Control of exocytosis in adrenal chromaffin cells by GTP-binding proteins studied using permeabilized cells and patch-clamp capacitance measurements

1994 ◽  
Vol 22 (2) ◽  
pp. 468-472 ◽  
Author(s):  
Robert D. Burgoyne ◽  
Susan E. Handel ◽  
Alan Morgan
Keyword(s):  
Patch Clamp ◽  
Chromaffin Cells ◽  
Binding Proteins ◽  
Adrenal Chromaffin Cells ◽  
Permeabilized Cells ◽  
Capacitance Measurements ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Adrenal Chromaffin

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 Patch clamp study on dopaminergic inhibition of adrenal chromaffin cells

1989 ◽  
Vol 49 ◽  
pp. 178
Author(s):  
Yasutake Mine ◽  
Akinori Akaike ◽  
Masashi Sasa ◽  
Shuji Takaori
Keyword(s):  
Patch Clamp ◽  
Chromaffin Cells ◽  
Adrenal Chromaffin Cells ◽  
Adrenal Chromaffin ◽  
Clamp Study

scholarly journals Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

1994 ◽  
Vol 126 (4) ◽  
pp. 1005-1015 ◽  
Author(s):  
J C Norman ◽  
L S Price ◽  
A J Ridley ◽  
A Hall ◽  
A Koffer
Keyword(s):  
Mast Cells ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Small Gtpases ◽  
Secretory Cells ◽  
Cortical Region ◽  
Permeabilized Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.

Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

2011 ◽  
Vol 301 (1) ◽  
pp. C86-C98 ◽  
Author(s):  
Juliana M. Rosa ◽  
Cristina J. Torregrosa-Hetland ◽  
Inés Colmena ◽  
Luis M. Gutiérrez ◽  
Antonio G. García ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Chromaffin Cells ◽  
Channel Blocker ◽  
Primary Cultures ◽  
Adrenal Chromaffin Cells ◽  
Cell Functions ◽  
Fluorescence Techniques ◽  
Voltage Dependent ◽  
Bovine Chromaffin Cells ◽  
Adrenal Chromaffin

Calcium (Ca2+)-dependent endocytosis has been linked to preferential Ca2+ entry through the L-type (α1D, CaV1.3) of voltage-dependent Ca2+ channels (VDCCs). Considering that the Ca2+-dependent exocytotic release of neurotransmitters is mostly triggered by Ca2+ entry through N-(α1B, CaV2.2) or PQ-VDCCs (α1A, CaV2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca2+ current ( ICa), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocytotic responses triggered by long depolarizing stimuli, in 1, 2, or 10 mM concentrations of extracellular Ca2+ ([Ca2+]e). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca2+ caused substantially smaller endocytotic responses compared with those produced by K+ depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clathrin was found; a 20–30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca2+ entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.

scholarly journals Stimulation of Ca2+-independent catecholamine secretion from digitonin-permeabilized bovine adrenal chromaffin cells by guanine nucleotide analogues. Relationship to arachidonate release

1990 ◽  
Vol 269 (2) ◽  
pp. 521-526 ◽  
Author(s):  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Arachidonic Acid ◽  
Chromaffin Cells ◽  
Binding Protein ◽  
Catecholamine Secretion ◽  
Arachidonic Acid Release ◽  
Adrenal Chromaffin Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Acid Release ◽  
Adrenal Chromaffin

The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5′-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.

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