765 sBRCM ACTIONS OF TYRAMINE AND COCAINE ON CATECHOLAMINE LEVELS IN SUBCELLULAR FRACTIONS OF THE ISOLATED CAT HEART H. A. CAMPOS, R. E. STITZEL AND F. E. SHIDEMAN Departm.ent of Pharm.acology, University of Wisconsin Medical School, Madison, Wisconsin Reprinted from THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICB Vol. 141, No. 3 September, 1963 Copyright. © 1963 by The Williams & Wilkins Co. Printed in U.S.A. H.eprintPd fn.1n 1 T11r_: .Jcn~H :\A L 01' 1-'1-t.\H\L.\f" OL O G Y .\ K D E X PERl~lF ::-.'TA L TrrEHAP~Tl'H·:-; Ynl. 1-11. :\o . ;) :-\1 ~ ptr111hPr. (!lü:l Copyrigh t- ='D 1 96 :_~ IJ y T I.e- Williaws &- Wilkins Co. 1~ r i 1 1 ted i 1i { ·.8.A. ACTIOl\S OF TYRA;\II~E AND COCAI::\E Ol\ CATECHOLA~Ill\E LEVELS Ii\' SUBCELLCLAR FRACTIONS OF Tl-IE ISOLATED CAT HEARTt H . A. CA.\'lPOS/ lL E. SlTl'ZEU AND F. E. ::-: IIIDE.\IA:'\é Depart111en/ of Phannacology , l ' nil'nsit¡¡ of Wisconsin !1ledirn l School, .llar/i.,on , W i3consú1 :\ ceeptcd fo r publical ion .\foy 20 , l!Jl;:) Burn ( 1930) ob>'crn:d that elerntion of diffcrential ('<' ntrifuµ:ation anc! ~hmrnd that in t.hc circulatinµ: lewb of cpinephrinc in the eat by reserpinized animal, thc uptake of infused a.dministration of thc amine cnha nced thc vasu­ norepinephrine \Yas greate:3t in the " soluble" all(I constridor ad.ivity of tyrnmine. Subscqucntly least in the "particulatc" frar tion. This find inµ; many :)tudies havc suµ:gc;:;ted that the 6ympathu­ suggestcd that restoration of r e spon~iven cs s to mimetic effect:; of tyrnmine are dependcnt, at tyraminc by adminis tration of norcpinephrine to lcast in part, on thc prcsenrc of eatccholamincs the rcserpine-trcated animal is a rcsult of in tissuc6 or bloud. One of the more significant re1::cnishrneí1f of _th¿.·'j'soluble'' fr_action. contributions of this naturc is t h.e hnciing by Bum J he hypothes1s tpat t~-rarmnc t>xcrts it:=< a nd R.and (1958) that admin~ :;tration of nor­ stim11latory cffect prfma rily through rdea;;c of cpinephrine to the catecholarnin,e-dcpk ted animal catcchula mines from storcs of norepincphrine in can rc:;tore the ,-tinrnlatory action of t.yramiuc. thc ":.;oluble" fraction of thc ccll has bern test.ed Such a re.-; toration of re~pon ;; i vf1i~t0 t~ramíne. in the present work ..· Sinee coeaine antagonizc ..; has abo bcen obsen·ed m the ITT' 1ílcted, 1sola·ted, ..the sti.mulatorv Úfferts of tvraminc (Taintcr and heart of thc doµ; ( Ucjrabla~ · a el al. , l ()fiS) a mi the Chanµ;, 1927;. th1·;;;·' ami .Tainter, 1931) , pre- rat (Axelrod et al., 1962). sumably by prevcntinµ: it. from relcasinµ: <'ate­ :\Iuschulh di scovcr~· (1900) t.hat re~erµi n c c hobmin e~ from their 4orage ,;ites (Flcekenstcin impairs the uptake of liorcpiucphrine by the rat and Storkle, l955; :.Iacmillan, 1959), its capaeity heart ~ u µ;gc,;te d that thc rcstoration of the t o antagonir.e tyr::iminc-indueed alteratiuns in stimulatory action of tyramine i. .; not relatcd to suhedlubr !"Oll<'t'ntrntion• of <'at eC' h o lami11 p~ \l·n:< repleni,;hmcnt of norepinephrine in ti~.-;uc :4ort>:<. examincd. H owever, further work ha" shom.i tha t re.,crpine does not eomplctely :ibolish the uptakc of l\IETH UDS. i\lalc a nd fernale <'at s weighing 1.8 tt• norepincphrinc by thc !'at kidrw.\· ur utcrus '1 .0 kg werc ancsthctizcd with et.her and ki ll ed b~· (Penncfathn all(I Ha nd, 196()) or b~· tlw doµ; cardicctomy 5 minutes after intravenuus adrninis· of (Campos and Shidcrnan, l ()()2) aud r::it heart tration hepa rin (500 17/ kg) . Aftcr remo val f rom thc anilllal, the heart was pl:1ced in warm Krebs · (Kopin et al., 19G2). Henscleit solution. This sulutiun was used as Catecholamines havc bcen d t>m cm~tr: t tccl in thc perfusion fl uid in a ll cxpcrirnents and cun­ particle~ "·ithin the erll w ~eH·ral t.i:-:-;ues t :üned in 1 liter G.n g :'\aCI, o.:l5 g h:CI, 0.28 g (Blaschko a nd \\-ek·h, l 95:3; YOll I:uh·r and CaCL, 2.0 g glucose, 0.1G2 g XalLPO, and 2.1 g; Hillarp. 1936 ; W cil-l\Iallwrbe and Bone, 19;') 7; NaHCn ,. Aftcr li gating t he hranchcs of t hc aorta O ~ tlunc! et al., HJGO), ineluclinµ: the rat hcart :md re n10v ing the pcrirardiurn, the heart was per­ (Pottcr and :\xclrod, l 9G2) ami tlw doµ; hcart fnsed through thc aort:i by means of an Auderson­ (W e¡.>; ma n a nd Kako. l\Jül ; Campo.-' ami Shide­ Langcnd11r ff a pparatus. Thc ¡)('rfnsion ílttid w:\;.; at n a man, l 9fl2). The la><t named author~ "eparatcd ma int.ained temperatnre of :lG cC :rnd p r<'>< · sure 11f üO nun Hg, and aerated with ,t. mixture of homogcna te.; of doµ; heart into thrce fractions !l5',o (), a nd f/ u CO,. Drugs we re di ssolvcd in th(• ("coar:=:e ," "pa.rticula.tP" and ··~o lu bk") by pcrfosion !luid and Clll]J luyed in tli<· foll11 wing con­ Tte ceivecl for publicatiun :\Ltrcl1 '.:!11, Hlfi:l . t·cntr:Lt ions : 1yramin c liydrne hl<lride , 1, 10 and 1 This investigation was support<'d in part ¡,~-a 100 µg / ml ; eoeaine liydruc lil "ridt', 10, 20 and ·lü research grant, ( II E-O I U:~!I) from t hP .:\ :tt innal µg / ml. Hcart;; " .l' rP pcrfnsPd fu r pni"d" of :m, lill Heart Insiit uie , U. S. l'ublic lle:ilt !i :'Prvicc. and no n 1i n ll t(':3 w i th tite;-; ~" snlur ion:-; 01' wit.h drn µ: ­ ' l'reseur addw s ~ : Dep"nmcnt , ;¡" Plt"rma - col og>-, Colleg;e of :\led!c:d SciPi1ces. l"niversity frec I\"rd i~- lfrns l' IPit sulnti .. n. of i\:l inm·:' or:<, :.\liimcapnli:=< 1 1. :.\li a m•,., 11 L\ . Tlií' w:1tPr c·t1n tl'llb of li1it!t :i t ri:tl :lll! l ldt 200 1963 TYHAMINE AXD CAHDTAC CATECIIOLAMIXES 291 vcntricul:tr t issue were detcrmined hy drying small fraction oí the atria iras less than 1.0 in the cat s:implcs (iipproximately 50 mg) to :i eonstant hea.rt (fig. l), wh ereas in the dog heart it 11·a,s weigl1 t. Tlic rcmaining atrial t iss 11 e and 1 g o J' the greater than 1.0. The " particulate" /"soluble" left vcntriclc were homogeni Y-e d separately in ratio in the 1·e1itricle 11·as greater than LO in both 0.075 M potassium phosphatc buffer, pH 7 .5. Homogeni zation was perforrncd in thc cole! using species. Since thc "coarse" fra.ction contains co ni<" al glass homogenizing tu bes . Time of homog­ seyeral element'i (nuclei, unbroken cells, mem­ enization at :L constant speed was 5 minutes in bra.ne fragments, etc.) , changes in its amine nwst ca.ses; however, homogenization for as long eontent are uninterpretable. Such valurs are as 15 minutes <lid not significantly alter the pat­ reported only i11 order to provide in fo rmation on tern of distribution of ca.techolamines. The vol11me altcrations in thc total concentration of cardiac of homogenate was brought l o 12 1111 by addition ratechola.mincs and the effect of the fractiona.tion of buffer. Six mi was ernploycd for fractiona.t ion proredure on recoveries. hy different ial centrifugation ami the remainder Spontaneous release of rateclwlamines. l'er­ was used for detcrm ination of catccholaminc con­ fusion of the hcart with Krebs-Henseleit solution ccntration in the whole lwmogenate. A "coarsc" resulted in a decrease in concentration (deplction) frnction was obtaincd by centrifugation of thc ltornogcnate in the cold at 2000 X g for 5 1n inuics. of eateeholamines. Depletion was more pro­ Thc supernatant was then ccntrifuged at 105,000 X nounced in the "soluble" than in the "particu­ u for (j0 minutes in a Spinco Model L Ultracentri­ late" fraction of both a. tria and ventricle. The fuge, providing two more fractions, "particulate" "particulate" fraction of the atria exhibited littlc anc.l "soluble." Adc.lition of norepinephrine to the change in catecholamine conccntration over a tissue sarnples before homogenization, in a con­ period of 00 minutes (fig. 1 and table 1) . Thc.-;c ccntration Himi lar to that found in the l1 eart, <l id ehanges can not be ascribed to alterations in the not changc the conccntration of catecholarnines in state of hydration of the hea.rt sinee no signiti cant thc "particulate" fra.ct.ion . Aclditional details of dcviation in water conC'entration from the normal the frn.ctionation are descrihed clsewhcre (Campos occurred over this period of time. ami í'hideman, l!Jfi2 ). Total ca.techolamincs, expressed as norepincphrinc, wcre dctermined Catecholamine depletion ind11ced by tyramine. spectrofiuorimet ically by the trihydroxyindole Perfusion oí the heart with tyramine (1 µµ; / mi) mcthod of f.ihore ami Olin (l!J58 ). Addition of for 30 minutes caused a signifieant depl etion of tyraminc or coeainc to standard solutions of catecholamines both in the "soluble" (l' < .01 ) cpinephrine or norepinephrine in co ncentrations and in the "partícula.te" (P < .02) fr:i c tion ~ of greatcr than those ernployed in t hese studies die! atria and ventricle (fig. 1 ami table 1). The ratc not interfere with the determinations of the of depletion was re<lu ced after 30 minutes of ,.,ltecholamines.