Tobacco Documents Online

! '" 41 THE COIINCIL FOR TOBACCO RESEARCH-U.S.A., INC. r_1 14LL- , s ~ri ,'J Re: Site visit with Dr. La:Ha,~'~i Albert Einstein College of Medicine, Bronx, N.Y. May 23, 1984 MEMORANDUM TO: Dr. S.C. Sonmers and Staff From: D.H. Ford and D. Stone -Grant No. 1126 BRl"Genetic differences in nicotine sensitivity in Drosophila mel.anogaster strains. " Goal: Zb use genetic analysis coupled with biochemi.cal characterization to dissect the interaction of nicotine with its binding ccanponent in the fruit fly CNS (head ganglia). Dr. Hall is using nicotine resitant mutants which she has developed to identify the genes involved in nicotine resitance. This is a trery labor intensive type of investigation, each phase of the study having to be repeated many tin-es to obtain sufficent data to make valid interpretations. Qverview: Past reports note that some nicotine resistant mutant flies showed alterations in alphaBungarotoxin (01 BT) binding as well as changes in the receptor to which theolBT bound. This generally accepted to be the nicotinic cholinergic receptor. Some flies, however, did not show the alteration in binding and further, while *JBT bunding overlapped that of nicotine, the nunbe.r of sites were not equivalent. This suggested that,when nicotine was used,it was binding to scet-ething other than to the Ach receptors to which the YBT bourui. Dr . Hall has recently developed a 3H-nicotine binding assay to further characterize the nature of nicotinic receptors. This assay is considerably more expensive than the qBT assay, the labelled nicotine y-?gting $1000 while an egtii.valent atrount of "tiurk could be done with $80 worth of I--4BT. For this new nicoti.ne binding assay she is using the nicotine resistant strain HF3, which is the one previously shown to have an alteration in the isoelectric point of the q-BT binding ccanponent. ' Recent data: Binding studies wit~ "H-nicotine in HR mutants (Table 1) showed that the dissociation constant KD for H-nicotine in the mutants was 88% of that observed in wild type Canton flies (not a significant difference). However, the total niunber of saturable binding sites Boax was 0.749 prol/mg protein as ocapa.red with 1.06 gnol/mg of protein in-wild flies. This is a significant reduction and suggests that the nicotine resitance in the HR mutants might be due to a decrease in nicotine binding sites. An analysis of nicotine binding sites in Canton wild type flies (Fig.1) shows the degree to which different agonist and antagonist ligands of nicotine block 3H-niootine binding. The effectiveness of the various ligands was d-tubocurarine O. acetylcholine ),eserine= carbamylcholine> neostigminer-` BT ^_decamethonitun*>- atropine. An interesting observation noted from these curves was that they did'not eorrespond to the smooth S-shaped curves-'usuaaly obtained with Scatchard plots of such binding -blocking studies, but were broken into two oontponents. This suggests the possibility that the 3H nicotine used is binding to at least two types of receptor. One may be cholinergic and oomparable to the at BT binding sites and the other may be r_on- 'cholinergic 'and comparable to the non-chol:inergic nicotine binding sites reporte4 by Abood and Lajtha in rodents. If this is.confirmed.; it would suggest that such non- cholinergic•-nicotinic binding sites..have been'bonserved phyolgenetically at least from the level of the fly Other studies: ~25i-a(BT alterations in twa behavioural mutants (bas and sesD2). The bas mutant maps on the X chr<xinsame. At 21°C it is stress sersitive,

2 beccaai.ng paralyzed for two to three minutes when shaken (no similar effect with wild flies). There is also a tenperature induced paralysis at 38°C. when caraoared with wild flies, the RD for binding was lower at 38eC. and higher at 0°C. (variable effect) and the Bmax (number of binding sites) was reduced at the higher temperature (table 2). The~other ~tress sensitive mutant (sesD ) was more consistent, showing a drop of both `b and max at both temperatures. Rids fly under normal tetperature conditions jumps and flies poorly, has an abnorlnal landing response•and, in r,ales courts abnormally). Since all these studies are preliminary, additional work must be done before conclusions are drawn. However, they suggest that the behaviour modificaYions in sesD2 may be associated with a decreases ino(BT cholinergic binding sites. H-nicotine binding sites have still to be determined in these two mutants. Since nACH binding sites on catecholaminergic (CA) autonomic neurons in rodents and the release of ACh to these receptors has been shown to influence differentation of the CA neurons, one is led to wonder if the alteration of ACh binding sites (conformational and total number) in such mutants as HR wnuld have any effect on the CA neurons present in the head aanglia (CNS) of th'ese flies. This was discussed with Dr. Hall who felt it might be profitable to undertake a simple pilot study (uptake of NE, DA or 5--HT in a crude synaptosome fraction), since CA neurons are known to modulate the functions of other neuronal systens and to have an effect on activity. An effect on peptide rgic neurons might also be of interest since substance P at least is a regulator of tyrosine hydroxylase, the rate limiting enzyme in the synthesis of NE and DA. Dr. Hall also indicated an interest in ecnploying her techniques to study Drosphila mutants which are deficient in choline acetyl transf?s'anse and in acetyl- cholinesterase. (Both mutants are lethal..) Camrent: Work in this program is progressing, but slowly due to the nature of the study. Each mutant created has to be testect several ways to detexm.ine the effects. HowevQ-r, Dr. Hall appears to be developing a concept of the nACh receptor and of nicotine a:. on such receptors (and possibly non-cholinerg~.c receptors) which should facilitate understanding in parallel studies being done in other laboratories with rodents. It is also interesting to note that her work is beginning to suggest that nicotine binds to at least tY9 kinds of receptor at this phylogenetic level, suggesting both cholinergic and non-cholinergic binding sites. This study, as it progresses, is still relevant to the goals of CTR and mPxits continued support through the R 2 year.. D.H.Ford and D. Stone mla