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Daniel T Oconnor - One of the best experts on this subject based on the ideXlab platform.
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novel peptide isomer strategy for stable inhibition of Catecholamine Release application to hypertension
Hypertension, 2012Co-Authors: Nilima Biswas, Jiaur R. Gayen, Manjula Mahata, Sushil K Mahata, Daniel T OconnorAbstract:Although hypertension remains the most potent and widespread cardiovascular risk factor, its pharmacological treatment has achieved only limited success. The chromogranin A–derived fragment catestatin inhibits Catecholamine Release by acting as an endogenous nicotinic cholinergic antagonist and can rescue hypertension in the setting of chromogranin A–targeted ablation. Here, we undertook novel peptide chemistry to synthesize isomers of catestatin: normal/wild-type as well as a retro-inverso (R-I) version, with not only inversion of chirality (L→D amino acids) but also reversal of sequence (carboxyl→amino). The R-I peptide was entirely resistant to proteolytic digestion and displayed enhanced potency as well as preserved specificity of action toward nicotinic cholinergic events: Catecholamine secretion, agonist desensitization, secretory protein transcription, and cationic signal transduction. Structural modeling suggested similar side-chain orientations of the wild-type and R-I isomers, whereas circular dichroism spectroscopy documented inversion of chirality. In vivo, the R-I peptide rescued hypertension in 2 mouse models of the human trait: monogenic chromogranin A–targeted ablation, with prolonged efficacy of the R-I version and a polygenic model, with magnified efficacy of the R-I version. These results may have general implications for generation of metabolically stable mimics of biologically active peptides for cardiovascular pathways. The findings also point the way toward a potential new class of drug therapeutics for an important risk trait and, more generally, open the door to broader applications of the R-I strategy in other pathways involved in cardiovascular biology, with the potential for synthesis of diagnostic and therapeutic probes for both physiology and disease.
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heritability and genome wide linkage in us and australian twins identify novel genomic regions controlling chromogranin a implications for secretion and blood pressure
Circulation, 2008Co-Authors: Daniel T Oconnor, Fangwen Rao, Madhusudan Das, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Gu Zhu, Maple M Fung, Lei Wang, Kuixing ZhangAbstract:Background— Chromogranin A (CHGA) triggers Catecholamine secretory granule biogenesis, and its catestatin fragment inhibits Catecholamine Release. We approached catestatin heritability using twin p...
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proteolytic cleavage of chromogranin a cga by plasmin selective liberation of a specific bioactive cga fragment that regulates Catecholamine Release
Journal of Biological Chemistry, 2001Co-Authors: Qijiao Jiang, Daniel T Oconnor, Lindsey A. Miles, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Robert J. ParmerAbstract: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.
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interaction of the Catecholamine Release inhibitory peptide catestatin human chromogranin a352 372 with the chromaffin cell surface and torpedo electroplax implications for nicotinic cholinergic antagonism
Regulatory Peptides, 2000Co-Authors: Laurent Taupenot, Robert J. Parmer, Manjula Mahata, Sushil K Mahata, Daniel T OconnorAbstract:Abstract The Catecholamine Release-inhibitory chromogranin A fragment catestatin (chromogranin A 344-364 ) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin’s likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [ 125 I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity ( K D =15.2±1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [ 125 I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [ 3 H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin’s non-competitive nicotinic mechanism. [ 125 I]-catestatin could be displaced from chromaffin cells by substance P (IC 50 ∼5 μM), though at far lower potency than displacement by catestatin itself (IC 50 ∼350–380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [ 125 I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [ 125 I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [ 125 I]-catestatin to Torpedo membranes suggested specific contacts of [ 125 I]-catestatin with the δ, γ, and β subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.
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primary structure and function of the Catecholamine Release inhibitory peptide catestatin chromogranin a344 364 identification of amino acid residues crucial for activity
Molecular Endocrinology, 2000Co-Authors: Arun R. Wakade, Daniel T OconnorAbstract:The novel chromogranin A fragment catestatin (bovine chromogranin A344−364; RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of Catecholamine Release (IC50, ∼0.2–0.3μ m) 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 A344−358. 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 th...
Robert J. Parmer - One of the best experts on this subject based on the ideXlab platform.
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the novel plasminogen receptor plasminogen receptorkt plg rkt regulates Catecholamine Release
Journal of Biological Chemistry, 2011Co-Authors: Hongdong Bai, Robert J. Parmer, Lindsey A. Miles, Nagyung Baik, William B Kiosses, Stan KrajewskiAbstract:Neurotransmitter Release by Catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-RKT, a novel plasminogen receptor, and its role in Catecholaminergic cell plasminogen activation and regulation of Catecholamine Release. Prominent staining with anti-Plg-RKT mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-RKT was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-RKT fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-RKT were consistent with Plg-RKT being an integral plasma membrane protein on the surface of Catecholaminergic cells. Cells stably overexpressing Plg-RKT exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [3H]norepinephrine Release from cells overexpressing Plg-RKT was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [3H]norepinephrine Release from non-transfected PC12 cells. In summary, Plg-RKT is present on the surface of Catecholaminergic cells and functions to stimulate plasminogen activation and modulate Catecholamine Release. Plg-RKT thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.
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proteolytic cleavage of chromogranin a cga by plasmin selective liberation of a specific bioactive cga fragment that regulates Catecholamine Release
Journal of Biological Chemistry, 2001Co-Authors: Qijiao Jiang, Daniel T Oconnor, Lindsey A. Miles, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Robert J. ParmerAbstract: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.
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interaction of the Catecholamine Release inhibitory peptide catestatin human chromogranin a352 372 with the chromaffin cell surface and torpedo electroplax implications for nicotinic cholinergic antagonism
Regulatory Peptides, 2000Co-Authors: Laurent Taupenot, Robert J. Parmer, Manjula Mahata, Sushil K Mahata, Daniel T OconnorAbstract:Abstract The Catecholamine Release-inhibitory chromogranin A fragment catestatin (chromogranin A 344-364 ) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin’s likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [ 125 I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity ( K D =15.2±1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [ 125 I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [ 3 H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin’s non-competitive nicotinic mechanism. [ 125 I]-catestatin could be displaced from chromaffin cells by substance P (IC 50 ∼5 μM), though at far lower potency than displacement by catestatin itself (IC 50 ∼350–380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [ 125 I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [ 125 I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [ 125 I]-catestatin to Torpedo membranes suggested specific contacts of [ 125 I]-catestatin with the δ, γ, and β subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.
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Formation of the Catecholamine Release-inhibitory Peptide Catestatin from Chromogranin A: DETERMINATION OF PROTEOLYTIC CLEAVAGE SITES IN HORMONE STORAGE GRANULES *
The Journal of biological chemistry, 2000Co-Authors: Carolyn V. Livsey Taylor, L Taupenot, Thomas Toneff, Sukkid Yasothornsrikul, Carlo Caporale, Qijiao Jiang, Robert J. ParmerAbstract:The catestatin fragment of chromogranin A is an inhibitor of Catecholamine Release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited Catecholamine Release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin Release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is Released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.
Sushil K Mahata - One of the best experts on this subject based on the ideXlab platform.
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novel peptide isomer strategy for stable inhibition of Catecholamine Release application to hypertension
Hypertension, 2012Co-Authors: Nilima Biswas, Jiaur R. Gayen, Manjula Mahata, Sushil K Mahata, Daniel T OconnorAbstract:Although hypertension remains the most potent and widespread cardiovascular risk factor, its pharmacological treatment has achieved only limited success. The chromogranin A–derived fragment catestatin inhibits Catecholamine Release by acting as an endogenous nicotinic cholinergic antagonist and can rescue hypertension in the setting of chromogranin A–targeted ablation. Here, we undertook novel peptide chemistry to synthesize isomers of catestatin: normal/wild-type as well as a retro-inverso (R-I) version, with not only inversion of chirality (L→D amino acids) but also reversal of sequence (carboxyl→amino). The R-I peptide was entirely resistant to proteolytic digestion and displayed enhanced potency as well as preserved specificity of action toward nicotinic cholinergic events: Catecholamine secretion, agonist desensitization, secretory protein transcription, and cationic signal transduction. Structural modeling suggested similar side-chain orientations of the wild-type and R-I isomers, whereas circular dichroism spectroscopy documented inversion of chirality. In vivo, the R-I peptide rescued hypertension in 2 mouse models of the human trait: monogenic chromogranin A–targeted ablation, with prolonged efficacy of the R-I version and a polygenic model, with magnified efficacy of the R-I version. These results may have general implications for generation of metabolically stable mimics of biologically active peptides for cardiovascular pathways. The findings also point the way toward a potential new class of drug therapeutics for an important risk trait and, more generally, open the door to broader applications of the R-I strategy in other pathways involved in cardiovascular biology, with the potential for synthesis of diagnostic and therapeutic probes for both physiology and disease.
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the Catecholamine Release inhibitory peptide catestatin chromogranin a344 364 modulates myocardial function in fish
The Journal of Experimental Biology, 2010Co-Authors: Sandra Imbrogno, Sushil K Mahata, Filippo Garofalo, Maria Carmela Cerra, Bruno TotaAbstract:SUMMARY Catestatin (CST), the 21-amino acid, cationic and hydrophobic peptide proteolytically derived from the ubiquitous chromogranin A (CgA), is an endogenous inhibitor of Catecholamine Release, a potent vasodilator in vivo and an anti-hypertensive agent in mammals, including humans. Recently, we discovered that CST also functions as an important negative modulator of heart performance in frog and rat. To gain an evolutionary perspective on CST cardiotropism in fish, we analysed the influence of bovine CST (CgA 344-364 ) on the eel heart, as well as the eventual species-specific mechanisms of its myocardial action. Experiments were carried out on fresh-water eels ( Anguilla anguilla L.) using an electrically paced isolated working heart preparation. Stroke volume and stroke work were used as measures of ventricular performance. Under basal conditions, CST (from 11 nmol l –1 to 165 nmol l –1 ) caused a concentration-dependent negative inotropism, which was abolished by inhibitors of either β 1 /β 2 (propranolol) or β 3 (SR 59230 ) adrenergic receptors, or by G i/o protein (PTx) or nitric oxide synthase (L-NMMA), or guanylate cyclase (ODQ) blockers. This suggests a β-adrenergic receptor-G i/o protein-NO-cGMP-dependent mechanism. By contrast, the CST-induced cardio-suppression was not influenced by atropine, unspecific muscarinic antagonist, thus excluding cholinergic receptor involvement. CST also counteracted the adrenergic (isoproterenol)-mediated positive inotropism. Under increased preload (i.e. Frank–Starling response) conditions, CST induced a significant increase of the Frank–Starling response, which was blocked by L-NMMA and thapsigargin, but independent from guanylate cyclase. In conclusion, this is the first report in fish that CST modulates myocardial performance under basal, as well as under increased preload, conditions and counteracts the adrenergic-mediated positive inotropism, which strikingly supports the evolutionary significance and establishes the cardioactive role of this peptide.
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heritability and genome wide linkage in us and australian twins identify novel genomic regions controlling chromogranin a implications for secretion and blood pressure
Circulation, 2008Co-Authors: Daniel T Oconnor, Fangwen Rao, Madhusudan Das, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Gu Zhu, Maple M Fung, Lei Wang, Kuixing ZhangAbstract:Background— Chromogranin A (CHGA) triggers Catecholamine secretory granule biogenesis, and its catestatin fragment inhibits Catecholamine Release. We approached catestatin heritability using twin p...
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proteolytic cleavage of chromogranin a cga by plasmin selective liberation of a specific bioactive cga fragment that regulates Catecholamine Release
Journal of Biological Chemistry, 2001Co-Authors: Qijiao Jiang, Daniel T Oconnor, Lindsey A. Miles, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Robert J. ParmerAbstract: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.
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interaction of the Catecholamine Release inhibitory peptide catestatin human chromogranin a352 372 with the chromaffin cell surface and torpedo electroplax implications for nicotinic cholinergic antagonism
Regulatory Peptides, 2000Co-Authors: Laurent Taupenot, Robert J. Parmer, Manjula Mahata, Sushil K Mahata, Daniel T OconnorAbstract:Abstract The Catecholamine Release-inhibitory chromogranin A fragment catestatin (chromogranin A 344-364 ) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin’s likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [ 125 I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity ( K D =15.2±1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [ 125 I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [ 3 H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin’s non-competitive nicotinic mechanism. [ 125 I]-catestatin could be displaced from chromaffin cells by substance P (IC 50 ∼5 μM), though at far lower potency than displacement by catestatin itself (IC 50 ∼350–380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [ 125 I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [ 125 I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [ 125 I]-catestatin to Torpedo membranes suggested specific contacts of [ 125 I]-catestatin with the δ, γ, and β subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.
Antonio G Garcia - One of the best experts on this subject based on the ideXlab platform.
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altered mitochondrial function calcium signaling and Catecholamine Release in chromaffin cells of diabetic and shr rats
European Journal of Pharmacology, 2017Co-Authors: Diego Castro Musial, Aron Jurkiewicz, Antonio G Garcia, Guilherme Henrique Souza Bomfim, Iago Mendezlopez, Juan Fernando Padin, Neide H Jurkiewicz, Regiane Mirandaferreira, Juan Alberto ArranztagarroAbstract:Comorbidity of diabetes and hypertension is frequent. Here, we have performed a comparative study in three animal models namely, normotensive Wistar Kyoto (WKY) rats, streptozotocin-induced diabetic rats (STZ), and spontaneously hypertensive rats (SHR). With respect WKY rats, we have found the following alterations in adrenal chromaffin cells from STZ and SHR rats: (1) diminished Ca2+ currents; (2) augmented [Ca2+]c elevations and Catecholamine Release in cells stimulated with angiotensin II or high K+; (3) unchanged expression of angiotensin II receptors AT1 and AT2; (4) higher density of secretory vesicles at subplasmalemmal sites; (5) mitochondria with lower cristae density that were partially depolarized; and (6) lower whole cell ATP content. These alterations may have their origin in (i) an augmented capacity of the endoplasmic reticulum [Ca2+] store likely due to (ii) impaired mitochondrial Ca2+ uptake; (iii) augmented high-[Ca2+]c microdomains at subplasmalemmal sites secondary to augmented calcium-induce calcium Release and to inositol tris-phosphate receptor mediated enhanced Ca2+ mobilization from the endoplasmic reticulum; and (iv) augmented vesicle pool. These alterations seem to be common to the two models of human hypertension here explored, STZ diabetic rats and SHR hypertensive rats.
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electrophysiological properties and augmented Catecholamine Release from chromaffin cells of wky and shr rats contributing to the hypertension development elicited by chronic etoh consumption
European Journal of Pharmacology, 2017Co-Authors: Guilherme Henrique Souza Bomfim, Aron Jurkiewicz, Iago Mendezlopez, Josecarlos Fernandezmorales, Juan Fernando Padin, Neide H Jurkiewicz, Antonio G GarciaAbstract:It is known that chronic ethanol (EtOH) consumption leads to hypertension development and has been associated with deleterious effects on the cardiovascular system. Whether this condition alters calcium (Ca2+) signaling and exocytosis in adrenal chromaffin cells (CCs) as the case is for genetic hypertension, is unknown. We explored this question in four randomized experimental groups, male Wistar Kyoto (WKY/EtOH) and Spontaneously Hypertensive (SHR/EtOH) rats were subjected to the intake of increasing EtOH concentrations (5-20%, for 30 days) and their respective controls (WKY/Control and SHR/Control) received water. WKY/EtOH developed hypertension and cardiac hypertrophy; blood aldehyde dehydrogenase (ALDH) and H2O2 were also augmented. In comparison with WKY/Control, CCs from WKY/EtOH had the following features: (i) depolarization and higher frequency of spontaneous action potentials; (ii) decreased Ca2+ currents with slower inactivation; (iii) decreased K+ currents; (iv) augmented K+-elicited cytosolic Ca2+ transients ([Ca2+]c); (v) enhanced K+-elicited Catecholamine Release. These cardiovascular, blood and CCs changes were qualitatively similar to those undergone by SHR/Control and SHR/EtOH. The results suggest that the hypertension elicited by chronic EtOH has pathogenic features common to genetic hypertension namely, augmented [Ca2+]c transients and Catecholamine Release from their CCs.
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the quantal Catecholamine Release from mouse chromaffin cells challenged with repeated ach pulses is regulated by the mitochondrial na ca2 exchanger
The Journal of Physiology, 2017Co-Authors: Angela Lopezgil, Luis Gandia, Mayte Montero, Iago Mendezlopez, Carmen Nanclares, Carmen Martinezramirez, Cristobal De Los Rios, Fernando J Padinnogueira, Antonio G GarciaAbstract:KEY POINTS Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. A subsequent K+ pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready Release-vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh-sensitive vesicle pool is refilled and maintained by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mitochondrial Na+ /Ca2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained Catecholamine Release is regulated by mitochondrial Ca2+ circulation through the mNCX, which couples respiration and ATP synthesis to long-term stimulation of chromaffin cells by endogenously Released ACh. ABSTRACT Using caged-Ca2+ photoRelease or paired depolarising pulses in voltage-clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve-CC synapse, is unknown. We have explored here whether an ACh-sensitive ready-Release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 μm, 2 s, every 30 s) plus a K+ pulse given at the end (75 mm K+ ). After the first 2-3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. The last K+ pulse, however, overcame such decay. Repeated ACh pulses to voltage-clamped cells elicited non-desensitising nicotinic currents. Also, the [Ca2+ ]c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+ /Ca2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mNCX.
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role of the endoplasmic reticulum and mitochondria on quantal Catecholamine Release from chromaffin cells of control and hypertensive rats
Journal of Pharmacology and Experimental Therapeutics, 2009Co-Authors: Regiane Mirandaferreira, Aron Jurkiewicz, Luis Gandia, Ricardo De Pascual, Afonso Caricatineto, Antonio G GarciaAbstract:Here, we present the first study on the effects of compounds that interfere with calcium (Ca2+) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal Catecholamine Release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K+ pulses triggered spike bursts of secretion by Ca2+ entry through Ca2+ channels. ER Ca2+ Release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K+, CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca2+ handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K+ were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca2+ entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.
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single vesicle Catecholamine Release has greater quantal content and faster kinetics in chromaffin cells from hypertensive as compared with normotensive rats
Journal of Pharmacology and Experimental Therapeutics, 2008Co-Authors: Regiane Mirandaferreira, Aron Jurkiewicz, Luis Gandia, Ricardo De Pascual, Antonio M G De Diego, Afonso Caricatineto, Antonio G GarciaAbstract:In a previous study performed in the intact adrenal gland (Lim et al., 2002), stimulation with acetylcholine (ACh) or high K + concentrations (K + ) produced greater Catecholamine Release in spontaneously hypertensive rats (SHR), as compared with normotensive animals. In this study, the time course of secretion was in the range of minutes. Hence, we do not know whether enhanced Release is due to greater quantal content and/or distinct kinetics in SHRs and control animals. To get insight into the mechanism involved in such enhanced Catecholamine secretory responses, we performed a single-vesicle Release study in primary cultures of adrenal chromaffin cells, recorded with amperometry. Cells were stimulated with 2-s pulses of 1 mM ACh or 70 mM K + . The secretory responses to ACh or K + pulses in SHR cells as compared with control cells had the following characteristics: 1) double number of secretory events, 2) 4-fold augmentation of total secretion, 3) cumulative secretion that saturated slowly, 4) 3-fold higher complex events with two to four superimposed spikes that may be explained by faster spike kinetics, 5) about 2- to 3-fold higher event frequency at earlier post stimulation periods, and 6) 2- to 5-fold higher quantal content of simple spikes. We conclude that SHR cells have faster and larger Catecholamine Release responses, explained by more vesicles ready to undergo exocytosis and greater quantal content of vesicles. This could have relevance to further understand the pathogenic mechanisms involved in the development of high blood pressure, as well as in the identification of new drug targets to treat hypertension.
Fangwen Rao - One of the best experts on this subject based on the ideXlab platform.
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heritability and genome wide linkage in us and australian twins identify novel genomic regions controlling chromogranin a implications for secretion and blood pressure
Circulation, 2008Co-Authors: Daniel T Oconnor, Fangwen Rao, Madhusudan Das, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Gu Zhu, Maple M Fung, Lei Wang, Kuixing ZhangAbstract:Background— Chromogranin A (CHGA) triggers Catecholamine secretory granule biogenesis, and its catestatin fragment inhibits Catecholamine Release. We approached catestatin heritability using twin p...
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Catecholamine Release inhibitory peptide catestatin chromogranin a352 372 naturally occurring amino acid variant gly364ser causes profound changes in human autonomic activity and alters risk for hypertension
Circulation, 2007Co-Authors: Fangwen Rao, Sucheta M. Vaingankar, Rany M. Salem, Brian P. Kennedy, Jiaur R. Gayen, Madhusudan Das, Brinda K. Rana, Gen Wen, Mats StridsbergAbstract:BACKGROUND - Chromogranin A, coReleased with Catecholamines by exocytosis, is cleaved to the Catecholamine Release-inhibitory fragment catestatin. We identified a natural nonsynonymous variant of catestatin, Gly364Ser, that alters human autonomic function and blood pressure. METHODS AND RESULTS - Gly364Ser heterozygotes and controls underwent physiological and biochemical phenotyping, including Catecholamine production, chromogranin A precursor, and its catestatin product. Case-control studies replicated effects of the gene on blood pressure in the population. Gly364Ser displayed diminished inhibition of Catecholamine secretion from cultured neurons. Gly/Ser heterozygotes displayed increased baroreceptor slope during upward deflections (by ≈47%) and downward deflections (by ≈44%), increased cardiac parasympathetic index (by ≈2.4-fold), and decreased cardiac sympathetic index (by ≈26%). Renal norepinephrine excretion was diminished by ≈26% and epinephrine excretion by ≈34% in Gly/Ser heterozygotes. The coalescent dated emergence of the variant to ≈70 000 years ago. Gly364Ser was in linkage disequilibrium with 1 major Chromogranin A promoter haplotype, although promoter haplotypes did not predict autonomic phenotypes. The 364Ser variant was associated with lower diastolic blood pressure in 2 independent/confirmatory groups of patients with hypertension; genotype groups differed by ≈5 to 6 mm Hg, and the polymorphism accounted for ≈1.8% of population diastolic blood pressure variance, although a significant gene-by-sex interaction existed, with an enhanced effect in men. CONCLUSIONS - The catestatin Gly364Ser variant causes profound changes in human autonomic activity, both parasympathetic and sympathetic, and seems to reduce risk of developing hypertension, especially in men. A model for catestatin action in the baroreceptor center of the nucleus of the tractus solitarius accounts for these actions.
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the Catecholamine Release inhibitory peptide catestatin chromogranin a chga 352 372 a naturally occurring amino acid variant gly364ser causes profound alterations in human autonomic activity
Clinical Pharmacology & Therapeutics, 2005Co-Authors: Fangwen RaoAbstract:Background Chromogranin A (CHGA), a protein co-stored and co-Released with Catecholamines by exocytosis, is cleaved to a Catecholamine Release-inhibitory fragment “catestatin” that acts as a nicotinic cholinergic antagonist. During systematic variant discovery, we identified a natural variant in catestatin: Gly364Ser. Methods N=17 Gly364Ser heterozygotes and controls were subjected to autonomic phenotyping, including measurements of Catecholamines, the CHGA precursor, and catestatin. Results Catestatin Gly364Ser displayed ~4.5-fold diminished inhibitory activity towards Catecholamine secretion. In autonomic monitoring, Gly/Ser heterozygotes displayed increased baroreceptor slope, increased cardiac parasympathetic index (by ~2.4-fold, p=0.032), and decreased cardiac sympathetic index (by ~26%, p=0.044). In the sympathetic system, renal norepinephrine excretion was diminished. Gly364Ser was in linkage disequilibrium with one major CHGA promoter haplotype, although a SNP used to “tag” the promoter haplotypes did not predict the autonomic phenotypes. The 364Ser variant did not occur in n=68 subjects with hypertension (χ2=5.45, p=0.0195). Conclusions The catestatin Gly364Ser functional variant causes profound changes in human autonomic nervous system activity, both parasympathetic and sympathetic. Clinical Pharmacology & Therapeutics (2005) 77, P6–P6; doi: 10.1016/j.clpt.2004.11.026