Rr.in Rc.r ~tdh.iin. Vol. ,~, pp. 355-]58 Prinled in II~e U.S.A

Effect of Cycloheximide on Temperature Regulation in Rats'

C H R I S T O P H E R C. B A R N E Y . e M I C H A E L J , K A T O V I C H :~ A N D M E L V I N J . F R E G L Y

Depar tme l l t o f PhysiohL~y, Utlivetwity ~[ 'Flor ida. Gai,t,s~,ille E L 32610

( R e c e i v e d 5 M a r c h 1979)

BARNEY. C. C.. M J. KATOVICfI AND M, J. FREGLY, Ej]).ct oj(ycIohe.villdth, t)n tepnpctatttlr t,,gHk,lion iH Itlt~ BRAIN RES. B U L L 4(3) 355-358, 1979.--The efl~ct of cyeloheximlde, an inhibitor of prolein synthesis, pal tempclTuure regulation in ~ffebrile rals was studied to determine whether its reported antipyretlc enact might be altribntahle to n non-specific antimetabolic el'feel. Within 60 rain :trier admlnfi/tmtion of cyelohexim~de (5 mgJkg IP) ~o fema]e tats lit ambient tenlpeallures of 34. 25 and 15~ a signific~nl decrease in colonic Iemperalure wlls observed as comp~red w6h control r~s administered s:dine IP. Measurement~ of nlte of uxygcn consnmpdo~l showed that cyclohgxlmlde (5 mg/k 6 IP) significz ntlv depressed lhe ~bil6y of rats to illcre~se their he~ prodttclion during :1 cold stress (15~ In the cohl tIae rifle or oxygen eo~su~pfion increased 8 7 = 0 8 Oll/mln/k~ "7~ iii the control I;~(s but only .8 • 0 9 n / in k6 'l z, n he eyclohexhnide.lreated ruls, Sillce the thermoregulalory changes accompnnyln 6 cold stress i I l ' ~ slmdar to those observed during die genesis of fever, Ihese data 5tlggesI that{ cyclohexingde May have :1 genel'~d dept'essnnt effecl on he~*t pFothlc[ion rather Ihan ~ specific ~nllipyrelic effect

Cyeloheximlde Tenlpernlure ~guhuion Cold stress

C Y C L O H E X I M I D E is a po len t inh ib i tor of prote in synthe- sis ill euknryo t i c o rgan i sms I81. Us in 6 cycloheximide, Siegert et aL {9,10] h a v e presented ev idence lha t induct ion o f fever in rnbbits by pyrogenlc agents is d e p e n d e n l on the synthes is of a specific f eve r -med ia t ing prole in . W h e n ~d- minis te red to rabbi ts 9fi ra in before pyrogen c h a l l e n r e , cy- c lohexlmide (5 mg/kg IV) p~evented ~be increase in body tem- pe ra tu re a ccompany ing admin i s t ra t ion of pyrogenic agenls (Newcas t l e d isease v i e s , E. Coil t ipopolysacchar ide or sytl- the t ie double-s t r :mded RNA) . However. cyc lohex imide has debi l i ta t ing side efl~c~s 13.111 pa r l i eu la r iy a t l h e doses used by Sieger t et td. [9.10j. Thus , w h e n cyc lohex imide was ad- min i s te red a lone , a fall ila body t e m p e r a l u r e of the rabbi~ was o b s e ~ e d I9.10]. A s imi lar f ind ing was repor ted w h e n rats w e r e admin i s t e red cyc lobex imide ( 10 mg/kg IP) [71. In these s tudies cyc lohex imide p reven ted the bype r tbe rmia and hype rac t iv i ty induced by s imul t aneous admin i s t r a t ion of m o n o a m i n e ox idase inhibi toJs and L- t ryp tophan 17]. Since cye lohex imide did not p reven t the hype r the rmla induced by exposure o f rabbi ts to heat [10J, S ieger t el aL discounted a nanspec i l i c effecl of cye [ohex lmide in p [ e v e n d n g fever , H o w e v e r , the metabol ic responses of m a m m a l s to hea t ex- posure and pyrogens are differel~(, Dur ing exposure to hea t . the an ima l a l t emp t s to m i n i m i z e hea t product ion and Io m a x i m i z e hea t loss to k e e p i ts in te rna l l e m p e r a t u r e f rom increas ing wh i l e dur ing induct ion of fever , the arl ima[ at- t empls to ra i se i ts in te rna l body t e m p e r a t u r e by inc reas ing hea t producl ion and conse rv ing hea t [21. In Ibis sense

physiologic responses observed du r ing fever are s imi la r to those obselwed dur ing cold exposure . The presen t exper - iments w e r e condncled to d e t e r m i n e Ihe efi~.:cl o f cy- clobeximide on the m a i n t e n a n c e of body temper~l lure of I'als dur ln 6 exposu re Io warm. neUlral and cold e n v i r o n m e n l s ~lnd on the r~te of oxygen consumpt ion dur ing exposure Io Ibe e o h h

METHOd)

l~al)eJh.t.nl I. f~f~,ct o f CychJhl..~iJiddl, f). Coh~.i( ~r tare

Thi r ty -s ix f ema le ra ts of Ihe Bhte Sprt lce Farms (Sprague -Dawley) s t rain we igh ing from 190-260 g w e r e used. T h e rats w e r e kept 4 to a c:lge in a room main ta i t l ed al

2 5 ~ I~ and i l luminated From 0700 to 1900 hrs. T h e rats w e r e g iven Pur ina Labora to ry C h o w and wa te r ad lib.

M e a s u r e m e n t s of colonic l e m p e r a t u r e fo l lowlng cy- c ]ohex imide w e r e made at ~lmbient t emper ; l tu res of 15 ~ I~ and 34 • I~C in a walk- in e n v i t o n m e n l a l chamber . For each e x p e d m e n t 12 rats w e r e each res t ra ined in u tunne l type cage cons is t ing of a wooden f loor and a wi re mesh cove r i l l . Co lon ic l e m p e r a t u r e was measu red wi th a . copper- cons t an t an the rmocoup le inser ted 6 cm beyond the anus and 1aped ~o the base of the taiL T e m p e r a t u r e s w e r e measured a t I m i n u t e in te rva ls by means of a recording pozent iometer .

Af t e r the ra ts w e r e placed in th~ res t ra in ing cages, they

ISupported by O ~ c r of Naval Research Contract N0914-75-C~0199 wi~h Ponds provided by the Naval Bureau of Medicine and Surgery. :Post-d~t oral fellow. Institutional End~r ine Tmlnlng Grant, National lnsdtule of Arlhrids and Metabolic Diseases Grant AM-07164-04. aFellow, American Henri Association. Suncoast Chapter.

C o p y r i g h t ~ 1979 A N K H O I n t e r n a t i o n a l Inc . - -0361-9230/79/030355-04500.90/0

B A R N E Y , K A T O V I C H A N D F R E G L Y

+ill +

~ C +o.,5.e

. . . . {-~I-} ~--~- t- ~ -~ -~~ -~ + + -~- ~ - ~

i ~mu~ts

PIG. I. ColollJr lempernlutCs of control (O)and cyclnheaimlde-lrealed (01 r;ll~ +~1 ambient tenlperatores of 34. 25 ;mtl 15~ At 0 (ime 5 mg eycloheximide/kg was administered IP. Showl] are lhe RIcIIII v~ducs z nile St~lnd;krd clror of the mean. *=p<0 f13. "+=l,<0.01.

Number in parenthesis represents number of rzsts in each group.

w e r e a l lowed I hr Io adapt . At the end of th is l i m e conl ro l measu remen t s w e r e made for 30 minutes . At the end of Ihe conlro] period, 6 of Ibe r~lts were admin i s t e red 5 mg cy- c lohexhn lde (S igma Chernicid Conlpany) /kg ]P. T h e remain- h i s 6 ra ts were g iven an eqttal vol t lme of the vebic le (0.15 M NtIC1. 1 ml/kg) IP. T e m p e r a t u r e me~ssurenlents w e r e Ihen con t inued for 2 hr.

' / 'he da la Wele ana lyzed by ca lcu la t ing m e a n colonic tea l - pera l t t res for each gronp eve ry 5 rnin. Means w e r e compared s ta t i s t ica l ly us ing a t - les t wi th s igni f icance set a t the 95% conf idence finlit 14}.

l~perimenl 2. E/~'~'t o f C?'cloh<,xlmi+le on Rt+tc o f O.x\vgen COttSIllllpl#MI

T w e l v e female ra ts of the Bhle Sprtlce Fa rms {.Sp~tgue- D a w l e y l s l raln we igh ing from 330 to 39O g w e r e used. T h e ra l s were main ta ined u n d e r the same condi t ions as d e s c 6 b e d ill E x p e r i m e n t 1.

Rale of oxygen cut, st+rapt{on was measu red us ing an open circui t sys t em and a B e c k m a n OM- I I oxygen ana lyze r . E a c h I'I1[ WILS placed in a cybndr ica l ahml in t /m cbamlmr wi th in a w a t e r bath. The Iqlte of moven)en l o f a i r p~ml lhe ruI was 350 ml /mln and lhe ef l ]uel l l oxygen concen t ra t ion was measured by the oxygen analyze+'. The i,qte o f oxygen consumpt ion was calcuhHed in t e rms of oxygen c o n s u m e d (STPD) pe r rain

per kg body wt t+r Colonic t cmpera lu re was m e a s u r e d wi th a thermis tor (Ye l low Springs [n s l rum e n t Co. No . 401) inser ted 6 cm beyond the nnus and t~ped Io Ihe base of the ta lk

For each study t w o ra ts w e r e p~aced in ~duminum cham- bers wi th in the s a m e w a t e r bath and t i le ra ts w e r e a l lowed Io adap t for 30 rain. T h e w a t e r bath t e m p e r a t u r e was in i t ia l ly a t 25 • I~ Dnr lng this t ime control m e a s u r e m e n t s w e r e made at 5 mln intervtds for 30 rnfn. T h e n one rat was adrninls tercd 5 mg cyc lobex imide /kg IP and the o ther ra l was admin i s t e red [ nd s~fine/kg IF. Mc a s u re m e n l s w e r e con t inued for 60 mln- u les , Then the w a t e r bath t empera tu re was ~ p i d l y reduced to 15 -+ I~ {o induce a cold s~ress and m e a s u r e m e n t s w e r e cont inued for an addi t iona l 60 minutes .

The data w e r e a n a l y z e d by ca lcu la t ing m e a n colonic tern+ pera lu re s and i~ltes of oxygen consumpt ion for each group. These were compared by means of a t - tes t wi th s igni f icance set a t the 95% conf idence l imi t [4}.

RESUI_'rs

Experimen~ /

Admin l s t r a don of cyc lohexi rn ide to r a t s s ign i f ican l ly de+ creased colonic t e m p e r a t u r e a l al l 3 a m b ie n l t e m p e r a t u r e s (Fig. I), At 34~ (Fig. 1A) colonic t empera tu re of the control

C Y C L O H E X 1 M I D E A N D T E M P E R A T U R E R E G U L A T I O N

5 ~

~o i

~ 1 7 6 t~ [ ~0 V,~-- ,~-g ~ i - , . ~.. ~ ............ ' i .r .........

+++I + ++++

FIG 2 Mean r,ue of oxygen consnmpPon (A) and mean colonic temperature tB} of control (�9 and cyc[oheximide treated tOI rats. At 0 time 3 mg r was udminiMcred IP. At 60 ngn the W~ller bath temperatule w;,s decreased from 25 to 15+C One S E is set off ut each me;in * =p <0.05. **=p<0 01, Number in puretuhesls repre

scnls number of rol~ in each group

group decreased 0.8~ wi th in 120 rain a f te r admin i s t r a t ion of sal ine whi le Ihe colonic t e m p e r a t u r e o f the cycloheximide- t rea led group decreased I .g~ dur ing the same time p e r i o d The m e a n colonic t empe ra tu r e s o f the cyc lohex imide - t rent ed group w e r e s igni f icant ly different f rom those of the control group from 20 to 120 mln a f le r admin i s t ra t ion of the drug, At 25~ (Fig. I B) the mean colonic temperature of the conl ro i group decreased 0,2~ whi le tha i of the cyc lohex imide - t rea ted g roup decreased 1.8~ 120 minu tes af ter admln i s l r a - l ion of cyc lohex imide . The m e a n colonic t e m p e r a t u r e s o f l h e egc lohex imide - t r ea t ed group w e r e s ignif icant ly d i f ferent f rom those of the control group f rom 6O to 120 rain a f te r admin i s t r a t ion o f cyc lohex imide . At 15~ the mean colonic t empera tu re or the control group increasgd 0.2~ wh i l e that of the cye lohex imide - t r ea t ed group decreased 3 0 ~ by the end of the e x p e r i m e n t a l period (Fig, ICL T h e m e a n colonic t empera tu re s o f the cycloheximide-treated group w e r e sig- n i f icant ly d i f ferent f rom these of the cont ro l g roup From 85 to 120 rain af ter admin i s t r a t ion of cyc loheximide .

A t the end of eacl~ s t u d y in E x p e r i m e n t I al l t rea ted ra ts appeared rela ive ly h e a l t h y . H o w e v e r , as t ime progressed the cyc lohex imide - t r ea t ed rats became m o r e and more de- bi l i ta ted and all of them died wi th in 48 h r of t r ea tmen t .

EMTerimem 2

Cye lohe• depressed oxygen consumpt ion and de- c reased colonic t e m p e l a l m e (Fig. 2). Dur ing the conl lo[ per iod, the m e a n colonic t e m pe ra tu r e s of both groups w e r e s table at 37.9 + 0.2~ for the control g roup nnd 37.8 _+ 0.3~ for the expe r im e n t a l g roup (Fig. 2R). D u l i n g dds t ime mean ra te of oxygen consumpt ion for the control group ~lveraged 21.2 • 0.6 ml /min /kg c' 7~ and that of the group lo be t rea ted averaged 2 1 5 § 0.8 mt /minfkg '+7~. Af te r a dm ln i s t r a 6on of sa l ine and eyc]oheximide , wi~h the wa te r bath l e m p e r a t u r e ma in t a ined a t 25~ ra te o f oxygen consumpt ion increased by an a v e r a g e of 1.0 ml /min/kg r'7~ for the eon[+ol group and decreased by an ave rage of 0.5 ml /min /kg ":a, for the cyclohe~. imide- t reated an imals . Dur ing th is 60 rain per iod. colonic t e m p e r a t u r e decreased 0.2~ in the control g roup and 1.4~ in the cycloheximlde+lrea ted grollp,

W h e n the wa te r hath t empera t t i r e was reduced to 15~ the ra te of oxygen consumpt ion of the eon l ro l g roup in- creased to 30 ml/mJnlkg ''7:~ wi th in ~0 rain whe re il r e m a i n e d (Pig. 2A). Rale of oxygen consumpt ion of the eyc lohex imide - t r emed group increased Io 25 ml /min /kg o.7~ during the firs~ I0 rain fo l lowing initiation of the cold s t ress

358

and then decreased to Ihe control level dur ing the n e x l 50 mil l . Dur ing the cold stres+., me~m ra te of oxygen consump- t lon or tile conlrol g roup was 29.9 m 0,8 m l l m i n / k g o7~ whi l e the inean r~lle of oxygen consumpt ion of the eyclobeximide+ I t ea l ed group was 23.3 • I. I ml/m~n/kg ''~;. T h e d i f fe rence in ru le o f oxygen consumpt ion was s igni l lcant f rom 70 to 120 rain fof lowing admin i s t ra t ion o f the cyc toheximlde . The dd fe rence in r a te of oxygen eonsumpt ior l be tween the two ga~u ps dur ing the cold s t ress was re l ]ec led in the i r respec t ive r tern peramte~, 'File co lon ic l e m p e r a t u r e o f Ihe control g roup decreased 2+9~ dar ing the cohl s t ress whi le the coi- n[lie tempelT[1ute o f tile c y c l o h e x i m i d e - l r e ~ l e d group de- cte; ised 5,8~C, Tbe colonic t emper l l lu rc of the cyc /oheximJde- l rea ted g roup wiss s igni f ican0y dil'f~rent from IhH~ of the r get, l ip ~d 411 ndn and f rom 5{) m 121) mil l a f l e r adminish 'a t ion of cyc lohex imide .

As in Expe r imen t 1. Ihe c y c l o h e x i m i d e 4 r e a l e d r;its died with[11 48 hr o[' I lcatnlenI ,

DISCUSNIOM

'/ ' /ell[inch[ with cyc~oheximide, a potent prote in syn lhes i s inh lb i to t . 5igidtSc;ll)lJy decreased cohmic t e m p e r a t u r e of fi l ls l es ted ill 15. 25 lind 34~C {Fig. I). T h e f inding of a cych lhex in r ide4eh l t ed deClease in colonic t emper l t tu re at an i ln lb lenl lClIIpCI~LIIIle (if 25~C is in a g r e e m e n l wi lh pre~ious f indings ill la ts 171 and rahbi ts 19,1(11, H o w e v e r , the decrease i l l colonic l e m p e r a t u r e of eyclohexiznide+lrcatcd rats duri t lg beat exposure does nul ilglCC wi th t i le obse lvu t lon of Sieger l et ~ll. I1(11 T h e s e invcs t iga lors noled thnt eyc lohex in l ide did 11ol p l cvcn l hype r the r ln i a du r ing hea t ex;postne in i l lbhi ts . I)d'l'el ences both il~ cXpelimenLM procedm e and species used inay cxphdn the [1{fl)rencc ill rest lhs, I Iowever , s ince cy- e lohex in l id r h:td a srilliJul effect on cl~lonie le n lpe l a lu r e at all 3 ambien t lettlperatttl 'er;, this pl 'oleln Syllthesis inh ih i (o r nl:ty h a v e a genera l eft'eel on tenlper l t l t l re i 'egtlbtllon ra then thal l It ~pecific effect hi p~evcnl lug the genes is of fever . T h e f indlng 11111[ tbc greatest dec rease i/t co/hide t empera l t l r e was oh- set ' re0 ;it ;in HmbJenl lenlpcr[t lnlC O]" lSeC hldlcnles th:lt the response Io cyclogexlmi[1e /i)[ly be passive in ni l l l l re . This s i igges t lon is suppor lcd by rile decrease in trite of oxygen c(lllStllllplio~l pl-r I1), this agent .

Cyclohcximi[1e a t t enua ted Ihe nbil i ty o f i~xts to inc rease

B A R N E Y , K A T O V I C H A N D F R E G L Y

the i r n~te of oxygen consumpt ion du r ing a cold s t ress (Fig . 2). T h e con t lo l ra ts sus ta ined a 35% inc rease in ra te o f oxy+ yen consumpt ion du r ing the cold stress, wh i l e the cyc lohex imide- t rea ted raLs increased the i r l a t e of oxygen consumpt ion on ly 2CcA at the ense t of cold s t ress and w e r e unable to ma in t a in the increase . The l ack o f a sus ta ined , cold induced increase in r a te o f oxygen consumpt ion by c y e l o h e x i m i d e q r e a l e d rats must con t r ibu te to the i r g r e a t e r decrease in colonic t empera tu re . The depressant effec~ of cyc lohex imide on ra te o f oxygen eonsua lp t ion m a y be due to tile debi l i ta t ing effects of the d rug [3, I I J or to Ihe effects of cyc lohex imide on subs t ra te uti l izat ion [51 or endocr ine re- sponses 16}, Al t e rna t ive ly . the mezabolic response to acu te cold exposure may requi re tbe syn lhes i s of a specific ther- moregu la lo ry p lo /e in in the brain such as has been postu- l a led for 1he response of rubbits to pyrogen [9,101.

T h e tmtonomlc responses to cold exposu re and the a d m i n - istn~tion of pyrogens are similur , f ioth t r e a lm e n t s induce an ac t iva t ion of hea t conserva t ion lind hea t product ion mech- uni~ms [21. In the present s ludy, cyc lohex imide depressed Ihe metabol ic response to cohl slress. "l'h u s cyc lohex imide f5 mg/kg) hns a ge ne l a l effect on ternper : l ture regula t ion thut nifty exphdn i ls an t ipy re t i c effect. Cyc lohe x im ide may pre+ ven t or a t l e n u a t e fever by a m e c h a n i s m s imi la r to its effects on t empera tu re regtnIation dur ing cold exposure r a the r than by inhihl r ion of tile ss'nt hesis o f a specific prote in medii l t Ol o f l?ver .

Cyc lohex im ide is ~e{hal when admin i s t e red to ra ts at high doses 13,111. 'ghis obviously l imits its usefu lness in exper i - men ta l inves t iga t iom +['he dose of c yc lohe x im k le Hsed here (5 my/ks) was a lso debBd:tt ing to rats in a s imi l a r s tudy [7]1 Siegel ' l gl a[. It), 101 did lust men t ion a n y side el'f~el~ o[ cy- c lohex imide accompany ing its admin i s tn l t ion to rabblzs at 5 rag/ks f i l l is not l e lha l to rabbit*, Ihe results r epo l t ed here for n i t s may be related In a specle~ di f ference hi to le rance ~o Ihe dl us.

ACKNOWLEPGEM EN1

Io~te Wdghz and Ihc techlgt2al assistance of Mr. Romdd Busch

I, Adolph. E F.. J, P Ih,rker and P, • Hoy, Multiple fact~rs in d[11st, ~ lm .I, Phy~i,,I 17g: 53"r 1954.

[. Bligh. J. It',llp,'/olJ.p Nl'ellh/Ihlll I,i {hlt,lm{lly .4ml Olhee Ver. h 'hr,m'~ New "York: Elsevier I'ublisldng COlllpllll~ t973.

3 Cb'ih. 3. J,. D. M. OSs~)[l~l ~tTitt ~'+ ~1. De, till. /dtenldon in phtsmt< ~llal cellular ella,'ill c ][~ld l intel n Ir162 ~lt~r r lethal and n(,lblr doses of eycloheximide gl the ml, 1got/era+ I'l.+e- m,/c. 25: 2407-24118, 1976

.1, I)nnir W. W, llio.,t,nisflcx: ..~ l.'olm,hlgl,tt I',~r Amdysis in the Ih',dOr S l i e m e ~ New York: John W[1cy luld So~s. 1974, pp.

5, G~w~ht-gain~. J, A., E. I'ina and V. C. 1)c g:lnr [lllp~rlnncc ol the esler[1~c~nlntl pl~cexs in adipose g$511r melahollsm ~ls evidenced b)' c~c]oheXill[1tle. /I/ochre,/. Pharmrw 27: t577- 1579. 1978,

~1 G; ~ I~ e ~1 , [~ , D , , R , [.. Ney ;tlld W. W. Davis. Studies on the role of prot t i l l ~ynttlesi~ in the regtli~lgon of curgcosterone pr~luc- [1Oll I,y tldrenec~lair [lOlll~ ~nc [11 .' v O f 'r , , , , t t l . ',1,1 ,~-(1 i br ~.l~, &l: la4~1450, 1~5

7, Gmhame-Sm[1h. D. G. The preventlon by inbib[1ors of bndt~ protcln synthesis of ~he hyperacdvi W and hyperpyrexla pro- dueed in nits by monoamine oxidase inhlbi[1o~ and the ad mlnlx- t~kl[on of L tr~,rptoph:m or 5-methox~..N+N Dimethyl tryptn- mine. ,/. Neurochr 19: 2-10%2-122. 1972.

8. Pestka, S Inbibltors of dboxome funct{osss. A. R,'v..~hcrtlbl,~l, 25: 487-562, 1971.

9. Siegert, R . W. K. Phill i~Dormsmn. K. R~ld~lk anti H blenzeL Inhlbifion of Newcastle dlsease vlms-lnouced fearer in rabbit~ by cy~loheximide. Atchs Viral 4/t: 367-373. 1975.

I~k Siegert, R,+ W. K Ph[11ip-Dormstom K. Radsakiind H MenzeL Mechanism of fever [ndtlcllon ia rabbits, htj'ecl. /mmum,L 14; 1130-tl37+ I976

1 I. Young. C. W., P+ F. Robinson and El. ,Slleklor, Inbibgioo of the synthesis of prolein in intac~ anlmMs by aeetoxu ~trld 11 metabolic tle~mgement concomitant with ISis bl~k:+de. lli, u.l+cm ph,lr.tl~r 12: 85g-g65. 1%3.