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A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells.
Ges IA, Brindley RL, Currie KP, Baudenbacher FJ
(2013) Lab Chip 13: 4663-73
MeSH Terms: Animals, Calcium, Carbachol, Catecholamines, Cattle, Cells, Cultured, Chromaffin Cells, Chromatography, High Pressure Liquid, Dimethylpolysiloxanes, Electrochemical Techniques, Electrodes, Kinetics, Microfluidic Analytical Techniques, Pituitary Adenylate Cyclase-Activating Polypeptide, Platinum, Potassium Chloride, Silicon, Stimulation, Chemical
Show Abstract · Added November 19, 2013
Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped "cell traps", each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion/repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time.
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18 MeSH Terms
Cytokine stimulation of epithelial cancer cells: the similar and divergent functions of IL-4 and IL-13.
Hallett MA, Venmar KT, Fingleton B
(2012) Cancer Res 72: 6338-43
MeSH Terms: Animals, Cytokines, Epithelial Cells, Humans, Interleukin-13, Interleukin-4, Models, Biological, Neoplasms, Glandular and Epithelial, Receptors, Interleukin-13, Receptors, Interleukin-4, Stimulation, Chemical
Show Abstract · Added March 7, 2014
The Th2 cytokines interleukin (IL)-4 and -13 are acknowledged regulators of lymphocyte proliferation and activation. They have also been well studied in the regulation of various myeloid-derived populations in tumor biology. It has become clear, however, that both cytokines can have direct effects on epithelial tumor cells expressing appropriate receptors. Changes in tumor proliferation, survival, and metastatic capability have all been ascribed to IL-4 and/or IL-13 action. Here, we evaluate the evidence to support direct tumor-promoting roles of these cytokines. We also identify the questions that should be addressed before proceeding with therapeutic approaches based on neutralization of IL-4 or IL-13 pathways.
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11 MeSH Terms
Odorant-specific requirements for arrestin function in Drosophila olfaction.
Merrill CE, Sherertz TM, Walker WB, Zwiebel LJ
(2005) J Neurobiol 63: 15-28
MeSH Terms: Acetates, Acetone, Animals, Animals, Genetically Modified, Arrestins, Behavior, Animal, Butanols, Dose-Response Relationship, Drug, Drosophila, Drosophila Proteins, Electrophysiology, Kinetics, Locomotion, Mutation, Odorants, Olfactory Pathways, Olfactory Receptor Neurons, Phosphoproteins, Receptors, Odorant, Smell, Stimulation, Chemical, Time Factors
Show Abstract · Added May 27, 2014
The ability to modulate olfactory sensitivity is necessary to detect chemical gradients and discriminate among a multitude of odor stimuli. Desensitization of odorant receptors has been postulated to occur when arrestins prevent the activation of downstream second messengers. A paucity of in vivo data on olfactory desensitization prompts use of Drosophila melanogaster genetics to investigate arrestins' role in regulating olfactory signaling pathways. Physiological analysis of peripheral olfactory sensitivity reveals decreased responsiveness to a host of chemically distinct odorants in flies deficient for arrestin1 (arr1), arrestin2 (arr2), or both. These phenotypes are manifest in odorant- and dose- dependent fashions. Additionally, mutants display altered adaptive properties under a prolonged exposure paradigm. Behaviorally, arr1 mutants are impaired in olfactory-based orientation towards attractive odor sources. As the olfactory deficits vary according to chemical identity and concentration, they indicate that a spectrum of arrestin activity is essential for odor processing depending upon the particular olfactory pathway involved. Arrestin mutant phenotypes are hypothesized to be a consequence of down-regulation of olfactory signaling to avoid cellular excitotoxicity. Importantly, phenotypic rescue of olfactory defects in arr1(1) mutants is achieved through transgenic expression of wild-type arr1. Taken together, these data clearly indicate that arrestins are required in a stimulus-specific manner for wild type olfactory function and add another level of complexity to peripheral odor coding mechanisms that ultimately impact olfactory behavior.
Copyright (c) 2004 Wiley Periodicals, Inc.
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22 MeSH Terms
Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets.
Gunawardana SC, Liu YJ, Macdonald MJ, Straub SG, Sharp GW
(2004) Am J Physiol Endocrinol Metab 287: E828-33
MeSH Terms: Acetyl Coenzyme A, Amino Acids, Amino Acids, Cyclic, Analysis of Variance, Animals, Calcium, Citric Acid Cycle, Enzyme Activation, Glucose, In Vitro Techniques, Insulin, Insulin Secretion, Islets of Langerhans, Ketoglutaric Acids, Male, Mitochondria, Rats, Rats, Wistar, Signal Transduction, Stimulation, Chemical, Up-Regulation
Show Abstract · Added November 1, 2012
Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the beta-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca(2+) concentration ([Ca(2+)](i)). The second phase requires increased [Ca(2+)](i) and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca(2+)-stimulated release via the K(ATP) channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K(+) channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased alpha-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of alpha-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP.
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21 MeSH Terms
Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake.
Knox CD, Belous AE, Pierce JM, Wakata A, Nicoud IB, Anderson CD, Pinson CW, Chari RS
(2004) Am J Physiol Gastrointest Liver Physiol 287: G533-40
MeSH Terms: Adenosine, Animals, Blotting, Western, Calcium, Calcium Channel Agonists, Calcium Channels, Calcium-Binding Proteins, Cell Membrane, Estrenes, Fluorescent Dyes, In Vitro Techniques, Inositol 1,4,5-Trisphosphate, Isoenzymes, Male, Microscopy, Fluorescence, Mitochondria, Liver, Phosphodiesterase Inhibitors, Phospholipase C delta, Pyrrolidinones, Rats, Rats, Sprague-Dawley, Spermine, Stimulation, Chemical, Type C Phospholipases
Show Abstract · Added January 10, 2014
Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.
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24 MeSH Terms
Engineering physiologically regulated insulin secretion in non-beta cells by expressing glucagon-like peptide 1 receptor.
Wu L, Nicholson W, Wu CY, Xu M, McGaha A, Shiota M, Powers AC
(2003) Gene Ther 10: 1712-20
MeSH Terms: Adenoviridae, Animals, Cells, Cultured, Diabetes Mellitus, Type 1, Gene Expression, Genetic Therapy, Genetic Vectors, Glucagon-Like Peptide-1 Receptor, Glucose, Humans, Insulin, Insulin Secretion, Male, Mice, Mice, Inbred NOD, Mice, SCID, Pituitary Gland, Pituitary Hormones, Rats, Rats, Sprague-Dawley, Receptors, Glucagon, Stimulation, Chemical, Transduction, Genetic
Show Abstract · Added December 10, 2013
Glucagon-like peptide 1 (GLP-1) is released from neuroendocrine cells in the intestine in the postprandial state and augments glucose-stimulated insulin secretion from pancreatic beta cells. To develop non-beta cells that exhibit physiologically regulated insulin secretion, we coexpressed the GLP-1 receptor and human insulin in primary rat pituitary cells using adenovirus-mediated gene transfer. The transduced cells were analyzed in a perifusion system and after transplantation into mice. Normal pituitary cells do not express the GLP-1 receptor as shown by the absence of GLP-1 receptor mRNA and the inability of GLP-1 to stimulate pituitary hormone secretion. Following transduction with an adenovirus carrying the GLP-1 receptor cDNA, the pituitary cells expressed functional GLP-1 receptors as reflected by the ability of GLP-1 to stimulate secretion of pituitary hormones. When both the GLP-1 receptor and human insulin were introduced, GLP-1 stimulated cosecretion of human insulin and endogenous pituitary hormones. GLP-1 was similar in potency to the hypothalamic-releasing hormones and stimulated hormone secretion in a dose-dependent fashion. In contrast to pancreatic beta cells, the hormone-releasing effect of GLP-1 on transduced pituitary cells was not dependent on the concentration of extracellular glucose. After transplantation of pituitary cells coexpressing human insulin and GLP-1 receptor into mice, enteral glucose stimulated insulin secretion. These results demonstrate a new approach to engineer physiologically regulated insulin secretion by non-beta cells.
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23 MeSH Terms
Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps.
Perriott LM, Kono T, Whitesell RR, Knobel SM, Piston DW, Granner DK, Powers AC, May JM
(2001) Am J Physiol Endocrinol Metab 281: E72-80
MeSH Terms: 3-O-Methylglucose, Adult, Aged, Biological Transport, Active, Cell Differentiation, Cells, Cultured, Female, Glucose, Hexokinase, Humans, Hypoglycemic Agents, Insulin, Kinetics, Male, Microscopy, Fluorescence, Middle Aged, Muscle, Skeletal, NADP, Phosphorylation, Pyruvic Acid, Stimulation, Chemical
Show Abstract · Added December 10, 2013
To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.
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21 MeSH Terms
Metabolism of ochratoxin A: absence of formation of genotoxic derivatives by human and rat enzymes.
Gautier J, Richoz J, Welti DH, Markovic J, Gremaud E, Guengerich FP, Turesky RJ
(2001) Chem Res Toxicol 14: 34-45
MeSH Terms: Animals, Biotransformation, Carcinogens, Cattle, Cytochrome P-450 Enzyme System, DNA, DNA Damage, Female, Glutathione Transferase, Humans, Isoenzymes, Kidney, Lipid Peroxidation, Male, Malondialdehyde, Microsomes, Liver, Mycotoxins, Ochratoxins, Prostaglandin-Endoperoxide Synthases, Protein Binding, Proteins, Rats, Rats, Inbred F344, Stimulation, Chemical
Show Abstract · Added May 26, 2014
Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10-25 pmol min(-1) (mg of protein)(-1)]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min(-1) (mg of protein)(-1)]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min(-1) (pmol of P450)(-1), respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min(-1) (pmol of P450)(-1)]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [(3)H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B(1), 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [(3)H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [(3)H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, (32)P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the (32)P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.
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24 MeSH Terms
Emotional responses to pleasant and unpleasant olfactory, visual, and auditory stimuli: a positron emission tomography study.
Royet JP, Zald D, Versace R, Costes N, Lavenne F, Koenig O, Gervais R
(2000) J Neurosci 20: 7752-9
MeSH Terms: Acoustic Stimulation, Adult, Brain, Brain Mapping, Data Display, Emotions, Female, Humans, Male, Odorants, Photic Stimulation, Pilot Projects, Reaction Time, Reference Values, Smell, Stimulation, Chemical, Tomography, Emission-Computed
Show Abstract · Added May 27, 2014
Neural correlates of responses to emotionally valenced olfactory, visual, and auditory stimuli were examined using positron emission tomography. Twelve volunteers were scanned using the water bolus method. For each sensory modality, regional cerebral blood flow (rCBF) during presentation of both pleasant and unpleasant stimuli was compared with that measured during presentation of neutral stimuli. During the emotionally valenced conditions, subjects performed forced-choice pleasant and unpleasant judgments. During the neutral conditions, subjects were asked to select at random one of a two key-press buttons. All stimulations were synchronized with inspiration, using an airflow olfactometer, to present the same number of stimuli for each sensory modality. A no-stimulation control condition was also performed in which no stimulus was presented. For all three sensory modalities, emotionally valenced stimuli led to increased rCBF in the orbitofrontal cortex, the temporal pole, and the superior frontal gyrus, in the left hemisphere. Emotionally valenced olfactory and visual but not auditory stimuli produced additional rCBF increases in the hypothalamus and the subcallosal gyrus. Only emotionally valenced olfactory stimuli induced bilateral rCBF increases in the amygdala. These findings suggest that pleasant and unpleasant emotional judgments recruit the same core network in the left hemisphere, regardless of the sensory modality. This core network is activated in addition to a number of circuits that are specific to individual sensory modalities. Finally, the data suggest a superior potency of emotionally valenced olfactory over visual and auditory stimuli in activating the amygdala.
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17 MeSH Terms
Protein kinase C activator PMA reduces the Ca(2+) response to antigen stimulation of adherent RBL-2H3 mucosal mast cells by inhibiting depletion of intracellular Ca(2+) stores.
Kuchtey J, Fewtrell C
(1999) J Cell Physiol 181: 113-23
MeSH Terms: Antigens, Calcium, Cell Adhesion, Cell Line, Enzyme Activation, Fluorescent Dyes, Fura-2, Logistic Models, Mast Cells, Patch-Clamp Techniques, Protein Kinase C, Stimulation, Chemical, Tetradecanoylphorbol Acetate, Thapsigargin
Show Abstract · Added February 19, 2015
Activation of protein kinase C has been shown to reduce the Ca(2+) responses of a variety of cell types. In most cases, the reduction is due to inhibition of Ca(2+) influx, but acceleration of Ca(2+) efflux and inhibition of Ca(2+) store depletion by protein kinase C activation have also been described. For adherent RBL-2H3 mucosal mast cells, results from whole-cell patch clamp experiments suggest that protein kinase C activation reduces Ca(2+) influx, while experiments with intact, fura-2-loaded cells suggest that Ca(2+) influx is not affected. Here we present single-cell data from Ca(2+) imaging experiments with adherent RBL-2H3 cells, showing that antigen-stimulated Ca(2+) responses of phorbol 12-myristate 13-acetate (PMA)-treated cells are more transient than those of control cells. PMA also reduced the response to antigen in the absence of extracellular Ca(2+), indicating that depletion of intracellular Ca(2+) stores is inhibited. If PMA was added after stores had been depleted by thapsigargin, a small decrease in [Ca(2+)](i) was observed, consistent with a slight inhibition of Ca(2+) influx. However, the major effect of PMA on the antigen-stimulated Ca(2+) response is to inhibit depletion of intracellular Ca(2+) stores. We also show that inhibition of protein kinase C did not enhance the Ca(2+) response to antigen, suggesting that inhibition of the Ca(2+) response by activation of protein kinase C does not contribute to the physiological response to antigen.
Copyright 1999 Wiley-Liss, Inc.
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14 MeSH Terms