Abstract
Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O(2)(-)) are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMECs) exposed to hyperglycaemia. CMECs were cultured under normal (5.5 mmol/l) or high glucose (22 mmol/l) concentrations for 7 days. The activity and expression (protein level) of endothelial NO synthase (eNOS), inducible NOS (iNOS), NAD(p)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were investigated by specific activity assays and Western analyses, respectively, while the effects of hyperglycaemia on nitrite and O(2)(-) generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p < 0.05) and increased the protein expressions and activities of p22-phox, a membrane-bound component of pro-oxidant NAD(p)H oxidase and antioxidant enzymes (p < 0.05). Free radical scavengers, namely, Tiron and mercaptopropionylglycine (MPG) (0.1-1 micromol/l) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p < 0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced pro-oxidant enzyme activity and diminished generation of antioxidant glutathione. By increasing the antioxidant enzyme capacity, CMEC may protect themselves against free radical-induced cell damage in diabetic conditions.
Highlights
Nitric oxide (NO) is generated from amino acid L-arginine by a class of enzymes termed as nitric oxide (NO) synthases (NOSs) which include two constitutively expressed isoforms, namely, endothelial NO synthases (NOSs) and neuronal NOS and an inducible isoform [1, 2]
Despite a lack of consensus on the expression and activity of endothelial NOS (eNOS) and generation of NO in diabetic conditions, an overwhelming body of evidence has in recent years indicated that reactive oxygen species (ROS) are released at greater levels in diabetic animals and cells cultured under high glucose conditions [11, 12]
The differences in protein expression were independent of both extracellular glycation and the increase in osmolarity as equimolar concentrations of L-glucose and mannitol did not alter the level of protein expressions, respectively
Summary
Nitric oxide (NO) is generated from amino acid L-arginine by a class of enzymes termed as NO synthases (NOSs) which include two constitutively expressed isoforms, namely, endothelial NOS (eNOS) and neuronal NOS (nNOS) and an inducible isoform (iNOS) [1, 2]. The activity and expression (protein level) of eNOS, iNOS, NAD(P)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutahione peroxidase (GPx) were investigated by specific activity assays and Western analyses, respectively while the effects of hyperglycaemia on nitrite and O2- generation were investigated by Griess reaction and cytochrome C reduction assay, respectively.
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