal conditions and after activation by pre-ganglionic stimuli. The G-462A variant altered a COUP-TF transcriptional control motif. The two alleles in transfected promoters differed in basal activity and in the responses to COUP-II-TF transactivation and to retinoic acid. In vitro findings of molecular heterosis were also noted with the transfected CHGA promoter wherein the diploid combination of the two G-462A alleles gave rise to higher luciferase expression than either allele in isolation. Our results suggest that common genetic variants in the CHGA promoter may regulate heritable changes in blood pressure.

The cholinesterases: analysis by pharmacogenomics in man.

We have undertaken a study on variations in cholinesterase (ChE) genes in relation to cardiovascular (CV) function and the metabolic syndrome. Peripheral and central nervous system control of cardiovascular (CV) function mediated through cholinergic pathways is critical in homeostatic maintenance of blood pressure and responsiveness to stress. For acetylcholinesterase (AChE; EC 3.1.1.7) our focus is to identify single nucleotide polymorphisms (SNPs) in the gene that are linked to cardiovascular function. For butyrylcholinesterase (BChE; EC 3.1.1.8) we examined whether BChE activity correlated with parameters of the metabolic syndrome and cardiovascular function. ChE can be found in whole blood enabling a characterization of biochemical phenotype in addition to correlating genotype with phenotypic physiologic responses. Analysis of enzymatic activity was determined spectrophotometrically in blood samples from twin and other subject registries. Correlation analysis revealed significant relationships between enzyme activity and certain CV endpoints. Linkage analysis with data from a dizygotic (DZ) twin set showed a suggestive linkage at the BChE locus, and statistical analysis revealed a high correlation between BChE activity and variables associated with cardiovascular risk and the metabolic syndrome. Pattern of within-pair twin correlations by zygosity and the ACE model-fitting findings suggest the major source of this variation (65%) is attributable to an additive genetic component. To date 19 SNPs have been identified by the re-sequencing of AChE including four nonsynonymous coding SNPs (cSNPs).

Radiological comparison of a FDNPP waste storage site during and after construction.

The clean-up effort that is occurring across the region affected by the 2011 Fukushima Daiichi Nuclear Power Plant accident is unprecedented in its magnitude as well as the financial cost that will eventually result. A major component of this remediation is the stripping of large volumes of material from the land surface, depositing this into large waste storage bags before placing these 1 cubic meter bags into specially constructed stores across Fukushima Prefecture. In this work, using an unmanned aerial vehicle to perform radiological surveys of a site, the time-resolved distribution of contamination during the construction of one of these waste storage sites was assessed. The results indicated that radioactive material was progressively leaching from the store into the surrounding environment. A subsequent survey of the site conducted eight months later revealed that in response to this survey and remedial actions, the contamination issue once existing on this site had been successfully resolved. Such results highlight the potential of low-altitude unmanned aerial systems to easily and rapidly assess site-wide changes over time - providing highly-visual results; therefore, permitting for prompt remedial actions to be undertaken as required. Use of UAV radiation mapping and airborne photogrammetry to produce a time-resolved assessment of remediation efforts within a Fukushima temporary storage facility.

Application of airborne photogrammetry for the visualisation and assessment of contamination migration arising from a Fukushima waste storage facility.

Airborne systems such as lightweight and highly portable unmanned aerial vehicles (UAVs) are becoming increasingly widespread in both academia and industry - with an ever-increasing range of applications, including (but not limited to), air quality sampling, wildlife monitoring and land-use mapping. In this work, high-resolution airborne photogrammetry obtained using a multi-rotor system operating at low survey altitudes, is combined with ground-based radiation mapping data acquired at an interim storage facility for wastes removed as part of the large-scale Fukushima clean-up program. The investigation aimed to assess the extent to which the remediation program at a specific site has contained the stored contaminants, as well as present a new methodology for rapidly surveying radiological sites globally. From the three-dimensional rendering of the site of interest, it was possible to not only generate a powerful graphic confirming the elevated radiological intensity existing at the location of the waste bags, but also to also illustrate the downslope movement of contamination due to species leakage from the large 1m3 storage bags. The entire survey took less than 1 h to perform, and was subsequently post-processed using graphical information software to obtain the renderings. The conclusions within this study not only highlight the usefulness of incorporating three-dimensional renderings within radiation mapping protocols, but also conclude that current methods of monitoring these storage facilities in the long term could be improved through the integration of UAVs within the standard protocol.

Chromogranin A in children with neuroblastoma. Serum concentration parallels disease stage and predicts survival.

Chromogranin A is an acidic protein costored and coreleased with catecholamines from storage vesicles. Its serum concentration is elevated in patients with peptide-producing endocrine neoplasia. We measured serum chromogranin A at the time of diagnosis in 34 children with all stages of neuroblastoma. With a sensitivity of 91% and specificity of 100%, serum chromogranin A emerged as a useful diagnostic tool for neuroblastoma, comparable to or better than other measurements such as neuron-specific enolase, ferritin, or dopamine-beta-hydroxylase. Mean serum chromogranin A correlated with disease stage (r = 0.76, P less than 0.01). The relationship of prognosis (progression-free survival) to baseline serum chromogranin A, age, and disease stage was determined in 34 patients at risk for relapse, with a median followup period of 18 mo (range, 1-48 mo). The survival rate for patients with lower serum chromogranin A levels (less than 190 ng/ml at the time of diagnosis) was 69%, whereas it was 30% for those with higher chromogranin A levels (P less than 0.05). Furthermore, when subjects were additionally stratified by either age or stage, chromogranin A was an effective prognostic tool in patients who either were older than 1 yr (P less than 0.005) or had more advanced disease (stage III or IV; P less than 0.05). We conclude that serum chromogranin A in neuroblastoma is (a) a valuable (sensitive and specific) diagnostic tool, (b) a correlate of tumor burden, and (c) a useful predictor of survival.

Cell type-specific gene expression in the neuroendocrine system. A neuroendocrine-specific regulatory element in the promoter of chromogranin A, a ubiquitous secretory granule core protein.

The acidic secretory protein chromogranin A universally occurs in amine and peptide hormone and neurotransmitter storage granules throughout the neuroendocrine system. What factors govern the activity of the chromogranin A gene, to yield such a widespread yet neuroendocrine-selective pattern of expression? To address this question, we isolated the mouse chromogranin A gene promoter. The promoter conferred cell type-specific expression in several neuroendocrine cell types (adrenal medullary chromaffin cells, anterior pituitary corticotropes, and anterior pituitary somatolactotropes) but not in control (fibroblast or kidney) cells. In neuroendocrine cells, analysis of promoter deletions established both positive and negative transcriptional regulatory domains. A distal positive domain (-4.8/-2.2 kbp) was discovered, as well as negative (-258/-181 bp) and positive (-147/-61 bp) domains in the proximate promoter. The proximate promoter contained a minimal neuroendocrine-specific element between -77 and -61 bp. Sequence alignment of the mouse promoter with corresponding regions in rat and bovine clones indicated that the mouse sequence shares over 85% homology with rat and 52% with bovine promoters. DNaseI footprinting and electrophoretic gel mobility shift assays demonstrated the presence of nuclear factors in neuroendocrine cells that recognized the proximate promoter. We conclude that the chromogranin A promoter contains both positive and negative domains governing its cell type-specific pattern of transcription, and that a small proximate region of the promoter, containing novel as well as previously described elements, interacts specifically with neuroendocrine nuclear proteins, and is thereby sufficient to ensure widespread neuroendocrine expression of the gene.

Peptidergic activation of transcription and secretion in chromaffin cells. Cis and trans signaling determinants of pituitary adenylyl cyclase-activating polypeptide (PACAP).

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. Chromogranin A is the major soluble core component in secretory vesicles. Since chromogranin A is secreted along with catecholamines, we asked whether PACAP regulates expression of the chromogranin A gene in PC12 rat chromaffin cells, so as to resynthesize the just-secreted protein, and whether such biosynthetic regulation is coupled mechanistically to catecholamine secretion. PACAP activated the endogenous chromogranin A gene by four- to fivefold. Proportional results (seven- to eightfold activation) were obtained with a transfected 1,200-bp mouse chromogranin A promoter/luciferase reporter construct. A series of chromogranin A promoter 5' deletion mutant/luciferase reporter constructs narrowed down the PACAP response element to a proximal region containing the cAMP response element (CRE box), at (-71 bp)5'-TGACGTAA-3'(-64 bp). Site-directed point mutations of the CRE site suppressed PACAP-induced trans-activation of the promoter. Thus, the proximal CRE box is entirely necessary for the chromogranin A promoter response to PACAP. Transfer of the CRE box to a neutral, heterologous promoter also conferred activation by PACAP, suggesting that the CRE domain is also sufficient to mediate the transcriptional response to PACAP. Expression of a dominant-negative mutant (KCREB) of the CRE-binding factor CREB markedly diminished trans-activation of the chromogranin A promoter by PACAP. Cotransfection of expression plasmids encoding the protein kinase A inhibitor, or an inactive protein kinase A (PKA) catalytic beta subunit, inhibited both forskolin and PACAP activation of chromogranin A transcription, revealing that PACAP-induced trans-activation is highly dependent on PKA. By contrast, inhibition of protein kinase C (by chronic exposure to phorbol ester) had no effect on transcriptional activation by PACAP. The potent PACAP/vasoactive intestinal peptide (VIP) type I receptor antagonist PACAP6-38 impaired both chromogranin A transcription or catecholamine secretion triggered by PACAP38, while the PACAP/VIP type II receptor antagonist (p-Chloro-D-Phe6, Leu17)-VIP had little or no ability to antagonize the PACAP38 effect. The agonist VIP was approximately 100- to 1,000-fold less potent than PACAP in stimulating either secretion or transcription. Thus, PACAP-evoked chromogranin A transcription and catecholamine secretion are likely mediated by the PACAP/VIP type I receptor isoform. Although the calcium channel antagonists Zn2+ (100 microM), nifedipine (10 microM), or ruthenium red (10 microM), or the cytosolic calcium chelator BAPTA-AM (50 microM) each strongly impaired PACAP-induced secretion, transcriptional activation of chromogranin A remained unaltered. Therefore, we propose that PACAP signals to chromogranin A transcription through the CRE in cis, and through PKA and CREB in trans. By contrast, a pathway involving cytosolic calcium entry through L-type voltage-dependent channels is required for PACAP to evoke catecholamine secretion.

Glucocorticoid activation of chromogranin A gene expression. Identification and characterization of a novel glucocorticoid response element.

Glucocorticoids regulate catecholamine biosynthesis and storage at several sites. Chromogranin A, an abundant protein complexed with catecholamines in secretory vesicles of chromaffin cells and sympathetic axons, is also augmented by glucocorticoids. This study reports isolation of the rat chromogranin A promoter to elucidate transcriptional regulation of chromogranin A biosynthesis by glucocorticoids in neuroendocrine cells. Endogenous chromogranin A gene expression was activated up to 3.5-fold in chromaffin cells by glucocorticoid, in time-dependent fashion. Inhibition of new protein synthesis by cycloheximide did not alter the rise in chromogranin A mRNA, suggesting that glucocorticoids directly activate the chromogranin A promoter; nuclear runoff assays confirmed a 3.3-fold increased rate of initiation of new chromogranin A transcripts after glucocorticoid. Transfected rat chromogranin A promoter/luciferase reporter constructs were activated 2.6-3.1-fold by glucocorticoid, and selective agonist/antagonist studies determined that dexamethasone effects were mediated by glucocorticoid receptors. Both rat and mouse chromogranin A promoter/luciferase reporter constructs were activated by glucocorticoid. A series of promoter deletions narrowed the region of glucocorticoid action to a 93-bp section of the promoter, from position -526 to -619 bp upstream of the cap site. A 15-bp sequence ([-583 bp] 5'-ACATGAGTGTGTCCT-3' [-597 bp]) within this region showed partial homology to a glucocorticoid response element (GRE; half-site in italics) consensus sequence, and several lines of experimental evidence confirmed its function as a GRE: (a) site-directed mutation of this GRE prevented glucocorticoid activation of a chromogranin A promoter/reporter; (b) transfer of this GRE to a heterologous (thymidine kinase) promoter/reporter conferred activation by glucocorticoid, in copy number-dependent and orientation-independent fashion; and (c) electrophoretic gel mobility shifts demonstrated binding of this GRE by ligand-activated glucocorticoid receptor, though at 2.75-fold lower affinity than the glucocorticoid receptor interaction with a consensus GRE. The rat chromogranin A GRE showed functional and structural similarities to GREs in other genes proportionally regulated by glucocorticoids. We conclude that a discrete domain of the chromogranin A promoter is both necessary and sufficient to confer glucocorticoid regulation onto the gene, and that the activity of this region also explains the degree of activation of the endogenous gene by glucocorticoid.

Stimulus coupling to transcription versus secretion in pheochromocytoma cells. Convergent and divergent signal transduction pathways and the crucial roles for route of cytosolic calcium entry and protein kinase C.

How do chromaffin cell secretory stimuli program resynthesis of secreted peptides and amines? We previously showed that the physiologic nicotinic cholinergic signal for secretion also activates the biosynthesis of chromogranin A, the major protein released with catecholamines. Here, we examine signal transduction pathways whereby secretory stimuli influence exocytotic secretion versus chromogranin A transcription. Both secretion and transcription depended on initial nicotinic-triggered sodium entry into the cytosol, followed by calcium entry through -type voltage-gated channels. When calcium entered through -type channels, activation of secretion paralleled activation of transcription (r = 0.897, P = 0.002). Calcium entry from intracellular stores or through calcium ionophore channels activated secretion, though not transcription. Nicotinic-stimulated transcription depended upon protein kinase C activation; nicotine caused translocation of protein kinase C to the cell membrane fraction, and inhibition of protein kinase C blocked activation of transcription, while activation of protein kinase C mimicked nicotine effects. Transcriptional responses to both nicotine and protein kinase C mapped principally onto the chromogranin A promoter's cAMP response element (TGACGTAA; CRE box). KCREB, a dominant negative mutant of the CRE-binding protein CREB, blunted activation of chromogranin A transcription by nicotine, phorbol ester, or membrane depolarization. We conclude that activation of chromogranin A transcription by secretory stimulation in chromaffin cells is highly dependent upon precise route of calcium entry into the cytosol; transcription occurred after entry of calcium through -type channels on the cell surface, and was mediated by protein kinase C activation. The trans-acting factor CREB ultimately relays the secretory signal to the chromogranin A promoter's CRE box in cis.

A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type specificity of the promoter. The functional activity of the CRE site was confirmed through studies of the endogenous chromogranin A gene. Northern mRNA analysis showed that expression of the endogenous chromogranin A gene was stimulated seven- to eightfold by cAMP in PC12 cells, whereas no induction occurred in the NIH3T3 cells. Similar cAMP induction was obtained with the transfected chromogranin A promoter in PC12 cells, and abolition of the CRE site (by deletion or point mutation) eliminated the induction. Thus, the CRE site in the chromogranin A proximal promoter is functional and plays a crucial, indeed indispensable, role in neuroendocrine-specific expression of the gene. These results also provide insight into transcriptional mechanisms governing acquisition of the neuroendocrine secretory phenotype.

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.

Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist.

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.

Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.

Chromogranins A and B and secretogranin II are a family of acidic proteins found in neuroendocrine secretory vesicles; these proteins contain multiple potential cleavage sites for proteolytic processing by the mammalian subtilisin-like serine endoproteases PC1 and PC2 (prohormone convertases 1 and 2), and furin. We explored the role of these endoproteases in chromogranin processing in AtT-20 mouse pituitary corticotropes. Expression of inducible antisense PC1 mRNA virtually abolished PC1 immunoreactivity on immunoblots. Chromogranin A immunoblots revealed chromogranin A processing, from both the NH2 and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells. After antisense PC1 induction, an approximately 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an approximately 50 kD NH2-terminal and an approximately 30 kD COOH-terminal fragment declined in abundance. Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [35S]Methionine/cysteine pulse-chase metabolic labeling in AtT-20 antisense PC1 and antisense furin cells revealed reciprocal changes in secreted chromogranin A COOH-terminal fragments (increased approximately 82 kD and decreased approximately 74 kD forms, as compared with wild-type AtT-20 cells) indicating decreased cleavage, while AtT-20 cells overexpressing PC2 showed increased processing to and secretion of approximately 71 and approximately 27 kD NH2-terminal chromogranin A fragments. Antisense PC1 specifically abolished regulated secretion of both chromogranin A and beta-endorphin in response to the usual secretagogue, corticotropin-releasing hormone. Moreover, immunocytochemistry demonstrated a relative decrease of chromogranin A in processes (where regulated secretory vesicles accumulate) of AtT-20 cells overexpressing either PC1 or PC2. These results demonstrate that chromogranin A is a substrate for the endogenous endoproteases PC1 and furin in vivo, and that such processing influences its trafficking into the regulated secretory pathway; furthermore, lack of change in chromogranin B and secretogranin II cleavage after diminution of PCl suggests that the action of PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.

Plasma chromogranin A: a marker of serotonin release and of emesis associated with cisplatin chemotherapy.

PURPOSE: Emesis is a common side effect of cancer chemotherapy. Serotonin released from gastrointestinal enterochromaffin cells (ECC) may mediate chemotherapy-induced emesis. Since chromogranin A (CgA) is colocalized in ECC storage granules with serotonin, we tested the hypothesis that plasma CgA could mark emesis and serotonin release from ECC. PATIENTS AND METHODS: The relationships between plasma CgA, serotonin release, and the development of vomiting following the first course of cisplatin chemotherapy were evaluated in 60 patients. RESULTS: CgA levels increased in 59 of 60 patients (245% +/- 18% increase above baseline levels, P < .001). The time course of the increase in plasma CgA matched that of emesis and of urinary 5-hydroxyindoleacetic acid (5-HIAA). Significant (P < .001) positive correlations were found between the dose of cisplatin and the increases in plasma CgA, and between the changes in plasma CgA and urinary 5-HIAA after cisplatin (r = .54, n = 39, P < .001). The increase in plasma CgA after cisplatin did not correlate with changes in serum lactic dehydrogenase (LDH) activity, a marker of cell toxicity or lysis. CONCLUSION: Plasma CgA marks emesis and serotonin release induced by cisplatin. Since both CgA and serotonin are costored in ECC granules, we suggest that the source of release of each may be the ECC. Increases in plasma CgA are not explained by drug cytotoxicity. Exocytosis appears as the main mechanism by which cisplatin releases serotonin. This work further supports the role of serotonin as a mediator of emesis associated with cisplatin and suggests that plasma CgA level is a valuable tool in studies of chemotherapy-induced emesis.

Contributions of the sympathetic nervous system, glutathione, body mass and gender to blood pressure increase with normal aging: influence of heredity.

Body mass and sympathetic activity increase with aging and might underlie blood pressure (BP) elevation. Increased body mass index (BMI) may elevate BP by increasing sympathetic activity. Glutathione (GSH) can decrease BP, and declines with aging. We measured systolic (SBP) and diastolic BP, BMI, plasma (NE(pl)) and urine norepinephrine (NEu), and plasma GSH in n=204 twins across the age spectrum. BP correlated directly with BMI, NEpl, and NEu, but inversely with GSH. Age correlated with BP, BMI, NEpl, and NEu. BP, BMI, NEpl, and NEu were higher in older subjects than younger subjects, whereas GSH was lower with aging. In older subjects with high (above median) NEpl, SBP was 8 mmHg higher than in those of comparable age with low NE. In younger subjects with high GSH, BP was significantly lower than in younger subjects having low GSH. NEu was significantly reduced in young high-BMI subjects vs young low-BMI subjects. The heritability (h2) of NEpl, NEu, and GSH ranged from approximately 50 to approximately 70%, and these biochemical quantities were considerably more heritable than BP. We conclude that increases in sympathetic activity contribute to aging-induced SBP elevations, especially in older females. GSH reductions apparently participate in aging-induced BP elevations, most strongly in males. BMI increases contribute to BP elevations, particularly in younger subjects. BMI elevations apparently raise BP mainly by peripheral mechanisms, with generally little sympathetic activation. Substantial h(2) for plasma GSH, NE, and urine NE suggests that such traits may be useful 'intermediate phenotypes' in the search for genetic determinants of BP.

Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A(344-364)): identification of amino acid residues crucial for activity.

The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A(344-364). Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A(344-364)) is sufficient to exert the peptide's typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.