Nitric oxide and the endothelium : characterisation of in vitro nitric oxide detection techniques and an ex vivo method of measuring endothelial function

[摘要] ENGLISH ABSTRACT:Introduction: Nitric oxide (NO) is an important chemical messenger in the cardiovascularsystem. Despite considerable progress in this field, there remains an on-going need foraffordable and user-friendly NO measurement techniques. Therefore, in this study weaimed to develop and characterise NO-detection techniques not previously used in ourlaboratory, and, in addition, characterise an ex vivo method to measure the functionaleffects of the endothelium and NO production in the vasculature.Methods: Three different NO-detection techniques were compared: (i) AmperometricNO sensors. Here, NO-increasing effects of known NO synthase (NOS) activatorswere investigated (insulin, acetylcholine and biosynthetic human insulin). Three differentNO sensors were evaluated on cultured endothelial cells and aortic tissue. Putative NOincreasingeffects of shear stress were also investigated; (ii) Nitrite (NO2-) + nitrate(NO3-) sensors. Here, I aimed to measure NO release from cultured endothelial cells;(iii) Colorimetric NO2- measurement assay with the Griess reagent. Here, NO2-production by endothelial cells was measured with a plate reader.In the second part of the study an organ bath - isometric tension technique wasestablished to measure endothelium-dependent function of aortic rings. Functionaldifferences in aortic rings isolated from diet-induced obese rats compared to lean ratswere investigated. Ring contraction was induced with phenylephrine and relaxation withacetylcholine. These investigations were further supported by western blot analyses ofselected critical proteins. Lastly, the effects of perivascular adipose tissue (PVAT) oncontraction and relaxation were investigated in endothelium-containing or denudedaortic ring segments.Results: Although some success was achieved with the amperometric sensorsregarding calibration, any experimental results obtained were difficult to repeat due toinstability of the sensors. With the NO2-/NO3- sensor we were not able to carry out anyplanned experiments due to failure to properly calibrate and standardise the sensors.Success was achieved with the Griess method. All the drugs used as positive controls(DEA/NO, fenofibrate, oleanolic acid and IL-1ß) proved to be potent inducers of NO2-release from endothelial cells.Interestingly, the isometric tension studies showed a higher % relaxation in high fat (HF)diet aortic rings compared to those from lean animals. Western blot data showeddownregulation of eNOS activation and iNOS expression in obese groups, which wassuggestive of endothelial dysfunction. Interestingly, proteins associated with oxidativestress (p22phox and nitrotyrosine) were downregulated in obese groups. The presenceof PVAT exerted anti-contractile effects on the rings from HF rats, however in denudedaortic rings, PVAT showed a significant pro-contractile response in both lean and HFgroups. PVAT also exerted anti-relaxation effects in aortic rings from both lean and HFrats.Conclusion: We managed to successfully establish two new techniques for ourlaboratory (Griess method and the organ bath – isometric tension method) which cancomplement the more established techniques in our laboratory in order to aid us infuture vascular research. Finally, the isometric tension technique used in the obese ratstudies generated interesting data, which further assisted in characterising the dietinducedobesity rat model in our laboratory.

[发布机构] Stellenbosch University