The dynamics of mitochondrial Ca2+ fluxes

AbstractWe have investigated the kinetics of mitochondrial Ca2+ influx and efflux and their dependence on cytosolic [Ca2+] and [Na+] using low-Ca2+-affinity aequorin. The rate of Ca2+ release from mitochondria increased linearly with mitochondrial [Ca2+] ([Ca2+]M). Na+-dependent Ca2+ release was predominant al low [Ca2+]M but saturated at [Ca2+]M around 400μM, while Na+-independent Ca2+ release was very slow at [Ca2+]M below 200μM, and then increased at higher [Ca2+]M, perhaps through the opening of a new pathway. Half-maximal activation of Na+-dependent Ca2+ release occurred at 5–10mM [Na+], within the physiological range of cytosolic [Na+]. Ca2+ entry rates were comparable in size to Ca2+ exit rates at cytosolic [Ca2+] ([Ca2+]c) below 7μM, but the rate of uptake was dramatically accelerated at higher [Ca2+]c. As a consequence, the presence of [Na+] considerably reduced the rate of [Ca2+]M increase at [Ca2+]c below 7μM, but its effect was hardly appreciable at 10μM [Ca2+]c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca2+]M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca2+ buffering, and comparison of our results with data on total mitochondrial Ca2+ fluxes indicate that the mitochondrial Ca2+ bound/Ca2+ free ratio is around 10- to 100-fold for most of the observed [Ca2+]M range and suggest that massive phosphate precipitation can only occur when [Ca2+]M reaches the millimolar range

Germline genetic findings which may impact therapeutic decisions in families with a presumed predisposition for hereditary breast and ovarian cancer

In this study, we aim to gain insight in the germline mutation spectrum of ATM, BARD1, BRIP1, ERCC4, PALB2, RAD51C and RAD51D in breast and ovarian cancer families from Spain. We have selected 180 index cases in whom a germline mutation in BRCA1 and BRCA2 was previously ruled out. The importance of disease-causing variants in these genes lies in the fact that they may have possible therapeutic implications according to clinical guidelines. All variants were assessed by combined annotation dependent depletion (CADD) for scoring their deleteriousness. In addition, we used the cancer genome interpreter to explore the implications of some variants in drug response. Finally, we compiled and evaluated the family history to assess whether carrying a pathogenic mutation was associated with age at diagnosis, tumour diversity of the pedigree and total number of cancer cases in the family. Eight unequivocal pathogenic mutations were found and another fourteen were prioritized as possible causal variants. Some of these molecular results could contribute to cancer diagnosis, treatment selection and prevention. We found a statistically significant association between tumour diversity in the family and carrying a variant with a high score predicting pathogenicity (p = 0.0003)

Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice

The study was partially presented as a poster in the 53rd Annual Meeting of the European Association for the Study of Diabetes, Lisbon 2017.The role of insulin-degrading enzyme (IDE), a metalloprotease with high affinity for insulin, in insulin clearance remains poorly understood. OBJECTIVE: This study aimed to clarify whether IDE is a major mediator of insulin clearance, and to define its role in the etiology of hepatic insulin resistance.[Methods] We generated mice with liver-specific deletion of Ide (L-IDE-KO) and assessed insulin clearance and action.[Results] L-IDE-KO mice exhibited higher (~20%) fasting and non-fasting plasma glucose levels, glucose intolerance and insulin resistance. This phenotype was associated with ~30% lower plasma membrane insulin receptor levels in liver, as well as ~55% reduction in insulin-stimulated phosphorylation of the insulin receptor, and its downstream signaling molecules, AKT1 and AKT2 (reduced by ~40%). In addition, FoxO1 was aberrantly distributed in cellular nuclei, in parallel with up-regulation of the gluconeogenic genes Pck1 and G6pc. Surprisingly, L-IDE-KO mice showed similar plasma insulin levels and hepatic insulin clearance as control mice, despite reduced phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1, which upon its insulin-stimulated phosphorylation, promotes receptor-mediated insulin uptake to be degraded.[Conclusion] IDE is not a rate-limiting regulator of plasma insulin levels in vivo.This work was supported by grants from the Ministerio de Economía, Industria y Competitividad: SAF2014-58702-C2-1-R and SAF2016-77871-C2-1-R to ICC; SAF2014-58702-C2-2-R and SAF2016-77871-C2-2-R to GP; supported by the EFSD European Research Programme on New Targets for Type 2 Diabetes supported by an educational research grant from MSD to ICC and GP; the National Institutes of Health: R01-DK054254, R01-DK083850 and RO1-HL-112248 to SMN, and R01-GM115617 to MAL; and the American Diabetes Association: Career Development Award 7-11-CD-13 to MAL.Peer reviewe

A novel regulatory mechanism of the mitochondrial Ca2+ uniporter revealed by the p38 mitogen-activated protein kinase inhibitor sb202190

It is widely acknowledged that mitochondrial Ca2+ uptake modulates the cytosolic [Ca2+] ([Ca2+]c) acting as a transient Ca2+ buffer. In addition, mitochondrial [Ca2+] ([Ca2+]M) regulates the rate of respiration and may trigger opening of the permeability transition pore and start apoptosis. However, no mechanism for the physiological regulation of mitochondrial Ca2+ uptake has been described. We show here that SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase, strongly stimulates ruthenium red-sensitive mitochondrial Ca2+ uptake, both in intact and in permeabilized HeLa cells. The [Ca2+]M peak induced by agonists was increased about fourfold in the presence of the inhibitor, with a concomitant reduction in the [Ca2+]c peak. The stimulation occurred fast and was rapidly reversible. In addition, experiments in permeabilized cells perfused with controlled [Ca2+] showed that SB202190 stimulated mitochondrial Ca2+ uptake by more than 10-fold, but only in the physiological [Ca2+]c range (1-4 mM). Other structurally related p38 MAP kinase inhibitors (SB203580, PD169316, or SB220025) produced little or no effect. Our data suggest that in HeLa cells, a protein kinase sensitive to SB202190 tonically inhibits the mitochondrial Ca2+ uniporter. This novel regulatory mechanism may be of paramount importance to modulate mitochondrial Ca2+ uptake under different physiopathological conditions.This work was supported by grants from the Spanish Ministry of Science and Technology (PM98/0142 and BFI2002-01397) and from Junta de Castilla y León (VA 005/02).Peer Reviewe

Monitoring mitochondrial [Ca2+] dynamics with rhod-2, ratiometric pericam and aequorin

9 páginas, 8 figuras.-- El pdf del artículo es el manuscrito de autor.The dynamics of mitochondrial [Ca2+] ([Ca2+]M) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca2+]M are fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with different methods. We have made here a systematic comparison of the response of several fluorescent dyes, rhod-2 and rhod-FF, and two Ca2+-sensitive proteins, aequorin and pericam. Our results show that measurements obtained with aequorin and pericam are consistent in terms of dynamic Ca2+ changes. Instead, fluorescent dyes failed to follow Ca2+ changes adequately, especially during repetitive stimulation. In particular, measures obtained with rhod-2 or rhod-FF evidenced the previously reported Ca2+-dependent inhibition of mitochondrial Ca2+ uptake, but data obtained with aequorin or pericam under the same conditions did not. The reason for the loss of response of fluorescent dyes is unclear. Loading with these dyes produced changes in mitochondrial morphology and membrane potential, which were small and reversible at low concentrations (1–2 μM), but produced large and prolonged damage at higher concentrations. In addition, cells loaded with low concentrations of rhod-2 suffered large changes in mitochondrial morphology after light excitation. Our results suggest that [Ca2+]M data obtained with these dyes should be taken with care.This work was supported by grants from Ministerio de Educación y Ciencia (BFU2008-01871) and from Junta de Castilla y León (VA103A08 and GR105). Sergio de la Fuente holds an FPI (Formación de Personal Investigador) fellowship from the Spanish Ministerio de Ciencia e Innovacion.Peer reviewe