Thesis (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Ischaemic preconditioning (PC) is the phenomenon whereby a short episode of
coronary occlusion followed by reperfusion protects the myocardium against a
subsequent period of prolonged (also called index or sustained) ischaemia. Even
though the exact mechanism of PC remains to be established, it implies that the heart
has an endogenous protective mechanism against ischaemia which, if identified, may
have important clinical implications. The importance of establishing the mechanism of
PC lies in the potential to convert this biological phenomenon into a therapeutic modality
to be used clinically. If mediated by certain components of a signal transduction
pathway, such a goal will be achievable.
Several triggers and signal transduction pathways have been implicated in the mechanism
of protection induced by PC: for example, receptor-dependent endogenous triggers (such
as adenosine and opioids) and receptor-independent endogenous triggers (such as free
radicals and calcium). However, the involvement of both the ~-adrenergic signalling
pathway as well as nitric oxide (NO) in PC has not been defined.
It has been suggested that all triggers are linked to a common final pathway, for
example, activation of protein kinase C (PKC) and/or the mitogen-activated kinases
(MAPKs), in particular p38 MAPK. However, the role of the latter is still controversial.
The aim of this study was to:
(A) characterize changes in the cyclic nucleotides, cAMP and cGMP, and p38 MAPK
occurring during the entire experimental procedure in an attempt to gain insights into
the possible mechanisms involved in ischaemie PC (Chapter 3);
(8) establish the significance of the changes observed in cAMP and cGMP by
pharmacological manipulation of their respective pathways (Chapters 4 and 5);
(C) establish the role of p38 MAPK in ischaemie PC: trigger or mediator involvement
(Chapter 6).
Isolated perfused working rat hearts were preconditioned by 3 x 5 min global ischaemia,
interspersed by 5 min reperfusion, followed by 25 min global ischaemia and 30 min
reperfusion. Functional recovery during reperfusion was used as end-point. Hearts
were freeze-clamped at different times during the PC protocol, sustained ischaemia, as well as during reperfusion. Tissue cyclic nucleotides (cAMP and cGMP), cyclic
nucleotide phosphodiesterase (cAMP- and cGMP-PDE) activities, adenylyl cyclase and
protein kinase A activities and p-adrenergic receptor characteristics were determined.
p38 MAPK activation was also assessed by Western blotting, using dual phospho-p38
MAPK (Thr180ITyr182) antibody as well as activating transcription factor 2 (ATF2)
activation. In addition, to evaluate the role of p38 MAPK in PC protection, the effect of
inhibition of p38 MAPK activation, by 8B203580, was determined in adult isolated rat
cardiomyocytes as well as in isolated perfused rat hearts.
Based on the results obtained, it is proposed that during a multi-cycle ischaemie PC
protocol triggers (presumably endogenous catecholamines and NO) are released which
induce cyclic changes in cyclic nucleotides, cAMP and cGMP. Both these cyclic
nucleotides transiently activate the downstream stress kinase, p38 MAPK, which may
trigger further downstream adaptive processes.
Furthermore, the sustained ischaemic period of PC hearts was characterized by
attenuated cAMP and elevated cGMP levels, as well as attenuated activation of p38
MAPK, which was associated with cardioprotection. In addition, pharmacological
attenuation of p38 MAPK activation during sustained ischaemia led to functional recovery.
It is concluded that the cardioprotection of PC is due to attenuation of ischaemia-induced
p38 MAPK activation. Pharmacological manipulation of this kinase should be considered
as a therapeutic modality in the future. / AFRIKAANSE OPSOMMING: Isgemiese prekondisionering (PK) verwys na die verskynsel waardeur 'n kort,
verbygaande episode van isgemie gevolg deur herperfusie, die miokardium teen 'n
daaropvolgende langdurige periode van isgemie beskerm. Die presiese meganisme
van beskerming van PK moet nog opgeklaar word, maar dit impliseer dat die hart oor 'n
endogene beskermingsmeganisme beskik wat, indien geïdentifiseer, belangrike kliniese
implikasies mag hê. Die belang van opklaring van die meganisme van PK lê daarin dat
'n biologiese verskynsel in 'n terapeutiese modaliteit vir kliniese gebruik, omgeskakel
kan word. Sou dit deur bepaalde komponente van 'n seintransduksiepad gemedieër
word, is so 'n doel bereikbaar.
Verskeie stimuli en seintransduksiepaaie is in PK betrokke: byvoorbeeld, reseptorafhanklike
endogene stimuli (soos adenosien en opioïde), asook reseptor-onafhanklike
endogene stimuli (soos vrye radikale en kalsium). Die betrokkenheid van die padrenerge
seintransduksiepad asook stikstofoksied (NO) in PK egter nog nie behoorlik
evalueer nie.
Dit is voorgestel dat alle stimuli op 'n finale algemene pad uitloop, soos byvoorbeeld die
aktivering van protein kinase C (PKC) en/of die mitogeen-geaktiveerde kinases
(MAPKs), spesifiek die p38 MAPKs. Laasgenoemde se rol in PK is steeds
kontroversieël.
Die doel van die studie was dus:
(A) karakterisering van die veranderinge in die sikliese nukleotiede, cAMP en cGMP,
en p38 MAPK wat tydens die hele eksperimentele prosedure plaasvind, in 'n
poging om meer insig te verkry aangaande moontlike meganismes betrokke in
isgemiese PK (Hoofstuk 3);
(8) bepaling van die belang van die waargenome veranderinge in cAMP en cGMP
deur hulonderskeie paaie farmakologies te manipuleer (Hoofstukke 4 en 5);
(C) bepaling van die rol van p38 MAPK in PK: betrokkenheid as stimulus of mediator
(Hoofstuk 6).
Geïsoleerde, geperfuseerde werkende rotharte is geprekondisioneer deur blootstelling
aan 3 x 5 min globale isgemie, afgewissel met 5 min herperfusie, gevolg deur 25 min globale isgemie en 30 min herperfusie. Funksionele herstel tydens herperfusie is as
eindpunt gebruik. Harte is op verskillende tye tydens die PK protokol, volgehoue
isgemie, asook herperfusie gevriesklamp. Weefsel sikliese nukleotiede (cAMP en
cGMP), die aktiwiteit van sikliese nukleotied fosfodiesterases (cAMP- en cGMP-PDE),
adeniel siklase en protein kinase A (PKA) asook die eienskappe van die p-adrenerge
reseptor is gemeet. p38 MAPK aktivering is met Westerse oordragtegnieke bepaal,
deur van dubbel gefosforileerde p38 MAPK (Thr180fTyr182) antiliggame asook
geaktiveerde transkripsie faktor 2 (ATF2) gebruik te maak. Die rol van p38 MAPK in PK
beskerming is evalueer deur die effek van inhibisie van p38 MAPK aktivering met SB
203580, in volwasse geïsoleerde rot kardiomiosiete asook in geïsoleerde geperfuseerde
rotharte, te bepaal.
Na aanleiding van die resultate, is voorgestel dat, tydens 'n multi-siklus isgemie PK
protokol, stimuli (moontlik endogene katekolamiene en NO) vrygestel word wat die
sikliese veranderinge in sikliese nukleotiede, cAMP en cGMP, veroorsaak. Beide
hierdie sikliese nukleotiede aktiveer die distale stres kinase, p38 MAPK, op 'n
betekenisvolle, maar verbygaande manier. Hierdie kinase mag verdere distale
aanpassingsprosesse stimuleer.
Die volgehoue isgemiese periode van PK harte is gekenmerk deur verminderde cAMP
en verhoogde cGMP vlakke, asook verminderde aktivering van p38 MAPK. Hierdie
veranderinge is met beskerming van die hart teen isgemie geassosieer.
Daarbenewens, farmakologiese vermindering van p38 MAPK aktivering tydens
volgehoue isgemie het tot verbeterde funksionele herstel gelei. Die gevolgtrekking is
gemaak dat die beskermende effek van PK die gevolg is van verminderde aktivering
van isgemies-geïnduseerde p38 MAPK. Farmakologiese manipulasie van hierdie
kinase moet in die toekoms as terapeutiese modaliteit oorweeg word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53039 |
| Date | 12 1900 |
| Creators | Marais, Erna |
| Contributors | Lochner, A., Moolman, J. A., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 210 p. : ill. |
| Rights | Stellenbosch University |
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