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

Loubser, Dirk Jacobus (2014-04)

Thesis (MScMedSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Introduction: Nitric oxide (NO) is an important chemical messenger in the cardiovascular system. Despite considerable progress in this field, there remains an on-going need for affordable and user-friendly NO measurement techniques. Therefore, in this study we aimed to develop and characterise NO-detection techniques not previously used in our laboratory, and, in addition, characterise an ex vivo method to measure the functional effects of the endothelium and NO production in the vasculature. Methods: Three different NO-detection techniques were compared: (i) Amperometric NO sensors. Here, NO-increasing effects of known NO synthase (NOS) activators were investigated (insulin, acetylcholine and biosynthetic human insulin). Three different NO sensors were evaluated on cultured endothelial cells and aortic tissue. Putative NOincreasing effects 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 was established to measure endothelium-dependent function of aortic rings. Functional differences in aortic rings isolated from diet-induced obese rats compared to lean rats were investigated. Ring contraction was induced with phenylephrine and relaxation with acetylcholine. These investigations were further supported by western blot analyses of selected critical proteins. Lastly, the effects of perivascular adipose tissue (PVAT) on contraction and relaxation were investigated in endothelium-containing or denuded aortic ring segments. Results: Although some success was achieved with the amperometric sensors regarding calibration, any experimental results obtained were difficult to repeat due to instability of the sensors. With the NO2 -/NO3 - sensor we were not able to carry out any planned 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 showed downregulation of eNOS activation and iNOS expression in obese groups, which was suggestive of endothelial dysfunction. Interestingly, proteins associated with oxidative stress (p22phox and nitrotyrosine) were downregulated in obese groups. The presence of PVAT exerted anti-contractile effects on the rings from HF rats, however in denuded aortic rings, PVAT showed a significant pro-contractile response in both lean and HF groups. PVAT also exerted anti-relaxation effects in aortic rings from both lean and HF rats. Conclusion: We managed to successfully establish two new techniques for our laboratory (Griess method and the organ bath – isometric tension method) which can complement the more established techniques in our laboratory in order to aid us in future vascular research. Finally, the isometric tension technique used in the obese rat studies generated interesting data, which further assisted in characterising the dietinduced obesity rat model in our laboratory.

AFRIKAANSE OPSOMMING: Inleiding: Stikstofoksied (NO) is ‘n belangrike chemiese boodskapper in die kardiovaskulêre sisteem. Ondanks vordering in die veld, bestaan daar ‘n aangaande behoefte aan bekostigbare en gebruikersvriendelike NO-metingstegnieke. Gevolglik het ons in hierdie studie daarna gemik om NO-metingstegnieke wat nie vantevore in ons laboratorium beskikbaar was nie, te ontwikkel en karakteriseer. Verder het ons ten doel gehad om ‘n ex vivo model te karakteriseer om die funksionele effekte van vaskulêre endoteel en NO produksie te meet. Metodes: Drie verskillende NO-metingstegnieke was ondersoek: (i) Amperometriese NO sensors. Hier het ons die verhogende effekte op NO van bekende aktiveerders van NO sintetase (NOS) ondersoek (Insulien, asetielcholien en biosintetiese menslike insulien). Drie verskillende NO-sensors was ge-evalueer in gekultuurde endoteelselle en aortaweefsel. Die vermeende NO verhogende effekte van die wrywingskragte opgewek deur laminere vloei (“shear stress”) is ook ondersoek. (ii) Nitriet (NO2 -) + nitraat (NO3 -) sensors. Hier het ons beplan om NO-vrystelling deur gekultuurde endoteelselle te meet. (iii) Kolorimetriese meting van NO2 - met die Griess reagens. Hier het ons m.b.v. ‘n mikroplaat leser die NO2 - - vrystelling deur endoteelselle gemeet. In die tweede deel van die studie het ons ‘n orgaan bad–isometriese spanningstegniek opgestel om endoteelafhanklike funksie van aortaringe te meet. Funksionele verskille in aortaringe van vetsugtige rotte is vergelyk met kontrole rotte. Ringkontraksie is met fenielefrien geïnduseer en verslapping met asetielcholien. Hierdie ondersoeke is verder ondersteun deur Western blot analises van sleutelproteïene in die aortaweefsel. Laastens het ons die effekte van perivaskulêre vetweefsel (PVAT) op kontraksie en verslapping in aortaringe met of sonder intakte endoteel ondersoek. Resultate: Alhoewel ‘n mate van sukses behaal was met die kalibrasie van die amperometriese sensors, was eksperimentele resultate moeilik om te herhaal a.g.v. sensor-onstabiliteit. Geen eksperimente kon met die NO2 -/NO3 - sensors uitgevoer word nie weens ‘n onvermoë om ordentlike kalibrasie en standardisering uit te voer. Ons het egter wel sukses behaal met die Griess-metode. Al die middels wat as positiewe kontroles gebruik was (DEA/NO, fenofibraat, oleanoliese suur and IL-1ß) het geblyk kragtige induseerders van NO2 - produksie vanaf endoteelselle te wees. Die isometriese spanningsstudies het ‘n hoer % verslapping getoon in die hoë vet (HF) dieet aortaringe in vergelyking met die kontroles. Western blot data het ‘n afregulering van eNOS en iNOS getoon in die HF diere, wat aanduidend is van endoteel disfunksie, terwyl proteïene geassosieer met oksidatiewe stress (p22phox en nitrotirosien) afgereguleer was in die HF groep. Die aanwesigheid van PVAT het ‘n anti-kontraktiele effek gehad op die ringe van die HF groep. Toe die endoteel egter verwyder was, het PVAT in beide kontrole en HF ringe ‘n beduidende pro-kontraktiele effek gehad. Verder het PVAT ook anti-verslappingseffekte op aortaringe beide kontrole en HF rotte uitgeoefen. Gevolgtrekking: Ons het daarin geslaag om twee nuwe tegnieke vir ons laboratorium suksesvol te vestig (Griess metode en die orgaanbad-isometriese spanningstegniek) wat in die toekoms die meer gevestigde tegnieke in ons laboratorium kan komplementeer. Laastens het die isometriese spanningstegniek wat in die dieetstudies gebruik is, data opgelewer wat ons verder sal help om die vetsug model in ons laboratorium te karakteriseer.

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