토끼 대동맥 혈관내피세포에서 저산소증이 내피세포성 이완인자의 분비에 미치는 영향

The Effect of Hypoxia on the Release of Endothelium-derived Relaxing Factor in Rabbit Thoracic Aorta

  • 최수승 (이화여자대학교 의과대학 흉부외과학교실)
  • Choi, Soo-Seung (Department of Thoracic and Cardiovascular Surgery, Ewha Womans University School of Medicine)
  • 발행 : 2009.10.05

초록

배경: 저산소증이 혈관평활근 수축성에 미치는 영향을 규명하기 위하여 저산소증이 혈관내피세포에서 내피세포성 이완인자의 분비에 미치는 영향과 그 기전을 규명하고자 하였다. 대상 및 방법: 토끼 대동맥에서 내피세포 의존성 이완과 관찰하고, 토끼 대동맥에서 내피세포성 이완인자 분비 정도를 내피세포를 제거한 경동맥의 수축에 미치는 영향으로 생물검증을 하였다. 마지막으로, 배양한 토끼 대동맥 혈관내피세포에서 세포내 $Ca^{2+}$ 변화를 측정하였다. 저산소증은 세포의 용액에 공급되는 산소를 질소로 대체하여 제거한 후 이 용액을 혈관 혹은 세포에 공급하여 유발시 거나, deoxyglucose 혹은 $CN^-$를 투여하여 화학적인 저산소증을 유발시켰다. 결과: 노에피네프린으로 토끼 대동맥을 수축시킨 다음 저산소증에 노출시키면 대동맥이 이완을 하였으며 저산소증에 반복하여 노출시키면 저산소증에 의한 이완이 더 크게 증가하였다. 이러한 저산소증에 의한 이완은 혈관내피세포를 제거한 대동맥에서는 관찰되지 않았다. 토끼 대동맥에서 분비되는 내피세포성 이완인자 분비를 내피세포를 제거한 경동맥을 이용하여 생물검증한 결과 저산소증에 의하여 내피세포성 이완인자의 분비가 증가하였는데 반복된 노출에 의하여 더 크게 증가하였다. 그리고 저산소증에 의한 내피세포성 이완인자 분비는 NO 생성을 억제하는 경우와 $K^+$ 통로 억제제인 tetraethyl ammonium (TEA)에 의하여 억제되었다. 배양한 혈관내피세포에서 ATP에 의하여 증가한 세포내 $Ca^{2+}$은 저산소증에 의하여 유의하게 증가하였으며 TEA에 의하여 억제되었다. Deoxyglucose에 의하여 세포내 $Ca^{2+}$이 증가하였으며 세포외 $Ca^{2+}$을 제거하면 감소하였다. $CN^-$ 역시 혈관내피세포 $Ca^{2+}$ 유입을 증가시켰다. 결론: 이러한 실험 결과로 미루어 토끼 대동맥에서 저산소증은 내피세포 의존성 이완을 유발하는데 이는 저산소증에 의한 세포내 $Ca^{2+}$ 유입 증가에 의하여 NO 생성이 증가되어 일어난 것으로 추정할 수 있었다.

Background: To clarify the effect of hypoxia on vascular contractility, we tried to show whether hypoxia induced the release of endothelium-derived relaxing factor (EDRF) and the nature of the underlying mechanism for this release. Material and Method: Isometric contractions were observed in rabbit aorta, and the released EDRF from the rabbit aorta was bioassayed by using rabbit denuded carotid artery. The intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in the cultured rabbit aortic endothelial cells was recorded by a microfluorimeter with using Fura-2/AM. Hypoxia was evoked to the blood vessels or endothelial cells by eliminating the $O_2$ in the aerating gases in the external solution. Chemical hypoxia was evoked by applying deoxyglucose or $CN^-$. Result: Hypoxia relaxed the precontracted rabbit thoracic aorta that had its endothelium, and the magnitude of the relaxation was gradually increased by repetitive bouts of hypoxia. In contrast, hypoxia-induced relaxation was not evoked in the aorta that was denuded of endothelium. In a bioassay experiment, hypoxia released endothelium-derived relaxing factor (EDRF) and the release was inhibited by L-NAME or the $K^+$ channel blocker tetraethylammonium (TEA). In the cultured endothelial cells, hypoxia augmented the ATP-induced increase of the intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) and this increase was inhibited by TEA. Furthermore, chemical hypoxia also increased the $Ca^{2+}$ influx. Conclusion: From these results, it can be concluded that hypoxia might induce the release of NO from rabbit aortic endothelial cells by increasing $[[Ca^{2+}]_i$.

키워드

참고문헌

  1. Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 2001;81:1415-59 https://doi.org/10.1152/physrev.2001.81.4.1415
  2. Inagami T, Naruse M, Hoover, R. Endothelium as an endocrine organ. Annu Rev Physiol 1995;57:171-89 https://doi.org/10.1146/annurev.ph.57.030195.001131
  3. Dobrina A, Rossi F. Metabolic properties of freshly isolated bovine endothelial cells. Biochim Biophys Acta 1983;762: 295-301 https://doi.org/10.1016/0167-4889(83)90084-8
  4. Cappelli-Bigazzi M, Battaglia C, Pannain S, Chiariello M, Ambrosio G. Role of oxidative metabolism on endothelium- dependent vascular relaxation of isolated vessels. J Mol Cell Cardiol 1997;29:871-9 https://doi.org/10.1006/jmcc.1996.0286
  5. Hashimoto M, Close LA, Ishida Y, Paul RJ. Dependence of endothelium-mediated relaxation on oxygen and metabolism in porcine coronary arteries. Am J Physiol 1993;265(1 Pt 2):H299-306 https://doi.org/10.1152/ajpcell.1993.265.1.C299
  6. Forman MB, Puett DW, Virmani R. Endothelial and myocardial injury during ischemia and reperfusion: pathogenesis and therapeutic implications. J Am Coll Cardiol 1989;13: 450-9 https://doi.org/10.1016/0735-1097(89)90526-3
  7. Suval WD, Duran WN, Boric MP, Hobson RW, Berendsen PB, Ritter AB. Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury. Am J Surg 1987;154:211-8 https://doi.org/10.1016/0002-9610(87)90181-4
  8. Flamant L, Toffoli S, Raes M, Michiels C. Hypoxia regulates inflammatory gene expression in endothelial cells. Exp Cell Res 2009;315:733-47 https://doi.org/10.1016/j.yexcr.2008.11.020
  9. Michiels C, Arnould T, Remacle J. Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions. Biochim Biophys Acta 2000;1497:1-10 https://doi.org/10.1016/S0167-4889(00)00041-0
  10. Rodman DM, Yamaguchi T, Hasunuma K, O'Brien RF, McMurtry IF. Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am J Physiol 1990;258(4 Pt 1):L207-14
  11. Johns RA, Linden JM, Peach MJ. Endothelium-dependent relaxation and cyclic GMP accumulation in rabbit pulmonary artery are selectively impaired by moderate hypoxia. Circ Res 1989;65:1508-15 https://doi.org/10.1161/01.RES.65.6.1508
  12. Brown IP, Thompson CI, Belloni FL. Role of nitric oxide in hypoxic coronary vasodilatation in isolated perfused guinea pig heart. Am J Physiol 1993;264(3 Pt 2):H821-9
  13. Archer SL, Tolins JP, Raij L, Weir EK. Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor. Biochem Biophys Res Commun 1989;164:1198-205 https://doi.org/10.1016/0006-291X(89)91796-8
  14. Aggarwal NT, Pfister SL, Gauthier KM, Chawengsub Y, Baker JE, Campbell WB. Chronic hypoxia enhances 15-lipoxygenase-mediated vasorelaxation in rabbit arteries. Am J Physiol Heart Circ Physiol 2009;296:H678-88 https://doi.org/10.1152/ajpheart.00777.2008
  15. Bajpai AK, Blaskova E, Pakala SB, et al. 15(S)-HETE production in human retinal microvascular endothelial cells by hypoxia: Novel role for MEK1 in 15(S)-HETE induced angiogenesis. Invest Ophthalmol Vis Sci 2007;48:4930-8 https://doi.org/10.1167/iovs.07-0617
  16. Rubanyi GM, Lorenz RR, Vanhoutte PM. Bioassay of endothelium-derived relaxing factor(s): inactivation by catecholamines. Am J Physiol 1985;249(1 Pt 2):H95-101
  17. Suh SH, Vennekens R, Manolopoulos VG, et al. Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and $Ca^{2+}$ signalling. Pflugers Arch 1999;438: 612-20 https://doi.org/10.1007/s004240051084
  18. Buckley BJ, Mirza Z, Whorton AR. Regulation of $Ca^{2+}$- dependent nitric oxide synthase in bovine aortic endothelial cells. Am J Physiol 1995;269:C757-65
  19. Winquist RJ, Bunting PB, Schofield TL. Blockade of endothelium-dependent relaxation by the amiloride analog dichlorobenzamil: possible role of $Na^+/Ca^{2+}$ exchange in the release of endothelium-derived relaxant factor. J Pharmacol Exp Ther 1985;235:644-50