Differential regulation by mitochondria of calcium currents, calcium transients and quantal catecholamine release from chromaffin cells of rat embryos and their mothers

S Vestring1,2, JC Fernández-Morales1, F Padín1, AM García de Diego1, M Maroto1, A García García1. 1Universidad Autónoma de Madrid, Departamento de Farmacología y Terapéutica, Facultad de Medicina, 28029, Spain, 2Technische Universität Dresden, Medizinische Fakultät, 01307, Germany

In chromaffin cells, the catecholamine release responses are shaped by a functional tetrad formed by the subtypes of voltaje-dependent Ca2+ channels (VDCCs), Ca2+ buffer proteins, mitochondria and the endoplasmic reticulum. We discovered that the L-subtype of VDCCs dominate the control of the quantal catecholamine release responses in rat embryo chromaffin cells (ECCs); in contrast, in their mother’s chromaffin cells (MCCs) this response was controlled by L- as well as N- and PQ-types of VDCCs1. Hence we have investigated the role of another component of the functional tetrad, namely the mitochondrion, on the secretion response elicited by depolarizing 30 mM K+ pulses using various pharmacological manipulations to interfere with the mitochondrial Ca2+ cycling occurring during cell stimulation.

Chromaffin cells were obtained from pregnant Wistar rats and from their 18-day-old rat embryos2. Changes of the cytosolic Ca2+ concentrations ([Ca2+]c) were measured using the calcium probe Fura-2 AM (10 µM). The setup for fluorescence recordings was composed of a Leica DMI 4000B inverted light microscope (Leica, Spain) equipped with an oil immersion objective (Leica 40x Plan Apo). Amperometry was used to measure catecholamine release at the single cell level. The experiments carried out were of a sandwich type where 30 mM K+ was applied during 10 s (P1); before and during the second high K+ pulse the drug was perifused (P2); finally the drug was washed out for 5 minutes with Tyrode solution and the recovery observed upon another 30 mM K+ pulse (P3).

Regarding [Ca2+]c data, statistical analyses were carried out with ANOVA one-way test and Tukey post-hoc analyses. For amperometric recordings, differences between means of group data fitting a normal distribution were assessed by using either analysis of variance or Kruskal-Wallis test for comparison among multiple groups, or Student’s t test for comparison between two groups. p<0.05 was taken as the limit of significance.

We discovered that the blocker of the Ca2+ uniporter, Ru360 unexpectedly produced a 57.9 ± 9.9% (n=14; N=3) blockade of the quantal release response in ECCs, but not in MCCs. Such retraction is accompanied by smaller cytosolic Ca2+ transients in ECCs. On the other hand, blockade with CGP37157 of the mitochondrial Na+/Ca2+ exchanger did not alter the quantal release of catecholamine in ECCs but blocked that of MCCs (74.5 ± 4.8%; n=13; N=4). As expected, the protonophore, FCCP enhanced the K+ secretory response although this effect seemed to be greater in ECCs (143.5 ± 16.5% of the electric charge; 212.7 ± 25.5% rise in the number of spike events; n=10; N=3), compared to MCCs (142.8 ± 17.8% of the electric charge; 128.6 ± 8.6% rise in the number of spike events; n=11; N=3). EM analysis indicated that mitochondria seemed to be closer to plasmalemmal exocytotic sites in ECCs; compared with MCCs.

We conclude that mitochondria seem to play a more relevant role in shaping Ca2+ and exocytotic signals in ECCs, as compared with mature and innervated MCCs; this may be due to different local distribution of mitochondria in both cell types. These results are relevant in the clinical context of fetal and neonate adaption to hypoxic and other stress responses, as well as to understand the pathogenesis of infant sudden death syndrome.

1 Fernandez-Morales JC et al., (2009). Am J Physiol Cell Physiol 297(2):C407-18.

2 Sorensen JB et al., (2003). Cell 114:75-86.