Nanomolar nitric oxide concentrations quickly and reversibly modulate astrocytic energy metabolism

Abstract

Nitric oxide (NO) is an intercellular messenger involved in multiple bodily functions. Prolonged NO exposure irreversibly inhibits respiration by covalent modification of mitochondrial cytochrome oxidase, a phenomenon of pathological relevance. However, the speed and potency of NO's metabolic effects at physiological concentrations are incompletely characterized. To this end, we set out to investigate the metabolic effects of NO in cultured astrocytes from mice by taking advantage of the high spatiotemporal resolution afforded by genetically encoded Förster resonance energy transfer (FRET) nanosensors. NO exposure resulted in immediate and reversible intracellular glucose depletion and lactate accumulation. Consistent with cytochrome oxidase involvement, the glycolytic effect was enhanced at a low oxygen level and became irreversible at a high NO concentration or after prolonged exposure. Measurements of both glycolytic rate and mitochondrial pyruvate consumption revealed significant effects even at nanomolar NO concentrations. We conclude that NO can modulate astrocytic energy metabolism in the short term, reversibly, and at concentrations known to be released by endothelial cells under physiological conditions. These findings suggest that NO modulates the size of the astrocytic lactate reservoir involved in neuronal fueling and signaling.In brain tissue and retina, NO contributes to both vasodilation and vasoconstriction (1, 2). The vasoactive effects of NO are mediated by the enzymes guanylate cyclase, cytochrome P450 epoxygenase, and -hydroxylase, which sense different NO concentrations. Guanylate cyclase is stimulated in the picomolar to low nanomolar range, whereas cytochrome P450 enzymes are inhibited at tens to hundreds of nanomolar NO (1, 3). However, there is more to NO than vasodilation. Endothelial cells express both endothelial NO synthase (eNOS) 3 and inducible NO synthase (iNOS) (4) resulting in NO production strong enough to sustain up to 70 nM extracellular NO in response to shear stress (5), to reach cells millimeters away in bicameral cultures (6), or to permeate across layers of astrocytes and myelin toward axons in vivo (7). A fourth well characterized molecular target of NO is mitochondrial cytochrome oxidase (EC 1.9.3.1), whose sensitivity to NO is similar to that of cytochrome P450 (8, 9). As inhibition of cytochrome oxidase reduces local oxygen consumption, endothelial NO has been proposed to extend the effective zone of oxygenation away from the vessel (10). A role for NO-dependent mitochondrial inhibition in brain tissue was suggested upon the observation that NO inhibits astrocytic respiration, resulting in stimulated glycolysis and lactate production (11, 12). Conceivably, inhibition of cytochrome oxidase may not only play a tonic distributive role but may also participate in activity-dependent neurometabolic coupling (13). However, there are aspects that need further exploring, notably the speed and potency of the metabolic effects in the face of actual NO concentrations found under...

Dual functions of AreA, a GATA transcription factor, on influencing ganoderic acid biosynthesis in Ganoderma lucidum

“…Recently, NO has been reported to function as an important cell signal, which functions over the entire lifespan. Studies in animals focus on NO due to not only its role as a potential target in cancer therapy, but also its functions in cell death, oxygen sensing and energy metabolism (Nunes et al, 2011;Kubo et al, 2016;San Martín et al, 2017). In plants, NO was shown to regulate seed germination, root and pollen tube growth, flowering, adaptive responses to abiotic stresses and pathogenesis (Bellin et al, 2013;Santolini et al, 2017).…”

MitoToxy Assay: a novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic

“…Binding of the test molecule, triggers a conformational change that affects the relative distance and/or orientation between the acceptor/donor fluorescent proteins, causing an increase or decrease in FRET efficiency (Miyawaki et al 1997). These genetically-encoded indicators have been instrumental to decipher molecular mechanism involved in fast modulation of oxidative and glycolytic metabolism in the brain (Baeza-Lehnert et al 2018;Fernandez-Moncada et al 2018;Lerchundi et al 2015;Machler et al 2016;San Martin et al 2017;San Martin et al 2014b;Valdebenito et al 2016). Being fluorescent and geneticallyencoded, these tools have a great potential to develop cell-based high-throughput methods for the pharmaceutical industry (Mendelsohn et al 2018;Schaaf et al 2018;Schaaf et al 2017;Zhao et al 2015;Zhao et al 2018).…”

“…Being fluorescent and geneticallyencoded, these tools have a great potential to develop cell-based high-throughput methods for the pharmaceutical industry (Mendelsohn et al 2018;Schaaf et al 2018;Schaaf et al 2017;Zhao et al 2015;Zhao et al 2018). Using a FRET-based lactate indicator Laconic (San Martin et al 2013), we have observed that the mitochondrial damage induced by sodium azide or physiological mitochondrial modulator signals such as nitric oxide, produces a fast and acute cytosolic lactate accumulation (San Martin et al 2017;San Martin et al 2013). A phenomenon explained primarily by a reduction of mitochondrial pyruvate consumption and secondary glycolysis activation (Bittner et al 2010;San Martin et al 2017;San Martin et al 2014a).…”

“…Using a FRET-based lactate indicator Laconic (San Martin et al 2013), we have observed that the mitochondrial damage induced by sodium azide or physiological mitochondrial modulator signals such as nitric oxide, produces a fast and acute cytosolic lactate accumulation (San Martin et al 2017;San Martin et al 2013). A phenomenon explained primarily by a reduction of mitochondrial pyruvate consumption and secondary glycolysis activation (Bittner et al 2010;San Martin et al 2017;San Martin et al 2014a). Both effects converge into increased cytosolic pyruvate and NADH levels, displacing the chemical equilibrium of LDH (Lactate Dehydrogenase) to lactate production within seconds.…”

Astrocytes take the stage in a tale of signaling-metabolism coupling

“…Thus, an alternative to the lactate shuttle hypothesis could be that both neurons and astrocytes produce lactate during brain activation and that this lactate pool can then be metabolized at a later point by both cell types (and maybe other cells) or dispersed within the brain parenchyma. However the story of neuronal coupling ends, these new data from San Martín et al (7) provide an interesting chapter that advances our understanding of astrocyte metabolism. …”