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Chromaffin Cells (chromaffin + cell)
Kinds of Chromaffin Cells Selected AbstractsUp-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-, in Adrenal Chromaffin CellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2000Involvement of Transcriptional, Translational Events Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12- O -tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-, (PKC-,) and novel PKC-, (but not atypical PKC-,) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-,. In the present study, chronic (,12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4,-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans -Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the ,-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor ,-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor ,-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-, up-regulates the density of the cell surface insulin receptor via transcriptional/translational events. [source] Reduction of Allodynia by Intrathecal Transplantation of Microencapsulated Porcine Chromaffin CellsARTIFICIAL ORGANS, Issue 3 2009Yu Mi Kim Abstract Bovine chromaffin cells (BCCs) are well known to have analgesic effect to reduce acute or chronic pain when transplanted in the subarachnoid space and have been considered as an alternative therapy for pain management. However, due to recent concerns over risks associated with prion transmission, porcine tissue is considered to be an alternate xenogeneic source for clinical use. In the present study, we investigated whether microencapsulated porcine adrenal medullary chromaffin cells (PCCs) also have analgesic effect to reduce allodynia caused by neuropathic pain in chronic constriction injury model of rat. PCCs were isolated from a porcine adrenal medulla and then microencapsulated with alginate and poly. In in vitro tests, the microencapsulated PCCs were investigated whether they have an ability to release catecholamines responding to nicotine stimulation. The levels of catecholamines released from the microencapsulated PCCs were significantly higher than from microencapsulated BCCs. In addition, the microencapsulated PCCs released catecholamines and met-enkephalin responding to cerebral spinal fluid (CSF) retrieved from a neuropathic pain model. In in vivo tests, implantation of microencapsulated PCCs reduced both mechanical and cold allodynia in chronic constriction injury model of a rat whereas the microencapsulated BCCs reduced only cold allodynia under the same conditions. The injection of antagonist of opioid peptides reversed the reduction of cold allodynia in microencapsulated PCC-received animal. The levels of catecholamines in the CSF of rats after implantation of microencapsulated PCCs were significantly higher than in the control group. These data suggest that microencapsulated PCCs may be another effective source for the treatment of neuropathic pain. [source] Immunoisolated Chromaffin Cells Implanted Into the Subarachnoid Space of Rats Reduce Cold Allodynia in a Model of Neuropathic Pain: A Novel Application of Microencapsulation TechnologyARTIFICIAL ORGANS, Issue 12 2004Yu Mi Kim Abstract:, Intrathecal transplants of adrenal medullary chromaffin cells relieve chronic pain by secreting catecholamines, opioids, and other neuroactive substances. Recently, macrocapsules with semipermeable membranes were used to isolate immunologically xenogenic chromaffin cells, but the poor viability in vivo of the encapsulated chromaffin cells limited the usefulness of this method. In this study, we used a novel method of encapsulation to increase the viability of chromaffin cells. We found that microencapsulated chromaffin cells that were implanted into the subarachnoid space of rats relieved cold allodynia in a model of neuropathic pain. Furthermore, microencapsulated chromaffin cells were morphologically normal and retained their functionality. These findings suggest that the intrathecal placement of microencapsulated chromaffin cells might be a useful method for treating chronic pain. [source] Regulation of Exocytosis in Chromaffin Cells by Trans -Insertion of Lysophosphatidylcholine and Arachidonic Acid into the Outer Leaflet of the Cell MembraneCHEMBIOCHEM, Issue 12 2006Christian Amatore Prof. Abstract Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans -insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans -insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA. [source] Dynamics of Full Fusion During Vesicular Exocytotic Events: Release of Adrenaline by Chromaffin CellsCHEMPHYSCHEM, Issue 2 2003Christian Amatore Prof. Abstract Vesicular exocytosis is important in the communication between cells in complex organisms. It controls the release of specific chemical or biochemical messengers stored in the emitting cell, which elicit a response upon detection by the target cells. Secretion of a messenger molecule (a neurotransmitter) was measured electrochemically, which allowed the quantification of cellular events and the validation of current physicochemical models. This model led us to formulate predictions about the occurrence and kinetics of vesicular exocytotic events based on the physicochemical meaning of its key parameters. These predictions were tested successfully through a series of experiments on chromaffin cells, involving changes of osmotic conditions, presence of trivalent ions and cholesterol-induced structuring of the cell plasmic membrane. [source] Oxygen-sensing pathway for SK channels in the ovine adrenal medullaCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2005Damien J Keating SUMMARY 1.,The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2.,The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3.,Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4.,One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5.,The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6.,These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state. [source] Modulation of calcium signalling by intracellular organelles seen with targeted aequorinsACTA PHYSIOLOGICA, Issue 1 2009M. T. Alonso Abstract The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+ -dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+ -signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell. [source] The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastomaINTERNATIONAL JOURNAL OF CANCER, Issue 3 2006Jasmien Hoebeeck Abstract Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p = 0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor ,. © 2006 Wiley-Liss, Inc. [source] Role of annexin A6 isoforms in catecholamine secretion by PC12 cells: Distinct influence on calcium responseJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2010Paulina Podszywalow-Bartnicka Abstract Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca2+] increases in response to cell excitation and triggers a signal-initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca2+ -regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock-down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6-1 and AnxA6-2 overexpression reduced ,[Ca2+]c upon depolarization by 32% and 58%, respectively, while AnxA6 knock-down increased ,[Ca2+]c by 44%. The mechanism of AnxA6 action on Ca2+ signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells. J. Cell. Biochem. 111: 168,178, 2010. © 2010 Wiley-Liss, Inc. [source] Munc18-1 as a key regulator of neurosecretionJOURNAL OF NEUROCHEMISTRY, Issue 1 2010Gayoung A. Han J. Neurochem. (2010) 115, 1,10. Abstract Munc18-1 plays essential roles in neurosecretion by interacting with syntaxin-1 and controlling the formation of the soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex. At least three important functions of Munc18-1 have been proposed: (i) molecular chaperone of syntaxin-1 for appropriate localization and expression of syntaxin-1, (ii) priming/stimulation of the SNARE-mediated membrane fusion, and (iii) docking of large dense-core vesicles to the plasma membrane. Similarly, at least two different binding modes have been proposed for the interaction between Munc18-1 and syntaxin-1: (i) binary binding to a ,closed' conformation of syntaxin-1, and (ii) binding to the N-terminal peptide of syntaxin-1, which is thought to enable an interaction with the quaternary SNARE complex and/or further stabilize the binary interaction between Munc18-1 and closed syntaxin-1. Recent structural analyses have identified critical Munc18-1 residues implicated in these different modes of binding. These have recently been tested functionally in rescue experiments using Munc18-1 null neurons, chromaffin cells and Munc18-1/-2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc18-1. In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic functions of Munc18-1 in neuroexocytosis. [source] Chromogranins as regulators of exocytosisJOURNAL OF NEUROCHEMISTRY, Issue 2 2010Ricardo Borges J. Neurochem. (2010) 114, 335,343. Abstract Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca2+. Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like amperometry, patch-amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca2+ in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells. [source] Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2010Pei-Chun Wu J. Neurochem. (2010) 112, 1210,1222. Abstract In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+]i) increase followed by two phases of [Ca2+]i decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K+ and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca2+ -induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+]i oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+]i increase which is further enhanced by the Ca2+ released from the ER mediated by Ca2+ -induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system. [source] Glycogen synthase kinase-3,: homologous regulation of cell surface insulin receptor level via controlling insulin receptor mRNA stability in adrenal chromaffin cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Hiroki Yokoo Abstract In cultured bovine adrenal chromaffin cells, 48 h-treatment with 20 mmol/L LiCl, 1 mmol/L valproic acid, 30 ,mol/L SB216763, 30 ,mol/L SB415286, or 100 nmol/L insulin, a condition that inhibits constitutive active glycogen synthase kinase-3 (GSK-3), decreased cell surface 125I-insulin binding capacity by ,39%, without altering the Kd value; LiCl, SB216763 or insulin decreased insulin receptor (IR) and IR precursor levels, attenuating insulin-induced Tyr-autophosphorylation of IR. LiCl increased inhibitory Ser9-phosphorylation of GSK-3, at 6 h, decreasing 125I-insulin binding at 24 h. SB216763-induced 125I-insulin binding reduction (IC50 = 3 ,mol/L) was preceded by ,-catenin level increase by SB216763 (EC50 = 11 ,mol/L), a hallmark of GSK-3 inhibition. Insulin-induced rapid (> 1 min) Ser9-phosphorylation of GSK-3, (Nemoto et al. 2006) was followed by ,48% decrease of IR level. LiCl did not stimulate endocytosis, nor proteolysis of IR. LiCl destabilized IR mRNA (t1/2 = 9.3 vs. 6.5 h), decreasing IR mRNA level by ,47%, without altering IR gene transcription. Decreases of 125I-insulin binding and IR level, as well as increased Ser9-phosphorylation of GSK-3, were restored to the control levels by washing the test compound-treated cells. Thus, GSK-3, regulates IR level via controlling IR mRNA stability. [source] Catecholamine exocytosis is diminished in R6/2 Huntington's disease model miceJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Michael A. Johnson Abstract In this work, the mechanisms responsible for dopamine (DA) release impairments observed previously in Huntington's disease model R6/2 mice were evaluated. Voltammetrically measured DA release evoked in striatal brain slices from 12-week old R6/2 mice by a single electrical stimulus pulse was only 19% of wild-type (WT) control mice. Iontophoresis experiments suggest that the concentration of released DA is not diluted by a larger striatal extracellular volume arising from brain atrophy, but, rather, that striatal dopaminergic terminals have a decreased capacity for DA release. This decreased capacity was not due to an altered requirement for extracellular Ca2+, and, as in WT mice, the release in R6/2 mice required functioning vesicular transporters. Catecholamine secretion from individual vesicles was measured during exocytosis from adrenal chromaffin cells harvested from R6/2 and WT mice. While the number of exocytotic events was unchanged, the amounts released per vesicle were significantly diminished in R6/2 mice, indicating that vesicular catecholamines are present in decreased amounts. Treatment of chromaffin cells with 3-nitropropionic acid decreased the vesicular release amount from WT cells by 50%, mimicking the release observed from untreated R6/2 cells. Thus, catecholamine release from tissues isolated from R6/2 mice is diminished because of impaired vesicle loading. [source] Calcium channel subtypes differentially regulate fusion pore stability and expansionJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Alvaro O. Ardiles Abstract Various studies have focused in the relative contribution of different voltage-activated Ca2+ channels (VACC) to total transmitter release. However, how Ca2+ entry through a given VACC subtype defines the pattern of individual exocytotic events remains unknown. To address this question, we have used amperometry in bovine chromaffin cells. L, N, and P/Q channels were individually or jointly blocked with furnidipine, ,-conotoxin GVIA, ,-agatoxin IVA, or ,-conotoxin MVIIC. The three channel types contributed similarly to cytosolic Ca2+ signals induced by 70 mmol/L K+. However, they exhibited different contributions to the frequency of exocytotic events and they were shown to differently regulate the final steps of the exocytosis. When compared with the other VACC subtypes, Ca2+ entry through P/Q channels effectively induced exocytosis, it decreased fusion pore stability and accelerated its expansion. Conversely, Ca2+ entry through N channels was less efficient in inducing exocytotic events, also slowing fusion pore expansion. Finally, Ca2+ entry through L channels inefficiently induced exocytosis, and the individual blockade of this channel significantly modified fusion pore dynamics. The distance between a given VACC subtype and the release sites could account for the differential effects of the distinct VACC on the fusion pore dynamics. [source] Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesisJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Larisa Bobrovskaya Abstract Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline. [source] Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretionJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Sergio D. Rosé Abstract The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine ,-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks. [source] Angiotensin II promotes the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanismJOURNAL OF NEUROCHEMISTRY, Issue 6 2001Martín Cammarota In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP-responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type-1 receptor (AT1)-dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H-89, SB203580 or KN-93, suggesting that it is mediated by the extracellular-regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP-dependent protein kinase (PKA), p38 mitogen-activated protein kinase (p38MAPK) or Ca2+/calmodulin-dependent protein kinases (CaMKs) dependent pathways. Gel-shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2-dependent upregulation of CRE-binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non-receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII-induced phosphorylation of ERK1/2, the CREB kinase p90RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src-mediated activation of ERK 1/2 and p90RSK. [source] Nitric Oxide-Sensitive Guanylyl Cyclase Activity Inhibition Through Cyclic GMP-Dependent DephosphorylationJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Rut Ferrero Abstract: The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter- and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine-3,,5,-monophosphate (cGMP)-dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C-type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP-permeant analogue (which also activates PKG), in the presence of a broad-spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)-dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT-5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the , subunit of sGC, and these changes correlated well with differences in SNP-elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP-stimulated cGMP rises without changing the SNP concentration required to produce half-maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its , subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the , subunit. [source] Up-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-, in Adrenal Chromaffin CellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2000Involvement of Transcriptional, Translational Events Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12- O -tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-, (PKC-,) and novel PKC-, (but not atypical PKC-,) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-,. In the present study, chronic (,12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4,-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans -Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the ,-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor ,-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor ,-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-, up-regulates the density of the cell surface insulin receptor via transcriptional/translational events. [source] Effect of Urotensin II on PC12 Rat Pheochromocytoma CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2010Y. Aita Urotensin II (U-II), initially identified as a cyclic peptide from fish urophysis, acts both as a strong vasoconstrictor and vasodilator in the vasculature via its receptor, G-protein coupled receptor 14. In addition, U-II and its receptor are co-expressed in the adrenal medulla, as well as in human pheochromocytomas, suggesting that this peptide may have some function in chromaffin cells. However, the precise role of U-II in these cells is unknown. In the present study, we initially demonstrate that U-II and its receptors mRNA are co-expressed in the rat pheochromocytoma cell line PC12. Moreover, U-II has not effect on tyrosine hydroxylase (TH), the rate-limiting enzyme involved in the biosynthesis of catecholamine, in terms of enzyme activity or at the mRNA level. However, U-II does induce an increase in the phosphorylation of TH specifically at Ser31 without affecting phosphorylation at the two other sites (Ser19 and Ser40). U-II also markedly activates extracellular signal-regulated kinases (ERKs) and p38, but not Jun N-terminal kinase. Blockade of the epidermal growth factor (EGF) receptor by AG1478 significantly reduces activation of ERK, suggesting that EGF receptor transactivation could act upstream of the ERK pathway in PC12 cells. Furthermore, U-II significantly increases dopamine secretion from PC12 cells. Finally, we show that U-II induced significant DNA synthesis in a ERKs and P38 mitogen-activated protein kinase-dependent manner. The results obtained indicate that U-II may exert its effects as a neuromodulator in chromaffin cells. [source] Reduction of Allodynia by Intrathecal Transplantation of Microencapsulated Porcine Chromaffin CellsARTIFICIAL ORGANS, Issue 3 2009Yu Mi Kim Abstract Bovine chromaffin cells (BCCs) are well known to have analgesic effect to reduce acute or chronic pain when transplanted in the subarachnoid space and have been considered as an alternative therapy for pain management. However, due to recent concerns over risks associated with prion transmission, porcine tissue is considered to be an alternate xenogeneic source for clinical use. In the present study, we investigated whether microencapsulated porcine adrenal medullary chromaffin cells (PCCs) also have analgesic effect to reduce allodynia caused by neuropathic pain in chronic constriction injury model of rat. PCCs were isolated from a porcine adrenal medulla and then microencapsulated with alginate and poly. In in vitro tests, the microencapsulated PCCs were investigated whether they have an ability to release catecholamines responding to nicotine stimulation. The levels of catecholamines released from the microencapsulated PCCs were significantly higher than from microencapsulated BCCs. In addition, the microencapsulated PCCs released catecholamines and met-enkephalin responding to cerebral spinal fluid (CSF) retrieved from a neuropathic pain model. In in vivo tests, implantation of microencapsulated PCCs reduced both mechanical and cold allodynia in chronic constriction injury model of a rat whereas the microencapsulated BCCs reduced only cold allodynia under the same conditions. The injection of antagonist of opioid peptides reversed the reduction of cold allodynia in microencapsulated PCC-received animal. The levels of catecholamines in the CSF of rats after implantation of microencapsulated PCCs were significantly higher than in the control group. These data suggest that microencapsulated PCCs may be another effective source for the treatment of neuropathic pain. [source] Immunoisolated Chromaffin Cells Implanted Into the Subarachnoid Space of Rats Reduce Cold Allodynia in a Model of Neuropathic Pain: A Novel Application of Microencapsulation TechnologyARTIFICIAL ORGANS, Issue 12 2004Yu Mi Kim Abstract:, Intrathecal transplants of adrenal medullary chromaffin cells relieve chronic pain by secreting catecholamines, opioids, and other neuroactive substances. Recently, macrocapsules with semipermeable membranes were used to isolate immunologically xenogenic chromaffin cells, but the poor viability in vivo of the encapsulated chromaffin cells limited the usefulness of this method. In this study, we used a novel method of encapsulation to increase the viability of chromaffin cells. We found that microencapsulated chromaffin cells that were implanted into the subarachnoid space of rats relieved cold allodynia in a model of neuropathic pain. Furthermore, microencapsulated chromaffin cells were morphologically normal and retained their functionality. These findings suggest that the intrathecal placement of microencapsulated chromaffin cells might be a useful method for treating chronic pain. [source] Short- and long-term differential effects of neuroprotective drug NS-7 on voltage-dependent sodium channels in adrenal chromaffin cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000Hiroki Yokoo In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced 22Na+ influx via voltage-dependent Na+ channels (IC50=11.4 ,M). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na+,K+ ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50=20.0 ,M) and catecholamine secretion (IC50=25.8 ,M). Chronic (12 h) treatment of cells with NS-7 increased cell surface [3H]-STX binding by 86% (EC50=10.5 ,M; t1/2=27 h), but did not alter the KD value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans -Golgi network, but was not associated with increased levels of Na+ channel ,- and ,1 -subunit mRNAs. In cells subjected to chronic NS-7 treatment, 22Na+ influx caused by veratridine (site 2 toxin), ,-scorpion venom (site 3 toxin) or ,-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced 22Na+ influx remained depressed even at higher concentration of veratridine; however, either ,- or ,-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced 22Na+ influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na+ channels, thereby reducing Ca2+ channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na+ channels via translational and externalization events, but persistently inhibits Na+ channel gating without impairing the cooperative interaction between the functional domains of Na+ channels. British Journal of Pharmacology (2000) 131, 779,787; doi:10.1038/sj.bjp.0703622 [source] Regulation of Exocytosis in Chromaffin Cells by Trans -Insertion of Lysophosphatidylcholine and Arachidonic Acid into the Outer Leaflet of the Cell MembraneCHEMBIOCHEM, Issue 12 2006Christian Amatore Prof. Abstract Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans -insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans -insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA. [source] Dynamics of Full Fusion During Vesicular Exocytotic Events: Release of Adrenaline by Chromaffin CellsCHEMPHYSCHEM, Issue 2 2003Christian Amatore Prof. Abstract Vesicular exocytosis is important in the communication between cells in complex organisms. It controls the release of specific chemical or biochemical messengers stored in the emitting cell, which elicit a response upon detection by the target cells. Secretion of a messenger molecule (a neurotransmitter) was measured electrochemically, which allowed the quantification of cellular events and the validation of current physicochemical models. This model led us to formulate predictions about the occurrence and kinetics of vesicular exocytotic events based on the physicochemical meaning of its key parameters. These predictions were tested successfully through a series of experiments on chromaffin cells, involving changes of osmotic conditions, presence of trivalent ions and cholesterol-induced structuring of the cell plasmic membrane. [source] Oxygen-sensing pathway for SK channels in the ovine adrenal medullaCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2005Damien J Keating SUMMARY 1.,The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2.,The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3.,Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4.,One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5.,The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6.,These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state. [source] | ||||||||||||||||