title: The quantal catecholamine release from mouse chromaffin cells challenged with repeated ACh pulses is regulated by the mitochondrial Na+/Ca2+exchanger.
abstract: Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. A subsequent K+pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release-vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh-sensitive vesicle pool is refilled and maintained by the rate of Ca2+delivery from mitochondria to the cytosol, through the mitochondrial Na+/Ca2+exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+]cclearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca2+circulation through the mNCX, which couples respiration and ATP synthesis to long-term stimulation of chromaffin cells by endogenously released ACh.
  
  
  
  Using caged-Ca2+photorelease or paired depolarising pulses in voltage-clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve-CC synapse, is unknown. We have explored here whether an ACh-sensitive ready-release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 μm, 2 s, every 30 s) plus a K+pulse given at the end (75 mm K+). After the first 2-3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. The last K+pulse, however, overcame such decay. Repeated ACh pulses to voltage-clamped cells elicited non-desensitising nicotinic currents. Also, the [Ca2+]ctransients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+/Ca2+exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+]cclearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+delivery from mitochondria to the cytosol, through the mNCX.
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.
authors.forename: Angela
authors.initials: A
authors.lastname: López-Gil
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Nanclares
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.
authors.forename: Iago
authors.initials: I
authors.lastname: Méndez-López
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Martínez-Ramírez
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, c/ Diego de León, 62, 28006, Madrid, Spain.
authors.forename: Cristóbal
authors.initials: C
authors.lastname: de Los Rios
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.
authors.forename: J Fernando
authors.initials: JF
authors.lastname: Padín-Nogueira
authors.affiliation: Instituto de Biologia y Genética Molecular, Universidad de Valladolid, c/ Sanz y Forés, 3, 47003, Valladolid, Spain.
authors.forename: Mayte
authors.initials: M
authors.lastname: Montero
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.
authors.forename: Luis
authors.identifier: 0000-0001-9597-8519
authors.initials: L
authors.lastname: Gandía
authors.affiliation: Instituto Teófilo Hernando, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/ Arzobispo Morcillo, 4, 28029, Madrid, Spain.|Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, c/ Diego de León, 62, 28006, Madrid, Spain.
authors.forename: Antonio G
authors.initials: AG
authors.lastname: García
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002395:Catecholamines / Q000502:physiology*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000502:physiology; D011188:Potassium / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000502:physiology*
chemical_list: D002395:Catecholamines; D019831:Sodium-Calcium Exchanger; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium
keywords: C57BL6J; GCP37157; ITH12662; acetylcholine; mitochondrial sodium/calcium exchanger; mouse chromaffin cells; quantal release of catecholamine