Grape Extract Inhibits the Ectopic ATP Synthase of Retinal Rod Outer Segments

Research Article

Austin Ophthalmol. 2016; 1(1): 1006.

Grape Extract Inhibits the Ectopic ATP Synthase of Retinal Rod Outer Segments

Calzia D¹, Traverso CE² and Panfoli I¹*

¹Department of Pharmacy, University of Genoa, Italy

²Clinica Oculistica, University of Genoa, Italy

*Corresponding author: Isabella Panfoli, Department of Pharmacy, University of Genoa, Viale Benedetto XV 3, 16132 Genova, Italy

Received: November 03, 2016; Accepted: November 23, 2016; Published: November 25, 2016

Abstract

We have previously shown that the retinal rod Outer Segments (OS), although devoid of mitochondria, express functional FoF1-ATP synthase and conduct oxidative phosphorylation. Similar to its mitochondrial counterpart, the rod OS ectopic FoF1-ATP synthase is inhibited by polyphenolic compounds. Grape, one of the first crops domesticated by humans, is currently used for a number of dietary products worldwide. As grape extract contains a mixture of polyphenols, we studied its effects on the OS ATP synthetic activity. ATP synthesis analysis was performed by spectrophotometry. Results demonstrated that commercial grape extract inhibits the OS ATP synthesis up to 98 % in a dose-dependent manner (final concentrations ranging from 0 to 500μg/ml). Presumably due to its elevated content in polyphenolic phytochemicals, grape extract can modulate the OS ATP synthase and subsequently lower the reactive oxygen species production by the ectopic respiratory chain coupled to FoF1- ATP synthase. Further studies may shed light on the molecular mechanism underlying the well-known beneficial effect of grapes and their extracts on the visual system; this could be beneficial in ocular conditions caused by oxidative stress such as age-related macular degeneration and diabetic retinopathy.

Keywords: Grape extract; F1Fo-ATP synthase; Quercetin; Resveratrol; Rod outer segment

Abbreviations

AMD: Age-Related Macular Degeneration; ATP Synthase; F1Fo-ATP Synthase; DR: Diabetic Retinopathy; ETC: Electron Transport Chain; OS: Rod Outer Segment; OXPHOS: Oxidative Phosphorylation; PUFA: Polyunsaturated Fatty Acids; ROI: Reactive Oxygen Intermediates; SD: Standard Deviation.

Introduction

Polyphenols are secondary metabolites widely distributed in plants, where they play metabolic roles and protect plants against UV, pathogen and herbivores [1]. Polyphenols include a wide variety of molecules with structural phenolic features that can be chemically classified according to the number of phenolic rings, and the substituents. The two main groups are the flavonoids, encompassing six major subgroups, and the non-flavonoids, comprising stilbenes [2]. Flavonoids, responsible for the colors of the flowers and fruits, are the most abundant polyphenols in human diet and widely studied. More than 8,000 different flavonoids have been identified. Phenolic compounds, especially anthocyanins, are abundant in Grape (Vitis spp.) [1]. These, abundantly included in the human diet, could alleviate the oxidative stress [3,4] by virtue of their ability to modulate several cellular processes such as proliferation, apoptosis and redox balance. Fruits like grapes and berries contain up to 300mg polyphenols per 100grams fresh weight [1]. Also resveratrol (a stilbene) is largely found in grape [5]. The complex variety of compounds present in grape has demonstrated to possess therapeutic properties. Recent studies have shown that the beneficial health effects promoted by grape can be attributed to its unique mix of polyphenolic compounds [3]. Epidemiological studies have shown that the consumption of grape and grape products lowers the risk of myocardial infarction [6]. Polyphenol intake seems to also act as neuroprotectant [7]. Oxidative stress is a major player in the pathogenesis of retinal degenerative diseases, such as Age Related Macular Degeneration (AMD) and Diabetic Retinopathy (DR) [8,9]. Photoreceptors consume about 4-folds more oxygen than the other retinal cells [10], indicating an active role of the rod OS in the O2 consumption of the outer retina [11], consistently to our previous proteomic and biochemical data reporting the ectopic presence and activity of FoF1-ATP synthase (ATP synthase) and the four respiratory chain complexes and cytochrome c in isolated rod OS [12–14]. It was demonstrated that the retinal rod Outer Segment (OS) more than the inner segment is the target of the oxidative stress-related cytotoxicity caused by exposure of mouse eyes to blue light [15,16]. We have also shown that the blue light-induced detrimental effects cause an impairment of the extra-mitochondrial oxidative phosphorylation in the rod OS, as a consequence of the oxidative damage [17], and that resveratrol, curcumin, quercetin and epigallocatechin gallate exert an inhibitory effect on both the ATPase and ATP synthase rod OS activity [18], consistent with the hypothesis that the OS express a functional ATP synthase [12]. This sheds light on the beneficial effect polyphenolic compounds exert on many retinal pathologies such as age related macular degeneration and diabetic retinopathy [4,7,19]. Here we tested the effect of a natural red grape extract on bovine purified rod OS.

Materials and Methods

Materials

All reagents were of analytical grade. MilliQ (Merck-Millipore) water was utilized throughout. Commercial red grape extract from a local Italian producer was utilized.

Extraction of retinas

Retinas were extracted from freshly enucleated bovine eyes (obtained from a local slaughterhouse) by a procedure we had developed [20] maximizing ROS yield. Briefly, eyecups deprived of vitreous and lens, are filled with Mammalian Ringer (0.157 MNaCl, 5 mMKCl, 7 mM Na2HPO4, 8 mM NaH2PO4, 0.5 mM MgCl2, 2 mM CaCl2 pH 6.9 plus protease inhibitor cocktail (Sigma-Aldrich, S. Louis, MO, USA) and 50μg/ml Ampicillin, for 10min. Then floating retinas are cut free of the optic nerve.

Purified bovine rod OS preparations

Purified bovine rod OS were prepared under dim red light from 20 retinas at 4°C, by sucrose/Ficoll continuous gradient centrifugation [18,21] in the presence of protease inhibitor cocktail (Sigma–Aldrich, S. Louis, MO) and ampicillin (50μg/ml). Rod OS preparation was routinely characterized for integrity of plasma membrane. ROS homogenates were obtained by mini glass-glass Potter homogenize on ice in 4:1 (w/v) hypotonic medium (5 mMTris-HCl pH 7.4, in MilliQ water, plus protease inhibitor cocktail and 50μg/ml ampicillin).

ATP synthesis assay in OS homogenates

The formation of ATP from ADP and inorganic phosphate was performed in rod OS according to our previous report [12]. Rod OS (0.04μg protein/ml) were incubated for 5 min at 37°C in 50mM Tris/ HCl (pH 7.4), 5mM KCl, 1mM EGTA, 5mM MgCl2, 0.6mM ouabain, 0.25mM di(adenosine)-5-penta-phosphate(Ap5A, adenylate kinase inhibitor), and 25μg/ml ampicillin. ATP synthesis was induced by adding 5 mM KH2PO4, 20mM succinate, 0.35mM NADH, and 0.3mM ADP at the same pH of the mixture. After stopping the reaction with 7% perchloric acid final concentration, the ATP concentration in each sample was measured using a spectrophotometrical method. Neutralized and clarified supernatant was added to a mixture containing 2mM MgCl2, 0.5mM NADP, 5mM Glucose, 100mM Tris/HCl pH 7.4 and 7U/ml of a mix of hexokinase and glucose-6- phosphate dehydrogenase (Roche Diagnostics Corp., Indianapolis, IN). NADP+ reduction was followed at 340nm using a dual-beam spectrophotometer (UNICAM UV2, Analytical S.n.c., Italy). Where necessary incubation medium contained 10μMoligomycin, 30μM resveratrol, 100μM quercetin or grape extract with final concentration between 1,25 to 500μg/ml.

Results

Purified rod OS synthesize ATP, through the ectopic expression of the mitochondrial ATP synthase in the disk membranes [12-14]. Considering that the OS ATP synthase is inhibited by polyphenolic phytochemicals [18] (such as resveratrol and quercetin, both abundant in grapes), here we tested a red grape crude extract. The purified bovine rod OS were previously extensively characterized, excluding contamination by mitochondria and IS organelle.ATP synthesis by OS homogenates is reported (Figure 1). A maximal activity of 0,5±0,03μmol/min/mg of protein was detected in the presence of 0.35m MNADH, 20mM succinate and 0.3mM ADP. ATP synthesis was specific, as shown by its inhibition by oligomycin (90%) and resveratrol (98%), inhibitors of Fo and F1 moiety respectively (Figure 1). Moreover, ATP production was inhibited by quercetin (85%), a phenolic compound abundant in grape extract together with resveratrol. Grape extract inhibited ATP synthesis in doseddependent manner by 9, 6% 41, 74, 91, 85, 88 and 100%. At the final concentrations 1.5, 2.5, 5, 10, 20, 125, 250, 500 μg /ml, respectively (Figure 2).

Citation:Calzia D, Traverso CE and Panfoli I. Grape Extract Inhibits the Ectopic ATP Synthase of Retinal Rod Outer Segments. Austin Ophthalmol. 2016; 1(1): 1006.