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1.
Cell Tissue Res ; 368(3): 487-501, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28220294

RESUMO

Chromogranin A (CgA) is a prohormone and a granulogenic factor that regulates secretory pathways in neuroendocrine tissues. In ß-cells of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. The impact of CgA deficiency on the formation and exocytosis of insulin vesicles is yet to be investigated. In addition, no literature exists on the impact of CgA on mitochondrial function in ß-cells. Using three different antibodies, we demonstrate that CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. CgA deficiency in Chga-KO islets leads to compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins and insulin genes, as well as increased insulin protein content. Ultrastructural studies of pancreatic islets revealed that Chga-KO ß-cells contain fewer immature secretory granules than wild-type (WT) control but increased numbers of mature secretory granules and plasma membrane-docked vesicles. Compared to WT control, CgA-deficient ß-cells exhibited increases in mitochondrial volume, numerical densities and fusion, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1 and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion observed in Chga-KO mice. We conclude that CgA is an important regulator for coordination of mitochondrial dynamics, secretory vesicular quanta and GSIS for optimal secretory functioning of ß-cells, suggesting a strong, CgA-dependent positive link between mitochondrial fusion and GSIS.


Assuntos
Cromogranina A/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Dinâmica Mitocondrial , Animais , Calreticulina/metabolismo , Diferenciação Celular , Cromogranina A/deficiência , Cromogranina A/metabolismo , Exocitose , Regulação da Expressão Gênica , Glucose/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial/genética , Fragmentos de Peptídeos/metabolismo , Vesículas Secretórias
2.
Cell Tissue Res ; 363(3): 693-712, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26572539

RESUMO

Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell.


Assuntos
Catecolaminas/metabolismo , Grânulos Cromafim/metabolismo , Cromogranina A/deficiência , Metabolismo Energético , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Animais , Western Blotting , Grânulos Cromafim/efeitos dos fármacos , Grânulos Cromafim/ultraestrutura , Cromogranina A/metabolismo , Dopamina/metabolismo , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Epinefrina/metabolismo , Exocitose/efeitos dos fármacos , Glucose/metabolismo , Glicogênio/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Insulina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Norepinefrina/metabolismo , Nervos Esplâncnicos/efeitos dos fármacos , Nervos Esplâncnicos/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo
3.
J Cardiovasc Transl Res ; 7(5): 483-493, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24821335

RESUMO

The prohormone chromogranin A (CHGA) is ubiquitously found in vesicles of adrenal chromaffin cells and adrenergic neurons, and it is processed to the hypotensive hormone peptide catestatin (CST). Both CHGA and CST regulate blood pressure and cardiac function. This study addresses their role in cardiac electrical activity. We have generated two genomically "humanized" transgenic mouse strains (Tg31CHGA+/+; Chga-/- (HumCHGA31) and Tg19CHGA+/+; Chga-/- (HumCHGA19)) with varied CHGA expression and the ability to rescue the Chga-/- phenotype (hypertensive, hyperadrenergic with dilated cardiomyopathy). The normotensive HumCHGA31 mice express CHGA at levels comparable to wild-type. In contrast, the hypertensive HumCHGA19 mice have low levels of CHGA. EKG recordings revealed that the QT interval, R-amplitude, and QRS time-voltage integral are markedly longer in HumCHGA19 compared to wild-type and HumCHGA31 mice. These differences are accompanied by increased heart rate and QT variability, indicating that ventricular assault happens in a status of low levels of circulating CST.


Assuntos
Arritmias Cardíacas/metabolismo , Cromogranina A/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Hipertensão/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Pressão Sanguínea , Cromogranina A/sangue , Cromogranina A/deficiência , Cromogranina A/genética , Modelos Animais de Doenças , Eletrocardiografia , Genótipo , Frequência Cardíaca , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fragmentos de Peptídeos/sangue , Fenótipo , Fatores de Tempo
4.
Eur Heart J ; 33(18): 2297-306, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22645192

RESUMO

AIMS: We here investigated the endothelial effects of the chromogranin A-derived peptide vasostatin-2 and its relation to coronary artery disease (CAD). METHODS AND RESULTS: We assessed the impact of recombinant vasostatin-1 and vasostatin-2 on tumour necrosis factor-alpha (TNFα)-, angiotensin II-, and oxidized low-density lipoprotein (oxLDL)-induced expression of adhesion molecules in human arterial endothelial cells. Vasostatin-1 and vasostatin-2 levels were examined in coronary endarterectomy specimens (n= 23), atherosclerotic aortas (n= 16), non-significant-atherosclerotic internal mammary arteries (n= 30), and non-atherosclerotic aortas (n= 10), as well as in peripheral blood mononuclear cells (PBMCs) from severe CAD patients (n= 50) and healthy volunteers (n= 21). Serum levels of vasostatin-2 were analysed in 968 consecutive patients undergoing coronary angiography. Vasostatin-1 and vasostatin-2 concentration-dependently inhibited TNFα-, angiotensin II-, and oxLDL-induced expression of adhesion molecules; and attenuated TNFα-induced adhesion of U937 monocytes to endothelial cells. Vasostatin-2 levels were significantly decreased in endarterectomy samples and atherosclerotic aortas compared with non-atherosclerotic internal mammary arteries and aortas, as well as in PBMCs of severe CAD patients compared with healthy controls (all P< 0.05). Serum vasostatin-2 levels were significantly lower in CAD patients (diameter stenosis ≥ 50%, n= 554) than in controls (normal arteries or diameter stenosis <30%, n= 281) (P< 0.001). Its concentrations correlated with the number of diseased coronary arteries and Syntax score in CAD patients (all P< 0.05). At multivariable regression analysis, decreased vasostatin-2 levels remained associated with CAD when other variables were taken into account. CONCLUSION: Vasostatin-2 has anti-inflammatory properties and is decreased in atherosclerotic plaque specimens and in PBMC of CAD patients. Decreased serum vasostatin-2 levels are associated with the presence and severity of CAD.


Assuntos
Cromogranina A/deficiência , Doença da Artéria Coronariana/etiologia , Fragmentos de Peptídeos/deficiência , Análise de Variância , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Cromogranina A/farmacologia , Doença da Artéria Coronariana/sangue , Células Endoteliais/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/farmacologia , Prognóstico , Proteínas Recombinantes/farmacologia , Análise de Regressão , Fator de Necrose Tumoral alfa/farmacologia
6.
Cell Host Microbe ; 7(4): 277-289, 2010 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-20413096

RESUMO

Stress mobilizes elements from the neuroendocrine system to modulate immune responses. Cholinergic stimulation via nicotinic receptor (nAchR) is a major neuroendocrine signaling axis associated with the stress response whose specific effects on the immune system are unknown. Here, we show that nAchR activation by topical agonist application or deletion of the nAChR antagonist catestatin (Chga(-/-)) reduced antimicrobial peptide (AMP) activity in skin extracts and increased susceptibility to methicillin-resistant Staphylococcus aureus and Group A Streptococcus infections. The adverse effects on AMP expression and infection were rescued by topical application of a nAChR antagonist. Stress-induced nAChR activation increased infection in wild-type, but not Chga(-/-) or cathelicidin-deficient, mice. These data identify a mechanism for the negative regulation of host-innate AMP response to infection through cholinergic activation and indicate nAChR-mediated cathelicidin dysregulation as a potential mechanism for increased susceptibility to infection following prolonged stress or nicotine use.


Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Regulação para Baixo , Agonistas Nicotínicos/imunologia , Antagonistas Nicotínicos/imunologia , Receptores Nicotínicos/efeitos dos fármacos , Dermatopatias Bacterianas/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Cromogranina A/deficiência , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Streptococcus/crescimento & desenvolvimento , Streptococcus/imunologia , Catelicidinas
7.
Physiol Genomics ; 41(1): 91-101, 2010 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-20009010

RESUMO

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.


Assuntos
Cromogranina A/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos Humanos Par 14/genética , Glândulas Suprarrenais/patologia , Animais , Sequência de Bases , Pressão Sanguínea , Catecolaminas/metabolismo , Cromogranina A/deficiência , Cromogranina A/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mutagênese Insercional/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Restrição Física , Estresse Fisiológico , Transgenes/genética
8.
Endocrinology ; 150(11): 5027-35, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19819970

RESUMO

We reported previously that chromogranin A (Chga) knockout (KO) mice are hypertensive and hyperadrenergic. Here we sought to determine the basis of such alterations by probing physiological, biochemical, and pharmacological responses to perturbations of the autonomic nervous system. In the conscious state, KO mice had substantially elevated basal high blood pressure (BP) and heart rate (HR); immobilization stress caused increments in systolic BP and HR in both wild-type (WT) and KO mice, with higher maxima but blunted increments in the KO state. Catestatin (CST; CHGA(352-372)) selectively diminished stress-induced increments in BP and HR in KO mice, implicating CST as an antihypertensive peptide, even in stressful conditions. Heightened plasma catecholamines in KO mice returned to WT level after CST. Stress caused further increments in catecholamines in WT mice but no change in KO mice. KO mice displayed diminished baroreflex sensitivity in response to either phenylephrine or sodium nitroprusside, accounting for exaggerated pressor and depressor responses to these compounds; baroreceptor function was normalized by CST. To probe the relative roles of endogenous/basal sympathetic vs. parasympathetic tone in control of BP and HR, we used the muscarinic-cholinergic antagonist atropine or the beta-adrenergic antagonist propranolol; HR and BP responses to each antagonist were exaggerated in KO animals. We conclude that ablation of Chga expression results in global disturbances in autonomic function, both sympathetic and parasympathetic, that can be abrogated (or rescued), at least in part, by replacement of CST. The results point to mechanisms whereby CHGA and its CST fragment act to control cardiovascular homeostasis.


Assuntos
Sistema Cardiovascular/fisiopatologia , Cromogranina A/deficiência , Cromogranina A/genética , Hipertensão/fisiopatologia , Animais , Anti-Hipertensivos/administração & dosagem , Pressão Sanguínea/efeitos dos fármacos , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Catecolaminas/sangue , Cromogranina A/administração & dosagem , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/sangue , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/administração & dosagem , Propranolol/administração & dosagem
9.
Regul Pept ; 151(1-3): 19-25, 2008 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-18722481

RESUMO

BACKGROUND: Chromogranin (Cg) A is expressed in neuroendocrine and neuronal tissues. It is involved in the generation of secretory granules and is cleaved to form biologically active peptides. Targeted ablation of the Chga gene resulted in increased plasma catecholamines, high blood pressure, and decreased size and number of adrenal medullary chromaffin granules. The aim of this study was to determine whether Chga null mice display changes in the morphology and function of the endocrine pancreas. MATERIALS AND METHODS: Sections of pancreata from Chga-/-, Chga+/- and Chga+/+ mice, were immunostained with antibodies against synaptophysin, CgA, CgB, secretogranin II and the four major pancreatic islet hormones. Plasma was analysed for glucose, insulin, glucagon, somatostatin and pancreatic polypeptide (PP). RESULTS: CgA epitopes were undetectable in the islets of Chga-/- animals. CgB and secretogranin II epitopes were expressed in the islets of all animal groups albeit with decreased expression in Chga-/- islets. The islet number and size were decreased in the Chga-/- animals compared with Chga+/+. The proportion of insulin cells was decreased but somatostatin and PP cells were increased in Chga-/- mice compared to Chga+/+ mice. The nuclear size was decreased in insulin cells and increased in somatostatin cells in Chga-/- mice. Plasma insulin level was markedly decreased in the Chga-/- mice although fasting plasma glucose and glucagon were normal. CONCLUSION: Ablation of the Chga gene affected the islet volume, the composition, distribution and nuclear size of islet cell types and plasma insulin concentration. Our data indicate decreased insulin cell function and increased glucagon cell function. Our study shows that CgA exerts a significant influence on the endocrine pancreas with importance in maintaining islet volume, cellular composition and function.


Assuntos
Cromogranina A/fisiologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/fisiologia , Animais , Contagem de Células , Tamanho Celular , Cromogranina A/deficiência , Cromogranina A/genética , Cromogranina B/metabolismo , Imuno-Histoquímica , Insulina/sangue , Ilhotas Pancreáticas/anatomia & histologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônios Pancreáticos/metabolismo , Secretogranina II/metabolismo
10.
J Neurosci ; 28(13): 3350-8, 2008 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-18367602

RESUMO

Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells from Chga null mice [chromogranin A knock-out (CgA-KO)] exhibited approximately 30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation with L-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed that L-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.


Assuntos
Células Cromafins/fisiologia , Cromograninas/fisiologia , Exocitose/fisiologia , Glândulas Suprarrenais/citologia , Animais , Catecolaminas/metabolismo , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Cromogranina A/deficiência , Dopaminérgicos/farmacologia , Eletroquímica/métodos , Exocitose/efeitos dos fármacos , Levodopa/farmacologia , Fusão de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Probabilidade , Tirosina 3-Mono-Oxigenase/metabolismo
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