The objective of the proposed research is to elucidate the mechanisms of inactivation, degradation, and turnover of neuronal nitric oxide synthase (NOS), a cytochrome P450-like hemoprotein, caused by exposure to xenobiotics, including drugs and environmental toxicants. The central hypothesis is that such effects diminish the ability to form NO, an important bioregulatory molecule involved in many physiological functions, and cause some forms of chemically-induced toxicities. They has been shown that certain xenobiotics, such as CBrCl3, can irreversibly inactivate the neuronal isoform of NOS in a time- and metabolism-dependent manner, similar to that found for this compound with liver microsomal cytochrome P450. Protection of NOS from inactivation is afforded by the L-, but not the D-, isomer of arginine suggesting an active site directed event. More recently it was shown that guanabenz (WytesinTM), an antihypertensive agent, inactivates penile neuronal NOS in vitro in a similar metabolism dependent manner and causes a loss of penile NOS protein and activity in vivo at pharmacologically relevant doses. This may be important since NO plays a key role in penile erection and since many antihypertensive agents, including guanabenz, are known to cause impotence as a toxic side effect. Thus, the aims of the current proposal are to understand how neuronal NOS is inactivated and how the steady state levels of NOS are subsequently affected. Guanabenz will be used as a model agent to develop methods for further study of other similarly toxic agents.
The specific aims are: (1) To determine the molecular mechanism of the inactivation caused by guanabenz with the use of purified recombinantly expressed neuronal NOS. (2) To characterize the decrease in the steady state levels of neuronal NOS caused by guanabenz in the T cell hybridoma 2B4 line. (3) To more fully characterize the effects of long term administration of guanabenz on NOS in vivo. (4) To utilize the in vitro, in vivo, and T cell line as they are developed to test other biomedically important chemicals, based on a computer based structure search as well as incidence of impotence, such as debrisoquin and bethanidine. These studies should lead to a better understanding of how drugs inactivate and regulate neuronal NOS, as well as the nature of the active site of the enzyme, and thereby lead to the design of safer drugs without undesired toxicological side effects, such as impotence. Conversely, the same knowledge could be used to design more effective inhibitors of NOS for pharmacological use in a variety of neurological diseases.
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