（論文

要様式３）

旨Mechanisms underlying the modulation of L-type Ca2+
channel by hydrogen peroxide in guinea-pig ventricularmyocytes楊磊【序論および目的】（適宜、項目をたてて、必ず２頁で記載する）

In the heart, ROS as highly reactive compounds accumulates in tissues during myocardialischemia/reperfusion and cause peroxidation of lipids and proteins which play an important role in
the pathogenesis of ischemia/reperfusion abnormalities, including myocardial stunning,irreversible injury, and reperfusion arrhythmias. The underlying mechanisms are not fully
understood. In this study, we investigated effects of hydrogen peroxide (H2O2) on the Ca2+channel using a patch-clamp technique in guinea-pig ventricular myocytes.
【材料および方法】

MgATP, Hydrogen peroxide (H2O2) tablet and 5,5’-dithiobis (2-nitrobenzoic acid) (DNTB) werepurchased from Sigma-Aldrich, KN-93 (CaMKII inhibitor) and KN-92 (inactive analog ofKN-93) from Calbiochem and Bay K 8644 (Ca2+ channel agonist) from Wako
Preparation of single cardiac myocytes and CaM and cell-attached and inside-out mode using thepatch-clamp technique.
【結果】

Fig. 1A and B, Ca2+ channel activity was rapidly increased without a change in the unitary
current amplitude. In average of 6 patches, Ca2+ channel activity was increased to 206 ± 32 % of
the control (Fig. 1D). This result confirmed the facilitating effect of H2O2 on LTCCs. Fig. 1C,
KN-93 significantly attenuatedH2O2-mediated facilitation (132 ± 15 % (n=9) vs 206 ±32 % withno drug, P<0.05), while KN-92, an inactive analog of KN-93, partially attenuated the facilitation
but it was not statistically significant (156 ± 13 % (n=5) vs 206 ± 32 % with no drug, P=0.30).
These results suggested that not only CaMKII-dependent but also independent pathways wereinvolved in H2O2-mediated facilitation of LTCCs.
Channel activity was changed by H2O2 from 144 ±32 % (control) to 272± 70 % in the absence ofKN-93 (n=5), whereas from 139 ±21 % to 231 ±29 % in the presence of KN-93 (n=6). Although
KN-93 seemed to slightly attenuate the increasing effect of H2O2 on channel activity, thisdifference was statistically insignificant. Thus KN93 did not significantly
affect theH2O2-mediated facilitation of the Ca channel in the inside-out mode.
in Fig. 3A and B, after Ca2+ channel activity was maintained by intact (untreated with H2O2)

CaM + ATP in the inside-out patches, we substituted the oxidized CaM for the untreated CaM.
Channel activity did not change significantly, suggesting that CaM was not oxidized or oxidized
CaM,
in Fig. 4, 1mM DNTB significantly increased Ca2+ channel activity maintained by CaM (from129 ±22 % up to 184 ± 21 %, n=5), suggesting that oxidation of cysteine residue(s) wasresponsible, at least partially, for the H2O2-mediated facilitation of LTCCs. we examined effectsof H2O2 on LTCC after application of DTNB. Application of H2O2 + CaM after DTNB + CaM
only slightly increased Ca2+ channel activity, which was statistically insignificant (Fig. 5)【結論及び考察】Thus our results indicate that, in addition to the CaMKII-dependent pathway, there is an additional
CaMKII-independent pathway for the H2O2-mediated facilitation of LTCC. Since most of
intracellular proteins are washed out in the inside-out patches, it is likely that direct oxidation ofLTCC or its associated proteins by H2O2 might be involved in the facilitation of LTCC. Since
H2O2-pretreated CaM does not mimic the facilitatory effect of H2O2, oxidation of CaM does notaccount for the mechanism of facilitation. It is consistent with that human CaM does not containcysteine residue. Thus, it seems most likely that the Ca2+ channel protein itself undergoes directoxidation by H2O2 as the CaMII-independent pathway of the LTCC facilitation.
Both cysteine and methionine residues are subject to oxidation by H2O2. The pore- forming
subunit α1C of the cardiac LTCC is rich in cysteine and methionine residues in the cytoplasmic
chains . Our findings that a specific cysteine oxidizing agent DTNB mimics the H2O2 effect andthat the effect of subsequently applied H2O2 is largely occluded suggest that cysteine residues areinvolved in the H2O2-mediated facilitation. However, our data do not exclude a possible
involvement of methionine residues. The future work should address to determine the oxidationsites responsible for the H2O2-mediated modulation of LTCC. In conclusion, LTCC may undergo
ROS-mediated modification via the direct oxidation of LTCC as well as the indirect pathways
involving CaMKII activation. This would be relevant for the understanding of ROS-mediatedregulation of ion channels and Ca overload and arrhythmogenesis during oxidation stress on theheart.

（The Journal of Physiological Sciences Vol.149, No.1

2003 年

in press）