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o~:_:t~:: the :;f:~i -~:.:_:::~I exposure, the lungs we: ::emr_-,,,~ :,. -inked, ~- ~ ~enized in I0 volumes of ,i ~ : : c_ont~:-i_ [11.25 . - . -., 0.07 M mannitol, and 0., .. :acid. All solutions wer,- h KO~. The ~suspension wa~ ]',' ~:,,q,,~, ~e at 600 i' 0.2'5 ', at ]7.~00@ g ~ith ~_1,/2 v~k;nme washes after the first ~pinso Ti:e uash ~oiut::_o~ contained 0.25 M sucrose: c~,.~, 0"~. , ',4. ., mannitol, bovinese~un-~ ~i~umin (5 mg/ml) and 0;005 Y¢:,:' :-~spirai:oJ:,~ ~v~;.~urements the mitochondria ,.,.~::~ :-,e. resusp~.ndc_d in a i~edJu_~u of 0.25 M sucrose, 0.07 M ~:a-~:.:J!:o~, and 0.000] J,IZ ~,i, gCl2 i~3 ih~.-,-~,-~',o of 2.5 gm of original tJ,v.~:::a t-o ] ,ml of." .madJ'dn~, Oxyga~: co~sunqgtion was measured at 30~C by i:h~ direct Warburg n~ei:hod wit]-~ ai:,: as the gas phase. The final Jn-~c-~fbation ~:<" lowing in micromoles where medium {~o~_~. {:ained ti~e : ~.._ _ indicaLe~ per total vol-~e of 3 ml: mannitol, I120~ sncrose~ 40007 ]<!qi~]~04 buffe~: at ~]~ 7.4, i0 as phosphate; tripho~_.4~"~e~ 6; ~,-~cuc2~utarate or succinate, 30; 50; i:O Ln :~its ~.<~.~ ,i~:,~,~ (Siva type III); MgCI2~ .~.~ ..... hond~ :, ~:~:,~nsion containing 0~8 to i :, :'~L ~.~-'~g, en. The ce:~~e~- well of th~ ~:~action vessel contained 0.2 50054854

~ lmilH) m) i lllml im~),~.ll i I |I 1 • mm m) il !:. i)(1:5 $6 55

7 transport cZ~=!:-.. .-~ 7~f>-linked substrate, ~-=i:cinat~, i-ypasses the first -_Z~:~::?-~ ~.~~io<.. site of the ele'.tr-:-, tran~ ...... chain, yieldin~ ~ ......... n~,Is of ATP per mol-a ,a of ¢~. ~,t~-n consumed. wfll be This antibiotic 6oe,~ not directly block electron transport, but inhibits phespkc::7-- transfer (5). This inhibition wJil suppress electron transpo<t Jn ~'tightly" coupled mitochondria. !n loosely coupled c::f,e~e].].es, electron transport in tP~e presence of oligomycir~ ,,.0~ be promoted to a degree depending upon the loss of coui~3i~~.i .<fiJc<e[~cy. Where oligomycin was ~sed it was added to the ~vd t<~c<~c,n<~rial suspension prior to addition to" the Warburg reactJo~ vessel, The antibiotic was added at a level of 0.8 mg per 0,5 .m] of suspension. The results, s-~u:::arized in Table 2, show that o]igomycin blocked all respi::ation with ~-ketoglutarate in both the sham controls anC~ r]-:c. ,~:<.c,~<~?rs~ but did not completely inhibJl respiratior~ J:.-~ i . c,-:urc, ls with succinate, i'he ind!,Jdual experiment:~- ::~a~ :< skown) for succinate re-veals ~., ~]lortionate lack of i.n]~ibitJe:< ::,\, o..~gomyc~n in those controls havir, g the greatest de<<eae ~ fr~ i~0 ratio compared to th<~ smokers, Similar i i 50054856

data from normal guinea pigs indicate com~iiue-¢- .... inhibition with either ~-ketoglutarate or _<~.~ir~- : :~ f J~-t~ . These findings with oligomycin sugge~- -hat • -~ ..... mitochondrial damage in sham control anim~ in the mitochondria from smoke exposed linked substrates in smokers, indic~ phosphorylation, g suppor~ phenomenon seen in the P:O data (Table~' I)$ oligomycin approach is apparent in the partisl iR]~£bition fo>n~J in the sham controls with succinate, but not wit!~ The authors are not aware of any comparable phenomenon demonstrated in vivo with animals .... physiological stress. Lowering of P:O ratio by stress is a fairly common finding (6-7). by administering uncoupling agents such as dinit::c,-, --,-, A rare clinical case in which uncoupling occurred 5n muscle oxidative phosphorylation accompanied by progre~a~v~ was reported by Lee et al (8). Whether the part5~ :,:eccv~p~:£ demonstrated.by us with cigarette smoke exposure effect upon the mitochondria, or by an indirc~.ct ::c~,i~: ~:! nc~ known, The possibi].ity that nicotine medi>tes through catecholamines is considered. We emphasize that while these data de~r~onst~at# beneficial tolerance to acute stress afforded cigarette smoke inhalation, the findings 50054857

9 ~pplied to h~,m~an smoking for a nu~_-.~r of reasons. The most are t!~e severe stress impos.~ by force mouth breathing in animals, the high concent-~ation~ of smoke inhaled (yielding up! to 257° saturation of hemoglobin by carbon monoxide), an~ the mech~ ~crlne an hosphoryl~tion:, by ~ivo. en ,'~ iJoseph L. Kyle! Ervin J. Hawrylewicz Life Sciences Research liT Research Institute i0 West 35th Street Chicago, Illinois 60616 The Council for Tobacco Research - U.S.A. 633 Third Avenue !~ew York, New York 10017 Joh~ H. Kreisher 500,-~.858

i. J. A. Braif:::d - 2__8, 836 (~7.! !i Consu Ita~t s I:-~ c: " ,_ , " .... ., Arch. Bicchem and " " .~ H Lardy~, J. L Conr:olly ~nd D. Johnson, .,:.,_oc,,e ,.~st~:'~:, .... L96!: ]969 (1964)', }q, A, o Mass., (Wi]ey, !qe,,,> h:o~:l<)p, 960 (L964), 9. Supporged ":-n p~::.:t i-,. ......... -,-c~,,:--.-,-~- ~_'::a:-,u~ 6<r' 2 x~.o:, Lhe Cou:ici-I for Tobacco Rescg,~:ch Li,S,A, The authors are indebLed [o