Organic nitrates (ORN) vasodilators have little intrinsic activity and must be bioactivated to liberate their vasoactive species, nitric oxide (NO). Our understanding of the enzymatic process involved remains incomplete despite over a century of clinical use. Mitochondrial aldehyde dehydrogenase (ALDH2) has been demonstrated to play a role in the bioactivation of nitroglycerin (NTG) but it is not likely to be the sole enzyme responsible. Interestingly, it has been shown to have little activity toward the metabolism of other clinically used ORN (i.e., isosorbide dinitrate (ISDN) and isosorbide-5-mononitrate (IS-5-MN)). Therefore, this thesis work was focused on the investigation of the role of ALDH1a1 and ALDH3a1 (two cytosolic forms of ALDH) on the bioactivation of ORN. Because the safety of chronic ORN therapy has recently come into question, this thesis also explored the role of cardiac ALDH2 inactivation as a possible mechanism of this potential toxicity.
Using purified enzyme and an NO-electrode, we demonstrated that ALDH1a1 was able to liberate NO from all three of the clinically used ORN, viz., NTG, ISDN and IS-5-MN. ALDH2, on the other hand, could bioactivate NTG and ISDN but not IS-5-MN. IS-5-MN was also a poor inactivator of ALDH2, consistent with the finding that it was a poor substrate for the isoform. NTG and ISDN inactivated ALDH2, while all three ORN were able to inactivate ALDH1a1, consistent with the metabolic profile of the isozymes. Using site-directed mutagenesis, we identified the catalytic residue of ORN bioactivation for ALDH1a1, at which the cysteine at position 303 was critical to the enzyme's activity while the cysteine at position 302 only modulated its activity.
Despite the suggestive in vitro metabolic results concerning ALDH1a1, experiments with an Aldh1a1 knock-out mice demonstrated that this enzyme was not substantially involved in mediating the ex vivo vasodilating effects of NTG or IS-5-MN. Further, mRNA quantification studies reveled that expression of the protein in the aortic tissue was not detectable. In the course of these studies, we found much higher expression of ALDH3a1 in murine aortic tissue. We expressed and purified recombinant ALDH3a1, and demonstrated that this isoform was able to liberate NO from NTG and ISDN but not IS-5-MN, suggesting that this enzyme could be involved in the bioactivation of these agents. Exploratory mathematical modeling of the bioactivation of NTG by ALDH3a1 was conducted to investigate the kinetics of NO production from NTG and subsequent mechanism-based enzyme inactivation. This modeling approach led to meaningful estimation of the relevant enzyme kinetic parameters involved in the bioactivation of ORN.
Using the Langendorff isolated mouse heart preparation, we compared the ability of 2 µM NTG and 16 mM IS-5-MN pre-exposure for 30 min to potentiate cardiac damage after ischemia and reperfusion (I/R) via the inactivation of cardioprotective ALDH2. IS-5-MN pre-exposure had no effect vs. control. On the other hand, NTG pre-exposure resulted in increased cardiac function but with an increase in the release of creatine kinase. These results suggest that while NTG may worsen tissue damage, its beneficial effects on coronary blood flow may preserve cardiac function.
In summary, this work investigated various aspects of ORN metabolism by three isozymes of ALDH, including bioactivation, enzyme inactivation and potential toxicity. It is hoped that our findings will contribute to a better understanding of the pharmacology and toxicology of ORN, thus leading to further improvement of the clinical efficacy and safety of these important cardiovascular agents.
|Commitee:||Balthasar, Joseph P., Blanco, Javier G.|
|School:||State University of New York at Buffalo|
|School Location:||United States -- New York|
|Source:||DAI-B 73/06, Dissertation Abstracts International|
|Subjects:||Pharmacology, Pharmacy sciences|
|Keywords:||Aldehyde dehydrognase, Cardiovascular pharmacology, Nitric oxide, Nitroglycerin, Organic nitrates|
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