Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the most
important constitutively expressed enzymatic source of nitric oxide (NO), a
major cardiac signalling molecule. On the whole, NO is regarded as a
cardioprotective molecule. The role of eNOS during ischaemia / hypoxia is
controversial; however, it is generally accepted that ischaemia / hypoxia
results in increased cardiac NO production. Most studies focus either on the
whole heart or isolated cell models. As yet, no study has compared findings
with regard to NO metabolism in these two distinct models, in a single study.
We hypothesise that observations in a whole heart model with regard to
increased NO production and eNOS involvement in ischaemia are the result
of events on cellular level and that the increase in NO production observed
during hypoxia in cardiomyocytes and endothelial cells is at least in part due
to the increase in expression and / or activation of eNOS. Furthermore, we
hypothesize that these effects are mediated via the PI3-K / PKB pathway. We
aimed to measure and compare NO-production and eNOS expression and
activation in the whole heart and isolated cardiac cells and measure PKB
expression and activation in the cells under normoxic and ischaemic / hypoxic
conditions. We also aimed to determine the effects of PI3-K / PKB pathway
inhibition on NO production and eNOS expression and activation in isolated
cardiac cells under normoxic and hypoxic conditions. Adult rat hearts were
perfused and global ischaemia induced for 15 and 20 min. Tissue
homogenates of perfused hearts were used for the measurement of nitrites
and determination of expression and activation of eNOS. Expression of eNOS
in the heart was also determined by immunohistochemical (IHC) analysis.
Cardiomyocytes were isolated from adult rat hearts by collagenase-perfusion,
and adult rat cardiac microvascular endothelial cells (CMEC) purchased
commercially. In the cells, hypoxia was induced by covering cell pellets with
mineral oil for 60 min. Cell viability was determined by trypan blue and
propidium iodide (PI) staining and intracellular NO production measured by
FACS analysis of the NO-specific probe, DAF-2/DA and by measurement of
nitrite levels (Griess reagent). Results show that in ischaemic hearts, nitrite
production increased by 12 % after 15 min ischaemia and 7 % after 20 min
ischaemia. Total eNOS expression remained unchanged (Western Blot and
IHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15
min ischaemia and decreased by 43% after 20 min ischaemia. In the cells,
both viability techniques verified that the hypoxia-protocol induced significant
damage. In isolated cardiomyocytes, NO-production increased 1.2-fold (by
DAF-2/DA fluorescence), total eNOS expression increased 2-fold and
activated eNOS increased 1.8-fold over control. In CMECs, NO-production
increased 1.6-fold (by DAF-2/DA fluorescence), total eNOS increased by 1.8-
fold and activated eNOS by 3-fold. With regards to our PI3-K / PKB
investigations, results showed an increase of 84 % and 88 % in expression
vii
and activation of PKB (phospho Ser473) in hypoxic cardiomyocytes,
respectively. In hypoxic CMECs, there was no change in PKB expression but
there was a 69 % increase in phosphorylated PKB. NO production in
wortmannin-treated hypoxic cardiomyocytes decreased by 12 % as compared
to untreated hypoxic cells. In treated hypoxic CMECs, NO production
decreased by 58 % as compared to untreated hypoxic cells. Treatment with
wortmannin did not change the expression of eNOS protein in the
cardiomyocytes, however, activated eNOS decreased by 41 % and 23 %
under baseline and hypoxic conditions in treated cells respectively. There was
a significant increase in NO production after exposure to O2 deficient
conditions in all models investigated, a trend similar to what previous studies
in literature found. However, the source of this NO is not fully understood
although it has been discovered that NOS plays a role. Our data reveals
similar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in the
cells; however, the trends observed at 20 min ischaemia are in conflict with
our cell data (i.e. decrease in activated eNOS). This may be due to the
severity of the ischaemic insult in whole hearts and/or the presence of other
cell types and paracrine factors in the whole heart. Hypoxia increased the
activation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKB
pathway reduced NO production and hypoxia-induced eNOS activation in
cardiomyocytes. In conclusion, we have, for the first time, demonstrated that
the increase in NO production during hypoxia is due (at least in part) to an
increase in eNOS phosphorylation at Ser1177 and that this is mediated via the
PI3-K / PKB pathway.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1917 |
| Date | 03 1900 |
| Creators | Chamane, Nontuthuko Zoleka Lynette |
| Contributors | Strydom, H., 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 | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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