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Influence of diabetes on cardiac nitric oxide synthase expression and activity
K Stockklauser-Färber1, T Ballhausen, A Laufer
1Clinical Biochemistry, Diabetesforschungsinstitut an der Heinrich-Heine-Universität, Auf'm Hennekamp 65, D-40225 Düsseldorf, Germany.
Unlabelled:
There is some evidence that the endothelium dependent vasodilatation of coronary arteries is impaired in both types of diabetes. The underlying mechanisms are not yet clear, in particular whether this defect is caused by a direct effect of diabetes on the activity and the expression of nitric oxide synthases (NOS) or indirectly by an enhanced inactivation of nitric oxide.
Methods:
To study this question we determined the activity (conversion of L-arginine to citrulline) and the mRNAs encoding the isoforms of NOS (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs by reverse transcriptase) in hearts of streptozotocin diabetic rats and in rat heart endothelial cells (RHEC). The formation of reactive oxygen intermediates (ROI) was measured by the dichloro-dihydro-fluorescein method.
Results:
The activity of total NOS and the amounts of mRNAs encoding ecNOS and iNOS were dependent on the duration of diabetes. After a diabetes duration of 4 to 6 weeks both the total activity as well as the mRNAs encoding ecNOS and iNOS were elevated. A reduction of NOS activity and the amounts of mRNAs of ecNOS and iNOS was only seen after a diabetes duration longer than 20 weeks, a time at which a loss of endothelium has been described. In RHEC, high glucose (22 mM) and H(2)O(2) (100 microM) were able to increase the mRNA encoding ecNOS, but not iNOS. This increase in ecNOS mRNA was inhibited by lipoic acid (1 microM). In addition, high glucose (22 and 30 mM) led to an enhanced formation of ROI and to activation of the transcription NF kappa B.
Conclusion:
These observations suggest that diabetes causes a temporary increase in NOS activity and ecNOS mRNA in the rat heart which is presumably the consequence of an enhanced oxidative stress exerted by hyperglycaemia. Together with previously published observations, our data suggest that the impairment of endothelium dependent vasodilatation in rat heart is not the consequence of a reduced activity and expression of NOS, but is caused by an enhanced inactivation of nitric oxide by ROI.
Insights
Diabetes impairs coronary artery vasodilation by increasing nitric oxide inactivation, not by reducing nitric oxide synthase (NOS) activity or expression. Oxidative stress from hyperglycemia plays a key role in this process.
Area of Science:
- Cardiovascular Physiology
- Endocrinology
- Molecular Biology
Background:
- Endothelium-dependent vasodilation in coronary arteries is often impaired in diabetes.
- The precise mechanisms, including the role of nitric oxide synthases (NOS) and nitric oxide (NO) inactivation, remain unclear.
Purpose of the Study:
- To investigate the impact of diabetes on NOS activity and expression in rat hearts.
- To determine whether impaired vasodilation is due to reduced NOS function or increased NO inactivation.
Main Methods:
- Assessed NOS activity and mRNA levels (ecNOS, iNOS) in streptozotocin-diabetic rat hearts and rat heart endothelial cells (RHEC).
- Measured reactive oxygen intermediate (ROI) formation using the dichloro-dihydro-fluorescein method.
- Utilized polymerase chain reaction and reverse transcription to quantify NOS mRNA.
Main Results:
- NOS activity and ecNOS/iNOS mRNA levels initially increased (4-6 weeks of diabetes) and then decreased ( >20 weeks).
- High glucose in RHEC increased ecNOS mRNA and ROI formation, activating NF-κB.
- Lipoic acid inhibited the high glucose-induced increase in ecNOS mRNA.
Conclusions:
- Diabetes temporarily elevates NOS activity and ecNOS mRNA in rat hearts, likely due to hyperglycemia-induced oxidative stress.
- Impaired vasodilation is attributed to enhanced NO inactivation by ROIs, rather than reduced NOS activity or expression.
- Oxidative stress is a critical factor in diabetes-related endothelial dysfunction.