G protein βγ Subunits Modulate the Number and Nature of Exocytotic Fusion Events in Adrenal Chromaffin Cells Independent of Calcium Entry

G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca2+ channels (Ca2+ channels) by G protein βγ subunits (Gβγ) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca2+-channel-independent effects of Gβγ on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gβγ on Ca2+ entry, we stimulated secretion using ionomycin (a Ca2+ ionophore) or direct intracellular application of Ca2+ through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gβγ significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge (“quantal size”) and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gβγ. We conclude that independent from effects on calcium entry, Gβγ can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gβγ might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.

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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

Biochemical Journal ◽

10.1042/bj2840321 ◽

1992 ◽

Vol 284 (2) ◽

pp. 321-326 ◽

Cited By ~ 27

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.

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A comparison of bradykinin, angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells

Bioscience Reports ◽

10.1007/bf01116001 ◽

1989 ◽

Vol 9 (2) ◽

pp. 243-252 ◽

Cited By ~ 58

Author(s):

A. J. O'Sullivan ◽

R. D. Burgoyne

Keyword(s):

Angiotensin Ii ◽

Chromaffin Cells ◽

Calcium Concentration ◽

Calcium Entry ◽

Free Calcium ◽

Adrenal Chromaffin Cells ◽

Free Calcium Concentration ◽

Adrenal Chromaffin ◽

External Calcium ◽

Stimulation Of

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca2+]i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca2+]i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca2+]i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca2+]i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca2+]i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.

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Colocalization of calcium entry and exocytotic release sites in adrenal chromaffin cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.7.2474 ◽

1995 ◽

Vol 92 (7) ◽

pp. 2474-2478 ◽

Cited By ~ 89

Author(s):

I. M. Robinson ◽

J. M. Finnegan ◽

J. R. Monck ◽

R. M. Wightman ◽

J. M. Fernandez

Keyword(s):

Chromaffin Cells ◽

Calcium Entry ◽

Adrenal Chromaffin Cells ◽

Adrenal Chromaffin

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Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0188 ◽

2015 ◽

Vol 370 (1672) ◽

pp. 20140188 ◽

Cited By ~ 3

Author(s):

Lin-Ling He ◽

Quan-Feng Zhang ◽

Lie-Cheng Wang ◽

Jing-Xia Dai ◽

Chang-He Wang ◽

...

Keyword(s):

Protein Kinase ◽

G Protein ◽

Acetylcholine Receptors ◽

Chromaffin Cells ◽

Sympathetic Neurons ◽

Second Messenger ◽

Muscarinic Acetylcholine Receptors ◽

Adrenal Chromaffin Cells ◽

Adrenal Chromaffin ◽

Ganglion Neurons

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide–Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

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SNAP-25 is present in a SNARE complex in adrenal chromaffin cells

FEBS Letters ◽

10.1016/0014-5793(94)00833-7 ◽

1994 ◽

Vol 351 (2) ◽

pp. 207-210 ◽

Cited By ~ 69

Author(s):

Dagmar Roth ◽

Robert D. Burgoyne

Keyword(s):

Chromaffin Cells ◽

Snare Complex ◽

Adrenal Chromaffin Cells ◽

Adrenal Chromaffin

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Morphological Observations on the Granule Types in Rabbit Extra-Adrenal Chromaffin Cells.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115213 ◽

1975 ◽

Vol 33 ◽

pp. 318-319

Author(s):

Joe A. Mascorro ◽

Robert D. Yates

Keyword(s):

Chromaffin Cells ◽

Sodium Phosphate ◽

Ganglion Cells ◽

Ultrastructural Level ◽

Osmic Acid ◽

Adrenal Chromaffin Cells ◽

Potassium Iodate ◽

Perfusion Fluid ◽

New Zealand White Rabbits ◽

Adrenal Chromaffin

Extra-adrenal chromaffin organs (abdominal paraganglia) constitute rich sources of catecholamines. It is believed that these bodies contain norepinephrine exclusively. However, the present workers recently observed epinephrine type granules in para- ganglion cells. This report investigates catecholamine containing granules in rabbit paraganglia at the ultrastructural level.New Zealand white rabbits (150-170 grams) were anesthetized with 50 mg/kg Nembutal (IP) and perfused with 3% glutaraldehyde buffered with 0.2M sodium phosphate, pH 7.3. The retroperitoneal tissue blocks were removed and placed in perfusion fluid for 4 hours. The abdominal paraganglia were dissected from the blocks, diced, washed in phosphate buffer and fixed in 1% osmic acid buffered with phosphate. In other animals, the glutaraldehyde perfused tissue blocks were immersed for 1 hour in 3% glutaraldehyde/2.5% potassium iodate buffered as before. The paraganglia were then diced, separated into two vials and washed in the buffer. A portion of this tissue received osmic acid fixation.

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