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THr•. Corarit. FON TGBACCO RESEARCH-U.S.A.. Iac: MmRAAIDZIlN Z10: W. U. Gardner FRLT'!: D. H. Ford H Ql 166083 June 10, 1980 SUBJECT: Grant 1186R2. Ferid Murad, M.D., Ph.D., University of Virginia School of Medicine, Qharlottesville. "Mechanism of Nitric Oxide Activation of Guanylate G1iclase." SI°I'PE VISIT on June 6, 1980. object of t':e Study. Zb deternLis:e the role of guanylate cyclase (GC) and cGP in normal lung function and in pathologic states as influenced by nitroso capowxis (NO) ; i.e., effect on relaxatiari of strdooth mascle in trachea and pulnbnauy arteries (potentially also in placental vessels); effects on secretions of neuoosal cells of lung and intestine; effects on pituitary gonadotrophin secretion; effects on cell growth and proliferation (cQ4P appears to provide a positive signal for cell growth while aAt4P provides a negative signal) - note cGW levels elevated in solid tunors (lung, hepatana and also in sane leukenias). Such elevated oGW levels in tinaar tissue paralleled by increases of cGw in sennn and urine, which can be readily measured. Fwrthiar, in proliferative tissue, particulate OC predaninates aver soluble levels, vhile in diabetes and during starvation the solubie levels GC are elevated and particulate levels are tmchzngefl. Questions. Are soluble and particulate DC different proteins? Are they regulated in different or interrelated ways? Are they associated with different kinds of cellular respon,se? Are their kinetics similar? Do they develop similar antibodies and will they cross-react with each other? Are either or both associated with riM synthesis? Do they act in similar or in different cellular oo~art~nts. He is making sane progress with the above questions in that the soluble CaC is now available in essentially a pure state and is being used to develop antibodies. The particulate fraction has been isolated and purified 200-fold. once it is essentially pure, the above c)uuest.ions may all be considered. Recent Develapnents. 1. Developrent of a shortened purification systen for OC soluble enz}me frcg lung and liver fran 2 weeks to 3-4 days using a G1P affinity column. Cbtain about 300-400 ug of enzyme/rwi. Now have a supply of several ng. 2. Are now using mice to obtain antibodies to soluble OC and have switched to a hybridana procedure to obtain the antibodies. Antigen injected into mice; spleen cells harvested and fused tbrmuse myelama cells to produce the hybridona, which in culture secretes the various iminuzoglobuL:.ns. Five to 8 hybridanas have been cloned and injected back into mice i.o. The subsequent ttmior

Hl;0" "1 1E6 081 i formation is then associated with formation of large amounts of ascites fluid loaded with antibody to the soluble C-C which can be readily aspirated. Further, different spleen cells appear to recoqnize different parts of the enzyme peptide chain. Thus, different clones synthesize antibodies recognizing different parts of the enzyme molecule. Plans to label each of these antibodies in future studies with ferritin for immmohistochemistry to identify cell types in lung associated with the GC ehzyme as well as to deteunine cell cxurartnent in which en.~zyme is located. 3. Working on ar.iino acid oarposition of soluble GC. 4. Studies with activation and inactivation of soluble GC usina cystine and cystamine suggest that active site of enzyme at a thiol group (see attached manuscript recently suYmitted for publication). igS-cystamine or cystine can also be used which permits one to follow the enzyme through the purification procedure. Note: incorporating the NH2CII2Qi2S- moiety inactivates the enzyme. Treatment of this inactivated carnpoimd with reducing agents restores its ability to be activated by IJO. 5. Is planning sorw studies with a colleague w?w works at a reactor site. will use radioactive nitrogen to label OC. Problem with this label is its short half life, thus requiring the work to be done at a reactor site. Would permit verification of purification process. ' Labora . 9na11 but well eqnipped. Space at this branch of the University of V i.-:ia is at a prenium. Have developed an apparatus for automatic processing of samples for WA studies (has been copied camiercially) . G1zrrently set up for thyzoxin, digitoxin, angiotensin I and 3 other peptides using 1251. Plan to use this setup for RIA work with the various antibodies obtained fran CC. Staff. Drs. H. Brandwein and J. Lewicki are new mmnbers of the group who startecast suamier. Dr. Lewic}:i is primarily involved in the hybri+3oma study and will be the member to direct the RIA and imrounohistochenistsy studies. Some RIA work planned to look for antibodies to OC in plasma and urine of patients with lung injury. Dr. Brandwein was not in, so vras unable to discuss his area of interest with him. Three other new staff mwibers since last year's in group, but not primarily associated with the project. Drs. Broiughler and Mittal have left the unit and have not oomzmaiicat.ed with Hurad oonoerning their research plans. Dr. Ito is returning this week to Japan. Evaluation. Active group with well defined goals proceeding first with obtaini.ng B-e-purMea enzyme before attaTting other studies. Soluble fE now essentially available and development of antibodies and RIA programs in progress. Particulate enzyme harder to purify, thus work has not proceeded as rapidly. Should be defining sites of soluble OC activity in various cell types in near future. Ultimate goal of understanding role of guanylate cyclas%(MP in norma]l and pathologic lung and liver tiasue (effects qf NO in smoke) clearly defined. Is basically cell biology ooncerned with GC/oC3P in relation to cell growth and proliferation, as potent.ially influenced by nitroso~unds. Reoamwdation. Oontinueci support for the new grant proposal (or inqui.ry), which will ~T"'crnunq a: Ziy early fall. D. H. Fbrd Mir: ek att.