EM and biochemical determinations of chromaffin cell ecto-ATPase activity

1990 ◽  
Vol 48 (3) ◽  
pp. 936-937
Author(s):  
V. Kriho ◽  
G. D. Pappas ◽  
R. P. Becker
Keyword(s):  
Plasma Membrane ◽  
Adrenal Gland ◽  
Atpase Activity ◽  
Extracellular Space ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Extracellular Atp ◽  
Bovine Chromaffin Cells ◽  
Local Metabolism

Exocytotic granules of bovine chromaffin cells contain both catecholamines and ATP. Upon stimulation, the granule contents are discharged into the extracellular space. Catecholamines are eventually hydrolyzed. The resultant choline is then taken up by the cell for recycling. The fate of the extracellular ATP has not been determined. Ecto-ATPase activity has been localized at the plasma membrane of the chromaffin cell in the adrenal gland. This ATPase activity may play a role in the local metabolism of the released ATP.Our present study further investigates this ecto-ATPase activity, using biochemistry and EM cytochemistry, on isolated, intact bovine chromaffin cells. Our biochemical results are seen in the histogram of Fig. 1. ATPase assays show considerable ATPase activity when both Ca++ and Mg++ are present in physiological concentrations in the incubating solution. Even when Ca++ is omitted from the incubating solution, a great deal of ecto-ATPase activity is still demonstrated. Omitting Mg++ from the medium, however, reduced the level of ATPase activity by 91%.

EM localization of chromaffin cell ATPase

1992 ◽  
Vol 50 (1) ◽  
pp. 816-817
Author(s):  
V. Kriho ◽  
G. D. Pappas
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Immunogold Labelling ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Cells In Culture ◽  
Membrane Bound ◽  
Bovine Chromaffin Cells

During exocytosis of the chromaffin granules, ATP is released. ATP can then be hydrolyzed by the ecto-ATPases of the plasma membrane to provide adenosine for reuptake or for activation of P1 purinoceptors. Chromaffin granule membranes also possess ATPase activity. This activity is linked to the uptake of catecholamines from the cytoplasm into the membrane-bound granule compartment.In this report we combine EM cytochemistry and immunogold labelling to provide further evidence for the presence of ATPase on both the plasma membrane and granule membranes of bovine chromaffin cells in culture.

scholarly journals Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

1991 ◽  
Vol 39 (11) ◽  
pp. 1485-1493 ◽  
Author(s):  
T Fujimoto ◽  
K Lee ◽  
S Miwa ◽  
K Ogawa
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Immunogold Electron Microscopy ◽  
Apparent Difference ◽  
Thin Filaments ◽  
Whole Cells ◽  
Immunocytochemical Techniques ◽  
Bovine Chromaffin Cells

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.

Short-Term Immunosuppression Enhances Long-Term Survival of Bovine Chromaffin Cell Xenografts in Rat Cns

1992 ◽  
Vol 1 (1) ◽  
pp. 33-41 ◽  
Author(s):  
John D. Ortega ◽  
Jacqueline Sagen ◽  
George D. Pappas
Keyword(s):  
Blood Brain Barrier ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Short Term ◽  
Term Survival ◽  
Long Term Survival ◽  
Bovine Chromaffin Cell ◽  
Bovine Chromaffin Cells ◽  
Rat Cns

Xenogeneic donors, a largely untapped resource, would solve many of the problems associated with the limited availability of human donor tissue for neural transplantation. Previous work in our laboratory has revealed that xenografts of isolated bovine chromaffin cells survive transplantation into the periaqueductal gray (PAG) of immunosuppressed adult rats. Electron microscopic analysis reveals that graft sites contain healthy chromaffin cells, but do not contain host immune cells typical of graft rejection. The aim of the current study was to assess the necessary conditions for long-term survival of bovine chromaffin cell xenografts in the central nervous system (CNS). In particular, the need for short-course vs. permanent immunosuppressive therapy with cyclosporine A (CsA) for the long-term survival of grafted bovine chromaffin cells was addressed. Grafts from animals receiving continuous CsA treatment for either 3, 6, or 12 wk contained large clumps of dopamines-β-hydroxylase (DBH) positive cells in contrast to the few surviving cells observed in nonimmunosuppressed animals. In addition, grafts from animals that had CsA treatment terminated at 3 or 6 wk contained similarly large clumps of DBH-positive cells. Furthermore, short-term immunosuppression (3 wk) appeared to enhance the long-term survival of grafted cells, since clumps of DBH staining cells could still be positively identified in the host PAG at least 1 yr after transplantation. Complete rejection of graft tissue depends on several factors, such as blood–brain barrier integrity, the presence of major histocompatability complex (MHC) antigens in either the host or graft, and the status of the host immune system. By using a suspension of isolated bovine chromaffin cells, potential MHC antigen presenting cells, such as endothelial cells, are eliminated. In addition, CsA treatment may negate the immunologic consequences of increased blood–brain barrier permeability following surgical trauma by attenuating the host cell mediated response. In summary, long-term survival of isolated chromaffin cell xenografts in the rat CNS may be attained by a short-term course of CsA.

Different contributions of L- and Q-type Ca2+ channels to Ca2+ signals and secretion in chromaffin cell subtypes

1997 ◽  
Vol 272 (2) ◽  
pp. C476-C484 ◽  
Author(s):  
R. B. Lomax ◽  
P. Michelena ◽  
L. Nunez ◽  
J. Garcia-Sancho ◽  
A. G. Garcia ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Channel Blocker ◽  
Cell Types ◽  
Bay K 8644 ◽  
High K ◽  
Voltage Dependent ◽  
Bovine Chromaffin Cells ◽  
P Type ◽  
Ca2 Channels

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.

scholarly journals Restriction of Secretory Granule Motion near the Plasma Membrane of Chromaffin Cells

2001 ◽  
Vol 153 (1) ◽  
pp. 177-190 ◽  
Author(s):  
Laura M. Johns ◽  
Edwin S. Levitan ◽  
Eric A. Shelden ◽  
Ronald W. Holz ◽  
Daniel Axelrod
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Chromaffin Cells ◽  
Botulinum Toxin A ◽  
Secretory Granules ◽  
Internal Reflection ◽  
Snare Proteins ◽  
Bovine Chromaffin Cells ◽  
Heterogeneous Matrix ◽  
Granule Motion

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the ∼300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

Electrical activity in chromaffin cells of intact mouse adrenal gland

1988 ◽  
Vol 254 (5) ◽  
pp. C675-C683 ◽  
Author(s):  
V. Nassar-Gentina ◽  
H. B. Pollard ◽  
E. Rojas
Keyword(s):  
Cell Membrane ◽  
Adrenal Gland ◽  
Electrical Activity ◽  
Action Potentials ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Resting Potential ◽  
Constant Field ◽  
Intact Mouse

Membrane potentials of medullary chromaffin cells of the adrenal gland of the mouse were measured in situ. Resting potential (-54.3 +/- 8.8 mV) depended on extracellular [K+] as predicted by the constant-field equation with a permeability ratio, PNa/PK, of 0.09. Current-voltage (I-V) relationships showed that the current is rectified across the chromaffin cell membrane. A rectification ratio of 0.4 was calculated from the slopes of the I-V curves for positive (41 +/- 26 M omega) and negative (103 +/- M omega) currents. Because input resistance for a resting chromaffin cell in isolation is approximately 5 G omega, the chromaffin cells in situ behave as if they were electrically coupled. Most cells responded to depolarizing current pulses with repetitive action potentials, but only 50% of them showed spontaneous electrical activity. Spontaneous activity was often seen in the presence of tetrodotoxin (3 microM). Although the application of the K+-channel blockers tetraethylammonium and Ba2+ greatly increased the amplitude of the action potentials, only Ba2+ induced continuous electrical activity. Application of acetylcholine (ACh) always depolarized the cell membrane. This effect was blocked by atropine but not by D-tubocurarine, suggesting that ACh stimulation of chromaffin cells in the mouse involves activation of muscarinic receptors.

A common molecular machinery for exocytosis and the ‘kiss-and-run’ mechanism in chromaffin cells is controlled by phosphorylation

2001 ◽  
Vol 114 (24) ◽  
pp. 4613-4620 ◽  
Author(s):  
Andreas W. Henkel ◽  
Guoxin Kang ◽  
Johannes Kornhuber
Keyword(s):  
Plasma Membrane ◽  
Protein Phosphorylation ◽  
Hot Spots ◽  
Extracellular Space ◽  
Chromaffin Cells ◽  
Kinase Inhibitor ◽  
Large Dense Core Vesicles ◽  
Molecular Machinery ◽  
Secretion Mode ◽  
Cell Capacitance

Exocytosis and ‘kiss-and-run’ secretion coexist in chromaffin cells. Our findings suggest that these mechanisms are closely related, based on their common molecular machinery. Here we present a model that describes how chromaffin cells regulate catecholamine release by switching the mode of secretion between the two pathways, a process controlled by phosphorylation. Stimulation-dependent vesicle-plasma membrane interactions in chromaffin cells were analysed by simultaneous ‘on-cell’ capacitance and conductance measurements, a technique that allows the monitoring of single vesicles. Capacitance steps represent fusions of large dense-core vesicles with the plasma membrane, whereas capacitance flickers correspond to transient connections of the vesicle lumen with the extracellular space. All these events require the presence of extracellular calcium in millimolar concentrations. ‘Kiss-and-run’ type of release is enhanced by the kinase inhibitor staurosporine, which suggests that this secretion mode is regulated by protein phosphorylation. We also observed capacitance bursts, which most probably represent ‘hot spots’ of secretion and we found that ‘kiss-and-run’ is the prevalent mechanism during these episodes. The significance of ‘kiss-and run’ for neurohormone release is even higher at physiological temperature, because up to half of all secretion events are mediated by this mechanism.

scholarly journals Anti-(14–3–3 protein) antibody inhibits stimulation of noradrenaline (norepinephrine) secretion by chromaffin-cell cytosolic proteins

1992 ◽  
Vol 285 (3) ◽  
pp. 697-700 ◽  
Author(s):  
Y N Wu ◽  
N D Vu ◽  
P D Wagner
Keyword(s):  
Sequence Analysis ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Significant Contribution ◽  
Secretory Activity ◽  
Bovine Brain ◽  
Cytosolic Proteins ◽  
Sequence Identity ◽  
Bovine Chromaffin Cells ◽  
Stimulation Of

Incubation of digitonin-permeabilized bovine chromaffin cells in the absence of Ca2+ results in a loss of both cytosolic proteins and Ca(2+)-dependent secretion. Addition of these leaked proteins prevents this loss of secretory activity. We have purified a protein from an extract of bovine adrenal medulla which can partially prevent this loss of Ca(2+)-dependent secretion. Antibody against this protein inhibited the ability of leaked chromaffin-cell proteins to prevent the loss of Ca(2+)-dependent secretion. Sequence analysis showed it to have sequence identity with bovine brain 14-3-3 protein. These results demonstrate that 14-3-3 protein makes a significant contribution to the ability of leaked chromaffin-cell proteins to maintain secretory activity.

scholarly journals Dynasore inhibits rapid endocytosis in bovine chromaffin cells

2009 ◽  
Vol 297 (2) ◽  
pp. C397-C406 ◽  
Author(s):  
Chia-Chang Tsai ◽  
Chih-Lung Lin ◽  
Tzu-Lun Wang ◽  
Ai-Chuan Chou ◽  
Min-Yi Chou ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Membrane Capacitance ◽  
Neuroendocrine Cells ◽  
Maximal Increase ◽  
Force Microscopy ◽  
Vesicle Recycling ◽  
Atomic Force ◽  
Bovine Chromaffin Cells ◽  
Patch Configuration

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all ∼110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis.

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