Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2+-shuttling event.

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 µm) in ASM cells does not induce Ca2+ signals but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+ rises and simultaneously increases Ca2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3ß-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the ß2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals.

Development of a high-throughput method for real-time assessment of cellular metabolism in intact long skeletal muscle fibre bundles.

KEY POINTS: We developed a method that allows for real-time assessment of cellular metabolism in isolated, intact long skeletal muscle fibre bundles from adult mice. This method can be used to study changes in mitochondrial function and fuel utilisation in live skeletal muscle fibre bundles. Our method enables flexibility in experimental design and high-throughput assessment of mitochondrial parameters in isolated skeletal muscle fibre bundles. Extensor digitorum longus (EDL) fibre bundles obtained from chronic high-fat diet fed mice had lower basal oxygen consumption under FCCP-induced maximal respiration, when compared to control chow-fed mice. EDL fibre bundles obtained from chronic high-fat diet fed mice had enhanced mitochondrial oxidation capacity under FCCP-induced maximal respiration, when compared to control chow-fed mice. ABSTRACT: Metabolic dysfunction in skeletal muscle contributes to the aetiology and development of muscle diseases and metabolic diseases. As such, assessment of skeletal muscle cellular bioenergetics provides a powerful means to understand the role of skeletal muscle metabolism in disease and to identify possible therapeutic targets. Here, we developed a method that allows for the real-time assessment of cellular respiration in intact skeletal muscle fibre bundles obtained from the extensor digitorum longus (EDL) muscle of adult mice. Using this method, we assessed the contribution of ATP turnover and proton leak to basal mitochondrial oxygen consumption rate (OCR). Our data demonstrate that the mitochondria in EDL fibres are loosely coupled. Moreover, in the presence of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), we show that palmitate exposure induced comparable peak OCR and higher total OCR in EDL fibre bundles when compared to pyruvate exposure, suggesting that fatty acids might be a more sustainable fuel source for skeletal muscle when mitochondria are driven to maximal respiration. Application of this method to EDL fibre bundles obtained from chronic high-fat diet fed mice revealed lower basal OCR and enhanced mitochondrial oxidation capacity in the presence of FCCP when compared to the chow-diet fed control mice. By using a 96-well microplate format, our method provides a flexible and efficient platform to investigate mitochondrial parameters of intact skeletal muscle fibres obtained from adult mice.

Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

Assessment of mitochondrial potential: implications for the correct monitoring of human sperm function.

Mitochondrial membrane potential (MMP) is an indicator of sperm functionality that can be assessed using specific fluorescent markers. However, the ability of distinct probes to dynamically evaluate sperm MMP has not been determined. In the present study, human sperm samples were independently labelled with MitoTracker Green, MitoTracker Red and JC-1. The ability of each probe to correctly monitor MMP was determined by incubating sperm with MMP disruptors (KCN, FCCP and valinomycin). Similarly, the effect of distinct fixatives (formaldehyde and methanol) was also tested. The three mitochondrial probes provided similar results, and were able to monitor changes in MMP when sperm had been previously incubated with MMP-disrupting agents. However, only JC-1 could, to a small extent, mirror MMP alterations after sperm labelling. Unexpectedly, the three probes were able to stain some pre-fixed sperm, even though this behaviour was very variable, especially for MitoTrackers. On the other hand, none of the probes was shown to be reliably fixable. Of the three probes tested JC-1 seems to be the most adequate, nevertheless, the choice of an MMP-specific probe may depend on the aim of each experimental setting and appropriate controls must always be performed.

Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation.

Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (DeltaPsim) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in DeltaPsim entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of DeltaPsim for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.