Ruthenium Red Modifies the Cardiac and Skeletal Muscle Ca2+ Release Channels (Ryanodine Receptors) by Multiple Mechanisms

Abstract

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca 2؉ The release and sequestration of Ca 2ϩ ions by the sarcoplasmic reticulum (SR), 1 an intracellular membrane compartment, is essential to the process of cardiac and skeletal muscle contraction and relaxation. The rapid release of Ca 2ϩ is mediated by Ca 2ϩ release channels, also known as ryanodine receptors (RyRs), because they bind the plant alkaloid ryanodine with high affinity and specificity (1-6). Skeletal and cardiac muscles express two major isoforms of the RyR, RyR1 and RyR2, respectively. In striated muscles, RyRs are concentrated in the junctional SR membrane near transverse tubular, voltage-sensitive L-type Ca 2ϩ channels (dihydropyridine receptors). A muscle action potential initiates dihydropyridine receptor conformational changes that activate the RyRs via a direct physical interaction in skeletal muscle or mediate the influx of Ca 2ϩ in cardiac muscle, leading to the release of Ca 2ϩ from the SR and subsequent muscle contraction. Both RyRs have been isolated as 30 S protein complexes composed of four 560-kDa (RyR polypeptide) and four 12-kDa (FK506-binding protein) subunits (1-5). The two channel activities are affected by endogenous and exogenous effectors, such as Ca 2ϩ , Mg 2ϩ , ATP, caffeine, ryanodine, and ruthenium red.Ruthenium red (RR) is one of the most potent inhibitors of SR Ca 2ϩ release (2). RR also inhibits mitochondrial Ca 2ϩ uptake. However, this activity was due to a contaminant (7) that may be related to an oxygen-bridged R360 complex that, at concentrations as high as 10 M, was without effect on SR Ca 2ϩ uptake or release (8). RR is a polycationic dye with a linear structure consisting of three ruthenium atoms with a net valence of 6. In SR vesicles, RR increased the rate of Ca 2ϩ uptake and decreased the rate of Ca 2ϩ release at concentrations ranging from 1 nM to 20 M (9 -18). In muscle fibers, RR concentrations greater than 20 M are required to inhibit SR Ca 2ϩ release because of RR binding to myoplasmic proteins (19,20). In intact single frog twitch muscle fibers, the estimated free RR concentration for half-block of SR Ca 2ϩ release was 2.4 M, in good agreement with the range reported for SR vesicle preparations (19). In [ 3 H]ryanodine binding measurements, RR decreased the B max value and increased the K D value, with the latter effect being due to a slower association rate (16). In single channel measurements, micromolar concentrations of RR decreased the channel open probability of the skeletal (21) and cardiac (22) RyRs by producing prolonged channel closings. A different effect was observed when ryanodine was used to lock the channels into a permanently open, Ca 2ϩ -insensitive subconductance state. RR blocked single ryanodine-modified skeletal RyRs by binding in a voltage-dependent manner to multiple sites located in the conductance pore of the channel (23).The present study was undertaken to clarify the effects of RR on ryanodine-unmodified ...

Evidence for a Functional Role of Endothelial Transient Receptor Potential V4 in Shear Stress–Induced Vasodilatation

“…This may also indicate that the rather nonselective blocker RuR does not exert gross unspecific effects or other effects caused by blocking ryanodine-sensitive Ca 2ϩ release channels in smooth muscle. 26 In CAs with a functionally active endothelium, inhibition of NO synthase alone or in combination with blockade of prostacyclin synthesis almost completely suppressed TRPV4-mediated vasodilatation, suggesting that this type of vasodilatation largely relies on the Ca 2ϩ -dependent synthesis and action of NO after TRPV4-mediated Ca 2ϩ -influx, whereas the other 2 major vasodilator systems (ie, the prostacyclin system or the EDHF system) do not seem to make a significant contribution in this CA. Regarding EDHF-mediated vasodilatation, Ca 2ϩ -dependent activation of endothelial K Ca channels of the IKCa1 and SKCa3 type and subsequent endothelial hyperpolarization have been considered a prerequisite for the generation of the EDHF signal in many vessels 27 and species including rat CAs.…”

Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4

“…Additionally, RuR also inhibited the H 2 O 2 -induced Ca 2ϩ influx in HLMVEC. However, it should be noted that RuR also inhibits other cation channels, and has been reported to both activate and inhibit sarcoplasmic reticulum and mitochondrial channels (16,57,71). To more firmly establish the role of TRPV4 in H 2 O 2 -induced Ca 2ϩ influx in HLMVECs, we inhibited TRPV4 with HC-067047, a putative specific inhibitor of TRPV4 previously shown to have minimal off-target effects (19,69 (23,46).…”

Caffeine-activated large-conductance plasma membrane cation channels in cardiac myocytes: characteristics and significance

“…When 10 M ruthenium red, another RyR inhibitor (27,51), was applied to the cell, together with caffeine, many openings showed smaller unitary currents (4 cells, Fig. 6).…”