Gebremedhin Debebe, Yamaura Ken, Harder David R
Cardiovascular Research Center, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
Am J Physiol Heart Circ Physiol. 2008 Jan;294(1):H107-20. doi: 10.1152/ajpheart.01416.2006. Epub 2007 Sep 28.
The mechanism of sensing hypoxia and hypoxia-induced activation of cerebral arterial Ca(2+)-activated K(+) (K(Ca)) channel currents and vasodilation is not known. We investigated the roles of the cytochrome P-450 4A (CYP 4A) omega-hydroxylase metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), and generation of superoxide in the hypoxia-evoked activation of the K(Ca) channel current in rat cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch-clamp analysis of K(+) channel current identified a voltage- and Ca(2+)-dependent 238 +/- 21-pS unitary K(+) currents that are inhibitable by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia (<2% O(2)) reversibly enhanced the open-state probability (NP(o)) of the 238-pS unitary K(Ca) current in cell-attached patches. This effect of hypoxia was not observed on unitary K(Ca) currents recorded from either excised inside-out or outside-out membrane patches. Inhibition of CYP 4A omega-hydroxylase activity increased the NP(o) of K(Ca) single-channel current. Hypoxia reduced the basal endogenous level of 20-HETE by 47 +/- 3% as well as catalytic formation of 20-HETE in cerebral arterial muscle homogenates as determined by liquid chromatography-mass spectrometry analysis. The concentration of authentic 20-HETE was reduced when incubated with the superoxide donor KO(2). Exogenous 20-HETE (100 nM) attenuated the hypoxia-induced activation of the K(Ca) current in CAMCs. Hypoxia did not augment the increase in NP(o) of K(Ca) channel current induced by suicide inhibition of endogenous CYP 4A omega-hydroxylase activity with 17-octadecynoic acid. In pressure (80 mmHg)-constricted cerebral arterial segments, hypoxia induced dilation that was partly attenuated by 20-HETE or by the K(Ca) channel blocker TEA. Exposure to hypoxia caused the generation of intracellular superoxide as evidenced by intense staining of arterial muscle with the fluorescent probe hydroethidine, by quantitation using fluorescent HPLC analysis, and by attenuation of the hypoxia-induced activation of the K(Ca) channel current by superoxide dismutation. These results suggest that the exposure of CAMCs to hypoxia results in the generation of superoxide and reduction in endogenous level of 20-HETE that may account for the hypoxia-induced activation of arterial K(Ca) channel currents and cerebral vasodilation.
目前尚不清楚大脑动脉中感受缺氧以及缺氧诱导脑动脉钙激活钾（K（Ca））通道电流和血管舒张的机制。我们研究了花生四烯酸的细胞色素P - 450 4A（CYP 4A）ω-羟化酶代谢产物20-羟基二十碳四烯酸（20-HETE）的作用，以及超氧化物的生成在大鼠脑动脉肌细胞（CAMCs）中缺氧诱发的K（Ca）通道电流激活和脑血管舒张中的作用。对K（+）通道电流进行膜片钳分析，确定了一种电压和钙依赖性的238±21 pS单位K（+）电流，该电流可被四乙铵（TEA，1 mM）或iberiotoxin（100 nM）抑制。缺氧（<2% O₂）可逆地增加了细胞贴附膜片中238 pS单位K（Ca）电流的开放概率（NP（o））。在从内向外或向外翻转的膜片上记录的单位K（Ca）电流上未观察到缺氧的这种作用。抑制CYP 4A ω-羟化酶活性增加了K（Ca）单通道电流的NP（o）。通过液相色谱-质谱分析测定，缺氧使脑动脉肌匀浆中20-HETE的基础内源性水平降低了47±3%，以及20-HETE的催化形成减少。与超氧化物供体KO₂孵育时，纯20-HETE的浓度降低。外源性20-HETE（100 nM）减弱了CAMCs中缺氧诱导的K（Ca）电流激活。缺氧并未增强用17-十八炔酸自杀抑制内源性CYP 4A ω-羟化酶活性所诱导的K（Ca）通道电流NP（o）的增加。在压力（80 mmHg）收缩的脑动脉节段中，缺氧诱导的舒张部分被20-HETE或K（Ca）通道阻滞剂TEA减弱。暴露于缺氧导致细胞内超氧化物的生成，这通过用荧光探针氢乙锭对动脉肌进行强烈染色、使用荧光HPLC分析进行定量以及超氧化物歧化对缺氧诱导的K（Ca）通道电流激活的减弱来证明。这些结果表明，CAMCs暴露于缺氧会导致超氧化物的生成和内源性20-HETE水平的降低，这可能解释了缺氧诱导的动脉K（Ca）通道电流激活和脑血管舒张。
