Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The Mechanism of -adrenergic preconditioning ( -PC)
Ischaemic preconditioning (IPC), a potent endogenous protective intervention against myocardial
ischaemia, is induced by exposure of the heart to repetitive short episodes of ischaemia and
reperfusion. The protective effects of this phenomenon have been demonstrated to be mediated by
release of autocoids such as adenosine, opioids and bradykinin. Release of endogenous
catecholamines and activation of the beta-adrenergic receptors (b-AR) have also been shown to be
involved in ischaemic preconditioning. However, the exact mechanism whereby activation of the -
adrenergic signal transduction pathway leads to cardioprotection, is still unknown.
In view of the above, the aims of the present study were to evaluate:
(i) the respective roles of the 1-, 2- and 3-AR receptors as well as the contribution of Gi
protein and PKA to -adrenergic preconditioning,
(ii) the role of the prosurvival kinases, PKB/Akt and ERK 44/p42 MAPKinase in -drenergic
preconditioning,
(iii) whether b-AR stimulation protect via ischaemia and the formation of adenosine; the
respective roles of the A1-, A2-, A3-adenosine receptors as well as the involvement of the
PI3-K/PKB/Akt and ERKp44/p42 signal transduction pathways, in the cardioprotective
phenomemon of -adrenergic preconditioning and
(iv) the contribution of the mitochondrial KATP channels (mKATP), reactive oxygen species and NO
to the mechanism of -AR-induced cardioprotection.
Methods: Isolated perfused rat hearts were subjected to 35 min regional ischaemia (RI) and
reperfusion. Infarct size (IS) was determined using tetrazolium staining (TTC) and data were
analyzed with ANOVA. Hearts were preconditioned with 5 min isoproterenol 0.1 μM ( 1/ 2-AR
agonist), or formoterol 1 nM ( 2-AR agonist) or BRL 37344 1 μM ( 3-AR agonist) followed by 5
min reperfusion. The roles of the 1-, 2- and 3-ARs as well as NO were explored by using the
selective antagonists CGP-20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) and NOS
inhibitors L-NAME (50 μM) or LNNA (50 μM) respectively. Involvement of ROS and the mK+
ATP
channels was studied by administration of N-acetyl cysteine (NAC, 300 μM) and the mitK+
ATP
iv
channel blocker 5-HD (100 μM) during the triggering phase. The role of PKA and PI3-K/Akt was
investigated by the administration of the blockers Rp-8-CPT-cAMPs (16 μM) and wortmannin (100
nM) respectively, prior to RI or at the onset of reperfusion. Pertussis toxin (PTX), 30 μg kg-1 was
administered i.p., 48 h prior to experimentation.
The role of adenosine and the adenosine A1, A3, A2A and A2B receptors was studied by using
adenosine deaminase and the selective antagonists DPCPX (1 μM), MRS 1191(1 μM), ZM241385
(1 μM) and MRS1754 (1 μM). Activation of PKB/Akt and ERKp44/p42 was determined by
Western blot.
Results: Infarct sizes of hearts preconditioned with isoproterenol of formoterol were significantly
smaller compared to those of non-preconditioned hearts. This was associated with an improvement
in postischaemic mechanical performance. However the 3-AR agonist BRL37344 could not reduce
infarct size. The 1- and 2-AR blockers CGP-20712A and ICI-118551 completely abolished the
isoproterenol-induced reduction in infarct size and improvement in mechanical recovery, while the
3-AR blocker was without effect.
Both Rp-8-CPT-cAMPs and wortmannin significantly increased infarct size when administered
before 1/ 2-AR preconditioning or at the onset of reperfusion while it reduced mechanical
recovery during reperfusion. PTX pretreatment had no significant effect on the reduction in infarct
size induced by 1/ 2-AR or 2-AR preconditioning, however it reduced mechanical recovery in the
latter. The NOS inhibitors had no effect on the reduction in infarct size induced by 1/ 2-AR
preconditioning, but depressed mechanical function during reperfusion.
The significant reduction in infarct size by 1/ 2-PC, was associated with activation of ERKp44/p42
and PKB/Akt during the triggering phase, as well as during reperfusion. DPCPX (A1-AdoR
antagonist) had no effect on the 1/ 2-PC-induced reduced infarct size or ERK p44/p42 and PKB
activation.
A2A-AdoR, but not A2b-AdoR, blockade during the trigger phase abolished the reduction in infarct
size of 1/ 2-PC. Both antagonists significantly reduced ERK and PKB activation in the trigger
phase. In addition, when applied at the onset of reperfusion they significantly reduced ERK p44 /
v
p42 MAPK and PKB/Akt activation to an even greater extent. MRS-1191 (A3-AdoR antagonist)
blocked 1/ 2-PC when applied prior to index ischaemia or when added during early reperfusion,
significantly inhibiting both ERK p44 and PKB activation.
Cardioprotection of 1/ 2-PC was abolished by inhibition of ROS generation with NAC in the
triggering phase as well as at the start of reperfusion. However, the mitoK+
ATP channel blocker 5-
HD was without effect.
Conclusions: Protection afforded by an acute transient stimulation of the -ARs, depends on the
activation of both 1-AR and 2-ARs but not the 3-AR. PKA as well as PI3-K activation prior to
sustained ischemia and at the onset of reperfusion were essential for cardioprotection. With
functional recovery as endpoint, it appears that NO is involved in 1/ 2-AR preconditioning, while
the Gi protein may play a role in 2-AR preconditioning.
The production of endogenous adenosine induced by transient b1/b2 stimulation of the isolated rat
heart is involved in b−AR preconditioning. Cardioprotection was shown not to be dependent on the
A1AdoR while activation of the A3-AdoR occurs during both the triggering and mediation phases.
Both the adenosine A2A and, to a lesser extent, the adenosine A2B receptors participate in the
triggering phase of b1/b2-PC. Generation of ROS during the triggering and reperfusion phases is
involved in eliciting protection, but no role for the mKATP channels could be demonstrated. Finally,
activation of the RISK pathway (PKB/Akt and ERKp44/p42) during the triggering phase is a
prerequisite for protection. In addition, cardioprotection by b-AR is characterized by activation of
the RISK pathway during reperfusion. / AFRIKAANSE OPSOMMING: Iskemiese prekondisionering (IPC) is ‘n kragtige endogene beskerming teen miokardiale iskemie,
wat deur blootstelling van die hart aan kort opeenvolgende episodes van iskemie en herperfusie,
ontlok word. Hierdie beskerming word medieer deur vrystelling van outakoïede soos adenosine,
opioïede en bradikinien. Vrystelling van endogene katekolamiene en aktivering van die betaadrenerge
reseptore (b-AR) is bewys om ook by hierdie proses betrokke te wees. Die presiese
meganismes waardeur aktivering van die -adrenerge seintransduksiepad tot miokardiale
beskerming lei, is nog onbekend.
In die lig van bogenoemde, was die doel van die huidige studie om die volgende te evalueer: (i) die
onderskeie rolle van die b1-, b2- en b3-AR sowel as die bydrae van die Gi proteïen en PKA in b-
adrenerge prekondisionering, (ii) of b-AR stimulasie beskerming ontlok via iskemie en vorming van
adenosien, die onderskeie rolle van die A1-, A2-, A3-adenosien reseptore (AdoRs) sowel as die PI3-
K/PKB/Akt en ERKp44/p42 seintransduksie paaie, (iv) die mitochondriale KATP (mKATP) kanale,
vry suurstof radikale en NO in b−AR prekondisionering.
Metodes: Geïsoleerde, geperfuseerde rotharte is aan 35 minute streeksiskemie en herperfusie
onderwerp. Infarktgrootte (IS) is deur die tetrazolium (TTC)-kleuringsmetode bepaal. Data is met
behulp van ANOVA analiseer. Harte is geprekondisioneer vir 5 min met isoproterenol 0.1 μM
( 1/ 2-AR agonist), of formoterol 1 nM ( 2-AR agonist) of BRL 37344 1 μM ( 3-AR agonist),
gevolg deur 5 min herperfusie, voor streeksiskemie. Die belang van die 1-, 2- en 3-ARs sowel
as NO is ondersoek, deur onderskeidelik gebruik te maak van selektiewe antagoniste nl CGP-
20712A (300 nM), ICI -18551 (50 nM), SR59230A (100 nM) en NOS inhibitore L-NAME (50μM)
of LNNA (50μM). Die rol van die mK+
ATP kanale en ROS is bepaal deur die toediening van die
mK+
ATP kanaal blokker 5-HD (100 μM) en die vrye-radikaal opruimer, N-asetiel cysteine (NAC,
300 μM). Die belang van PKA en PI3-K/Akt is bepaal deur toediening van die PKA blokker Rp-8-
CPT-cAMPs (16μM) en wortmannin (100nM) respektiewelik. Pertussis toxin (PTX), 30 μg kg-1 is
i.p toegedien, 48 uur voor eksperimentasie.
vii
Die rol van adenosien en die adenosien A1, A2A, A2B en A3 reseptore is bestudeer, deur gebruik te
maak van adenosien deaminase en die selektiewe antagoniste DPCPX (1 μM), MRS 1191(1 μM),
ZM241385 (1 μM) and MRS1754 (1 μM),repektiewelik. Die middels is deurgaans toegedien tydens
die prekondisioneringsprotokol (“snellerfase”) of tydens vroeë herperfusie. Aktivering van PKB/Akt
en ERK p44/p42 is deur Western blot analise bepaal.
Resultate: Infarktgrootte van harte wat geprekondisioneer is met of isoproterenol ( 1/ 2-PC) of
formoterol ( 2-PC), was beduidend kleiner as díe van ongeprekondisioneerde harte. Dit is
geassosieer met ‘n toename in postiskemiese meganiese herstel. Die 3-AR agonis BRL37344 ( 3-
PC) het egter geen effek op infarktgrootte gehad nie. Die selektiewe 1- en 2-AR blokkers CGP-
20712A en ICI-118551 het die afname in infarktgrootte heeltemal opgehef, asook die verbetering in
meganiese herstel tydens herperfusie terwyl die 3-AR blokker geen effek getoon het nie. Beide Rp-
8-CPT-cAMPs en wortmannin het infarktgrootte beduidend vergroot en meganiese herstel
beduidend verlaag, wanneer dit net voor 1/ 2-prekondisionering of tydens die begin van
herperfusie toegedien is. PTX voorafbehandeling het geen beduidende effek op die vermindering
van infarktgrootte (geïnduseer deur 1/ 2-PC of 2-PC) gehad nie. Meganiese herstel is egter
verminder in die geval van 2-PC. Die NOS inhibitore het geen effek op die vermindering in
infarktgrootte geïnduseer deur b1/b2 gehad nie, maar het ook meganiese herstel onderdruk.
Die beduidende afname in infarktgrootte deur b1/b2 prekondisionering is gekenmerk deur
aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase. Soortgelyke aktivering van hierdie
kinases is ook tydens herperfusie van b-AR geprekondisioneerde harte waargeneem.
DPCPX (A1-AdoR antagonis) het geen effek op die infarkt-verminderde effek van 1/ 2-
prekondisionering of op ERK p44/p42 en PKB aktivering gehad nie. A2A-AdoR, maar nie A2b –
AdoR, blokkade tydens die snellerfase, het die effek van b-AR prekondisionering op infarktgroottee
opgehef. Beide antagoniste het die aktivering van ERKp42/p44 en PKB/Akt tydens die snellerfase
onderdruk. Wanneer toegedien tydens herperfusie, het dit die aktivering van hierdie kinases tot ‘n
groter mate onderdruk. MRS-1191 (A3-AdoR antagonis) het infarktgrootte beduidend verhoog en
1/ 2-prekondisionering geblokkeer, beide wanneer dit voor indeks-iskemie toegedien is of tydens
vroeë herperfusie, tesame met inhibisie van PKB en ERK p44/p44 aktivering.
viii
Die kardiobeskerming van 1/ 2-prekondisionering is opgehef deur middel van opruiming van vry
suurstof radikale deur NAC in die snellerfase sowel as aan die begin van herperfusie. Die mK+
ATP
kanaal blokker 5-HD het geen effek op b-AR prekondisionering gehad nie.
Gevolgtrekking: Kardiobeskerming teweeggebring deur ‘n kort periode van stimulasie van die -
ARs, is afhanklik van die aktivering van beide 1-AR en 2-ARs, maar nie 3-AR nie. PKA sowel
as PI3-K aktivering, net voor volgehoue iskemie en tydens vroeë herperfusie, is aangedui om
noodsaaklik vir 1/ 2-AR prekondisionering te wees. Waar funksionele herstel as eindpunt gebruik
is, blyk dit dat NO wel betrokke is by 1/ 2-AR prekondisionering, terwyl die Gi protein ‘n rol mag
speel in 2-AR prekondisionering.
Vorming van endogene adenosien tydens b-adrenerge stimulasie is betrokke by b-AR
prekondisionering. Hierdie beskerming is nie van die A1-AdoR afhanklik nie, maar aktivering van
die A3-AdoR is nodig tydens beide die sneller en herperfusie fases. Beide die A2A-AdoR, en tot ‘n
mindere mate die A2B–AdoR, is ook betrokke by die snellerfase. Vorming van vry suurstof radikale
is nodig vir b-AR prekondisionering, nterwyl die mKATP kanale nie betrokke is nie. Ten slotte,
aktivering van die RISK seintransduksiepad (ERKp42/p44 en PKB/Akt) tydens die snellerfase is ‘n
voorvereiste vir die ontlokking van beskerming. Daarbenewens word b-AR prekondisionering
gekarakteriseer deur aktivering van hierdie pad tydens herperfusie. / South African Medical Research Council / University of Stellenbosch
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/6630 |
| Date | 03 1900 |
| Creators | Salie, Ruduwaan |
| Contributors | Lochner, A., Moolman, J. A., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical physiology |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxxiv, 304 p. : ill. |
| Rights | University of Stellenbosch |
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