title: Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C.
abstract: From experiments performed at room temperature, we know that the buffering of Ca(2+) by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca(2+)]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca(2+) transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca(2+) buffering to the shaping of [Ca(2+)]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca(2+) uniporter (mCUP) with Ru360 or the mitochondrial Na(+)/Ca(2+) exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca(2+) cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca(2+)]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca(2+)]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca(2+) uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca(2+)]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh-induced secretion, with minor changes of the ACh-induced [Ca(2+)]c transients. Combined Ru/CGP did not alter the resting membrane potential in current-clamped cells. Additionally, Ru/CGP did not increase basal [Ca(2+)]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh-elicited exocytotic response through its efficient Ca(2+) removal by the mCUP, M2 shapes the lower [Ca(2+)]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.
authors.affiliation: Departamento de Farmacología, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Afonso
authors.initials: A
authors.lastname: Caricati-Neto
authors.affiliation: Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, Madrid, 28029, Spain ; Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029Madrid, Spain.
authors.forename: Juan-Fernando
authors.initials: JF
authors.lastname: Padín
authors.affiliation: Departamento de Farmacología, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Edilson-Dantas
authors.initials: ED
authors.lastname: Silva-Junior
authors.affiliation: Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, Madrid, 28029, Spain ; Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029Madrid, Spain.
authors.forename: José-Carlos
authors.initials: JC
authors.lastname: Fernández-Morales
authors.affiliation: Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, Madrid, 28029, Spain ; Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029Madrid, Spain.
authors.forename: Antonio-Miguel G
authors.initials: AM
authors.lastname: de Diego
authors.affiliation: Departamento de Farmacología, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Aron
authors.initials: A
authors.lastname: Jurkiewicz
authors.affiliation: Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, Madrid, 28029, Spain ; Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029Madrid, Spain ; Servicio de Farmacología Clínica, Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, C/Diego de León, 62, Madrid, 28006, Spain.
authors.forename: Antonio G
authors.initials: AG
authors.lastname: García
keywords: CGP37157; Calcium homeostasis; FCCP; Ru360; catecholamines; chromaffin cells; exocytosis; mitochondria; oligomycin; rotenone
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