非損傷檢測胎兒肺上皮細胞微環境中Cl-流的變化情況
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《JEB》 肺上皮細胞離子流與疾病發生 | |
| 非損傷檢測胎兒肺上皮細胞微環境中Cl-流的變化情況
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  上圖： 使用非損傷微測技術檢測Cl-流的實時圖和Cl-流的變化情況 肺上皮細胞中體液運輸由上皮細胞膜兩側滲透壓產生的Na+和Cl-流調節支配，而離子轉運過程中所需的能量是由活性Na+/K+ATP酶所提供，Na+和Cl-的跨膜分布是由配體調節的離子通道和交換器所控制，所以如果Cl-的轉運發生缺陷將導致與體液運輸失調相關的疾病。 囊性纖維病的發生就是由于Cl-轉運的缺失而造成。對于這種疾病的研究，先前采用的檢測方法，如同位素示蹤法檢測到的并不是單一的Cl-在胞內外的轉運情況，而是進出細胞的所有離子轉運的總體情況，所以得到的結論并不能精確的說明Cl-在細胞內外的變化情況。而非損傷微測技術（SrE）通過選用Cl-自參比電極檢測胞外Cl-流的變化的方法克服了這個問題。 在該研究中通過選擇性Cl-自參比電極實時、高分辨率、非損傷性的方式來檢測胎兒遠端肺上皮細胞基底或頂端在激動劑作用下Cl-流的變化方向和胞外Cl-數量變化。研究結果表明肺遠端頂膜生理學微環境中體液轉運與Cl-梯度存在密切關系，Cl-的動力學梯度能夠作為質膜微環境中真實的Cl-指示者。這對于理解通氣管表面液體中的鹽濃度調控細胞和分子過程與上皮體液轉運的關系非常重要。 關鍵詞：自參比電極(self-referencing electrode,SrE);lung(肺);囊性纖維化(cystic Fibrosis);β2腎上腺素受體(β2 adrenoreceptor);氯離子流(Cl- flux)
參考文獻：LAND SC, et al. The Journal of Experimental Biology, 2001, 204, 785-795
全文下載：http://jeb.biologists.org/cgi/reprint/204/4/785
A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (JCl) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmolcm-2 s-1). A Cl- effusion gradient was used to confirm that empirical measurements of JCl using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical JCl was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50mmoll-1, an apical-tobasolateral (inward) flux, maximal at 400 pmolcm-2 s-1, was observed; this reverted to a continuous basolateralto-apical (outward) flux of 203 pmolcm-2 s-1 at [Cl]e> 100 mmoll-1. At [Cl-]e>100mmoll-1, isoproterenol (basolaterally applied, 10 mmol l-1) activated a Cl- influx of 561 pmolcm-2 s-1, whereas UTP (apically applied, 100 mmol l-1) stimulated a Cl- efflux of 300 pmolcm-2 s-1. In all cases, 50–70 % of JCl was abolished by Cl- channel blockade using 10 mmol l-1 diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to β2 adrenoreceptor and P2Y receptor agonists. Key words: self-referencing electrode, fluid transport, lung, cystic fibrosis, β2 adrenoreceptor, P2Y receptor, Cl- flux. | |
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