Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro.
Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro.
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DOI：
10.1016/s0021-9258(18)47024-x
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发表时间：
1994-11
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影响因子：
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通讯作者：
Jean-Luc BalligandS;Dan Ungureanu-LongroisSg;William;-W.;Simmons;David Pimental;Tadeusz A. Malinskin;Matthias Kapturczakn;Ziad Tahan;Charles;Lowensteinll;Amy;DavidoP;Ralph;Kelly;Thomas;Smith;Thomas MichelSS
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文献类型：
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作者：
Jean-Luc BalligandS;Dan Ungureanu-LongroisSg;William;-W.;Simmons;David Pimental;Tadeusz A. Malinskin;Matthias Kapturczakn;Ziad Tahan;Charles;Lowensteinll;Amy;DavidoP;Ralph;Kelly;Thomas;Smith;Thomas MichelSS
心肌的细胞成分包含结构性和诱导性一氧化氮(NO)信号通路，这些信号通路调节心肌细胞的收缩特性。心肌中表达的诱导型一氧化氮合酶亚型(S)以及表达诱导型一氧化氮合酶活性的特定细胞类型的一致性尚不清楚。我们用逆转录聚合酶链式反应从炎性细胞因子处理的成年大鼠心室肌细胞(ARVM)原代培养中扩增出与其他iNOS基因序列几乎相同的217个碱基对的cDNA.在Northern blotts中，我们没有发现对照心肌细胞中有iNOS基因的表达，但IL-1β和干扰素-γ都能单独增加原代培养的ARVM细胞中iNOS基因的丰度，在12h达到最高表达，在放线菌素C_1处理的细胞中iNOS基因的半衰期为4h，地塞米松和转化生长因子-β均可减弱IL-1β和干扰素-γ诱导的iNOS基因的丰度和酶活性。地塞米松可阻断脂多糖诱导的大鼠心肌细胞诱导型一氧化氮合酶的表达，但不影响心肌细胞GTP环水解酶的表达。为了进一步表征产生NO的特定细胞类型，我们使用了NO特异性的卟啉/Nafion包被的微传感器，记录了在L精氨酸耗竭的培养液中，经IL-1β和干扰素γ处理的单个分离的ARVM释放NO的情况。在与NO微型传感器并列的细胞附近微量注射L精氨酸后，可检测到NO的释放，但不能检测到D-精氨酸的释放，也不能检测到L-N-单甲基精氨酸预处理的Arm的释放。在L-精氨酸耗竭的培养液中维持的细胞因子处理的血管畸形在加入L-精氨酸后，对异丙肾上腺素的收缩反应也受到抑制，但D-精氨酸的作用不明显。这些结果表明，心肌细胞在暴露于特定炎性细胞因子后收缩功能的改变是由于心肌细胞诱导型一氧化氮合酶的诱导。
Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocytes, but both interleukin-1 beta and interferon-gamma individually increased iNOS mRNA abundance in primary cultures of ARVM, with maximal expression at 12 h. The half-life of iNOS mRNA in actinomycin C1-treated cells was 4 h. Both dexamethasone and transforming growth factor-beta attenuated the induction of iNOS mRNA abundance and enzyme activity by IL-1 beta and INF gamma. Pretreatment with dexamethasone also abolished the induction of iNOS mRNA, but not the increase in GTP cyclohydrolase mRNA in purified cardiac myocytes from lipopolysaccharide-injected rats. In order to further characterize the specific cell type producing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1 beta and IFN gamma in L-arginine-depleted medium. NO release could be detected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjection of D-arginine, and not from ARVM pretreated with L-N-monomethylarginine. Cytokine-pretreated ARVM that had been maintained in L-arginine-depleted medium also exhibited a depressed contractile response to isoproterenol after addition of L-arginine, but not D-arginine. These results indicate that altered contractile function of cardiac myocytes following exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.