Calcium-dependent activation of mitochondrial metabolism in mammalian cells

Abstract

Endogenous fluorophores provide a simple, but elegant means to investigate the relationship between agonist-evoked Ca 2+ signals and the activation of mitochondrial metabolism. In this article, we discuss the methods and strategies to measure cellular pyridine nucleotide and flavoprotein fluorescence alone or in combination with Ca 2+ -sensitive indicators. These methods were developed using primary cultured hepatocytes and neurons, which contain relatively high levels of endogenous fluorophores and robust metabolic responses. Nevertheless, these methods are amendable to a wide variety of primary cell types and cell lines that maintain active mitochondrial metabolism.

In Vivo Tissue-wide Synchronization of Mitochondrial Metabolic Oscillations

“…This is in contrast with what has been previously reported in both ex vivo and in vitro model systems where oscillations are triggered by the application of exogenous stimuli (Aon et al, 2003; Bruce et al, 2004; Gaspers and Thomas, 2008; Merrins et al, 2010; Mironov and Richter, 2001; Voronina et al, 2002). Our findings underscore the fact that in the native tissue the complex integration of signals coming from the vasculature, the nervous system, the tissue microenvironment, and the surrounding cells, ensure the proper conditions to maintain mitochondrial metabolism in an oscillatory modality (Kim et al, 2010).…”

“…No correlation was observed among the period of the oscillations and heartbeat, blood pressure, or blood glucose levels (Figure S2). Moreover, whereas in these systems oscillations were triggered by specific stimuli such as glucose (Merrins et al, 2010), laser-induced ROS (Aon et al, 2003), or receptor stimulation (Bruce et al, 2004; Gaspers and Thomas, 2008; Voronina et al, 2002), in vivo they were spontaneous and constitutive, and were impaired by high laser power (>25 mW) (Figure S3). The in vivo NADH oscillations were not due to ischemia, as reported for the intact brain (Mayevsky and Ziv, 1991; Mironov and Richter, 2001), since they occurred when blood flow and perfusion were within physiological levels (Baveja et al, 2002; Oyanagi-Tanaka et al, 2001), and they were completely abolished upon interruption of the circulation (Figure S3).…”

Presynaptic Mitochondria in Functionally Different Motor Neurons Exhibit Similar Affinities for Ca2+But Exert Little Influence as Ca2+Buffers at Nerve Firing RatesIn Situ

“…The slower rate of rhod-2 fluorescence decay in both terminal types (Fig. 4D), relative to rhod-FF or rhod-5N, may be due to a contribution of rhod-2 to the Ca 2+ buffering capacity of the mitochondrial matrix, which can slow the kinetics of mitochondrial Ca 2+ loss (Gaspers and Thomas, 2008). …”

Adrenoceptors in brain: Cellular gene expression and effects on astrocytic metabolism and [Ca2+]i

“…Stimulation of glycogenolysis matters, because brain glycogenolysis no longer is regarded as primarily an emergency function, but as an integral part of glucose breakdown via a substantial ‘glucose-glycogen shunt’ (Swanson et al, 1992; Dienel et al, 2007; Hertz et al, 2007). A noradrenaline-induced increase in mitochondrial Ca 2+ has been observed in astrocytes (Simpson et al, 1998), but stimulation of oxidative metabolism by increased [Ca 2+ ] i , followed by increased intramitochondrial Ca 2+ , has mainly been studied in muscle and liver, where a direct stimulation was demonstrated of the mitochondrial dehydrogenases pyruvate dehydrogenase, isocitrate dehydrogenase (which stimulates degradation of isocitrate, an intermediate between citrate and α-ketoglutarate in the TCA cycle) and α-ketoglutarate dehydrogenase (McCormack and Denton, 1990; Rutter et al, 1996; Gaspers and Thomas, 2008; Griffith and Rutter, 2009). Fluxes through the metabolic steps catalyzed by two of these enzymes were shown above to be stimulated by noradrenaline, whereas flux catalyzed by the isocitrate dehydrogenase has not been studied in astrocytes, because no convenient substrate entering the mitochondria exists for this reaction.…”