Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor. Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O(2) in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F(o)F(1)-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS. Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.

Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments.

PANFOLI, ISABELLA;CALZIA, DANIELA;RAVERA, SILVIA;CALZIA, DANIELA;TACCHETTI, CARLO;CANDIANI, SIMONA;MORELLI, ALESSANDRO;
2011

Abstract

Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor. Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O(2) in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F(o)F(1)-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS. Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/282629
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