A Mechanism of Action for Morphine-Induced

8/9/2019 A Mechanism of Action for Morphine-Induced

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vo l 270, No . 3Prin ted in U .S .A .

A BBR EV IA TiON S: N K, na tu ra l k ille r ; HPA , hypothalamic-pitu itary-adrenal; FBS , fe ta l bov in e serum ; NBCS , n ewbo rn ca lf se rum ; RPM I , Roswel l ParkMemo r i a l In st i tu te; HBSS , H anks b alanced salt so lu t ion ; DMSO , d ir ne th yl s utf ox id e; P BS , p ho sp ha te -b uf fe re d sa l ine ; P1 , prop ld lum io d ide ; R U486 ,R ousse l-U cla f 38 48 6.

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0022-3565/94/2703-1127$03.O0/OT us Jo uw .L O F P HA RM AC OL OG Y A ND E XP ER IM E NT A L T HE R AP EU T IC SC opy rig ht C 19 94 by T h e Am eric an Soc iety for Ph arm aco lo gy and Exper imenta l The r apeu t ic sJPET 27 1127-1133, 1994

A M echan ism o f A c tio n fo r M orph in e -In ducedIm munosupp ress ion : C o rtico s te rone M ed ia tes M orph ine -InducedS upp ress ion o f N a tu ra l K ille r C e ll A c tiv ity 1DAV ID 0. FRE IER 2 and BRUCE A . FUCHSDepar tmen t o f P ha rm ac olo gy a nd T ox ic olo gy , M ed ic al C olle ge o f V irg in ia , V irg in ia Commonwea l th U nW e rs lt y, R ic hm o nd , W r gin iaA cc ep te d fo r pu blic adon M ay 5 , 1994

ABSTRACTM orph ine is a d rug of abuse w ith an ab i l i ty t o d ow n -r eg ul at eimmune responsiveness th a t cou ld have po term a lly se rio us con -s equences in bo th he ro in add ic ts and in the c lin ic al en vironm en t.The ex ac t m echan ism o f ach on by w h ich m orph ine ind ucesimmuno su p p re s s i o n has ye t to b e d ear ly d e term in ed . A d irectmech an i sm of action is suggested to ope rate th rough lym pho-cy te op ia te recep tors , bu t the natu re o f su ch re ce pto rs is st ill inquest ion . The a lternative , an ind irec t m echan ism of act ion isproposed to be med ia t ed by tw o po ssib le pa thw ay s, hyp o tha l-amlc-pituitary-adrenal ax is ac tiva tion w ith inc rea sed produc t ionof ad ren al co rticoste ro ids , o r ac tiva tion o f the sym path etic na ry -ou s system and con com itan t ca techo lam in e re lea se . Na tu ra lk ille r (N K) cell ac tiv ity was used to de te rm ine p o ten tia l ind irec tmech an i sms of ac tio n fo r m orp h ine . NK act iv i ty in th e B 6C 3F1mous e w as sup pressed be tw een 1 2 an d 48 h r after im plan ta tiono f 7 5 mg tim ed-ra lea se m o rph ine p elle ts . M orph in e sup pres sedN K ac tiv ity in a dose -responsiv e m anner. T he op ia te an tagon is ts

na lo xon e and na ltrex on e com p lete ly b lo ck ed m orp hin e- in du ce dsuppression of NK ac tiv ity , w he reas na loxo ne m e th iod ide , acongener th a t cros ses th e blo od -bra in ba rrie r m uch m o re slow lythan na lox one , p rod uced ve ry lfttle b lo ck ad e. Im p lan ta tion o f th e75 -m g m orph ine pe llets p rod uced a sign ifican t s lev a tion in serumco rtico ste ro ne lev els . In vitro expo su re to cor ticosterone isknow n to suppress NK a ctiv ity d ire ctly , w he rea sin vitro morphinew as unab le to a lter d irec tly NK ac tiv ity . T h e g lucocortico id recap -to r an tagon ist R ous seR icla f 3 8486 b lOC ked morphine - I nducedsuppression o f NK ac tiv ity in a dose-re sp ons ive fa sh ion . Nalt rex-one (1 0-m g p alle t) an tagon ized th e m orphin e-Induced e leva tionin serum cor ticosterone. These resu lts sug gest th a t th e p r inC ip lemech an i sm of ac tion fo r morphine - I nduced suppression o f NKact iv i ty is th rou gh a cen tra lly located , o pi at e r ec ep to r- m ed ia te d,elevat ion in d rcu la ting co rtico steron e w hich d irec tly su pp re sse sNK act iv i ty .

Op ia tes have been sh ow n to ex er t an imm uno suppressiv eeffec t in labo ra to ry an im a ls , an d data suggest th ey m ay haves im ila r e ffects in m an . The ab ility o f NK cells to ly se tum ortargets is o n e of th e im mun e pa ram ete rs th a t is suppressed bym o rph ine adm in istra tion . S hav it et a t. (1984) sh ow ed a do se -re spo nsive su ppre ssion o f N K ac tiv ity in fem a le F isch er ra tsa f ter da ily in jection o f 10 , 30 an d 50 m g/kg /d a y for a 4-dayper iod . Direct cen tra l adm in istra tio n o f m orph in e to ra ts ha ssh ow n that an op ia te recep tor w ith in th e cen tra l n ervou s sys-tem is in vo lv ed in m orph ine -in duced imm uno sup pre ssio n(Shav i t e t a t., 1986 ). D irec t cen tra l adm in istra tion of m orph in eto th e p er iaqu educta l g ra y m atter o f th e hypotha lam us of m aleFischer ra ts sh ow ed th is as th e p r in c ip a l site fo r th is recep to r

Received fo r p ub lic atio n D ec em be r 2, 1993 .1 Work suppor ted by N ID A G rants DA 00490 and R 01DA07292 to B . F .2 Present add ress : M inneapo lis M ediC al R esearch Foundation , 914 S . 8 th St .

D - 3 , Minneapolis, MN 55408.

in regu la tin g m orph in e s e ffects on NK activ ity (W eber an dPert, 19 89) . T h e strong cen tra l n ervou s sys tem -m ed ia ted e ffec to f m or ph in e su gg es ts th a t suppressio n of NK activ ity by m or-ph ine is pr im arily m edia ted by an ind irec t pa thw ay o f ac tion ,beg inn in g w ith cen tra l op ia te recep to rs . T o d eterm in e th em echan ism o f actio n of m orp h ine -indu ced imm uno supp re ssio n ,stud ies m u st b e under tak en to estab lish th e ex ac t pa thw ay bywh ich m orph in e w ork s.

M orph in e-in duced im m unosuppression by a d irect m ech a-n ism of a c tion th rough op ia te recep tors upon lym phocytes isn ot con sisten tly suppor ted by expe rim en ta l ev id ence (S ib in gaand G o ldste in , 198 8) . I t ha s been sug gested t ha t g lu co co rt ic oi dsand /o r ag en ts o f th e sym path etic n ervou s system (i.e., ca te -ch o l am in es ) m ay serve a s th e m ed ia to rs o f m orph in es im m u -nosupp ress iv e ac tions (B ryan t e t aL , 1990; Shav i t et aL , 1986) .In trace reb roven tr icu la r adm in istra tion o f m orph in e inducesinc rea sed h ypo tha lam ic co rtico trop in -re lea sin g fac to r leve lsand in tu rn p rod uces an in crease in ad ren ocortica l ac tiv ity


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1 12 8 F rIar an d Fuc hs(S u emaru et at., 1986) . The ab ility o f th e p er iaqu educta l graym atter to regu la te m orph in e-indu ced suppression o f NK activ -it y su gg ests th at ac tiv a tion o f th e H PA ax is is im po rtan t inm ed ia tin g m orph in e -indu ced im munosuppressio n .

Im p lanta tion o f 75-m g m orph in e p e lle ts indu ces g lu co co r ti-co id -m ed ia ted thym ic apop tos is th at is re spon sib le fo r th edram a tic th ym ic atrophy seen in m orphin e -d ep enden t m ice(Fuchs and Pruett , 19 93 ). T h is effec t w as com p le te ly b lock edby th e op ia te an ta gon ist n a loxon e and th e g lu coco rtico id recep -tor an ta gon is t, RU 486 . T he thym ic a trophy w as show n to b eprimari ly due to th e induction o f apop tosis (F reier and Fuch s,1993 ) and thym ocyte apop to sis is a g lu cocor tico id -sen sit iveprocess (McConkey et a t., 1989 ). A com par ison study of ad re -n a l ec tomy and RU 486 after im p lan ta tion of 75 -m g m orph in ep elle ts in C 3H /H eN m ice ind ica ted that ad rena l ac tiv a tio n w asa cr itica l com ponen t in th e m echan ism of ac tio n of m orph in e -indu ced im m unosuppression (B ryan t e t a t., 1991) . T o d ete rm in ec learly the m echan ism o f ac tio n of m orph in e in m ed ia tin g th esuppres sion of NK activ ity , th e d irect a ction s o f ad rena l cor ti-costero ids and the action of m orph ine on centra l op ia te recep -tor s m u st b e link ed .

O ur purpose w a s to d e term in e th e pharm aco lo g ic param eterso f m orph in e -indu ced suppress ion of NK activ ity in m ice andto eva lua te the ab ility o f g lu co co rtico id horm ones to serve asth e d irec t m ed ia to rs o fth is m o rp h ine -indu ced supp re ssio n . Th eop ia te an ta gon ists na lox on e and na ltrex on e w ere u sed to eva l-uate th e im portan ce of an op ia te receptor in th is ph enom enonand na loxon e m eth iod id e w a s u sed to show th e cen tra l loca tionof th e op ia te recep to r in vo lved . F u rth erm ore , th e g lu co co r ti-co id -spec ific an tagon ist, RU 486 , w as used to ev a lu ate th e ro leo f adrena l cor tico stero id s in m orph in e -indu ced im m uno-suppres sion . S erum leve ls o f cor tico steron e and th e ch rom iumrelea se as say for NK activ ity w ere u sed to assess th e e ffec t ive -n ess o f th ese trea tm en ts.

M ate r ia ls and M ethodsA nim als . F em a le B 6C 3F1 m ice (v iru s free, T acon ic F arm s, G er-

man town , NY ), 6 to 12 w eek s o ld , w ere housed fou r pe r cag e on a 12 -hr lig ht/dark schedu le ( ligh t on at 6 A .M . ) . M ice w ere quarantin ed for1 week an d tested to ensure th e ir sp ecific pa thogen -free status be foreuse. Food and w ater w ere p rov id ed a d lib itu m. A ll anim als w ere caredfor a s d escr ib ed in G u id e for Care an d U se of Laboratory Anim als asadopted by th e N atio na l In st itu tes o f H ea lth . A ll m ic e w ere sa crif ic edbe tween 7 and 10 A .M . to m ain tain experim enta l consistency .

D rug s/chem icals. T im ed-relea se m orphin e p ellets (75 , 25 and 8m g), p lacebo p e lle ts , n a ltrex on e pel lets (10 m g) an d m or ph in e su lfa tew er e o bta in ed from th e N ationa l In stitu te on D rug A bu se (N IDA ) inRockv ille , M D. The drug cor tico steron e and all m ed ia an d supplementsw ere p urch ased from S igm a C hem ical C o. (S t. Lou is , MO ). R U 38486(R U 486) w as a g ift of R ou sae l-U c la f(R om ain ev ille , F ran ce) . N a loxon ehydroch lo r ide and na lox on e m eth iod id e w ere purchased from ResearchB io ch em ica l s In c. (N atick , M A). PBS w as p urch ased from Hyclon e(Log an , UT ). N BC S w as pu rchased from IC N B io ch em iC als In c . (C am -bridg e, M A). C u ltu re m ed ia was R PM I 1640 + 10% FBS supp lementedwith 2 n M g lu ta .m in e, 10 0 U /m l of p en ic illin , 10 0 g /m l o f strep tom y-cm , 2 .25 m g /m l o f sod ium b ica rbon ate and 25 mM H EPE S buffer .

Im plantatio n ofpellets and osm o tic pumps. P ellet im p lan tation swe r e done as p rev iou sly descr ibed (F reie r an d Fuch s , 1 99 3). B rie fly ,m ice w ere an esth e tized , an in cisio n w as m ade in th e low er ba ck of th emouse an d th e p elle t w a s in serted . T he w oundw as c losed w ith a su rg icalstaple and sw abbed w ith a 2% poly v iny l p yrro lid on e iod ine so lu tionused in p la ce o f B etad in e , a s p rev iou sly descr ibed .

Micro-osmot ic pum ps (m ode l 1007D ; lo t #046101; m ean pum p ingra te , 0 .4 9 jsl/h r , m ean fill vo lum e, 98 ) ob ta in ed from A iza co rpo ratio n

V o l. 2 70(Pa lo A lto , CA ) w ere u sed for t imed release o f n al ox on e hydrochlor idean d na loxone m eth iod ide (1 or 5 m g/kg /d ay ). B o th d rugs w ere preparedin R inger s so lu tio n (veh icle ) . T h e pum ps w ere im p lan ted u sin g th es ame su rg ic al p rocedure s descr ibed for th e p e llets . A ll m ice for pum pstud ies w eigh ed at least 2 0 g . B efore im plan ta tio n , th e m icro -osm o ticpum ps w ere in cubated fo r 4 h r i n p hy si ol og ic al sal ine at 37C toel iminate th e lag tim e w h ich w ou ld have occurred had th e pum ps beenim p lan ted directly . Th is allow ed for im med ia te re lea se of an tagon is tinto t he a nim a l.

Sp lenocy te cultu re. Sp leens w ere asep tica lly rem oved f rom naivemice an d placed into HBSS . Sp leen s w ere th en m ash ed be tween th efros ted end s of microscope slid es to p roduce a sin g le ce ll su sp en sion .Splenocytes were w ash ed once in H BSS and tw ice in RPM I 1640supp lem en ted w ith 10% PBS , pen ic i l l in /s t rep tomycin , HEPE S an dsodium b ic ar bo na te . S ple no cy te s w ere su sp ended in 5 m l of R PM I in25 -cm2 cu lture f lasks at a con cen tra tion o f 1 x iO cells /m i and p lacedin to an incubator (5% C 0 2; 3 7# {1 76 }C )o r 3 h r . M orph in e su lfate preparedin R inger s so lu tion w a s added to cultures ju st befo re incubat ion .Corticosterone w as prepared in DMSO , a nd ne ce ssa ry d ilu tion w asdon e to b r in g th e fina l DM SO leve l in culture to less than 0 .1%.Vehicle-cultured ce lls receiv ed bo th R inger s and DM SO . After 3 hrce lls w ere wash ed in R PM I 1640 1- 10% N BC S, counted an d d ilu ted toap propria te co ncen tra tio n fo r u se in the ch rom ium -relea se procedure.

C hrom ium -release assa y . T he ch rom ium -re lease assay w as don eas de sc ribe d (P uc ce tti e t a L , 1980) w ith som e modif icat ions. Yac-1thym ic lym phom a ce lls (Am erican T ype Culture Collec t ion , Rockv il le ,MD ) are grow n in con tinuou s cu ltu re (R PM ! 1640 + 10% PBS ) andkep t in log g row th phase fo r u se as ta rg et ce lls . O n th e da y of assayce ll num ber is determined by hem acytom eter coun t and 1 x iO Y ac-1ce lls are p laced in to a con ica l tub e a nd p el le te d at 17 5 x g. Supernatan tis re mo ve d and th e cells are resu sp ended in 50 0 C i o f [5N a]C rO 4 in avo lum e of 0 .5 m l. Y ac -1 ce lls are th en incubated w ith th e chromiumfor 90 mm in a 3 7# {1 76 }Ca ter bath . C ells w ere w ash ed fiv e tim es in R PM !1640 + 10% NBCS and th en coun ted an d dilu ted to w ork in g c on cen -tra tions. Exper im ental re lea se w as d eterm in ed by measur ing rad ioac-tiv ity from a 100 -l sam p le of sup ernatant u sing the a verage of fourrep lica tio ns fo r each treatm ent a t each effec to r/ta rge t ra tio . Max imumrelease w as d eterm in ed u sin g 10 0 M l of Y ac ce lls (1 x 10 p lu s 100 dof 2 N H C1. Spon tan eous release was 10% or le ss fo r a ll exp er im ents.On ly Y ac ce ll p repa ra tio ns >90% viab le w ere u sed for th e d e term ina -t ion o f ch rom ium relea se. T o ca lcu la te th e p ercen tage of release th efo llow ing form ula w as u seth% S pec if ic R ele ase

- Experim en tal R e lease - Spon taneous R e lea se < #{174}M axim um R elease - Spontaneous R elease

Da t a were ana lyzed for e ffec tor /ta rg et ra tios o f 400:1 , 200:1 , 100:1 ,50:1 , 25 :1 and 12 .5 :1 . E ach sam p le is run in quad rup lica te and ser ia ld ilu tion is d on e to p rov id e th e d esired e ffec to r/targe t ra tios. O nce ser ia ld ilu tion s a re com pleted , chrom ium -labeled Yac-1 cells are added . E f-fector / target ra tio cu rves a re analyzed u sin g th e A llf it program (D c -Lean e t aL , 1978). T h is p rog ram f its a n eq ua tio n to th e sigm oid effecto r /ta rge t re sponse curv es. Th is equatio n is th en u sed to d eterm in e ly ticunits. For ou r purpo ses a ly tic u n it is defin ed as th e num ber of ce llsnecessa ry to in du ce 1 0% sp ec ific rele ase and is ex pressed as lytic units,io splenocytes.

linm uno flu orescen t sta in in g for flow cytom etr ic analysis. A naliquot of 1 .5 x i0 cells w a s rem oved from ea ch sp leno cy te sam p lebefo re prepa ra tio n for th e NK assay . E ach a liquo t w a s p laced in to aw ell o f a 9 6-w e ll p la te in a vo lum e of 20 0 z l o f R PM !. P la te s w ere th encen tr i fuged and ce lls w a sh ed twice in 1 50 l o f bu ffer (PBS + 1%bovine serum a lb umin an d 0 .1% sodium a sid e). C ells w ere pe lleted bycentri.fu ga tion fo r 2 mm at 300 x g . S upe rn ata nts w ere a spirate d vi a a25-gauge need le, th e p la te b r ie fly v or texed to resuspend th e ce ll p e lle tand th en 5 o f m ou se IgG (S igm a C hem ica l Co.) dilu ted 1: 5 f roms tock , was added to each w e ll. T h is w as don e to e lim inate non sp ec ificstain ing o f NK 1.1 on sp lenocytes . T h e p la te w as a llow ed to s it o n ice


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1994 Activity 1129fo r 5 mm , w ash ed on ce in 150 l o fPB S buffe r and 150 o f b io tiny la tedNK 1 .1 a nt ib od y a dd ed (d ilu te d 1:150 from stock). T h is w as incuba tedon ice , in th e dark , for 20 mm , w a sh ed th ree tim es in PBS bu ffer an d150 L l o f ph ycoe ry th r in -co n jug ated strepav id in (d ilu ted 1 :150 fromstock) w as added and a llow ed to in cubate on ice for 20 mm . C ells a reth en w a sh ed th ree tim es in 150 o f PBS bu ffer and 150 l o f P1 (0 .1mg /m l; d ilu ted 1:2 0 in PB S) w as added and th e p late incub ated on icefor 4 mm . A fter a fina l PB S w ash , the pe llet w as resuspen ded in 2 00 o f PB S and ce lls ana lyzed on a FACSCAN f low cytom ete r (B ecton /Dick inson , Sa n Jose , C A ). A gate w as se t up to exc lud e nonv iab le cellsfrom the ana lysis based upon P1 sta in and 5000 viab le c ells w ereanalyzed. P 1 s ta in in g w as a lso u sed to d e term in e v iab ility for in v itr o-cultured splenocyte s. S ta in in g w as done a s d escrib ed abov e, but o nlyP1 w as u sed to sta in th e ce lls .

RU 486 prepara tion an d adm in is tra tion . M ice w ere g iven RU48 6 a t 5 , 2 5 or 100 mg /kg in a susp en sion of 0 .2% Tween 80 and 0 .25%m ethy lcel lu lose in R in gers so lu t ion . T he m ixtu re w as v ortexed ov er-n igh t to p ro du ce a co nsis ten t su spensio n of RU 486 f o r a dm in i st ra ti o n.RU 486 w a s adm in istered by ora l ga vag e a t 1 h r before pe lle t im plan-ta t ion at a v o lum e of 0 .2 m l. V eh ic le-trea ted an im a ls rece ived a 0 .2 -m ivo lum e o f the 0 .2% Tw een 80 , 0 .25% m ethy lce llu lose in R ingersso lu tion w ithou t RU 486 . Th is preparat ion ha s been prev ious ly de-sc r ibed b y o th ers (P h ilibe rt, 198 4) .

C ortico steron e rad io imm uno assay . A ll as say s w ere cond uc tedusing stand a rd ized k its pu rchased from D iagno stic Produ cts Co rpo ra-tion (L os Ange les, CA ) . Serum sam ples w ere taken by card iac pun c tu rea t th e tim e o f sacrif ice. S acr ifice w as conducted by CO 2 inha la tio n .Blood samp le s w ere p la ced in to g lass te st tub es and a llow ed to sit a troom tem pera tu re fo r 1 hr . T h is a llow ed c lo t form ation to begin . Clotsw ere freed from th e tub e w all u sin g w ooden app lica to r stick s. T ubeswere th en re fr igera ted for a t leas t 2 h r , bu t no m ore than 24 hr to a llowc lo ts to con trac t. S am ples were th en cen tr ifu g ed at 500 x g an d serumrem oved from red blo od ce lls a nd p la ced in to m icrocen tr ifu ge tub es.Thbes w ere then stored fro zen at -70 C a nd d ef ro st ed on da y o f a ss ay .A ll samp le s w ere assa yed w ith in 1 m onth of in itia l freez ing . U nknow nse rum concen tra tion s w ere com pared to stand ard cu rv es d ete rm inedfrom k it p rov id ed know n sam p les. F in a l cor tico steron e lev els are as-se ssed as nanog ram s pe r m illilite rs o f co rticoste rone .

Sta tist ica l analysis. A p a ram e tric one -w ay an aly sis o f v ar iance asper fo rmed to de te rm ine w he the r the re w ere sig n if ican t d iffe rencesbetw een tre atm en t gro ups . W hen such diffe ren ces w e re found po st-hocanalysis w as done u sin g e ither D unn etts t te st o r D un can s N ewM ultip le R ange test to determ in e sign if ican ce . D unn etts t te s t w asused in stud ies to d eterm ine w hether m orph ine w as ind uc ing a sig n if i-can t suppres sion of con tro l (p lacebo) respon ses. D uncan s M ultip leRange te s t w as used to de te rm in e the eff icacy ofan tag on ists in b lock in gthe ef fects o f m orphin e. Resu l ts were con sid ered sign if ican t a t P s .05 .

Resu l t sM ice w ere im p lan ted w ith eith er 7 5-m g m orph in e tim ed -

re lea se pe lle ts or p laceb o p e lle ts a t th e ind icated tim e b eforesacrif ice. Sp leen s w ere a sep tica lly rem ov ed and a sin g le ce llsu sp en sion p repa red . NK cyto tox ic a ctiv ity w as m ea su red byth e 5 1C r-re lea se m ethod . T he ea rlie st sign if ican t e ffect o f m or-ph in e on NK activ ity w as at 12 h r and a sign if ican t suppress ionw a s m ain ta in ed th rough 48 hr (fig . 1 ) . N K activ ity b eg an toreco ver by 96 h r after m orph in e , and by 7 days (16 8 hr) NKac tiv ity had re tu rn ed to p lacebo con tro l v a lu es. M orph in e se ffect on sp len ic ce llu lar ity fo llow ed a sim ilar pattern , bu trecovery was only abou t 75% of p lacebo con tro ls by 7 day s(d a ta not shown ). Im p lan ta tion of 8 - , 25 - and 75-m g m orph in ep e lle ts d ose -re spon siv ely suppressed NK activ ity a t 48 h r (fig .2) . To determ in e w heth er th e lo ss in NK activ ity w as th e resu lto f a redu ctio n in to ta l sp len ic NK ce lls , th e NK 1.1 an tibodyw as u sed to as se ss sp len ic NK ce ll p opu la tion s . M orph in e

Fig . 1. T ime course of e ffec t o f morph ine on sp len ic NK cel l cy to tox icactivity. M ice w ere im p lan ted w ith m orph ine (75 -m g ) o r p ceb o pe lletsan d sacrificed at t im e zero . X ax is represen ts tim e in hou rs af te r implan-ta tion , w h ereas y ax is rep re sen ts ly tic un its . Lyt ic units a re d ef in ed asth e num ber o f cells need ed to p rodu ce 1 0% lys is o f Y ac-1 tum or targetsin a 4-h r ch rom iu m re lea se a ssay . D a ta a re p re se nt ed as ly t ic un its p er1 00 m illio n sp lenocy tes . Each ba r rep re sen ts poo led resu lts o f m ultip leanimals, where n is the n um ber o f an im als , w he re n = 1 1 ( na iv e) , n = 5(1 68 , 48 h r a nd f or p la ce bo -t re ate d at 96 h r) and n = 4 (2 4 , 1 2 an d 3 hrand fo r morph ine-t reated a t 96 h r) . Exper iment rep re sen ts on e of fou rseparate repetitions. E rro r ba rs rep resen t S .E .M .; P < .0 1 d ifferencefrom na ive t rea tment .

Fig . 2. Dose-response o f e ffec t o f morph ine on NK cell c yt ot ox ic a ct iv ft y.M orphine (8 -, 25 - 75 -m g ) o r pe llets w ere im plan ted in mice48 h r be fo re sac r ifice . R esu lts a re ex pressed as lytic un its pe r 1 0 m I ll io nsp lenocy tes . Lytic un its a re def ined a s th e num ber o f cells n eed ed toprodu ce 10% lys is o f Y ac-1 tum or ta rge ts ; n = 3 fo r e ac h tr ea tm en tgrou p and expe rim en t rep re sen ts one of th ree rep etitio ns. ** < .0 1dif fe rence from placeb o-trea ted g roup .cau sed a tim e-depend en t dec rea se in N K 1 .1 k sp len ocy te s , bu tthe dec rease in to tal sp len ic N K cells w as o n ly sign if ican t at48 hr a fte r m orph in e pe llet im p lan ta tion (fig . 3 ). T h is sugg es tstha t a los s in sp len ic NK ce ll n um ber is on ly p a rtia lly re sp on-sib le fo r the dec rease in sp len ic NK ac tiv ity .

Sp lenocy tes w e re expo sed to in creas ing con cen tra tion s o fm orph in e in in v itro cu ltu re to de te rm in e w he the r m o rp h inew as d irectly cau sing the supp re ssio n of N K activ ity . A t co ncen -tra tion s o f 0 .1 , 1 .0 , 10 and 100 zM o f m orph in e th ere w as nosupp re ssio n o f N K ac tiv ity (f ig . 4 ) . A slig h t en hancem en tapp eared to b e in ev iden ce , but th is w as n ot s ign ifican t. C e llv iab ilitie s , m easu red afte r the 3 -h r cu ltu re p eriod , w ere n od iffe ren t from un trea ted o r v eh icle - trea ted con tro ls (da ta n o tsh ow n). Th is sug gests tha t m orp h ine is ac ting th roug h anin d irec t m echan ism to ind uce the ob se rved supp re ssio n of NKact iv i ty .


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I 1 30 Fre ler and Fuch s Vo l . 27 0

T ime (hours )Fig . 3 . A ltera tio n s o f sp ien ic NK ce ll p opu la tion by m orph in e. T he e ffec tof th e 75-m g m orphin e p elle t o n sp ien ic NK cells w a s ev a lua ted u s in g ab io tiny la ted NK 1 . 1 an tib ody and strep av id in -phy co ery thnn , fo llow ed byFAC S ana lysis . R aw percen tage da ta w ere correc ted fo r to tal sp len iccellularity. B ars rep resen t g roup s o f poo led data (n = 5 fo r all g ro upsexcep t 48 - and 24-h r m orphin e in wh ich n = 4 an d 1 2-h r p la ce bo inwh ich n = 3) . S ta tis tic al s ign ific anc e d ete rm ine d b y t wo -w ay ana lyS iS ofva rian ce . E rro r b ars rep resen t S .E .M . < . 01 d if fe re nc e f rom naiv econ tro ls .

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Fig . 4. In v itr o NK ce l l cy to tox ic response after m orph in e o r cortico ste r-o ne p ra in ou ba ti on . N aiv e sp lenocy te s w ere cu ltu red fo r 3 h r a t 1 x 10 cells /m I in a vo lum e of 5 m l w ith th e ind ica ted concen tra t ion of d rug .Drug w as added in a vo lum e of 5 0 M I. Co rticosteron e w as p repared in1% DM SO , w here the f ina l con cen tra tion o f DM SO in cu ltu re w as 0 .1%or less . Th e s ame co ncen tratio n o f DM SO was added to a ll m o rp h ine -trea ted cu ltu res . T h ree sepa ra te fla sk s w ere used fo r each trea tm en t.E xpe rim en t rep re se nts on e of th ree rep etitio n s. E rro r bars rep resen tS .E .M . < .0 1 d ifte ren ce from veh icle tre atm en t.

The invo lvem en t o f op ia te recep to rs in m orph in es im m u -nosuppressive effec ts w as assessed us in g the op ia te antagon istsna lo xone and na loxon e m eth iod lid e . N a loxon e m eth iod id e, aquaternary cong en er of na lo xon e w hich p enetrates th e b lood -bra in ba rrier a t a m uch s low er rate , w as u sed to d e term in e th epossib le location of th e op ia te recep tor , e ith er centra l or p e-r iph era l. C on tinu ou s in fu sion of na lo xon e com plete ly antag o-n ized th e suppressiv e effec ts o f m orph in e (f ig . 5 ) and im p lan -

Fig . 5. I n v o lv emen t o f op ia te recep tors in m orph ine-indu ced suppressionof NK cell cy to to x ic ity . N alo xon e and na lox one m e th iod ide w ere ad rn in -is te red fo r 16 hr using the A iz et m rn io sm o ti c pum p, im plan ted sim ulta -neous ly w ith p lacebo or m orph k e (7 5 -m g) p elle tS . E ach trea tm en t g rou pha s n = 5. F igu re rep resen ts one o f tw o r ep etitio ns. L ytic un its a redef ined as the number of ce lls needed t o p ro du ce 10% ly sis o f Y ac -1ta rge ts . #{149} P .0 1 ; < .0 5 diffe rence from p lacebo + veh icle.

2000

Fig . 6 . E ffect o f m o rph ine an d RU 486 on s er um c or ti co st er on e levels.Groups of fo u r m ice w ere im plan ted w ith m orph in e (75 -m g ) o r p laceb opellets 16 hr be fore sacr ifice . RU 486 w as g iven oral ly I h r b e fo re p e lle timplan ta t ion . B lo od w as d raw n by card iac punctu re at th e tim e ofsacrifice. E rro r ba rs rep re sen t S .E .M . S ign if ican ce d eterm in ed by on e -way ana lysis o f v ar ian ce. < .0 1 dif fe rence from pla ce bo + veh iclede term in ed by D unn e tts te st.ta t ion o f 10 -m g na ltrexon e p e llets produced a com parab lean tag on ism (da ta n o t show n). T he adm in istra tion o f n alo xon em eth iod ide produ ced on ly a p ar tia l b lockade o f m orph ine-in du ced supp ression , w hich w as not d ose resp on sive (fig . 5 ).This supp orts an in d irec t m echan ism of ac tion th ro ug h acen tra lly lo cated op ia te recep tor .

Morphine is a k now n ac tiva to r o f the H PA axis, and indu cesin crea sed ad reno co r tica l ou tpu t. C or tico steron e has b een im -plica ted as th e m edia to r fo r m orph ines im m unosuppressiveeffec ts an d w as ab le to do se -d ep en den tly su ppre ss N K ac tiv ityin vitro ( fig . 4 ) . M easu rem en t o f s erum co rticoste rone leve ls inm ice im p lanted w ith th e 7 5-m g m orph ine p e lle ts sh ow ed asign if ican t in crease in c ircu la tin g cor ticosteron e (f ig . 6 ) .

To determ ine th e im po rtance of th e increase in serum corti-costeron e in m ed ia tin g m orph in es suppressive effec ts , th e g lu -


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1 9 9 4 INKAC t IVI tY 1131cocor tico id recep tor an tagon ist R U 486 w as u sed . R U 48 6 ha dno e ffect on th e m orph in e -indu ced e leva tion in serum co rticos-teron e (f ig . 6 ). T h is is n ot an unexpec ted ou tcom e becau se RU48 6 preven ts feedba ck inh ib it ion and th erefore w ou ld p rev en ta d ecrease in co rticosteron e ou tpu t a fter m orph in e -inducede levation . RU 48 6 dose -respons ive ly an tag on ized th e in v ivom orph in e -indu ced suppres sion of NK activ ity (f ig . 7) . Th i ssuppor ts ad reno co rtica l ac tiva tion as the m echan ism o f mo r -ph in e -indu ced suppres sion o f NK activ ity . N a ltrex on e w as ab leto b lo ck th e induced eleva tio n in serum co rticosteron e bym orph in e, fu rth er suppo rtin g co r ticosteron e as th e m ed ia tin ga g en t an d supportin g th e in vo lvem en t of an op ia te recep tor inth e ad renocor tica l ac tiv a tion (fig . 8 ) .

D iscuss ionM orph in e ha s strong im m unosuppressive poten tia l a nd th e

con sequ en ces o f its e ffec ts in clin ica l u se and in op ia te abuseneed to b e c lear ly understo od . Suppression o f NK ce ll cy to to x icac tiv ity by m orph in e w a s cho sen fo r ev a lu atin g a m echan ismof action fo r m orph ine-in du ced im m uno supp ress io n . I t hasbeen shown that m orph in e can suppress NK activ ity (Shav ite t a t., 1983 ; Web e r e t a L , 1987 ; H o e t aL , 1986) , bu t th em echan ism o f a ction is still unc lear . T im e-d ep enden t e ffec ts o fm orph in e on sp len ic param eters have b een dem on stra ted a sea rly as 2 4 h r (B ryan t e t aL , 1988) and m axim um effec ts w ereseen at 3 to 5 day s a fter p e llet im p lan ta tion w ith fu ll recov eryby day 28 (A rora et a L , 1990 ). Im p lan ta tion o f 75 -m g m orp h inepe lle ts a t 168 , 96 , 48 , 24 , 12 and 3 hr b e fo re sa cr if ice w as don eto exam in e th e k in etic s o f m orph in e s effec t on NK activ ity .T h e s trongest suppressive effec t w as seen a t 12 , 24 and 48 hraf ter m orph in e p e llet im p lan ta tion , w ith recovery by 7 d ay s(168 hr ) after m orph in e p elle t im p lan ta tion . T h is tim e cou rsew as sh o rte r than th ose fo r m ito gen ic p ro life rativ e e ffec ts(Bryan t et a t., 1988 ) an d alte ra tio ns in thym ic subpopu la tio ns(F re i er an d Fuch s, 199 3). M orph in e dose -d ep enden tly sup -pressed NK activ ity in m ice im p lan ted w ith 8- , 25 - or 75-m gm orph in e p elle ts . T h e tim e- and dose -d ep enden t stud ies p ro-

Fig . 7 . RU 486 dose-re sp onsiv e an tago n ism of morph ine-inducedsupp re ssio n of NK caN cy to tox ic ity . G ro ups o f fou r m ice w ere im plan tedwith m o rph ine (75 -m g ) o r p lacebo pe llets 16 h r be fore sac lific e . R U 486w as g iven o ra lly at th e ind ica ted dose, 1 h r b e fore p elle t implantation .Ly tic u n its are de fined as th e number o f c ells necessary to produ ce 10%ty sis . E xp erim en t rep re sen ts o ne of th ree rep e tit ion s. E rror ba rs rep re-sen t S .E .M . S ig nific an ce determined by one-w ay ana iysis o f v an an ce .** P < .01 and P < .0 5 d iffe rence from m orp h ine and veh icle and fromm o rph ine + RU 486 5 mg/kg; P < .0 5 d iffe ren ce from m o rp h ine + RU486 25 m g /k g , d et er mi ne d b y D u nc an s Mult ip le R ange te st .

Fig . 8. Naltrex one an tag on ize s mO rph in e-ind uced inc rea se s o f se rumco rticoste rone . G rou ps of fou r m ice w ere p Ian ted w ith p laceb o , m or-phk e (75 -m g) o r n altrexon e (1 0 -m g) pe lle ts 16 h r b e fo re sac iiflc e . B ioods amp le s were taken by card iac punctu re at th e tim e o f sacrif ice. E xp er -im en t rep re sen ts o ne of th ree repe tition s. E rro r bars rep resen t S .E .M.S ig nific an ce d eter min ed by one-w ay ana lysis o f variance. < .05 and** P < .0 1 d iffe rence from p lacebo + morph ine de te rm ined b y D un ca nsM ultip le R ang e te st.v id ed th e n ecessary param eters fo r fu r th er in vestig a tion s onth e m echan ism of ac tion of m orph in e .

M orphine causes a rap id lo ss in sp len ic cellu la rity , and thelo s s in sp len ic N K ce lls cou ld b e th e rea so n fo r the o bse rveddecrea se in NK activ ity . T h e NK 1 .1 m ono clona l an tib ody w a schosen to m easu re NK ce ll num ber in sp leen s o f m orph in e-an d placeb o-treated m ice .Th is an tib ody recogn izes a ce ll su r-face p rote in found on th e m ajor ity o f sp len ic NK ce lls (K ooan d H atzfe ld , 19 80 ). A liquots o f sp len ic NK cells w ere a sse ssedus ing th e b io tin NK 1.1 an tib ody and strepav id in -phyco -e ry th r in . A s ig n if ican t decrease in NK 1.1 sp lenocytes w as inev idence a t 48 hr after m orp h ine pe lle t im p lan ta tion (fig . 3 ) . Itis im portan t to note th at m orph in e suppres sed NK activ ity a t12 h r (fig . 1 ), bu t NK 1 .1 ana lysis ind ica te s th at NK ce ll num berw as not d ifferen t from p lacebo contro ls a t th is t im e. T h issu ggests the observ ed supp ress io n of NK activ ity is on ly par-t ia lly du e to a los s in NK ce lls from th e sp leen . B ecau se NKactiv ity is strong ly suppressed a t 12 h r , w hen NK 1 .1k ce llnumber s are st ill com parab le to p lacebo con tro l lev els , th esuppres sion of NK activ ity is m o st lik e ly to b e due to an e ffec to f m orph ine on th e NK cell fun c tion , as oppo sed to a d irectlo ss in sp len ic NK ce ll num ber.

Sp leno cy tes w ere in cu ba ted fo r 3 hr in v itro a t c o nc e nt ra ti on so f 0 .1 , 1 .0 , 10 and 10 0 jM of m orph in e. T h is w a s don e tode te rm ine w heth er m orph ine h ad a d irec t imm unosupp ress iv ee ffec t on NK activ ity . T h ere w as no e ffec t o f m orph in e at anycon cen tra tio n in com parison to un trea ted or veh icle con tro ls(fig . 4 ) . S im ila r resu lts h a ve b een ob serv ed dur ing in v itroexposu re of pu rif ied hum an per iph era l b lood lym pho cyte s tom orph in e . A 4 -h r cu ltu re had no sign ifican t effec t on NK


6/7

I 1 32 Frs la r and Fuch s Vol . 27 0a ctiv ity ex cep t a t th e h igh est con cen tra tio n o f 1 x iO M (100M ) (Yeager e t a L , 1992) . C ell v iab ility w a s not a sse ssed byY eag er and co-w orkers , b u t w e observed no effec t on sp leno cy tev iab ility in ou r in v itro cu ltu re sy stem . T here fo re it seem sun like ly tha t m o rph ine has the capac ity to d irec tly supp ressN K ac tiv ity .

T h e op ia te receptor an tag on ist n a lo xon e w as used to deter -min e w heth er an op ia te recep to r-m ed ia ted m echan ism wa sin vo lved . Its cong en er , n a lo xon e m eth iod id e , w h ich has a lim -i ted ab ility to cross th e b lood -b ra in barr ier (Io rio and F rig en i,1 984 , R usse ll e t a t.,, 1982 ) w as used to d eterm in e th e loca tionof op ia te recep tor s in v o lved . C on tinuou s adm in istra tion o f 1or 5 m g/kg /d a y o f na lo xon e com p lete ly an ta gon ized m orph in e -indu ced suppression of NK activ ity . Im p lan ta tio n of 1 0-m gnaltrex one p e llets a lso anta gon ized m orphin e- in duced suppres-sion of NK activ ity (d ata not show n ). N a loxon e m eth iod id ew as only ab le to an ta gon ize pa rtia lly th e e ffec ts o f m orph in e( f ig . 5) and th e doses o f 1 and 5 m g/kg /d ay p roduced anequ ivalen t leve l o f an tag on ism . T h is suppor ts th e in terp re ta -t ion o f a cen tra l op ia te recep tor by w h ich m orph in e ac ts toindu ce a suppression of NK activ ity . In trap er iton ea l in jec tio no f N -m ethy l m orph in e , a quaternary form of th e agon ist , fai ledto p roduce a s ign ifican t suppression of NK activ ity (Shav it etzL , 1986) . T h is suggests m orph in e is ac tin g on a cen tra l op ia terecep to r to suppress NK activ ity . D irec t in jec tion o f m orph ineinto th e p eriaqu educta l gray m atter indu ced suppres sion of NKactiv ity in m ice (W ebe r and P e rt, 19 89) and m orph in e ste reos -pacifically su ppre ssed an tibod y prod uc tion to th e an tigen TN P -ova lbum in (W eber et aL , 1987 ). R ecen tly th e m ito gen ic re -sponse o f p eriph era l b lo od lym phocyte s w a s suppressed byacu te m orph in e adm in istra tion (H ernandez et a t. 1993 ). T h esuppres sive e ffec t o f m orph in e w a s cen tra lly m ed ia ted , bu t w a snot link ed to g lu ocor tico id e lev ation . T hese stud ie s suppor t aro le for a cen tra l op ia te recep tor in m ed ia tin g m orph in e -in -d uc ed i m m un os up pr es si on .

A ctiva tion o f th e H PA ax is and production of ad renocor tica lste ro id s or ac tiv ation of the sym pathe tic nervo us sys tem hav eb een sug gested a s a possib le ind irect p athw ay for m orph in e sim m unosuppressive e ffec ts (B ryan t et aL , 1987; Shav it e t a t.,1986) . H PA ax is ac tiva tio n in creases cen tra l cor tico trop in -relea sing facto r and produ ctio n o f ad reno co rtico trop ic ho rm oneand produces an in crease in serum cor tico steron e leve ls . M or-ph in e can ac tiv a te this pa thw ay th rough cen tra l op ia te recep -to rs (S uem aru e t aL , 1986) and adrenocor tica l ac tiv a tio n hasb een link ed to m orph in e s im m unosuppressive effec ts (B ryan te t aL , 1991 ). O the r stud ie s hav e also sh ow n a strong ro le fo rg lu cocor tico id s in m ed ia tin g m orph in e -indu ced e ffects onsup pres sion o f an tib ody p ro duc tion and thym ic ap op to sis , re -sp ective ly (P rue tt e t aL , 1992 ; Fu ch s and Pruett , 1 993 ).

T o a ssess th e im po rtan ce o f ad rena l cor ticostero id s in m or-ph in e -indu ced suppres sion of NK activ ity severa l m ethod s w ereu sed . The 75 -m g m orph in e p e lle ts p roduced a sign if ican t in -crea se in circu la tin g cor ticosteron e 16 h r after pe llet im plan -tation (fig . 6 ) and cor tico steron e d irectly suppres sed NK activ -it y in a do se-respon sive fash ion (f ig . 4 ). A num ber of stud ie s inm ice and in hum an s (u sin g p er iph era l b lood lym phocyte s) ,sh ow reduction in NK activ ity d irec tly indu ced by g lu cocor ti-co id s (M ate ra e t aL , 1988 ; G atti e t a t., 1987 ; P ed ersen andB eyer , 19 86; C ox e t aL , 1982 ) . O ra l adm in istra tion of 5 , 2 5 and10 0 m g /kg o f R U 486 do se -d ep enden tly an tagon ized m orph in e -indu ced suppression of NK activ ity (f ig . 7) . RU 486 (5 m g /kg)d id not sig n ifican tly d iffer from m orph in e and v eh ic le , ind ica t-

in g a no e ffec t leve l. R U 486 d id not a lter e levated serumco rticoste rone leve ls . T hese re su lts sup po rt the conc lu sion tha tmorph ine s tim ula tes increased adrena l co rticostero id ac tiva -tion to p roduce th e suppress ion of NK activ ity .

T h e induced e levation in serum co rtico steron e w as a lso an -tagon ized by im plan ta tio n of 10-m g na ltrexon e p elle ts (fig . 8 ).T h is is a k ey resu l t b ecau se it l inks th e ab ility o f m orph in e toopera te a t a cen tra l op ia te recep to r w ith m orp h ine-inducedeleva tio n in serum co rticosteron e. Becau se naltrexon e corn-p lete ly an tag on izes th e m orph in e-indu ced e lev ation o f serumco rticosterone and th e suppression of NK activ ity , th is strong lysu ppo rts an in d irect m echan ism of ac tio n th rough cen tra l op i-at e recep to rs to a ctiva te th e H PA axis and in crease ad renocor -tica l p roductio n o f co rticosteron e .

A drena lectom y is ano th er a lterna tive fo r asse ssin g th e ro leo f adrenocor tica l a ctiva tion in m orph in e s im muno suppress ivem echan ism , bu t rem oval o f the ad rena l g land also rem ov es asou rce of ca techo lam in es, as w e ll a s o th er ho rm ones, and thu sl imits th e ab ility to ru le ou t th ese o th er horm ones as b e in gin v o lved in m orph in e s suppressiv e ac tion s. T h e thym ic in vo -lu tion p roduced by m orphin e w a s ab roga ted by ad rena lectom y(Sei e t aL , 1991) , bu t B ryan t and co llea gu es (19 91) sta te th atth e sym pa th e tic n ervou s sy stem cannot b e ru led ou t on th ebasis of ad rena lectom y stu d ie s a lone.

Tw o recen t repo rts have suggested tha t th e m echan ism sin vo lved in m ed ia tin g m orph in e s effects on imm une fun ctio nm ay be ev en m ore com plica ted than hypo th esized . U sin g a doseo f 15 m g /kg of m orph in e in m ale Lewis ra ts and exam in in g anumber of im m une param eters 1 h r after m orph in e, a dose-d ep en den t su ppre ssion of C on A ind uced p ro life ra tion o fsp len ic and b lood lym pho cy te s, p roductio n o f IL -2 and IF N bysp leen and lym ph nodes and sp len ic NK ce ll a c tiv ity (L ysle eta L , 1993 ). Suppression ofth ese pa ram eters w ere a ll an ta gonizedby na ltrexon e adm in istra tio n . In th e com pan ion paper th e be tarecep to r an tagon ists n ado lo l, a teno lo l and IC I-11 8 ,551 w ereused to as ses s th e ro le o f th e sym pathe tic n ervou s sys tem inmediat ing m orph in e s suppress ive e ffects . A ll th ree were ab leto an ta gon ize m orph in e -indu ced suppressio n of C on A - andPHA- in d u ced sp len ic lym phocyte p ro lifera tio n bu t w ere unab leto b lock th e suppression of NK activ ity (F echo e t a L , 1993) .Lysle and co -w ork ers (1993) su ggest th a t m orph in e ha s co rn -p artm en t spec ific e ffec ts on im mun e fun c tio n , dep end ing uponth e or ig in o f th e lym phocytes . T h e beta antagonists used byFech o e t a L (1993) w ere unab le to antag on ize m orph in e -indu cedsuppression of NK activ ity . C on sid erin g th e resu lts o fou r w orkand tha t o f F echo and co -w ork ers (1993) , th e sugg es ted co rn -p artm en t spec ific ity may opera te in su ch a w ay that d ifferen timm une func tions are effec ted by d ifferen t regu la tory pa th -way s , e.g., NK cell ac t iv ity seems to b e con tro lled by th e H PAax is and cor ticos tero id p roduction , w herea s C on A lym pho cy tep ro lifera tion is under sym pa th e tic n ervou s sy stem regu la tion .T hese stud ies have im portan t con sid era tion s fo r fu tu re w orkeva lua ting th e e ffec ts o f m orph in e upon im m une pa ram e te rs .

M orph in e -induced suppression of NK activ ity is m ax im a lwith in 12 to 4 8 h r a fte r ex pos ure and is a dose -d ep endent e ffect .This suppression is med ia t ed by a cen tra lly lo ca ted op ia terecep to r, w hich ind uces ac tiva tio n of the H PA axis and in-c r ea se s th e c ircu la tin g leve ls o f cor ticos teron e in serum . T heseh igh lev e ls o f serum co rticosterone para llel th e suppressio n o fNK activ ity by m orph in e, and th e e ffec ts are do se d ep enden tlyb lock ed by th e stero id an tagon ist R U 486 . F rom a ll o f th esestu d ie s , a p ic tu re of th e m echan ism of m orphin e s im m unosup -


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1994 INkActivily 1133press ive pa thw ay can be f o rmed . In suppressin g NK activ ity ,m o rph in e is ac tin g th ro ugh an ind irec t path , b y ac tiv a tin g th eH PA ax is th rou gh a cen tra l op ia te recep tor and induc in g h ighco rt icosterone leve ls th a t p roduce th e ob serv ed suppres sion .Aeknow led gmen t s

The au tho rs w ould lik e to thank D r. L ouis S . Harr is fo r the g ift o f the t imed-re le as e p el le ts ofmorphine sulfa te and both D r. H arris and D r. A lbert E . Munsonfo r ad vic e an d d iscu ssion dur ing th e c ou rs e of th es e s tu di es .Re fe r en cesARORA , P . K ., FR IDE , E ., P wrrrro , J ., W AG GlE , K ., A ND S KO LN IC K, P. : Morph in e -

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e ffects ofch ron ic morph in e treatm ent in m ice . Life Sc i. 41: 17 31-1 738 , 1987 .BRYANT, H . U ., Bw eroN , E . W . A ND H OL AD AY , J . W .: M orp hin e p elle t-in du ce d

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Send rep rin t reque sts to : D avid 0. Freier , Ph.D . , M inneapolis M edical R e-s ea rc h F ou nd at io n, 91 4 S. 8t h Street , D -3 , Minneapolis, M N 5540 4.

A Mechanism of Action for Morphine-Induced

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