scholarly journals Origin of Afferent Projections into Bovine Chromaffin Cell Implants in the Rat Periaqueductal Gray Determined by Retrograde and Anterograde Tracing

1994 ◽  
Vol 5 (1) ◽  
pp. 31-48 ◽  
Author(s):  
John D. Ortega ◽  
Jacqueline Sagen ◽  
George D. Pappas
Keyword(s):  
Substantia Nigra ◽  
Lateral Hypothalamus ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Cingulate Cortex ◽  
Periaqueductal Gray ◽  
Term Survival ◽  
Bovine Chromaffin Cell ◽  
Bovine Chromaffin Cells ◽  
Anterograde Tracer

We have previously described long-term survival of isolated bovine chromaffin cell suspension grafts in the periaqueductal gray of adult rats. Electron microscopic analysis of the graft sites revealed synapses on the transplanted chromaffin cells. The origin of these synapses is not known, but they are probably derived from the host since the initial grafts were suspensions of chromaffin cells that were essentially free of other cell types.In order to determine the origin of the observed synapses, retrograde and anterograde tracer analyses, were performed on grafted rats at 4 and 8 weeks after transplantation. Following injection of the retrograde tracer (Fluoro-Gold) into graft sites, four major host sites were labeled: hindbrain reticular formation, substantia nigra, lateral hypothalamus, and cingulate cortex. Injection of anterograde tracer (rhodamine-conjugated dextranamine) into the substantia nigra, lateral hypothalamus, and cingulate cortex produced labeled fibers and terminals in and around 4 and 8 week old chromaffin cell graft sites. An increase in both the number of retrogradely labeled cells, as well as in the density of anterogradely labeled fibers and terminals within the graft site, was observed from 4 to 8 weeks.This study shows that graft innervation from the host is primarily from areas that normally project afferent fibers to the periaqueductal gray. The increase in labeled fibers, and terminals over 8 weeks suggests thatde novosynapse formation on grafted bovine chromaffin cells is a continuous process that is dependent on the regenerative capacity and plasticity of the host neuronal network and the grafted bovine chromaffin cells.

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.

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%.

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.

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 Molecular study of the Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

1998 ◽  
Vol 336 (2) ◽  
pp. 305-310 ◽  
Author(s):  
Chien-Yuan PAN ◽  
Yeh-Shiu CHU ◽  
Lung-Sen KAO
Keyword(s):  
Protein Kinase ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Variable Region ◽  
Molecular Study ◽  
Intracellular Loop ◽  
Bovine Chromaffin Cell ◽  
Exchange Activity

To identify the Na+/Ca2+ exchanger expressed in bovine chromaffin cells, the ncx gene was cloned from a bovine chromaffin cell cDNA library. Five partial clones were obtained and their nucleotide sequences showed that there were at least three isoforms containing different intracellular loops. The 3´-untranslated region was the same in all the clones. To examine the Na+/Ca2+ exchange activity of the clones, full-length ncx1 genes were constructed by replacing the corresponding region of bovine cardiac ncx1 clone p17 with the different regions from two bovine chromaffin cell clones; these were designated p17c and p17h. p17h, but not p17c, showed Na+/Ca2+ exchange activity when expressed in Chinese hamster ovary cells and Xenopus oocytes. The expressed exchange activity of p17 was inhibited by 8-bromoadenosine 3´:5´-cyclic monophosphate (8-Br-cAMP) but was not affected by PMA. However, the activity of p17h was inhibited by PMA but enhanced by 8-Br-cAMP. The agents that changed the activity of protein kinase C and cAMP-dependent protein kinase modulated the endogenous Na+/Ca2+ exchange current of chromaffin cells in a manner similar to that of p17h. Our results suggest that the p17h clone is the major isoform of the exchanger in chromaffin cells and is similar to the major ncx1 isoform in kidney. The exchange activity could be regulated by phosphorylation, and the variable region in the intracellular loop is important for the different effects of phosphorylation on the different isoforms.

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.

Permeation and inactivation by calcium and manganese of bovine adrenal chromaffin cell calcium channels

1992 ◽  
Vol 263 (4) ◽  
pp. C818-C824 ◽  
Author(s):  
R. I. Fonteriz ◽  
J. Garcia-Sancho ◽  
L. Gandia ◽  
M. G. Lopez ◽  
A. G. Garcia
Keyword(s):  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Divalent Cations ◽  
Receptor Activation ◽  
Adrenal Chromaffin Cell ◽  
High K ◽  
Bovine Chromaffin Cells ◽  
Adrenal Chromaffin ◽  
Ca2 Channels ◽  
Stimulation Of

Stimulation of fura-2-loaded bovine chromaffin cells with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 10 microM) or depolarization with high [K+] (50 mM) accelerated the entry of both Ca2+ and Mn2+, used here as a Ca2+ surrogate for Ca2+ channels. Removal of extracellular Na+ prevented the effects of DMPP but did not modify the effects of K+, indicating that Na+ is necessary for coupling of Ca2+ entry to the nicotinic receptor activation and that the ionophore associated with it is functionally impermeable to divalent cations. DMPP- as well as K(+)-evoked Ca2+ and Mn2+ influx were blocked completely by Ni2+ but only partially by dihydropyridines, suggesting that, in addition to L-type Ca2+ channels, other Ca2+ entry pathways may be present. Inactivation of Ca2+ channels, followed by comparing the rates of Mn2+ uptake at different time periods after the addition of DMPP or high K+, did not happen in the absence of extracellular Ca2+. When 1 mM Ca2+ was present, a delayed inhibition (half time, 10-20 s) was observed, suggesting that it is not due to the entry of Ca2+ itself but to the increase of the cytoplasmic Ca2+ concentration ([Ca2+]i) that takes a few seconds to develop. The influx of Ca2+, estimated from the increase of [Ca2+]i, was also impaired in a time-dependent fashion by previous entry of Mn2+. Inactivation of Ca2+ entry was achieved at estimated mean intracellular Mn2+ concentrations as low as 10(-9) M.

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