Primary culture of bovine chromaffin cells

O’Connor, Daniel T.; Mahata, Sushil K.; Mahata, Manjula; Jiang, Qijiao; Hook, Vivian; Taupenot, Laurent

doi:10.1038/nprot.2007.136

Cited by 31 publications

(34 citation statements)

References 24 publications

(10 reference statements)

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“…Pheochromocytoma PC12 cells and primary chromaffin cells isolated from fresh bovine medulla were cultured as described previously (22,23). In some experiments, PC12 cells were treated with nerve growth factor (NGF; 2.5 S, 100 ng/ml; Invitrogen) 48 h prior to transfection and further differentiated by NGF for an additional 48 h.…”

Section: Cell Culturementioning

confidence: 99%

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

Courel

Vasquez

Hook

et al. 2008

Journal of Biological Chemistry

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Section: Cell Culturementioning

confidence: 99%

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

Courel

Vasquez

Hook

et al. 2008

Journal of Biological Chemistry

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“…Chromaffin cells were prepared by collagenase digestion of medullary tissue followed by further separation from debris and erythrocytes by centrifugation on a Percoll gradient (GE-Amersham Biosciences, Uppsala, Sweden) as described. 19 Details of transfection, preparation of carbon fiber electrodes, and amperometric recordings are described in appendix e-3.…”

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confidence: 99%

Mutant SNAP25B causes myasthenia, cortical hyperexcitability, ataxia, and intellectual disability

et al. 2014

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Objective: To identify and characterize the molecular basis of a syndrome associated with myasthenia, cortical hyperexcitability, cerebellar ataxia, and intellectual disability.Methods: We performed in vitro microelectrode studies of neuromuscular transmission, performed exome and Sanger sequencing, and analyzed functional consequences of the identified mutation in expression studies.Results: Neuromuscular transmission at patient endplates was compromised by reduced evoked quantal release. Exome sequencing identified a dominant de novo variant, p.Ile67Asn, in SNAP25B, a SNARE protein essential for exocytosis of synaptic vesicles from nerve terminals and of dense-core vesicles from endocrine cells. Ca 21 -triggered exocytosis is initiated when synaptobrevin attached to synaptic vesicles (v-SNARE) assembles with SNAP25B and syntaxin anchored in the presynaptic membrane (t-SNAREs) into an a-helical coiled-coil held together by hydrophobic interactions. Pathogenicity of the Ile67Asn mutation was confirmed by 2 measures. First, the Ca 21 triggered fusion of liposomes incorporating v-SNARE with liposomes containing t-SNAREs was hindered when t-SNAREs harbored the mutant SNAP25B moiety. Second, depolarization of bovine chromaffin cells transfected with mutant SNAP25B or with mutant plus wildtype SNAP25B markedly reduced depolarization-evoked exocytosis compared with wild-type transfected cells. Conclusion:Ile67Asn variant in SNAP25B is pathogenic because it inhibits synaptic vesicle exocytosis. We attribute the deleterious effects of the mutation to disruption of the hydrophobic a-helical coiled-coil structure of the SNARE complex by replacement of a highly hydrophobic isoleucine by a strongly hydrophilic asparagine. Neurology ® 2014;83:2247-2255 GLOSSARY AChR 5 acetylcholine receptor; cDNA 5 complementary DNA; CMS 5 congenital myasthenic syndrome; EP 5 endplate; MEPP 5 miniature endplate potential; SNAP25B 5 synaptosomal-associated protein, 25B; SNARE 5 soluble N-ethylmaleimide-sensitive factor attachment protein receptor; t-SNAREs 5 target membrane-attached SNAP25B and syntaxin; v-SNARE 5 synaptic vesicle-attached synaptobrevin.The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are core components of the synaptic vesicle fusion machinery. Synaptobrevin attached to the synaptic vesicles is referred to as a v-SNARE, and SNAP25 (synaptosomal-associated protein of 25 kD) and syntaxin attached to the target plasma membrane are designated as t-SNAREs.1 The assembled complex is a coiled-coil in which a-helices are held together by strong hydrophobic interactions. The complex is stabilized by complexin, a small soluble neuronal protein.2 In the resting state, Munc18 binds to a closed form of syntaxin and blocks formation of the SNARE complex but assists the SNARE complex to effect exocytosis in the active state. 3 SNAP25 also participates in endocytosis at hippocampal synapses 4 and negatively modulates the neuronal voltage-gated calcium channel during intense activity.5 Also, ...

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“…Activity-dependent secretion of neurotransmitters from neuronal-like chromaffin cells was stimulated by KCl depolarization which represents regulated secretion and by nicotine which activates the nicotinic cholinergic receptor [38, 40–42]. KCl depolarization and nicotine activation of the cholinergic receptor of neuronal-like chromaffin cells are utilized in the field to assess activity-dependent, regulated secretion [35, 40, 43–50].…”

Section: Resultsmentioning

confidence: 99%

“…Chromaffin cells were prepared from fresh adrenal medulla tissue (bovine) as we have previously described [25, 35, 38]. Briefly, chromaffin cells were dissected from fresh adrenal glands, dissociated in a collagenase/DNase solution at 37°C, filtered, and centrifuged.…”

Section: Methodsmentioning

confidence: 99%

Pyroglutamate-Amyloid-β and Glutaminyl Cyclase Are Colocalized with Amyloid-β in Secretory Vesicles and Undergo Activity-Dependent, Regulated Secretion

Cynis¹,

Funkelstein

Toneff

et al. 2014

Neurodegener Dis

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Background and Aims: N-truncated pyroglutamate (pGlu)-amyloid-β [Aβ(3-40/42)] peptides are key components that promote Aβ peptide accumulation, leading to neurodegeneration and memory loss in Alzheimer's disease. Because Aβ deposition in the brain occurs in an activity-dependent manner, it is important to define the subcellular organelle for pGlu-Aβ(3-40/42) production by glutaminyl cyclase (QC) and their colocalization with full-length Aβ(1-40/42) peptides for activity-dependent, regulated secretion. Therefore, the objective of this study was to investigate the hypothesis that pGlu-Aβ and QC are colocalized with Aβ in dense-core secretory vesicles (DCSV) for activity-dependent secretion with neurotransmitters. Methods: Purified DCSV were assessed for pGlu-Aβ(3-40/42), Aβ(1-40/42), QC, and neurotransmitter secretion. Neuron-like chromaffin cells were analyzed for cosecretion of pGlu-Aβ, QC, Aβ, and neuropeptides. The cells were treated with a QC inhibitor, and pGlu-Aβ production was measured. Human neuroblastoma cells were also examined for pGlu-Aβ and QC secretion. Results: Isolated DCSV contain pGlu-Aβ(3-40/42), QC, and Aβ(1-40/42) with neuropeptide and catecholamine neurotransmitters. Cellular pGlu-Aβ and QC undergo activity-dependent cosecretion with Aβ and enkephalin and galanin neurotransmitters. The QC inhibitor decreased the level of secreted pGlu-Aβ. The human neuroblastoma cells displayed regulated secretion of pGlu-Aβ that was colocalized with QC. Conclusions: pGlu-Aβ and QC are present with Aβ in DCSV and undergo activity-dependent, regulated cosecretion with neurotransmitters.

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Primary culture of bovine chromaffin cells

Cited by 31 publications

References 24 publications

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

Mutant SNAP25B causes myasthenia, cortical hyperexcitability, ataxia, and intellectual disability

Pyroglutamate-Amyloid-β and Glutaminyl Cyclase Are Colocalized with Amyloid-β in Secretory Vesicles and Undergo Activity-Dependent, Regulated Secretion

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