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.

scholarly journals Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells

1988 ◽  
Vol 256 (2) ◽  
pp. 343-350 ◽  
Author(s):  
S Cockcroft ◽  
J Stutchfield
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dimethyl Sulphoxide ◽  
Hl60 Cell ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Hl60 Cells ◽  
Permeabilized Cells ◽  
Streptolysin O

The promyelocytic HL60 cell can be differentiated with dimethyl sulphoxide or dibutyryl cyclic AMP leading to the appearance of fMetLeuPhe receptors on the cell surface. G-protein-stimulated polyphosphoinositide phosphodiesterase (PPI-pde) activity was assessed in membranes prepared from both differentiated and non-differentiated HL60 cells. Both the extent of the response and the rank order of potency of the GTP analogues to stimulate PPI-pde activation (guanosine 5′-[gamma-thio]triphosphate (GTP[S]) greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG) remains unchanged after differentiation with dimethyl sulphoxide. In comparison, differentiation by dibutyryl cyclic AMP leads to diminution of PPI-pde activity when stimulated by GTP[S] or fluoride, but not by millimolar concentrations of Ca2+. GTP[S]-stimulated PPI-pde in membranes is sensitive to the presence of Ca2+ (pCa 8-5). Pertussis-toxin pretreatment of intact HL60 cells leads to inhibition of both the secretory response and the formation of inositol phosphates when stimulated by fMetLeuPhe. In contrast, pertussis-toxin pretreatment has no effect on either GTP[S]- or fluoride-stimulated PPI-pde. Neomycin in a concentration-dependent manner inhibits both GTP[S] plus Ca2+ (pCa 5)-stimulated secretion and PPI-pde activation in streptolysin-O-permeabilized cells. The extent of PPI-pde activation in membranes compared with streptolysin-O-permeabilized cells reveals that the membrane preparation does not possess all the components that make up the inositide signalling system.

scholarly journals Exocytosis in chromaffin cells: evidence for a MgATP-independent step that requires a pertussis toxin-sensitive GTP-binding protein

1994 ◽  
Vol 300 (1) ◽  
pp. 217-227 ◽  
Author(s):  
N Vitale ◽  
D Thiersé ◽  
D Aunis ◽  
M F Bader
Keyword(s):  
Arachidonic Acid ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Chromaffin Cells ◽  
Potent Inhibitor ◽  
Secretory Granules ◽  
Neurosecretory Cells ◽  
Catecholamine Secretion ◽  
Heterotrimeric G Proteins ◽  
Permeabilized Cells

We have previously described that mastoparan, an amphiphilic tetradecapeptide that activates heterotrimeric G-proteins, inhibits Ca(2+)-induced MgATP-dependent secretion from streptolysin-O-permeabilized chromaffin cells [Vitale, Mukai, Rouot, Thiersé, Aunis and Bader (1993) J. Biol. Chem. 268, 14715-14723]. Our observations suggest the involvement of an inhibitory G(o)-protein, possibly located on the membrane of secretory granules, in the final stages of the exocytotic pathway in chromaffin cells. Here, we demonstrate that mastoparan is also able to stimulate the Ca(2+)-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally blocked by tetanus toxin, a potent inhibitor of exocytosis in all neurosecretory cells so far investigated, suggesting that the mastoparan target is a component of the exocytotic machinery. Mas17, a mastoparan analogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AlF4-, which selectively activates trimeric G-proteins, triggered MgATP-independent secretion. Non-hydrolysable GTP analogues (GTP[S] and p[NH]ppG) mimicked the dual effects of mastoparan on secretion: they inhibited exocytosis in the presence of MgATP and stimulated MgATP-independent secretion. The different potencies displayed by these two analogues suggest the involvement of two distinct G-proteins. Accordingly, the mastoparan-induced MgATP-independent secretion is highly sensitive to pertussis toxin (PTX) whereas the inhibition by mastoparan of secretion in the presence of MgATP is resistant to PTX treatment. When permeabilized cells were incubated with mastoparan, the release of arachidonic acid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoic acid, a potent inhibitor of intracellular phospholipase A2, inhibited both the arachidonate release and the MgATP-independent catecholamine secretion evoked by mastoparan. In contrast, neomycin, an inhibitor of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mastoparan. We conclude that two distinct heterotrimeric G-proteins act in series in the exocytotic pathway in chromaffin cells: one controls an ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca(2+)-dependent step which does not require MgATP but possibly involves the generation of arachidonic acid.

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 Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

1994 ◽  
Vol 3 (1) ◽  
pp. 45-51
Author(s):  
M. Gollasch ◽  
T. Kleppisch ◽  
D. Krautwurst ◽  
D. Lewinsohn ◽  
J. Hescheler
Keyword(s):  
Cyclic Amp ◽  
Guinea Pig ◽  
G Protein ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Guinea Pig Heart ◽  
Ventricular Cells ◽  
Inwardly Rectifying

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+currents (presumably of the IK1type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

scholarly journals Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

1994 ◽  
Vol 304 (2) ◽  
pp. 531-536 ◽  
Author(s):  
H Ohnishi ◽  
T Mine ◽  
I Kojima
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

scholarly journals Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production

2002 ◽  
Vol 172 (1) ◽  
pp. 95-104 ◽  
Author(s):  
AM Ronco ◽  
PF Moraga ◽  
MN Llanos
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Leydig Cells ◽  
Inhibitory Effect ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Camp Production

We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of PLA(2) and G proteins in the release of AA from hCG-stimulated Leydig cells, and under particular conditions, regulation of cAMP production by this fatty acid in these cells.

Pertussis toxin facilitates secretagogue-induced catecholamine release from cultured bovine adrenal chromaffin cells

1987 ◽  
Vol 144 (2) ◽  
pp. 907-914 ◽  
Author(s):  
Teruo Tanaka ◽  
Hiromitsu Yokohama ◽  
Manabu Negishi ◽  
Hideya Hayashi ◽  
Seiji Ito ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Chromaffin Cells ◽  
Catecholamine Release ◽  
Adrenal Chromaffin Cells ◽  
Adrenal Chromaffin

scholarly journals Erythropoietin stimulates G-protein-coupled phospholipase D in haematopoietic target cells

1996 ◽  
Vol 314 (3) ◽  
pp. 853-860 ◽  
Author(s):  
Sanda CLEJAN ◽  
Conrad MALLIA ◽  
David VINSON ◽  
Robert DOTSON ◽  
Barbara S. BECKMAN
Keyword(s):  
G Protein ◽  
Phospholipase D ◽  
Second Messengers ◽  
Haematopoietic Stem Cell ◽  
Target Cells ◽  
High Concentration ◽  
Guanine Nucleotide Binding Protein ◽  
Maximal Increase ◽  
Permeabilized Cells ◽  
Early Peak

A murine haematopoietic stem-cell line, B6SUt.EP, responsive to erythropoietin (EPO), has been found to exhibit both early and late changes in diacylglycerol (DAG) and phosphatidic acid (PA) as measured by HPLC and TLC. DAG levels peaked at 5 s with a 28.1% increase compared with control levels (from 17.3 to 22.2 pmol/106 cells) with a later peak at 30 min (84.2% increase from 17.3 to 31.9 pmol). These changes were concentration-dependent from 0.025 to 10 units/ml EPO (5 s, EC50 = 0.82 unit/ml; 30 min, EC50 = 0.10 unit/ml). In addition, PA levels increased 752.3% compared with control levels (from 8.6 to 64.7 μg/106 cells) with an early peak at 20 s, as measured by both HPLC and TLC (5 s, EC50 = 0.07 unit/ml). G-protein regulation was investigated by studying the effects of the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate (GTP[S]) on PA synthesis. The addition of GTP[S] (10 μM) in permeabilized cells increased PA content from 6.3 μg to 48.6 μg per 106 cells. In the presence of EPO and GTP[S], PA levels increased to 64.8 μg. An antagonist of G-proteins, guanosine 5´-[β-thio]diphosphate (GDP[S]), had no effect on control levels of PA (5.9 μg/106 cells) but blocked the effect of EPO on PA (30.6 μg/106 cells). Thus, EPO stimulated both lipid second messengers, DAG and PA. Our results demonstrate DAG kinetics to be biphasic, as observed with a high concentration of EPO, or monophasic, as observed with low concentrations of EPO. The PA accumulation preceding that of DAG in the slower and sustaining phase suggests that PA was not derived from DAG. This was confirmed by the stimulation of PA (without ATP) by GTP[S], effectively excluding phosphorylation of DAG by DAG kinase in the formation of PA. In addition, phospholipase D (PLD) activation was demonstrated with a maximal increase in phosphatidylethanol at 5 min, suggesting that EPO increases PA via a guanine nucleotide-binding protein coupled to PLD. The temporal relationship of the evolution of PA and DAG is further strengthened by experiments with ethanol and propranolol as inhibitors of the DAG/PA phosphohydrolase reaction and R59022 as an inhibitor of the DAG kinase reaction.

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