Biphasic Modulation of Ryanodine Receptors by Sulfhydryl Oxidation in Rat Ventricular Myocytes

To understand better the modulation of ryanodine receptors (RyRs) during oxidative stress, the effect of 4,4′-dithiodipyridine (DTDP), a cell-permeant and thiol-reactive oxidant, on global Ca2+ signal and spontaneous Ca2+ sparks of rat ventricular myocytes was investigated. It was shown that a brief Ca2+ transient was elicited by DTDP, when its concentration was raised to 100 μM DTDP. In addition a dose-dependent increase of cytoplasmic free Zn2+ concentration was induced by DTDP. An increase of the frequency of spontaneous Ca2+ sparks appeared at 3 μM DTDP, whereas higher concentration of DTDP caused a biphasic change of the frequency in both intact and permeabilized myocytes. Consistent with the biphasic effect, caffeine-induced Ca2+ transients were similarly affected. Because DTDP did not reduce the free Ca2+ concentration in the sarcoplasmic reticulum lumen, it is likely that the effects of DTDP on the frequency and caffeine-induced Ca2+ transients are due mainly to sulfhydryl oxidation-induced activation and subsequent inactivation of RyRs. Unlike the frequency, the spatio-temporal properties of Ca2+ sparks were not influenced by DTDP. The finding that DTDP does not affect the duration of Ca2+ sparks is inconsistent with that the DTDP-induced increase of the open time of reconstituted RyR channels. The mechanism underlying this discrepancy, especially the possible role of the interaction between arrayed RyRs in myocytes, is discussed. This study suggests that, even if oxidative stress is mild enough not to cause intracellular Ca2+ accumulation, it may affect signaling pathways through directly modulating the RyR or its complex and in turn changing the frequency of spontaneous Ca2+ sparks. Thus, the functional importance of moderate oxidative stress should not be overlooked.