scholarly journals Synaptotagmin isoforms confer distinct activation kinetics and dynamics to chromaffin cell granules

2017 ◽  
Vol 149 (8) ◽  
pp. 763-780 ◽  
Author(s):  
Tejeshwar C. Rao ◽  
Zuleirys Santana Rodriguez ◽  
Mazdak M. Bradberry ◽  
Alexandra H. Ranski ◽  
Peter J. Dahl ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Functional Difference ◽  
Activation Kinetics ◽  
Fast Kinetics ◽  
Permeabilized Cells ◽  
Fusion Site ◽  
Kinetics And Dynamics ◽  
Synaptotagmin 1 ◽  
Dense Core Granules

Adrenomedullary chromaffin cells respond to sympathetic nervous system activation by secreting a cocktail of potent neuropeptides and hormones into the circulation. The distinct phases of the chromaffin cell secretory response have been attributed to the progressive fusion of distinct populations of dense core granules with different activation kinetics. However, it has been difficult to define what distinguishes these populations at the molecular level. Functional segregation of granule pools may depend on selective sorting of synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7), which our previous work showed are rarely cosorted to the same granule. Here we assess the consequences of selective sorting of Syt isoforms in chromaffin cells, particularly with respect to granule dynamics and activation kinetics. Upon depolarization of cells expressing fluorescent Syt isoforms using elevated K+, we find that Syt-7 granules fuse with faster kinetics than Syt-1 granules, irrespective of stimulation strength. Pharmacological blockade of Ca2+ channels reveals differential dependence of Syt-1 versus Syt-7 granule exocytosis on Ca2+ channel subtypes. Syt-7 granules also show a greater tendency to fuse in clusters than Syt-1 granules, and granules harboring Syt-1 travel a greater distance before fusion than those with Syt-7, suggesting that there is spatial and fusion-site heterogeneity among the two granule populations. However, the greatest functional difference between granule populations is their responsiveness to Ca2+. Upon introduction of Ca2+ into permeabilized cells, Syt-7 granules fuse with fast kinetics and high efficacy, even at low Ca2+ levels (e.g., when cells are weakly stimulated). Conversely, Syt-1 granules require a comparatively larger increase in intracellular Ca2+ for activation. At Ca2+ concentrations above 30 µM, activation kinetics are faster for Syt-1 granules than for Syt-7 granules. Our study provides evidence for functional specialization of chromaffin cell granules via selective expression of Syt isoforms with different Ca2+ sensitivities.

scholarly journals Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules

2014 ◽  
Vol 25 (16) ◽  
pp. 2416-2427 ◽  
Author(s):  
Tejeshwar C. Rao ◽  
Daniel R. Passmore ◽  
Andrew R. Peleman ◽  
Madhurima Das ◽  
Edwin R. Chapman ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Secretory Granules ◽  
Fusion Pore ◽  
Cargo Proteins ◽  
Granule Membrane ◽  
Synaptotagmin 1 ◽  
Physiological Homeostasis ◽  
Dense Core Granules ◽  
Adrenal Chromaffin ◽  
Granule Membrane Proteins

Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca2+-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.

scholarly journals Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins

1992 ◽  
Vol 284 (2) ◽  
pp. 321-326 ◽  
Author(s):  
G Ahnert-Hilger ◽  
U Wegenhorst ◽  
B Stecher ◽  
K Spicher ◽  
W Rosenthal ◽  
...  
Keyword(s):  
G Protein ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Prolonged Incubation ◽  
Permeabilized Cells ◽  
Alpha Subunits ◽  
Cytoplasmic Material ◽  
Streptolysin O ◽  
Adrenal Chromaffin

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

scholarly journals Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin.

1991 ◽  
Vol 113 (5) ◽  
pp. 1057-1067 ◽  
Author(s):  
M L Vitale ◽  
A Rodríguez Del Castillo ◽  
L Tchakarov ◽  
J M Trifaró
Keyword(s):  
Actin Filament ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Catecholamine Secretion ◽  
Cortical Region ◽  
Cortical Surface ◽  
Filamentous Actin ◽  
Cell Stimulation ◽  
Rhodamine Phalloidin ◽  
Filament Networks

Immunofluorescence and cytochemical studies have demonstrated that filamentous actin is mainly localized in the cortical surface of the chromaffin cell. It has been suggested that these actin filament networks act as a barrier to the secretory granules, impeding their contact with the plasma membrane. Stimulation of chromaffin cells produces a disassembly of actin filament networks, implying the removal of the barrier. The presence of gelsolin and scinderin, two Ca(2+)-dependent actin filament severing proteins, in the cortical surface of the chromaffin cells, suggests the possibility that cell stimulation brings about activation of one or more actin filament severing proteins with the consequent disruption of actin networks. Therefore, biochemical studies and fluorescence microscopy experiments with scinderin and gelsolin antibodies and rhodamine-phalloidin, a probe for filamentous actin, were performed in cultured chromaffin cells to study the distribution of scinderin, gelsolin, and filamentous actin during cell stimulation and to correlate the possible changes with catecholamine secretion. Here we report that during nicotinic stimulation or K(+)-evoked depolarization, subcortical scinderin but not gelsolin is redistributed and that this redistribution precedes catecholamine secretion. The rearrangement of scinderin in patches is mediated by nicotinic receptors. Cell stimulation produces similar patterns of distribution of scinderin and filamentous actin. However, after the removal of the stimulus, the recovery of scinderin cortical pattern of distribution is faster than F-actin reassembly, suggesting that scinderin is bound in the cortical region of the cell to a component other than F-actin. We also demonstrate that peripheral actin filament disassembly and subplasmalemmal scinderin redistribution are calcium-dependent events. Moreover, experiments with an antibody against dopamine-beta-hydroxylase suggest that exocytosis sites are preferentially localized to areas of F-actin disassembly.

The cytoskeleton as a barrier to exocytosis in secretory cells

1988 ◽  
Vol 139 (1) ◽  
pp. 253-266 ◽  
Author(s):  
D. Aunis ◽  
M. F. Bader
Keyword(s):  
Chromaffin Cells ◽  
Binding Proteins ◽  
Secretory Granules ◽  
Actin Binding ◽  
Bacterial Toxin ◽  
Secretory Cells ◽  
Free Access ◽  
Actin Binding Proteins ◽  
Permeabilized Cells ◽  
Cytoskeletal Network

Chromaffin cells of the adrenal medulla synthesize, store and secrete catecholamines. These cells contain numerous electron-dense secretory granules which discharge their contents into the extracellular space by exocytosis. The subplasmalemmal area of the chromaffin cell is characterized by the presence of a highly organized cytoskeletal network. F-Actin seems to be exclusively localized in this area and together with specific actin-binding proteins forms a dense viscoelastic gel; fodrin, vinculin and caldesmon, three actin cross-linking proteins, and gelsolin, an actin-severing protein, are found in this subplasmalemmal region. Since fodrin-, caldesmon- and alpha-actinin-binding sites exist on secretory granule membranes, actin filaments can also link secretory granules. Chromaffin granules can be entrapped in this subplasmalemmal lattice and thus the cytoskeleton acts as a barrier preventing exocytosis. When cells are stimulated, molecular rearrangements of the subplasmalemmal cytoskeleton take place: F-actin depolymerizes and fodrin reorganizes into patches. In addition, introduction of monospecific antifodrin immunoglobulins into digitonin-permeabilized cells blocks exocytosis, demonstrating the crucial role of this actin-binding protein. In bacterial toxin-permeabilized chromaffin cells, experiments using actin-perturbing agents such as cytochalasin D and DNAase I suggest that exocytosis is in part controlled by the cytoskeleton. The intracellular signal governing the cytoskeletal reorganization (associated with exocytosis) is calcium. Calcium inhibits some and activates other actin-binding proteins and consequently causes dissolution of the subplasmalemmal cytoskeleton. This dissolution of cytoskeletal filaments should result in granule detachment and permit granules free access to exocytotic sites on the plasma membrane.

2′,3′-Cyclic nucleotide 3′-phosphodiesterase is associated with mitochondria in diverse adrenal cell types

1997 ◽  
Vol 110 (23) ◽  
pp. 2979-2985
Author(s):  
B. McFerran ◽  
R. Burgoyne
Keyword(s):  
Cell Cultures ◽  
Cyclic Nucleotide ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Pcr Amplification ◽  
Cell Types ◽  
Adrenal Cell ◽  
Immunofluorescence Studies ◽  
Mitochondrial Staining ◽  
Intracellular Localisation

In this study, we have examined the expression and intracellular localisation of the myelin protein 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in bovine adrenal medullary chromaffin cell cultures. By immunoblotting, using two distinct anti-CNP monoclonal antibodies, CNP was detected in medullary cell cultures and expression of CNP was confirmed by reverse transcription and PCR amplification. CNP did not leak from digitonin-permeabilised chromaffin cells, suggesting that there is no cytosolic pool of this protein. Immunofluorescence studies with both antibodies showed that all cells in the medullary chromaffin cell culture were stained with a punctate appearance consistent with an intracellular localisation for CNP. More specifically it was demonstrated that CNP is co-localised with mitochondria. Various cell types in chromaffin cell cultures were stained with a mitochondrial pattern and CNP staining was co-localised with mitochondrial staining. These results show that CNP is a widely expressed protein that is associated with mitochondria and provides new clues as to its cellular function outside of myelin structures.

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.

scholarly journals A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers

1991 ◽  
Vol 274 (2) ◽  
pp. 339-347 ◽  
Author(s):  
J M Sontag ◽  
D Thierse ◽  
B Rouot ◽  
D Aunis ◽  
M F Bader
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Chromaffin Cells ◽  
Second Messengers ◽  
Catecholamine Release ◽  
Secretory Process ◽  
Permeabilized Cells ◽  
Arachidonic Acid Concentration

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.

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.

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