生理学报
生理學報
생이학보
ACTA PHYSIOLOGICA SINICA
2008年
6期
709-714
,共6页
叶红%马万里%金肆%刘声远%王迪浔%胡清华
葉紅%馬萬裏%金肆%劉聲遠%王迪潯%鬍清華
협홍%마만리%금사%류성원%왕적심%호청화
支气管%平滑肌%香烟提取物%钾通道%蛋白激酶C
支氣管%平滑肌%香煙提取物%鉀通道%蛋白激酶C
지기관%평활기%향연제취물%갑통도%단백격매C
bronchus%myocyte%cigarette smoke extract%potassium channel%protein kinase C
大电导的钙活化钾通道(large-conductance calcium-activated potassium channel,BKCa)和电压依赖性钾通道Kv1.5在气道高反应性的发牛机制中具有重要作用.已知吸烟可致气道高反应,但钾通道的变化在其发病中的作用尚需进一步阐明.本文旨在研究香烟提取物(cigarette smoke extract,CSE)对培养的大鼠支气管半滑肌细胞(bronchial smooth muscle cels,BSMCs)钾通道BKCa和Kv1.5表达的直接作用,以及蛋白激酶C(protein kinase C,PKC)在其中的作用.实验采用原代培养人鼠BSMCs,用5%CSE刺激,免疫印迹检测PKC亚型的表达和转位,半定量RT-PCR、免疫印EIJ迹实验检测BKca和Kv1.5的mRNA和蛋白表达,然后用PKC抑制剂BIM和G6e6983与CSE共作用,检测其对BKCa 和Kv1.5的mRNA和蛋门表达的影响.结果显示,5% CSE使PKCε、η、θ发生明显的膜转位,并使BKCa和Kv1.5的蛋白和mRNA表达明显降低;选择性PKC抑制剂BIM或Goe6983与CSE共同作用,均可使BKCa和Kv1.5的蛋白和mRNA表达部分恢复.上述结果提示,CSE可引起BSMCs的BKCa和Kv1.5表达下调,PKC￡、T1、ε、η、θ参与其信号转导.
大電導的鈣活化鉀通道(large-conductance calcium-activated potassium channel,BKCa)和電壓依賴性鉀通道Kv1.5在氣道高反應性的髮牛機製中具有重要作用.已知吸煙可緻氣道高反應,但鉀通道的變化在其髮病中的作用尚需進一步闡明.本文旨在研究香煙提取物(cigarette smoke extract,CSE)對培養的大鼠支氣管半滑肌細胞(bronchial smooth muscle cels,BSMCs)鉀通道BKCa和Kv1.5錶達的直接作用,以及蛋白激酶C(protein kinase C,PKC)在其中的作用.實驗採用原代培養人鼠BSMCs,用5%CSE刺激,免疫印跡檢測PKC亞型的錶達和轉位,半定量RT-PCR、免疫印EIJ跡實驗檢測BKca和Kv1.5的mRNA和蛋白錶達,然後用PKC抑製劑BIM和G6e6983與CSE共作用,檢測其對BKCa 和Kv1.5的mRNA和蛋門錶達的影響.結果顯示,5% CSE使PKCε、η、θ髮生明顯的膜轉位,併使BKCa和Kv1.5的蛋白和mRNA錶達明顯降低;選擇性PKC抑製劑BIM或Goe6983與CSE共同作用,均可使BKCa和Kv1.5的蛋白和mRNA錶達部分恢複.上述結果提示,CSE可引起BSMCs的BKCa和Kv1.5錶達下調,PKC￡、T1、ε、η、θ參與其信號轉導.
대전도적개활화갑통도(large-conductance calcium-activated potassium channel,BKCa)화전압의뢰성갑통도Kv1.5재기도고반응성적발우궤제중구유중요작용.이지흡연가치기도고반응,단갑통도적변화재기발병중적작용상수진일보천명.본문지재연구향연제취물(cigarette smoke extract,CSE)대배양적대서지기관반활기세포(bronchial smooth muscle cels,BSMCs)갑통도BKCa화Kv1.5표체적직접작용,이급단백격매C(protein kinase C,PKC)재기중적작용.실험채용원대배양인서BSMCs,용5%CSE자격,면역인적검측PKC아형적표체화전위,반정량RT-PCR、면역인EIJ적실험검측BKca화Kv1.5적mRNA화단백표체,연후용PKC억제제BIM화G6e6983여CSE공작용,검측기대BKCa 화Kv1.5적mRNA화단문표체적영향.결과현시,5% CSE사PKCε、η、θ발생명현적막전위,병사BKCa화Kv1.5적단백화mRNA표체명현강저;선택성PKC억제제BIM혹Goe6983여CSE공동작용,균가사BKCa화Kv1.5적단백화mRNA표체부분회복.상술결과제시,CSE가인기BSMCs적BKCa화Kv1.5표체하조,PKC￡、T1、ε、η、θ삼여기신호전도.
Large-conductance calcium-activated potassium channel (BKCa) and voltage-gated potassium channel Kv 1.5 play an important role in the pathogenesis of bronchial hyperresponsiveness (BHR). It is known that cigarette smoke can induce BHR, however, the role of BKCa and Kv1.5 expression in it remains to be further elucidated. The purpose of the present study was to investigate the direct effects of cigarette smoke extract (CSE) on BKCa and Kv1.5 expression, and the role of protein kinase C (PKC) isoforms activation in primary cultured rat bronchial smooth muscle cells (BSMCs). Primarily cultured rat BSMCs were treated with 5% CSE, the expression and translocation of PKC isoforms were measured by Western blot, and the mRNA and protein levels of BKCa and Kv1.5 a-subunits were determined by semi-quantitative RT-PCR and Western blot, respectively. The results showed that 5% CSE induced the translocation of PKCε, PKη, PKCθ from soluble fraction to particulate fraction, and reduced mRNA and protein expressions of BKCa and Kv1.5 a-subunits. The decreased expression of potassium channels was partly restored by PKC inhibitor, BIM or Goe6983. In summary, CSE may activate PKC isoforms ε,η,θ, thereby down-regulate the expressions of BKCa and Kv1.5 in BSMCs.
