Thesis (MScMedSc)--Stellenbosch University, 2014. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/86496 |
| Date | 04 1900 |
| Creators | Loubser, Dirk Jacobus |
| Contributors | Strijdom, Hans, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 129 p. : ill. |
| Rights | Stellenbosch University |
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