Green tea catechins are potent sensitizers of ryanodine receptor type 1 (RyR1)

Abstract

Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5–10 nM) increases channel open probability (Po) twofold, by lengthening open dwell time. The degree of channel activation is concentration-dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((−)-epigallocatechin) and EC ((−)-epicatechin) showed a rank order of activity toward RyR1 (EGCG > ECG ≫ EGC >>> EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by ≤100 μM cytoplasmic Ca²⁺ without altering inhibitory potency by >100 μM Ca²⁺. EGCG as high as 10 μM in the extracellular medium potentiated Ca²⁺ transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca²⁺ release or slowing Ca²⁺ transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities.

Introduction

Catechins, a group of polyphenols extracted from green tea leaves, are a source of pharmacologically active compounds that have been proposed to confer protective, palliative and therapeutic remedies for human health to combat human diseases. EGCG, ECG, EGC and EC collectively constitute about 30% of the dry weight of green tea leaves [1]. EGCG is a major catechin constituent, accounting for ∼50% of the total catechins in green tea, and has received the most experimental attention due to its broad biological activities [2]. Green tea polyphenols are generally regarded as antioxidants [3]. Chemically they all possess multiple hydroxyl substituents on the A ring, C ring, B ring (gallo-) and/or D ring (gallate) [4]. Polyphenol moieties act as scavengers of reactive oxygen species including superoxide radical, singlet oxygen, hydroxyl radical, peroxyl radical, nitric oxide, nitrogen dioxide and peroxynitrite [4], [5]. Catechins are also known to chelate nutritive metal ions such as iron [6]. On the other hand, results from several studies on the redox properties of green tea polyphenols reveal paradoxical properties in that they act as pro-oxidants by autooxidizing to generate superoxide and semiquinone radicals [4], [7]. In addition to their anti-oxidative or pro-oxidative activities, additional biological activities have been attributed to green tea polyphenols that are apparently not directly related to their redox properties [4].

One biological action attributed to green tea polyphenols is their ability to influence intracellular Ca²⁺ in both non-excitable and excitable cells [8], [9], [10]. However, the principle mechanisms responsible for affecting changes in intracellular Ca²⁺ by green tea polyphenols remain unanswered. One major limitation to identifying molecular targets by which catechins mediate changes in Ca²⁺ dependent cellular signaling events is that most of the published studies use exceedingly high concentrations of EGCG (typically >50 μM). Because of their chemical properties, green tea polyphenols have high affinity for membrane phospholipids, they are capable of damaging membrane structure or even fragment lipid bilayers when present at high concentrations (>30 μM) [11], [12], [13]. Since it is unlikely that tissue concentrations reach such high levels [14], the experimental use of high concentrations of polyphenols in cellular and biochemical studies are likely to produce several non-specific interactions, making data analysis and interpretation difficult.

Here, we report that RyR1, a broadly expressed intracellular Ca²⁺ release channel, presents a very sensitive biochemical target of two of the major components of green tea polyphenols, EGCG and ECG. Sub-micromolar EGCG or ECG is sufficient to significantly sensitize activation of RyR1 channels by its physiological modulator Ca²⁺. Importantly, when EGCG is applied to skeletal myotubes or adult FDB fibers at concentrations that should saturate sensitizing activity toward RyR1 (5–10 μM) it does not elicit spontaneous rise in Ca²⁺ (release from stores or Ca²⁺ entry) in resting cells or cells undergoing stimulation. Rather, EGCG potentiates the Ca²⁺ transient amplitude evoked by electrical stimuli without slowing Ca²⁺ transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction may contribute to their biological activities.