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1.
Rev. esp. anestesiol. reanim ; 63(1): 48-53, ene. 2016. ilus
Article in English | IBECS | ID: ibc-150076

ABSTRACT

Pheochromocytoma is a tumour of the chromaffin tissue. It may, through catecholamine release, have deleterious effects on myocardial structure. A 48-year-old woman with a history of hypertension and type II diabetes mellitus (ASA II) was diagnosed of pheochromocytoma-induced myocarditis, which caused severe cardiogenic shock, with an ejection fraction of 20%. Extreme blood pressure swings required aggressive therapy with vasoactive drugs (norepinephrine and dopamine) and an intra-aortic balloon pump, despite which severe haemodynamic instability persisted. Finally, the use of magnesium sulphate allowed for cardiovascular stabilization and weaning off vasoactive drugs prior to surgery. 123I-metaiodobenzylguanidine scintigraphy helps not only to functionally confirm tumour tissue, but also to assess severity and prognosis of cardiac failure. Prognosis of pheochromocytoma-induced heart failure can be very poor. The use of these two well-known and relatively simple ‘tools’ for treatment and prognosis is a helpful option to keep in mind (AU)


Los feocromocitomas son tumores del tejido cromafín. Pueden, a través de la secreción de catecolaminas, causar efectos deletéreos sobre el miocardio. Una mujer de 48 años, con antecedentes de hipertensión arterial y diabetes mellitus tipo II (ASA II) fue diagnosticada de feocromocitoma, con miocardiopatía, y shock cardiogénico con fracción de eyección del 20%. Las extremas oscilaciones hemodinámicas requirieron tratamiento con fármacos vasoactivos (noradrenalina y dopamina) así como el uso de un balón de contrapulsación intra-aórtico, a pesar de lo cual persistía la inestabilidad. Finalmente, el uso de sulfato de magnesio permitió la estabilización de la paciente, pudiéndose retirar las drogas vasoactivas previo a la cirugía. El uso de la gammagrafía con 123-metayodobenzilguanidina sirve no sólo para la clasificación funcional del tejido tumoral, sino también para evaluar la severidad y pronóstico del fallo cardíaco. El pronóstico de la insuficiencia cardíaca inducida por feocromocitoma puede ser grave. El uso de estas conocidas y relativamente sencillas ‘herramientas’ para el tratamiento y el pronóstico son una opción útil a tener en cuenta (AU)


Subject(s)
Humans , Female , Adult , Pheochromocytoma/metabolism , Pheochromocytoma/pathology , Radionuclide Imaging/methods , Magnesium Sulfate/administration & dosage , Chromaffin System/injuries , Chromaffin System/metabolism , Pheochromocytoma/complications , Pheochromocytoma/diagnosis , Radionuclide Imaging/instrumentation , Magnesium Sulfate , Chromaffin System/abnormalities , Chromaffin System/cytology
2.
Pak J Biol Sci ; 11(7): 1032-6, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18810974

ABSTRACT

In C. auratus the adrenal chramaffin tissue is situated around the posterior cardinal veins, in the head kidney. Chromaffin tissue consists of two types of cells containing secretory granules, adrenaline and nor adrenaline cells. The cells produced catecholamine hormones. Adrenaline cell contains electron-lucent granules, whereas nor adrenaline cells possesses electron-dense granules. Cholinergic fibers embedded in the head kidney innervated the chromaffin cell. Two types of secretory structures, synaptic vesicles and secretory granules are found within the presynaptic terminal. Secretory granules discharge their contests, as neuropeptide in non synaptic area of nerve terminal by exocytosis, whereas synaptic vesicles discharge their contents as neurotransmitters at the synaptic thickening (active zone) in the presynaptic terminal by exocytosis.


Subject(s)
Adrenal Medulla/cytology , Adrenal Medulla/metabolism , Chromaffin System/cytology , Chromaffin System/innervation , Goldfish/metabolism , Adrenal Medulla/ultrastructure , Animals , Chromaffin Cells/metabolism , Chromaffin Cells/ultrastructure , Chromaffin System/metabolism , Chromaffin System/ultrastructure , Cytoplasmic Granules/ultrastructure , Epinephrine/metabolism , Exocytosis , Norepinephrine/metabolism , Secretory Vesicles/metabolism , Secretory Vesicles/ultrastructure , Synapses/ultrastructure , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure
3.
Peptides ; 28(9): 1871-82, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17651866

ABSTRACT

Initial PACAP-regulated transcriptomes of PACAP-treated cultured chromaffin cells, and the adrenal gland of wild-type versus PACAP-deficient mice, have been assembled using microarray analysis. These were compared to previously acquired PACAP-regulated transcriptome sets from PC12 cells and mouse central nervous system, using the same microarray platform. The Ingenuity Pathways Knowledge Base was then employed to group regulated transcripts into common first and second messenger regulatory clusters. The purpose of our meta-analysis was to identify sets of genes regulated distinctly or in common by the neurotransmitter/neurotrophin PACAP in specific physiological contexts. Results suggest that PACAP participates in both the basal differentiated expression, and the induction upon physiological stimulation, of distinct sets of transcripts in neuronal and endocrine cells. PACAP in both developmental and acute regulatory paradigms acts on target genes also regulated by either TNFalpha or TGFbeta, two first messengers acting on transcription mainly through NFkappaB and Smads, respectively.


Subject(s)
Adrenal Glands/metabolism , Chromaffin System/drug effects , Gene Expression Regulation/drug effects , Oligonucleotide Array Sequence Analysis/methods , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Animals , Cattle , Cell Line , Chromaffin System/cytology , Chromaffin System/metabolism , Gene Expression Profiling , Mice , Models, Genetic , PC12 Cells , Pituitary Adenylate Cyclase-Activating Polypeptide/deficiency , Rats
4.
Development ; 129(20): 4729-38, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12361965

ABSTRACT

The sympathoadrenal (SA) cell lineage is a derivative of the neural crest (NC), which gives rise to sympathetic neurons and neuroendocrine chromaffin cells. Signals that are important for specification of these two types of cells are largely unknown. MASH1 plays an important role for neuronal as well as catecholaminergic differentiation. Mash1 knockout mice display severe deficits in sympathetic ganglia, yet their adrenal medulla has been reported to be largely normal suggesting that MASH1 is essential for neuronal but not for neuroendocrine differentiation. We show now that MASH1 function is necessary for the development of the vast majority of chromaffin cells. Most adrenal medullary cells in Mash1(-/-) mice identified by Phox2b immunoreactivity, lack the catecholaminergic marker tyrosine hydroxylase. Mash1 mutant and wild-type mice have almost identical numbers of Phox2b-positive cells in their adrenal glands at embryonic day (E) 13.5; however, only one-third of the Phox2b-positive adrenal cell population seen in Mash1(+/+) mice is maintained in Mash1(-/-) mice at birth. Similar to Phox2b, cells expressing Phox2a and Hand2 (dHand) clearly outnumber TH-positive cells. Most cells in the adrenal medulla of Mash1(-/-) mice do not contain chromaffin granules, display a very immature, neuroblast-like phenotype, and, unlike wild-type adrenal chromaffin cells, show prolonged expression of neurofilament and Ret comparable with that observed in wild-type sympathetic ganglia. However, few chromaffin cells in Mash1(-/-) mice become PNMT positive and downregulate neurofilament and Ret expression. Together, these findings suggest that the development of chromaffin cells does depend on MASH1 function not only for catecholaminergic differentiation but also for general chromaffin cell differentiation.


Subject(s)
Chromaffin Cells/physiology , Chromaffin System/embryology , DNA-Binding Proteins/metabolism , Drosophila Proteins , Transcription Factors/metabolism , Adrenal Medulla/embryology , Adrenal Medulla/metabolism , Adrenal Medulla/pathology , Animals , Apoptosis/genetics , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation/physiology , Chromaffin Cells/ultrastructure , Chromaffin System/cytology , DNA-Binding Proteins/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Knockout , Mice, Mutant Strains , Nerve Tissue Proteins , Neurofilament Proteins/metabolism , Neurons/cytology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ret , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Transcription Factors/genetics , Tyrosine 3-Monooxygenase/metabolism
5.
J Physiol ; 517 ( Pt 2): 495-505, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10332097

ABSTRACT

1. Gastrointestinal endocrine cells produce biogenic amines which are transported into secretory vesicles by one of two proton-amine exchangers, vesicular monoamine transporters type 1 and 2 (VMAT1 and 2). We report here the presence of VMAT1 in rat gastrin (G) cells and the relevance of VMAT1 function for the modulation of progastrin processing by biogenic and dietary amines. 2. In immunocytochemical studies VMAT1, but not VMAT2, was localized to subpopulations of G cells and enterochromaffin (EC) cells; neither was found in antral D cells. The expression of VMAT1 in antral mucosa was confirmed by Northern blot analysis, which revealed an mRNA band of approximately 3.2 kb, and by Western blot analysis, which revealed a major protein of 55 kDa. 3. In pulse-chase labelling experiments, the conversion of the amidated gastrin G34 to G17 was inhibited by biogenic amine precursors (L-DOPA and 5-hydroxytryptophan). This inhibition was stereospecific and sensitive to reserpine (50 nM), which blocks VMAT1 and VMAT2, but resistant to tetrabenazine, which is a selective inhibitor of VMAT2. 4. Dietary amines such as tyramine and tryptamine also inhibited G34 cleavage. This effect was associated with a loss of the electron-dense core of G cell secretory vesicles. It was not stereospecific or reserpine sensitive, but was correlated with hydrophobicity. 5. Thus rat antral G cells can express VMAT1; transport of biogenic amines into secretory vesicles by VMAT1 is associated with inhibition of G34 cleavage, perhaps by raising intravesicular pH. Dietary amines also modulate cleavage of progastrin-derived peptides, but do so by a VMAT1-independent mechanism; they may act as weak bases that passively permeate secretory vesicle membranes and raise intravesicular pH.


Subject(s)
Digestive System/metabolism , Endocrine Glands/metabolism , Gastrins/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Neuropeptides , Amino Acids/administration & dosage , Amino Acids/physiology , Animals , Chromaffin System/cytology , Chromaffin System/metabolism , Diet , Digestive System/cytology , Endocrine Glands/cytology , Immunohistochemistry , Male , Membrane Glycoproteins/biosynthesis , Pyloric Antrum/metabolism , Rats , Rats, Wistar , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport Proteins
6.
Rev Neurol ; 26(154): 1032-9, 1998 Jun.
Article in Spanish | MEDLINE | ID: mdl-9658489

ABSTRACT

INTRODUCTION: The progress made by contemporary neurobiology opens new horizons both for study and for treatment of disorders of the nervous system. At the present time we are in the age of growth factors. These are molecules which affect survival, development and the normal functioning of cell populations. DEVELOPMENT: One of the most widely studied growth factors is nervous growth factor (NGF) which is necessary for normal life of various types of neurones, including sensory nerves and nerves derived from the neural crests. Diabetic neuropathy, worldwide a major neurological disorder, is primarily characterized by involvement of the fine fibres for temperature and pain perception and also by a variety of autonomic disorders. The great dependence of sensory and sympathetic nerves on NGF, the quantity of results which show alterations in the levels of neurotrophic factors in diabetic neuropathy (DN), and the encouraging experimental and clinical results of using NGF as a new alternative to treatment with DN, were the basic reasons which led us to do this study. CONCLUSIONS: We started by considering neurotrophic factors, especially NGF and its connection with DN disorders and its clinical applications, we made a summary of the main findings in this field to date.


Subject(s)
Diabetic Neuropathies/physiopathology , Nerve Growth Factors/physiology , Alzheimer Disease/therapy , Animals , Calcitriol/pharmacology , Cell Transplantation , Chromaffin System/cytology , Clinical Trials as Topic , Diabetes Mellitus, Experimental/physiopathology , Diabetic Neuropathies/drug therapy , Diabetic Neuropathies/prevention & control , Gene Expression Regulation/drug effects , Graft Survival/drug effects , Humans , Mice , Multicenter Studies as Topic , Nerve Growth Factors/pharmacology , Nerve Growth Factors/therapeutic use , Nerve Regeneration/drug effects , Neural Crest/cytology , Neurons, Afferent/drug effects , Neurons, Afferent/physiology , Parkinson Disease/therapy , Rats , Receptors, Nerve Growth Factor/drug effects , Receptors, Nerve Growth Factor/physiology , Recombinant Fusion Proteins/therapeutic use , Streptozocin , Sympathetic Nervous System/cytology , Sympathetic Nervous System/drug effects , Sympathetic Nervous System/physiology
7.
Am J Physiol ; 272(2 Pt 1): C476-84, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9124290

ABSTRACT

In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.


Subject(s)
Calcium Channels/physiology , Calcium/metabolism , Chromaffin System/metabolism , Signal Transduction , omega-Conotoxins , Animals , Calcium Channel Blockers/pharmacology , Cattle , Chromaffin System/cytology , Cytosol/metabolism , Dihydropyridines/pharmacology , Epinephrine/metabolism , Norepinephrine/metabolism , Osmolar Concentration , Peptides/pharmacology , Potassium/pharmacology , omega-Conotoxin GVIA
8.
Surgery ; 120(6): 1026-32, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8957490

ABSTRACT

BACKGROUND: Although somatostatin is recognized as an inhibitor of neuroendocrine cell secretion, its effect on cell proliferation has not been well defined. Generation of low acid and hypergastrinemia through irreversible H2-receptor blockade (loxtidine) in the African rodent mastomys results in gastric carcinoids (ECLomas) within 4 months. This study was undertaken to evaluate and characterize the precise somatostatin receptor (SSTR) subtype on the mastomys enterochromaffin-like (ECL) cell and to define its role in the regulation of ECL cell secretion and proliferation. METHODS: A pure preparation (approximately 90%) of ECL cells was derived by a combination of pronase digestion and density gradient separation. We assessed the effect of somatostatin (10(-15) to 10(-7) mol/L) on gastrin-stimulated ECL cell histamine secretion and DNA synthesis (bromodeoxyuridine uptake). SSTR2 subtype was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) using gene specific primers and mRNA isolated from normal and hypergastrinemia-induced ECLoma. The polymerase chain reaction product was confirmed by Southern analysis, subcloned, and sequenced. RESULTS: Somatostatin inhibited both gastrin-stimulated histamine secretion (IC50, 5 x 10(-13) mol/L) and DNA synthesis (IC50, 10(-10) mol/L). SSTR2 was identified in the mastomys' brain, and both normal and tumor ECL cells and comparison of the brain and ECL cell SSTR2 nucleotide sequences revealed homology of 99%. CONCLUSIONS: The SSTR2 is expressed by the mastomys' ECL cell and ECLoma. Receptor activation inhibits both ECL cell secretory and proliferative functions.


Subject(s)
Carcinoid Tumor/pathology , Cell Transformation, Neoplastic , Chromaffin System/cytology , Receptors, Somatostatin/physiology , Stomach Neoplasms/pathology , Stomach/cytology , Animals , Base Sequence , Chromaffin System/metabolism , Chromaffin System/physiology , DNA/biosynthesis , Gastric Mucosa/metabolism , Histamine Release , Muridae , Receptors, Somatostatin/genetics , Stomach/physiology
9.
Endocr Res ; 22(4): 839-43, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8969947

ABSTRACT

Previous morphological and physiological evidence indicates that the adrenal medulla can modulate adrenocortical steroidogenesis, most likely via paracrine or neuronal interactions. To study directly chromaffin-adrenocortical cellular interactions, we previously developed co-cultures of frog (Rana pipiens) adrenal (interrenal) cells. Importantly, chromaffin cells in these co-cultures extend processes that project toward or onto adrenocortical cells, thereby providing the substrate for direct autonomic regulation of adrenocortical function and also mimicking the organization in vivo. To test whether chromaffin cells in our co-cultures affect adrenocortical steroidogenesis, we used veratridine, a sodium ionophore, to depolarize chromaffin cells. Chronic veratridine (50 microM) results in increased corticosterone secretion on days 3 (950%), and 4 (350%). These results indicate that chromaffin cell activation results in the modulation of corticosteroidogenesis.


Subject(s)
Adrenal Cortex Hormones/biosynthesis , Adrenal Cortex/cytology , Chromaffin System/cytology , Animals , Autonomic Nervous System/physiology , Cell Communication , Coculture Techniques , Rana pipiens , Veratridine/pharmacology
10.
Hypertension ; 28(3): 414-20, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8794826

ABSTRACT

Vesicular monoamine transport (VMAT) inhibitors, such as reserpine and tetrabenazine, impair vesicular catecholamine storage in chromaffin cells and sympathetic neurons, thereby lowering blood pressure. Here we describe a novel action of VMAT inhibitors-blockade of L-type voltage-gated calcium channels-that may also influence catecholamine release from both PC12 rat pheochromocytoma cells and bovine adrenal chromaffin cells. When given alone, VMAT inhibitors acutely release catecholamines from chromaffin cells in a dose-dependent fashion. However, VMAT inhibitors block catecholamine secretion stimulated by either nicotinic cholinergic agonists or cell membrane depolarization, each of which rely on the opening of L-type channels; the inhibition was more potent after long-term exposure to VMAT inhibitors (IC50 < 100 nmol/L). Reserpine blocked nicotinic-stimulated catecholamine release from neurite-bearing PC12 cells. Reserpine also antagonized catecholamine release triggered by combined membrane depolarization and the dihydropyridine L-type channel agonist Bay K8644, and reserpine blocked cellular uptake of extracellular 45Ca2+ in response to nicotine. Taken together, these results indicate that VMAT inhibitors are also antagonists at L-type voltage-gated calcium channels. Classic L-type channel antagonists (verapamil or nifedipine) also exhibited the reciprocal actions; acutely, they released norepinephrine from chromaffin cells, and chronically, they depleted cellular catecholamine stores, albeit with inferior molar potency to reserpine (IC50 < 1 nmol/L). We conclude that VMAT inhibitors and L-type calcium channel antagonists exert reciprocal inhibitory actions on each other's more classic pharmacological targets. Furthermore, these novel actions are seen at concentrations of these compounds frequently taken to be specific in vitro and likely to occur during antihypertensive treatment in vivo.


Subject(s)
Calcium Channels/drug effects , Membrane Glycoproteins/antagonists & inhibitors , Membrane Transport Proteins , Neuropeptides , Norepinephrine/antagonists & inhibitors , Reserpine/pharmacology , Tetrabenazine/pharmacology , Adrenal Glands/cytology , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Animals , Biological Transport/drug effects , Calcium Channel Blockers/pharmacology , Cattle , Chromaffin System/cytology , Chromaffin System/metabolism , Norepinephrine/metabolism , PC12 Cells , Rats , Vesicular Biogenic Amine Transport Proteins
11.
Anal Chem ; 68(18): 3180-6, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8797378

ABSTRACT

Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-micron diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that high-pass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo.


Subject(s)
Catecholamines/analysis , Animals , Cattle , Chromaffin System/chemistry , Chromaffin System/cytology , Electrochemistry , Male , Microelectrodes , Rats , Rats, Sprague-Dawley
12.
J Neurochem ; 67(3): 1056-62, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8752112

ABSTRACT

An increase in extracellular Ca2+ concentration from 0.25 to 10 mM enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K+ concentration (75 mM). The increment in extracellular Ca2+ concentration also increased the observed peak inward Ca2+ current in response to long (10-s) depolarizing pulses from a holding potential of -55 mV to +5 mV, from about -26 to -400 pA. However, the total amount of Ca2+ influx into the cell only increased when the extracellular Ca2+ concentration was raised from 0.25 to 1 mM and then remained constant up to 10 mM extracellular Ca2+. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 mM extracellular Ca2+, of approximately 4 and 41 s, being the fast component of secretion produced by the exocytosis of approximately 220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca2+ concentration.


Subject(s)
Calcium/pharmacology , Chromaffin System/cytology , Chromaffin System/physiology , Adenosine Triphosphate/metabolism , Animals , Calcium/metabolism , Calcium Channels/physiology , Cattle , Cells, Cultured/metabolism , Cells, Cultured/physiology , Electric Conductivity , Electrophysiology , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology
13.
J Neurochem ; 67(3): 1217-24, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8752129

ABSTRACT

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.


Subject(s)
Chromaffin System/cytology , Phenylethanolamine N-Methyltransferase/metabolism , Adrenal Glands/cytology , Adrenal Glands/enzymology , Animals , Antibody Specificity , Cattle , Cell Separation , Cells, Cultured/chemistry , Cells, Cultured/cytology , Cells, Cultured/enzymology , Centrifugation , Chromaffin System/chemistry , Chromaffin System/enzymology , Colloids , Cycloheximide/pharmacology , Dexamethasone/pharmacology , Epinephrine/analysis , Epinephrine/metabolism , Gene Expression/drug effects , Immunohistochemistry , Norepinephrine/analysis , Norepinephrine/metabolism , Phenylethanolamine N-Methyltransferase/genetics , Phenylethanolamine N-Methyltransferase/immunology , Povidone , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/analysis , Silicon Dioxide , Tyrosine 3-Monooxygenase/metabolism
14.
Neuron ; 17(2): 287-96, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8780652

ABSTRACT

The cortical F-actin cytoskeleton represents a negative control for secretion, and it must be locally disassembled to allow chromaffin vesicle exocytosis. Recombinant scinderin (a Ca(2+)-dependent F-actin-severing protein) potentiated Ca(2+)-evoked F-actin disassembly and exocytosis in permeabilized chromaffin cells, an effect blocked by peptides Sc-ABP1 and Sc-ABP2 (with sequences corresponding to two actin-binding sites of scinderin), exogenous gamma-actin, or phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 effect was blocked by peptide Sc-PIP2BP (with sequence corresponding to a PIP2-binding site of scinderin). Truncated scinderin254-715 (lacking actin-severing domains) did not potentiate exocytosis. Sc-ABP1, Sc-ABP2, and gamma-actin also inhibited exocytosis in the absence of recombinant scinderin, suggesting an inhibition of endogenous scinderin. Results suggest that scinderin-evoked cortical F-actin disassembly is required for secretion and that scinderin is an important component of the exocytotic machinery.


Subject(s)
Exocytosis/drug effects , Microfilament Proteins/pharmacology , Phosphatidylinositol Phosphates/pharmacology , Actins/drug effects , Actins/metabolism , Actins/pharmacology , Adrenal Glands/cytology , Animals , Calcium/pharmacology , Cattle , Cells, Cultured/cytology , Cells, Cultured/physiology , Chromaffin System/cytology , Gelsolin , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/metabolism , Microscopy, Fluorescence , Microscopy, Video , Phosphatidylinositol 4,5-Diphosphate , Recombinant Proteins/pharmacology
15.
J Neurochem ; 67(1): 155-62, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8666986

ABSTRACT

We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+ -free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 microM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 microM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 microM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 +/- 0.38 s) or muscarine (2.2 +/- 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 +/- 0.8 s) was significantly shorter than that evoked by muscarine (33.1 +/- 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An approximately 20-s refractory period was found between repeated caffeine-evoked exocytosis bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland.


Subject(s)
Caffeine/pharmacology , Calcium/metabolism , Chromaffin System/cytology , Exocytosis/physiology , Muscarine/pharmacology , Adrenal Glands/cytology , Animals , Cells, Cultured/cytology , Cells, Cultured/metabolism , Chromaffin System/drug effects , Electrophysiology , Exocytosis/drug effects , Fluorescent Dyes , Indoles , Kinetics , Potassium Chloride/pharmacology , Rats , Rats, Sprague-Dawley , Ryanodine/pharmacology
16.
J Neurochem ; 66(6): 2264-71, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8632147

ABSTRACT

In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c-fos, c-jun, and junB mRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE-2 element of the proenkephalin promoter was sufficient for basal and second messenger-induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE-2 and AP-1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP-1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c-fos, but not of c-jun or CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP-1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovine chromaffin cells.


Subject(s)
Chromaffin System/cytology , Enkephalins/genetics , Protein Precursors/genetics , Transcription Factor AP-1/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/drug effects , Binding Sites/genetics , Cattle , Cells, Cultured/enzymology , Cells, Cultured/physiology , Colforsin/pharmacology , Cyclic AMP Response Element-Binding Protein/drug effects , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/genetics , Genes, Immediate-Early/genetics , Genes, Reporter/physiology , Kinetics , Molecular Sequence Data , Nuclear Proteins/drug effects , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotides/metabolism , Peptide Fragments/drug effects , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylation , Potassium Chloride/pharmacology , Protein Kinase C/genetics , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-fos/drug effects , Proto-Oncogene Proteins c-fos/genetics , RNA, Messenger/genetics , Second Messenger Systems/genetics , Tetradecanoylphorbol Acetate/pharmacology , Time Factors , Transcription Factor AP-1/biosynthesis , Transcription Factor AP-1/metabolism
17.
J Neurochem ; 66(6): 2517-22, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8632177

ABSTRACT

KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced 45Ca2+ influx, which is essential for functional responses. Carbachol-stimulated 22Na+ influx, veratridine-induced 22Na+ influx, and 56 mM K(+)-evoked 45Ca2+ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Adrenal Medulla/cytology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Chromaffin System/cytology , Enzyme Inhibitors/pharmacology , Isoquinolines/pharmacology , Piperazines/pharmacology , Adolescent , Adrenal Medulla/chemistry , Adrenal Medulla/enzymology , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Carbachol/pharmacology , Catecholamines/metabolism , Cattle , Chromaffin System/drug effects , Chromaffin System/enzymology , Humans , Ion Channels/drug effects , Nicotinic Agonists/pharmacology , Sodium/metabolism , Tyrosine 3-Monooxygenase/drug effects , Tyrosine 3-Monooxygenase/metabolism
18.
Gastroenterology ; 110(6): 1835-46, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8964409

ABSTRACT

BACKGROUND & AIMS: Peripheral regulation of acid secretion depends mainly on stimulation or inhibition of the three major gastric endocrine cells (enterochromaffin-like, gastrin, and somatostatin). The aim of this paper was to define physiological responses of enterochromaffin-like, gastrin, and somatostatin cells in a mixed endocrine cell population by measuring ligand-selective changes of intracellular calcium ([Ca2+]i) in individual cells. METHODS: Endocrine cells were enriched from a rat gastric cell suspension by elutriation, a density-gradient fractionation, and a 48-hour short-term culture. [Ca2+]i responses of individual cells to various ligands such as gastrin/carboxy-terminal cholecystokinin octapeptide and selective cholecystokinin antagonists, carbachol, and gastrin-releasing peptide were monitored using video imaging in a perfusion chamber. Characteristic [Ca2+]i changes distinguished the three cell types, confirmed by immunostaining. RESULTS: All enterochromaffin-like cells respond to cholecystokinin-B receptor stimulation, but only a few respond to carbachol. Gastrin cells respond to both gastrin-releasing peptide and carbachol but not to cholecystokinin-receptor agonists. Somatostatin cells have both stimulatory cholecystokinin-A and cholecystokinin-B receptors and inhibitory muscarinic receptors. All cells have inhibitory somatostatin receptors. CONCLUSIONS: Calcium-signaling responses of gastric endocrine cells are distinctive. This allows individual cell types in a mixed population to be characterized and permits an analysis of the hormones and transmitters that act directly on a specific cell type.


Subject(s)
Calcium/metabolism , Endocrine Glands/metabolism , Gastric Mucosa/metabolism , Intracellular Membranes/metabolism , Animals , Chromaffin System/cytology , Chromaffin System/metabolism , Endocrine Glands/cytology , Female , Ligands , Rats , Rats, Sprague-Dawley , Receptors, Bombesin/metabolism , Receptors, Cholecystokinin/metabolism , Receptors, Muscarinic/metabolism , Receptors, Somatostatin/metabolism , Stomach/cytology
19.
Cell Tissue Res ; 284(2): 303-16, 1996 May.
Article in English | MEDLINE | ID: mdl-8625396

ABSTRACT

Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release. Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis. Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event. However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release. Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity. For example, cell size and numbers of "empty' granules and vesicles remained constant and attempts to label "any' organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful. In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace. An alternative hypothesis, therefore, is outlined which better accommodates existing data.


Subject(s)
Chromaffin System/cytology , Exocytosis/physiology , Potassium Chloride/pharmacology , Adrenal Medulla/cytology , Animals , Catecholamines/metabolism , Cattle , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cell Size , Chromaffin System/ultrastructure , Cytoplasmic Granules/physiology , Cytoplasmic Granules/ultrastructure , Exocytosis/drug effects , Intracellular Membranes/physiology , Intracellular Membranes/ultrastructure , Microscopy, Electron
20.
J Neurochem ; 66(5): 1914-23, 1996 May.
Article in English | MEDLINE | ID: mdl-8780018

ABSTRACT

Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic beta-cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for < 6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine-containing vesicles at later days in culture is responsible for this alteration.


Subject(s)
Chromaffin System/physiology , Electrophysiology/methods , Exocytosis/physiology , Islets of Langerhans/physiology , Mast Cells/physiology , PC12 Cells/physiology , Animals , Cattle , Chromaffin System/cytology , Electric Conductivity , Epinephrine/metabolism , Male , Models, Biological , Norepinephrine/metabolism , Osmolar Concentration , Potassium/pharmacology , Rats , Rats, Sprague-Dawley
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