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'ri-Ir•. COUNCIL FOR'FU$ACCO RESEARCII- U.A.A.', INC. MEMGRANDUM TO: W. U. Gardner and Staff NIf3l1hEi111 May 15, 1979 FROM: D. H. Ford SUB,AECT: Grant 1166R1. Ferid Murad, M.D., Ph.D., University of Virginia School of Medicine, Charlottesville. "Mechanism of Nitric Oxide Activation of Guanylate Cyclase." SITE VISIT on May 10, 1979. Dr. Mur,ad is an enthusiastic young investigator who has been involved with cyclic nuclebtide research since its early days. He has a well-equipped laboratory (including the new additions requested) and adequate space. He generally has up to six postdoctoral or other postgraduate fellows associated with him, as well as being associated with others in the Medical School (i.e., Dr. Paul Lacey in Pathology who is an electronmicroscopist). Dr. Murad's interest in cGMP is essen tially that of a cell biologist, being concerned with how cGMP may function in regulating cell activity, though perhaps not in the same sense the cAMP is c.onsi dered as a "second" messenger. I Guanylate cyclase, the enzyme synthes izing cGMP exists in soluble and iii particulate forms, which are antigerdically different from each other. The particulate form has been associated primarily with the RER, Golgi apparatus and cell menbrane and to a lesser degree with the nucleus. The observation that nitric oxide and other free radicals activate guanylate cyclase (rather than inhibit them, as Cccurs with heme compounds) was of particular interest to Murad and lead to a currently investigated question; How do free radicals in smoke reguiate guanylate cyclase and thus influence cGM1'? it should further be noted that Murad has observed that activation of guanylate cyclase with hydroxyl radical changes the properties of the enzyme (obtained from liver) to permit it to catalyze the formation of cAMP Since it appears that cGMP may be involved in growth, differentiation, smooth muscle activity and mucus secretion in intestines, trachea and bronchi, factors such as hormones and environmental factors which influence cGMP bvcome important in the design of Murad's experimental protocols (i.e., the role of nitroglycerin via conversion to nitric oxide) to activate guanylate cyclase. Murad has successfully purified the soluble guanylate cyclase and has started 'o prepare antigens to it, using rabbits. So far his antigens are of low activity and not suitable for immunocytochemical studies. However, he hopes to obtain high activity antigens in the near future and will then undertake the immunocytochemistry with Dr. Paul Laceyr using ferritiu as a label to identify the location of the antigen in electronmicrographs. At this point he will attempt to determine if physiologic manipulations will alter the localization of the soluble enzyme. He also plans to produce antibodies to the particula enzyme and determine if its particula localizations can be manipulated. over ....

NK01166112 Since Murad now has several 100 ug of purified enzyme from liver and lung of rat, he is also investigating how guanylate cyclase is activated by free radicals. Murad has developed a small glass system of tubes and filters through which he can pass cigarette smoke to obtain a gas phase with which he has exposed some of his enzymes preparations. Levels of nitric oxide cFm be regulated in the gas phase by adding it to the system. The degree of activation of the enzynbe and the amount of bGlip produced was shown to be dependent on the amount of nitric oxide present. He hopes to be able to apply this to studying the effects on airway smooth tauscle activity and mucus secretion. (Note: ACh was also observed to activate guanylate cyclase with subsequent increases in cGMP, but it inhibits formation of cAMP.) For the current year Murad has an excess of $1,200 in personnel funds, but a deficit in his supply budget. He will be writing us for permission to transfer the $1,200 to supplies. Essentially, Murad is a cell biologist concerned with the role of cGMP in cells and factmrs which activate br suppress it under normal and abnormal conditions. In relation to the latter, he is interested in the effects of many of the products in smoke, such as nitric oxide, nitrosamines as well as in hormones, etc. Since the inception of the grant three papers and one abstract have been submitted for publication. One of the papers has been published: Braughler, et al. 1979. Purification of soluble guanylate cyclase from rat liver. Proc. Nat. Acad. Sci. 76:219-222. An article of special interest: Mittal and Murad 1977 Mini-Review. Properties and oxidative regulation of guanylate cyclase. J. Cyclic Nucleotide Research 3:381-391. D. H. Ford DHF:ek