Measurement of exocytosis by amperometry in adrenal chromaffin cells: Effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics

Graham, Margaret E.; Fisher, Richard; Burgoyne, Robert D.

doi:10.1016/s0300-9084(00)00196-6

Cited by 94 publications

(107 citation statements)

References 53 publications

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“…However, partial fusion events (kiss and run) that might alter neurotransmitter release have been proposed. In neuroendocrine cells, large dense-core vesicles can transiently fuse with the membrane (12) to vary release (13,14) an effect that may be caused by G␤␥ (15). Synaptic vesicles may also partially fuse (16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

Photowala

Blackmer

Schwartz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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mentioning

confidence: 99%

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

Photowala

Blackmer

Schwartz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…To overcome this limitation, fusion constructs between enhanced green fluorescent protein (EGFP) (5) and PKC␥ (6), PKC␣ (7), PKC␦ (8), and PKC␤II (9) have recently been used to monitor the dynamics of membrane translocation of PKCs in a number of non-excitable cell types and appear faithfully to reflect the behavior of the endogenous PKC isoforms. However, while PKC may play an important role in agonist stimulation of exocytosis from neurosecretory cells (10), no data are presently available on the dynamics of conventional PKCs in any excitable cell type.…”

mentioning

confidence: 99%

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Pinton¹,

Tsuboi²,

Ainscow³

et al. 2002

Journal of Biological Chemistry

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The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet ␤-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (␤II), novel (␦), or atypical () PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 ␤-cells.

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“…Maximum transfection efficiency was 45 % at 1,400 V (Fig. 2c) requires 10 7 cells while achieving only 2-5 % transfection efficiency (Graham et al 2000). Electroporations for all experiments below were done at 1,400 V. We next determined the extent to which the developed electroporation method allows co-transfection of individual electroporated cells with multiple plasmids.…”

Section: Resultsmentioning

confidence: 99%

“…Electrochemical amperometric recordings employed 5 lm carbon fiber electrodes (CFE) (ALA, Farmingdale, NY, USA), as previously described (Barclay et al 2003;Graham et al 2000). Cells were stimulated by perfusion for 2 min with 75 mM K ?…”

Section: Electrophysiologymentioning

confidence: 99%

“…Since first proposed (Alesci et al 2007;Graham et al 2000), electroporation has become a potent and useful tool mediating prokaryote plasmid transduction and efficient eukaryote transfection. Unfortunately, its conventionally large cell number requirement has precluded its use when limited tissue mass, as with mouse adrenal glands, results in few isolated primary cells.…”

Section: Introductionmentioning

confidence: 99%

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Efficient transfection of dissociated mouse chromaffin cells using small-volume electroporation

et al. 2014

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We have developed an improved procedure for isolating and transfecting a chromaffin cellenriched population of primary cells from adult mouse adrenal glands. Significantly, the parameters of a novel electroporation transfection technique were optimized to achieve an average transfection efficiency of 45 % on the small number of cells derived from the mouse glands. Such transfection efficiency was previously unachievable with the electroporation protocols conventionally used with bovine chromaffin cells, even with use of large cell numbers. Our small scale technique now makes feasible the use of genetically homogenous inbred mouse models for investigations on the exocytotic pathway without the time, expense, and cellular changes associated with viral approaches. High fidelity co-expression of multiple plasmids in individual cells is a further advantage of the procedure. To assess whether the biophysical characteristics of mouse adrenal chromaffin cells were altered by this process, we examined structural integrity using immunocytochemistry and functional response to stimuli using calcium imaging, amperometry, and whole-cell capacitance and current clamp recordings. We conclude these parameters are minimally affected. Finally, we demonstrate that high transfection efficiency makes possible the use of primary mouse adrenal chromaffin cells, rather than a cell line, in human growth hormone secretion assays for high throughput evaluation of secretion.

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Measurement of exocytosis by amperometry in adrenal chromaffin cells: Effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics

Cited by 94 publications

References 53 publications

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C βII in Living Pancreatic Islet β-Cells

Efficient transfection of dissociated mouse chromaffin cells using small-volume electroporation

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