Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C

Physiological Reports

Afonso Caricati-Neto²  Juan-Fernando Padín¹  Edilson-Dantas Silva-Junior²  José-Carlos Fernández-Morales¹  Antonio-Miguel G. de Diego¹  Aron Jurkiewicz² 

[1] Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain;Departamento de Farmacología, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

关键词: Calcium homeostasis;    catecholamines;    CGP37157;    chromaffin cells;    exocytosis;    FCCP;    mitochondria;    oligomycin;    rotenone;    Ru360;   

DOI  :  10.1002/phy2.182

来源: Wiley

【 摘 要 】

Abstract

From experiments performed at room temperature, we know that the buffering of Ca²⁺ by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca²⁺]_c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca²⁺ 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 Ca²⁺ buffering to the shaping of [Ca²⁺]_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 Ca²⁺ uniporter (mCUP) with Ru360 or the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca²⁺ cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca²⁺]_c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca²⁺]_c clearance and a mild decrease in secretion. The mCC resulting in continued Ca²⁺ 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 [Ca²⁺]_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 [Ca²⁺]_c transients. Combined Ru/CGP did not alter the resting membrane potential in current-clamped cells. Additionally, Ru/CGP did not increase basal [Ca²⁺]_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 Ca²⁺ removal by the mCUP, M2 shapes the lower [Ca²⁺]_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.

【 授权许可】

CC BY   
© 2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

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