Mitochondrial sodium/calcium exchanger (NCLX) regulates basal and starvation-induced autophagy through calcium signaling.

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporters and efflux by Na+ /Ca2+ exchangers (NCLX). Here, we describe a novel relationship among NCLX, intracellular Ca2+ , and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa.

Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca(2+) and protein tyrosine phosphorylation. The sites that regulate intracellular Ca(2+) concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca(2+) influx and efflux. Our aim was to study the involvement of mitochondrial Ca(2+) cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45 min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca(2+) efflux; (c) H+RU 360, a specific inhibitor of Ca(2+) influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20 µM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5 µM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.

Hyposmolarity evokes norepinephrine efflux from synaptosomes by a depolarization- and Ca2+ -dependent exocytotic mechanism.

Osmolarity reduction (20%) elicited 3H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na+-dependent and La3+-, Gd3+- and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca2+ ([Ca2+]i) rise with contributions from external Ca2+ influx and internal Ca2+ release, probably through the mitochondrial Na+-Ca2+ exchanger; and (iii) activation of a [Ca2+]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca2+]i rise by preventing depolarization with La3+, and by the inhibitory effects of Ca2+-free medium (EGTA; 50%), CGP37157 (the mitochondrial Na+-Ca2+ exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na+ omission, 75% by La3+, 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca2+ release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K+-stimulated NE release, studied in parallel, was unaffected by Na+ omission, or by La3+, Gd3+ or ruthenium red. It was fully dependent on external Ca2+, insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K+-evoked depolarization and [Ca2+]i rise mechanisms, are able to trigger a depolarization-dependent, Ca2+-dependent and TeTX-sensitive mechanism for neurotransmitter release.