The Korean Journal of Malacology (한국패류학회지)

The Malacological Society of Korea (한국패류학회)
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Quantification of nitric oxide concentration in the hemocytes of Manila clam Ruditapes philippinarum by using 4,5-diaminofluorescein diacetate (DAF-2) detection method

바지락 (Ruditapes philippinarum) 혈구의 일산화질소 (nitric oxide) 정량

  • Nam, Ki-Woong (Department of Aquatic Life Medicine, Kunsan National University) ;
  • Yang, Hyun-Sung (East Sea Research Institute, Korea Institute of Ocean Science and Technology (KIOST)) ;
  • Park, Kyung-Il (Department of Aquatic Life Medicine, Kunsan National University)
  • 남기웅 (군산대학교 해양과학대학 수산생명의학과) ;
  • 양현성 (한국해양과학기술원 동해연구소) ;
  • 박경일 (군산대학교 해양과학대학 수산생명의학과)
  • Received : 2013.03.10
  • Accepted : 2013.03.20
  • Published : 2013.03.31
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Abstract

Nitric oxide (NO) is an important intra-intercellular signaling molecule that regulates many physiological processes and participates in the development some pathological conditions in animals. In this study, we compared different methods for determining NO concentration in the hemocytes of Manila clam Ruditapes philippinarum. For measuring the intracellular NO levels, we used the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA), and the quantification methods that were compared were based on image analysis, spectrophotometry, and flow cytometry. NO concentration could be determined using all the 3 methods, and the concentration varied significantly depending upon the presence of NO regulators in the hemocytes; NO concentration increased in the presence of L-arginine, while it decreased in the presence of N-nitro-L-arginine methyl ester. In particular, it is found that estimation of NO using a flowcytometry is more economical, reliable and accurate compared to image analysis and spectrophotometry. Accordingly we believe that determining NO concentration by using flowcytometry will be useful in evaluating physiological and pathological conditions in marine bivalves.

일산화질소 (NO) 는 면역계에서 세포내 외의 신호전달에 관여하는 물질로 생물의 생리적, 병리학적 기작을 조절한다. 본 연구는 바지락 혈구의 NO 농도 측정을 위해 4,5-diaminofluorescein diacetate (DAF-2 DA) 를 이용한 DAF assay의 적용이 가능한지 확인하고자 화상분석법, 형광흡광도 측정법 및 유세포분석 기법 등을 이용하였다. 연구결과 인위적인 바지락 혈구의 NO 생성을 위해 L-arginine을 첨가한 경우 대조구에 비하여 NO 생성이 유의적으로 증가하였고, 반대로 NO 저해제인 L-NAME를 첨가한 경우 NO 생성은 급격히 감소하였다. 이러한 결과는 본 조사에 이용된 화상분석법, 형광흡광도 측정법 및 유세포분석 기법 등 모든 조사 방법에서 동일하게 확인되었다. 특히 3가지 측정 방법 중 유세포 분석법은 측정의 신속성, 신뢰성 및 정확성을 담보할 수 있는 유용한 방법으로 판단된다. 따라서 유세포 분석기를 이용한 NO 측정은 향후 바지락의 생리적 병리적 특성을 확인하는데 유용한 마커로써 이용될 수 있을 것으로 기대된다.

Keywords

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