The effect of hypoxia on nitric oxide and endothelial nitric oxide synthase in the whole heart and isolated cardiac cells: the role of thePI3–K / PKB pathway as a possible mediator.

[摘要] In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the mostimportant constitutively expressed enzymatic source of nitric oxide (NO), amajor cardiac signalling molecule. On the whole, NO is regarded as acardioprotective molecule. The role of eNOS during ischaemia / hypoxia iscontroversial; however, it is generally accepted that ischaemia / hypoxiaresults in increased cardiac NO production. Most studies focus either on thewhole heart or isolated cell models. As yet, no study has compared findingswith regard to NO metabolism in these two distinct models, in a single study.We hypothesise that observations in a whole heart model with regard toincreased NO production and eNOS involvement in ischaemia are the resultof events on cellular level and that the increase in NO production observedduring hypoxia in cardiomyocytes and endothelial cells is at least in part dueto the increase in expression and / or activation of eNOS. Furthermore, wehypothesize that these effects are mediated via the PI3-K / PKB pathway. Weaimed to measure and compare NO-production and eNOS expression andactivation in the whole heart and isolated cardiac cells and measure PKBexpression and activation in the cells under normoxic and ischaemic / hypoxicconditions. We also aimed to determine the effects of PI3-K / PKB pathwayinhibition on NO production and eNOS expression and activation in isolatedcardiac cells under normoxic and hypoxic conditions. Adult rat hearts wereperfused and global ischaemia induced for 15 and 20 min. Tissuehomogenates of perfused hearts were used for the measurement of nitritesand determination of expression and activation of eNOS. Expression of eNOSin 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) purchasedcommercially. In the cells, hypoxia was induced by covering cell pellets withmineral oil for 60 min. Cell viability was determined by trypan blue andpropidium iodide (PI) staining and intracellular NO production measured byFACS analysis of the NO-specific probe, DAF-2/DA and by measurement ofnitrite levels (Griess reagent). Results show that in ischaemic hearts, nitriteproduction increased by 12 % after 15 min ischaemia and 7 % after 20 minischaemia. Total eNOS expression remained unchanged (Western Blot andIHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15min ischaemia and decreased by 43% after 20 min ischaemia. In the cells,both viability techniques verified that the hypoxia-protocol induced significantdamage. In isolated cardiomyocytes, NO-production increased 1.2-fold (byDAF-2/DA fluorescence), total eNOS expression increased 2-fold andactivated eNOS increased 1.8-fold over control. In CMECs, NO-productionincreased 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 / PKBinvestigations, results showed an increase of 84 % and 88 % in expressionviiand activation of PKB (phospho Ser473) in hypoxic cardiomyocytes,respectively. In hypoxic CMECs, there was no change in PKB expression butthere was a 69 % increase in phosphorylated PKB. NO production inwortmannin-treated hypoxic cardiomyocytes decreased by 12 % as comparedto untreated hypoxic cells. In treated hypoxic CMECs, NO productiondecreased by 58 % as compared to untreated hypoxic cells. Treatment withwortmannin did not change the expression of eNOS protein in thecardiomyocytes, however, activated eNOS decreased by 41 % and 23 %under baseline and hypoxic conditions in treated cells respectively. There wasa significant increase in NO production after exposure to O2 deficientconditions in all models investigated, a trend similar to what previous studiesin literature found. However, the source of this NO is not fully understoodalthough it has been discovered that NOS plays a role. Our data revealssimilar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in thecells; however, the trends observed at 20 min ischaemia are in conflict withour cell data (i.e. decrease in activated eNOS). This may be due to theseverity of the ischaemic insult in whole hearts and/or the presence of othercell types and paracrine factors in the whole heart. Hypoxia increased theactivation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKBpathway reduced NO production and hypoxia-induced eNOS activation incardiomyocytes. In conclusion, we have, for the first time, demonstrated thatthe increase in NO production during hypoxia is due (at least in part) to anincrease in eNOS phosphorylation at Ser1177 and that this is mediated via thePI3-K / PKB pathway.

[发布机构] Stellenbosch University