Tobacco Documents Online

THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. 000 11IIRD AVENUE NEW YORK. N.Y. 10022 Memorandum To: Dr. J.F.Glenn and Staff From: D.H.Ford Re: Site visit with Dr. R. Lewis, University of Wyoming, Laramie, wy, March 29, 1989. Grant No. 2244R1 entitled "Physiologic effects of Proenkephalin peptides." Goal: To determine the biologic role, if any, of proenkephalin peptides synthesized and release4by the adrenal medulla into the circulating blood stream.He feels that they may be involved in adaptiation to stress. Once he has concluded his rabbit work, he plans to extend his preliminary work on these peptides in man under normal and stress conditions. Also plans to determine the effect of nicotine on the stress response in rabbits. Personnel: Aside ffom a technician, Dr. Lewis has three graduate students assist3,ng n the program. One (Lisa Boddie), who does much of the peptide separation work, has just returned from a pregnancy leave. Mr. Katzenstein will be completing his part of the program in another year, while Mr. Hiddinge has just started this past January. Results: Progress was delayed at the initiation of the program due to difficulty in cannulating the rabbit jugular or carotid vessels for maintained periods of withdrawal. The ear vein, however, has been shown to be satisfactory. They are now also using an electric foot shock stress instead of a swim stress in ice water, since the cold water caused the ear veins to constrict. They have now isolated three proenkephalin peptides, as determined by radioimmune assay. These three peptides, E, F and B appear to all be synthesized in the adrenal medulla.The sensitivity of their assays is in the nanamolar range. These same peptides have also been identified in the guinea pig and rat. The distribution of all three peptides has been determined for a large number of organs, suggesting biological conservation.The amount of each peptide present in an organ varied with specie and organ. The highest levels were generally in the pituitary and lowest in brain and muscle.. However, since the brain was=analyzed as a whole, includ- ing white matter, it is conceivable that there might be significant concentrations in the grey matter. The work performed by Mr.Hiddinge shows that the F form of the peptide exists as an d-helix, the E form as a4 -pleated sheet, while the B form may exist in either of these two configurations. Each peptide differs in its lipophility, which probably influences the degree of penetrance from the blood stream into a particular organ, where it may bind to a receptor.The binding affinities of the three peptides varies considerably. Peptide E binds 200 times more effectively than peptide B and 300 times more so than peptide F. All bind more effectively than endorphin, but less than dynorphin Question: What is responsible for the different binding affinities? Is there a specific region of the peptide involved in binding? Is

2 ^ the different uptake in different organs , or different species related to the binding affinity? It was further noted that in solution, the peptides are structure- less and only assume a helical or pleated structure after binding to a receptor. All the organ uptake studies were done with 1311-labelled peptides, which only provide an overall concept of localization. They have been attempting to create an 3H-labelled peptide, but without success. When they accomplish this, they plan to do a radio- autographic study by light and EM microscopy to determine a more specific localization. It is also not clear that the 131I-labelled yet peptides which accumulate in the organs represent the entire peptide or only a portion of it. Thus, this too remains to be determined. Perhaps it is only the enkephalin portion of the peptide. The receptor binding studies completed so far suggest that it is the r'(-receptor which is most involved. A further interest of Dr. Lewis is to determine if the release and possible role of these peptides in stress or severe exercise have any effect on immune function, somewhat along the lines of investigation pursued by Blalock and by Stanisz in relation to neuroendocrine-immune interrealtionships. Comment: While progress has been slow, many of the initial difficulties have been overcome and they appear to be nearing the end of the preliminary investigations. Two manuscripts are undergoing preparation and should be completed in time for his `Continuation' application in May. They are also planning a couple of presentations for the Neuroscience meeting in Phoenix in Nov. Their further studies appear directed toward a more refined localization of the three peptides in rat, rabbit and guinea pig and a further clarification of possible receptors of the opiate binding class, as well as of the molecular form of peptide which is bound in specific organs. They also appear to be nearing the point where they will be attempting to determine what biologic effect these peptides may have. Thus, while there are still many basic issues to be resolved, Dr. Lewis should soon be able to start to evaluate if these circulating opioid peptides are of significance in the response to stress and do they have any effect on the function of the various organs which accumulate them (i.e., pituitary, lung, liver, kidney, etc.). Considering all the possible ramification of how these peptides (with half lives of up to 20-30 minutes) may influence a wide variety of functions, this program should be of increasing interest as it evolves. DHF :a.