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Effect of tobacco smoke on nitric oxide synthesis

Institution: University of California, Davis
Investigator(s): Patrick Wong, Ph.D.
Award Cycle: 1999 (Cycle 8) Grant #: 8FT-0150 Award: $70,000
Subject Area: Cardiovascular Disease
Award Type: Postdoctoral Fellowship Awards

Initial Award Abstract
Smoking may lead to hypertension and stroke due to the decreased ability for the body to synthesize nitric oxide (NO). NO is a small molecule which has been implicated in a variety of physiological processes. One of its more important functions involves the relaxation of blood vessels. NO has been shown to dilate blood vessels and thus, allow more blood to flow. The human body takes advantages of this system to regulate the amount of blood flow to various parts of the body by adjusting the amount of NO produced. NO is also involved in the thinning of the blood by decreasing the stickiness of blood platelet cells. This process decreases the chance of these cells forming aggregates which may block small arteries and lead to strokes.

NO is produced in the body by an enzyme called nitric oxide synthase (NOS). NOS produces NO from L-arginine, one of the 20 essential amino acids. When NOS does not function property, hypertension can occur since the blood vessels are more constricted. Also, there is an increased chance of stroke because of these narrow vessels and thickening of the blood. It is by this process by which we believe that smoking can cause hypertension and strokes.

It has been shown by us and other investigators that components of cigarette smoke can react with L-arginine to form an L-arginine adduct. This reaction may have two possible effects. First, it may decrease the amount of L-arginine in the body and thus, less NO is produced since there is less of its precursor. Secondly, the adduct formed may bind to the NOS itself and inhibit its function. In order to investigate these possibilities further, both cultured cells and laboratory animals will be exposed to cigarette smoke. Afterward, levels of L-arginine and its possible cigarette smoke adduct will be measured and correlated with possible decreases in NOS function. The benefits of such a study include not only establishing a link between smoking and various cardiovascular diseases, but also a possible treatment program for smokers who may be hypertensive through L-arginine replacement therapy.

Final Report
Cigarette smoking is associated with an increased risk of cardiovascular disease through increased hypertension and platelet aggregation. Both symptoms of which may be caused by decreased endogenous nitric oxide (NO) formation. NO has been shown to be an Important regulator of blood flow and smooth muscle relaxation. It has been previously shown that cigarette smoking reduces the production of endogenous NO. However, the mechanism of the decrease in NO levels has yet to be determined. Nitric oxide synthase (NOS) is the enzyme responsible for converting L-arginine (one of the 20 essential amino acids) to NO and L-chrulline. Previous studies have shown that NOS activity is decreased in the presence of cigarette smoke extract. Also, it has been demonstrated that smokers have lower NOS activity than non-smokers. The mechanism of these observations is not known, however, a possible target includes the formation of adducts of L-arginine, the main substrate for NOS, with components of cigarette smoke. The formation of such aforementioned adducts may lead to decreased L-arginine for NO synthesis or the formation of a compound which inhibits NOS. Both possibilities of which are supported by studies which show that endothelial dysfunction by cigarette smoke is prevented through dietary L-arginine supplementation.

Experiments in our laboratory have focused on the direct reaction between cigarette smoke extract and L-arginine. In these studies, a solution of L-arginine was exposed to filtered cigarette smoke. We observed the formation of a possible L-arginine adduct The structure of this adduct was elucidated by ultraviolet and mass spectroscopy which gives information on the structural relationships between atoms in a molecule and basic molecular weights. Based upon these results, we hypothesized that cigarette smoke components react with L-arginine to form an L-arginine adduct This adduct can then decrease the substrate availability for NOS or even Inhibit NOS.

The next step was to identify which of the more than 4000 components of cigarette smoke may be reacting with L-arginine. We focused on acetaldehyde (a major cigarette smoke component) since the adduct formed between cigarette smoke and L-arginine. appears to have a similar molecular weight and ultraviolet spectrum as a product as a possible acetaldehyde-L-arginine compound. Direct synthesis of this adduct with acetaldehyde and L-arginine and further analytical analysis confirms that this complex is definitely formed and acetaldehyde appears to be the major compound Involved. The synthesized acetaldehyde- L-arginine adduct was then used In subsequent studies.

The final studies of this project focused on the ability of the acetaldehyde-L-arginine adduct to inhibit NOS in both a purified enzyme system and biological system. In the first study, the acetaldehyde-L-arginine adduct was able to decrease NO synthesis by commercially obtained NOS In a concentration dependent manner. This study demonstrated the ability of this adduct to block NOS activity and revealed the adduct as a potent inhibitor. The final study was to test the ability of the acetaldehyde -L-arginine adduct to inhibit a NOS mediated response in a vascular smooth muscle system similar to the blood vessels in the heart. Incubation of the adduct in preconstricted tissues decreased the tissue response to the biochemical acetylcholine. Acetylcholine mediated relaxation has been shown in previous studies to be a NO mediated response. This in vitro study may provide a model for acetaldehyde (an thus cigarette smoke) induced hypertension.

The significance of information obtained though this study is not only elucidation of the mechanism of smoking Induced cardiovascular disease, but also provide a possible better method of treatment and diagnosis for smokers who have developed cardiovascular disease symptoms through the possibility of L-arginine replacement therapies.