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REACT IO NS O F STR IPS OF RABB IT AORTA TO EP INE PH R IXE ,

ISO PRO PYLARTERENO L , SO D IUM N ITR ITE AND

O TH ER DRUG S ’

RO BERT F . FU RCH G OTT AN D SU CH IN BHA D RA KOM ’

Depa rtm en t o f Ph arm aco lo gy , W a sh ing ton U niv ers ity Sch oo l o f M ed ic ine , S I. L ou is, M o.

R eceiv ed fo r pu blicat ion D ecem ber 2 9 , 19 52

T he in vitro re spo nses o f ring s, s trip s o r segm en ts o f a rte rie s to v ariou s d rug s

and physical chang es h ave been the sub jec t o f num ero us inv es tiga tions since

the p io nee r w o rk of 0 . B . M eye r (190 5). In m o st ca ses the so u rce o f a rterie s

has b een slaug h te rh ouse an im a ls . H ow eve r, in th e co urse o f stu d ie s in th is lab o ra-

to ry on the re la tion be tw een m etabo lism and co n trac tion of sm oo th m usc les it

w as fou nd th at sp ira lly cu t str ip s o f rab b it tho racic ao rta w ere adm irab ly su ited

fo r bo th q ua lita tiv e and quan tita tive inv estiga tions o f th e e ffec ts o f d rugs o n

a rte ria l sm oo th m usc le . Th e pre sen t p ape r is co nce rned prim arily w ith the

re spo nse o f such str ip s to va riou s d ru gs, an d a lso w ith th eir gene ra l ch aracte r is-

tic s a s p ha rm aco lo g ical test ob jec ts .

ME THOD S . F or e ach exp erim ent a rabb it w e igh ing 2 .5 to 3 .5 kgm . w a s rap id ly dec ap ita te d .

The d esc end ing th ora cic aorta w as rem ov ed an d plac ed in a Pe tri d ish con tain in g K reb s-

b ica rbo nate so lu tion (K rebs and H etis ele it, 193 2) at room tem pera ture . Ex cess fa t a nd

conn ective tis sue w ere trim m ed off. Th e w ho le le ng th of ao rta w as then cu t alo ng a clo se

sp ira l. D uring the cu ttin g , w hich w as don e w ith a sm a ll, sh arp -po in te d scis sors , the u ncu t

p ort ion of the aorta , h eld gen tly be tw een the thum b and finge rs of th e o perato r’s fre e han d ,

w as g rad ua lly ro ta ted and m ov ed fo rw a rd tow a rd the scis sors in such a m ann er as to perm it

a co n tin uo us sp ira l in cis ion . T he resu ltin g s trip w as usua lly ab ou t 0 .4 mm . th ic k , 2 m m .

w ide and 20 cm . long , and con tained sm oo th m u scle fib ers (th e circu lar fib ers o f the in ta ct

ao rta) orie n te d at abo ut 15 deg ree s rela tiv e to i ts long ax is. F rom th is s trip sh orter strip s

of 2 to 4 cm . leng th (un stre tch ed ) w ere cu t fo r use in exp erim ents. D u rin g the w ho le p ro-

cedure o f prepar ing th e str ips , th e tis sue w as k ept m ois tened w ith K reb s so lu tion .

Th e strips w ere m oun ted in m usc le ch am bers of 2 0 m l. w ork ing vo lum e w ith th e a id o f

sta in less steel S -h oo ks. T he m usc le ba th a ssem b ly , m ain tained a t 37 .5#{176} ,w as of the typ e

prev ious ly d esc rib ed (F urchgo tt, 1 950 ). R epla cem en t o f so lu tio n in a m usc le cham ber w as

ac com plish ed by the ov er- flow pr inc ip le , w ith en ough fre sh so lu tion f rom th e rese rvo ir

fla sk to d ilu te any prev io us ly ad ded dru g a t leas t a tho usand fo ld . Th e so lu tio n w as K reb s-

b ic arb ona te so lu tio n con tain in g 0 .01 3! g lucose . Th rou gh th is so lu tion 95 p er cen t 02-5

per cen t CO 2 w as bubbled , b o th in th e m uscle cham be rs and reserv o ir f lask , g iv ing a pH

of 7 .4 . T he iso to n ic le vers w ere of the in k-w rit ing g rav ity - typ e, ad justed to g ive 9 fo ld am -

plific atio n , and counte rw e igh ted to exer t 4 gm . ten sio n on the str ips . In m any exp erim en ts

in ord er to m in im ize the lag in re spo nse du e to th e sm a ll am oun t of fric tion be tw een w rit ing

po in ts an d kym ograph paper , the leve rs w e re su b je cted to co ntin uo usv ib ratio n of sm all

am pli tud e by m ean s of a sm a ll v ib ratin g m otor co nnected to the iron sta nd su ppo rting

t h em .

‘Th is inv est iga tion w as sup po rted b y a gran t f rom the L ife Insurance M ed ica l R esea rch

Fund . Tw o p relim in ary repor ts on som e o f th e w ork in clud ed in th is p ape r h av e alread y

been m ade (F urchgo tt, 1 952 a,b ).

2 P resen t ad dres s: D epar tm ent o f P hys io lo gy , C hulalo ngkorn H o spita l M ed ical Schoo l,

B angkok , Th aila nd .

12 9

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13 0 ROBERT F . FL ’RCHGOT F AND SUCH IN BHADRAKOM

Stock so lu tio ns o f l -ep inephr ine b itar tra te and m onohyd rated l-no rep ineph rine b itar-

tra te (W in th ro p-S tea rns ) w ere k ep t fro zen p rio r to use . S to ck so lu tions of isoprop y lar -

te reno l hydro ch lo ride (W in th rop-S tea rn s Isupre l con ta in ing an ti-ox id an ts), h is tam ine

d iph osp ha te (van C am p) and ace ty lcho line brom ide (E as tm an) w ere k ep t re frige ra ted ,

and in the case o f th e la st tw o dru gs, w ere m ade up fre sh ly abo u t eve ry w eek . W ork in g

so lu tions of des ired concen tra tion fo r experim en ta l use w ere m ade by d ilu tin g s tock so lu -

t ions w ith iso ton ic sa line . In ex pe rim ents w ith the sym pathom im etic dru gs th e sal ine

co nta ined 0 .0 1 M H C I, and the w o rk ing so lu tion s w ere fresh ly p repared b e fo re each ex pe ri-

m ent and kep t in an ic e-b ath th rou ghout. Ind iv id ual add itio ns of so lu tion s o f d rugs to a

m uscle ch am ber w e re 0 .2 m l. o r less. T he con cen tration figu res used in th is pap er a lw ays

re fe r to the con cen trations o f the sa lt ra the r than the free base . In kym og raph records,

2 3 4

T IM E IN HOURS

F IG . 1 . Change in leng th an d sensitiv ity o f rab b it a ort ic strips w ith t im e. A bsc issa is

t im e a fter str ip w a s at tached to lev er exe rtin g 4 gm . ten sion . O rd in ate is chang e in leng th

in pe r c en t o f in itia l len g th u nd er tens ion (fo llow ing im m ed iate stre tch in g o n attachm ent

to le ver ). C u rve s w ere con structed from m ea surem ents m ad e on o rig ina l kym ograph reco rd s.

A . Th ree exp erim en ts show in g gradu al e lon gation u nde r ten sio n . B . Typic al e xpe rim ent

show ing gradu a l inc rease in sensitiv ity to ep in ephrine w ith tim e , w ith a ttainm en t o f m ax i-

m al sens itiv ity in 2 to 3 h ours . A t each ho riz on tal b ar, the strip w as expo sed to 3 X 10

l-ep ineph rine b ita rtra te .

the concen tra tio n f ig ure a t the po in t o f add ition represen ts the to ta l concen tra tio n o f the

dru g a fte r th e p art icu lara d d ition .

R E SULTS . Chang es in L eng th an d D rug S en sitiv ity w ith T ime . W hen ao rtic

strip s a re f irs t a ttach ed to leve rs u nde r 4 gm . ten sion th ey imm edia tely stre tch

ab ou t 4 0 pe r cen t. T h ey th en u nd ergo a fu rthe r g radu al inc rea se in leng th (fig .

1A ), w h ich usu ally am oun ts to ab ou t 5 p er cen t, b u t v arie s from 2 to 1 0 pe r

cen t. T h is g radua l inc rea se approx im a tely fo llow s an exponen tia l cu rv e w ith a

ha lf-tim e of 10 to 25 m in u te s, so tha t e lo ng atio n is e ssen tia lly com ple te in 1 .25

to 3 h ours.

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REACT IO NS OF AORT IC ST RIPS TO DRUGS 13 1

T o de te rm ine w he the r the g radua l e lon ga tion is a ssoc ia ted w ith a g radu al

lo ss o f sp on taneou s ton e,3 use w as m ade of N aNO2 , w hich , a s w ill b e show n in alate r sec tion of th is p ape r, is ab le to su ppre ss com ple tely low to m oderate tone

o f ao rtic strip s . A dd itio ns o f th is d ru g a t con cen tra tion s o f 1 0 to 10 to str ip s

d u ring e lon ga tion produ ced on ly slig h t relax atio n and d id n o t alte r the tim e-

ch aracte r is tics o f the g rad ua l elo nga tion itself (f ig . 7C ). It is the re fo re con -

c lu ded tha t the g radu a l elo nga tio n do es n o t re su lt from a gradua l lo s s o f spon -

tan eo us to ne , bu t ra the r tha t it re su lts from a physica l p rocess in w hich ce rtain

und efined struc tu ra l elem en ts o f th e ao rtic strip s slow ly leng th en und er tensio n .

If a standa rd sm a ll con cen tra tion of ep in ephrine (10 to 10-8) is te sted a t

in te rva ls a fte r m oun ting a str ip in a m u scle ch am ber, the co n trac tile re spo nse

p rogre ssive ly in creases an d reach es a m ax im um in 2 .5 ho urs to 3 h ours (f ig .

1B ). O nce m ax im al sensitiv ity has been a tta in ed , the re spon se to a sm all s tan d -

a rd te st dose o f ep ineph rin e usua lly rem a ins essen tially con stan t fo r 4 to 6

hours. B y the p rop er type of expe rim en ts the p rog re ssive inc rea se in sens itiv ity

o f s trip s to ep in ep hrine w as show n to be sim ply depend en t o n the pe rio d o f

incub atio n a t 37 .5# {1 76}n K rebs so lu tion , rath e r than on any chang es re la ted to th e

g radua l elo nga tion , o r o n any sensitiz ing e ffects o f su ccessive add itions o f ep in ep h-

rin e . The p ro gress ive inc rea se in sen sitiv ity is no t pecu liar to ep ineph rine , bu t

a lso occurs w ith o the r s tim u la ting drugs such as no rep in ep hrin e , h is tam in e an d

acety lcho lin e ove r the sam e pe riod o f tim e.

T im e C ha rac te ris tic s o f C on trac tion w ith E p in ep hrine a nd R e laxa tion a fte r ite

R emova l . O n add ition of 10 to 10 0 tim es the m in im al e ffec tive concen tratio n of

ep ineph rin e shorten ing b eg ins w ith in 5 seco nds an d is com ple ted w ith in 2 to 6

m inu te s.4 O n w ash in g o u t ep in ep hrine from the m usc le cham ber, re lax atio n

beg ins rap id ly d uring the w ash in g b u t g radua lly becom es m uch s low er, so tha t

even 5 m in u te s af te r ch an g ing so lu tion it is o n ly 75 to 90 pe r cen t com ple te

(f ig . 2A ). F rom th is tim e on relaxa tion app ro x im a te ly fo llow s an exponen tia l

cu rve w ith a ha lf- tim e of 10 to 1 5 m inu tes .

O n add ition of h ig h co ncen tra tion s o f ep in ep hrin e (o f the o rde r o f 10 -s) ,

capab le o f p ro duc ing m ax im a l con tractio n , ab ou t 10 m in u te s a re requ ired fo r

com ple tion of sho rten in g . H ow eve r , re laxa tion a fter w ash ing is m uch slow er

th an w ith m od era te con cen tra tion s (fig . 2B ). In deed , ove r the f irs t 5 m inu te s

a fte r w ash ing no re laxa tion is usua lly de tec tab le . R elaxa tion th en se ts in at a

In the p resen t paper the term “to ne” h as been used to define a sta te o f m ain tained

co n trac tion (o r con trac tu re? ). W e believe th at su ch a sta te is dep end en t on co ntin uo us

activity of th e co ntract ile e lem ents o f a rter ia l sm ooth m u scle , and that su ch act iv i ty

requ ire s en ergy d erived from m etab o lic p rocesse s.

T he m ain tenance o f tone af ter ep inephr ine ad dit ion depen ds on the m ain ten an ce of

the ep ineph rine co ncen tra tion in th e so lu tion ba th in g th e arte ria l strip . F or th is reason

the d is tilled w ate r and reagen ts used in m aking the so lu tio n need to b e free of traces o f

heavy m eta ls w hich m ight cataly ze the oxidat ion of ad ded ep inep hr ine. O ccas ion ally , it

w as fo und th at ar teria l strips in on e pa rticu la r m usc le ch am ber cou ld not m ain tain to ne

afte r e p in ep hrin e add ition . Th is w as tra ced to cor ros ion of th e m etal s tock of th e h ypo -

derm ic need le used as a bubb le r in the m u sc le ch am ber in ques tio n . C hang ing the n eed le

co rrec ted the situa tio n .

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13 2 ROBERT F . FURCHGOPT AND SUCH IN BHADRAKOM

ra te w h ich g radu ally inc rea se s o ve r a pe riod of abo u t 5 m in u te s and th en beg in s

to dec rease g rad ua lly . A dd itio na l w ashes w ith in the firs t 5 m inu te s do no t hasten

the on set o f re laxa tion . A bou t 1 5 m in u te s a re requ ired fo r 5 0 pe r cen t re laxa tion ,

and ab ou t ano th er 60 m inu tes fo r e ssen tia lly com ple te re laxa tion . Th e la st ha lf

o f the re laxa tion is approx im a tely an exponen tia l fun ctio n o f tim e , w ith a h alf-

tim e o f abo u t 1 0 m in u te s. Th e de layed o nse t o f re laxa tion a fte r flu sh in g ou t

ep ineph rin e so lu tio n g iv ing m ax im a l re spon se is p ro bab ly due in p a rt to the

tim e requ ired fo r o u tw ard d iffu sion to reduce the concen tratio n of th e d rug in the

(B )

I

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I I 1 I I-

F IG . 2 . Resp onse to v ary ing concen tra tions o f l-ep ineph rin e b ita rtra te . A rrow ind ica tes

add ition of d ru g and W ind ica tes change of f lu id . T im e m arks are a t 5 m inu te in te rva ls.

A . C on trac tion o n add ition an d imm edia te relaxa tion o n w ashou t o f low concen tra tio n of

ep in eph rin e. S trip (un der ten sio n of leve r) w a s abou t 6 cm . in len g th . B . C ontrac tion o n

additio n and d elayed rela xa tion on w ashou t o f h ig h co ncen tration of ep ineph rine . S tr ip

w as ab o u t 3 cm . in leng th an d sh ortened approx im a tely 2 7 p er cen t. C . G raded contract ion

resu ltin g from progressive inc rease in co ncen tration of ep inephrine (ep inephrine concen tra -

tion se ries ). S trip w as abou t 3 cm . in len g th , an d m axim a l sh orte n in g w as approx im a tely

22 p er cen t.

reg io n o f th e sm oo th m usc le recep to rs be low a lev el cap ab le o f p rodu c ing es -

sen tia lly m ax im a l co n trac tion .

T he fin al g radua l ph ase o f re laxa tion a fte r the w asho u t o f b o th m od era te

an d h igh concen tratio ns o f ep in ep hrine is n o t due to a g radua l lo ss o f d rug -

sus tain ed to ne o r spo n taneous to ne . T h is is ind ica ted b y the fac t tha t th e add i-

tio n o f N aNO 2 (10 to 1 0-s ) to str ip s d u rin g th is p hase p rodu ces o n ly a sligh t

ex tra re lax atio n , w h ich is o f s im ila r m agn itud e to tha t p rod uced w ith strip s

w h ich have firs t been a llow ed to com ple te th eir re laxa tion (f ig . 7D ).R ela tion o f R espon se to C oncen tra tion o f E p ineph rine . The m in im a l e ffec tive

con cen tra tion of ep ineph rine (tha t con cen tra tion g iv ing a 1 to 2 m m . rise on

the k ym ograp h reco rd ) w as de te rm ined on 32 ao rtic str ip s w hich h ad firs t been

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REACT IO NS O F AORT IC ST RIPS T O DRUGS 13 3

a llow ed to a tta in m ax im a l sensitiv ity ( tab le 1 ). Th e ob served va lues ranged

from 1 to 2 0 X 10-’# {176}m ean : 5 .9 X 10-10 ± 0 .9 X 10-10 (SEM )). In th e con cen -

tra tion range up to ab ou t 5 tim es the m in im a l effec tive con cen tra tion , th e degree

o f con tractio n is usu ally app rox im a te ly p ropo rtio na l to the am oun t o f ep ineph -

rif le added ; how ev er , w ith m any strip s a sign ifican tly g rea te r d eg ree o f co n trac -

tio n pe r m ic ro g ram o f ep in ephrine occu rs in th e up pe r ha lf o f th is low rang e

th an a t the m in im a l e ffectiv e con cen tra tion itse lf .

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F IG . 3 . T yp ical co ncen tration -ac tiv ity curv es ob ta ined w ith l-ep in ep hrine b itar trate .

A and B . P lo t o f da ta from firs t ep inephrine con cen tration se ries w ith fu lly sensitized

strips. N ote d ev iatio n of ac tua l (so lid ) cu rves th roug h poin ts f rom th eoretic al (d o tted)

cu rves at in te rm ed iate h igh er leve ls o f co n traction . T h eo re tica l cu rves w ere con struc ted

on th e b asis o f E qu atio n 1 us ing K valu es of 1 .4 X 10 8 and 1 .9 X 10 respec tiv e ly fo r A

an d B . C and D . P lo t of d ata from second ep in eph rin e co ncen tration se rie s. S am e strip

w as u sed fo r C as fo r A , an d fo r D as for B . N ote in ea ch ca se the dec rea sed sens itiv ity

a t low er le ve ls of con trac tion , a nd the clo se approx im a tio n o f th eo retical a nd observ ed

curv es. Th eo retical curves in C and D have K va lues of 4 .2 X 10 and 1 .4 X 10 re spe c-

t ively .

The resp onses o f s trip s to p ro g res sive ly h ig he r co ncen tra tion s o f ep in ep hrine ,

add ed in ser ie s w ith n o w ash ou t (f ig . 2 ) , w e re u tiliz ed to ob tain com ple te con -

cen tra tion -ac tiv ity cu rves (fig . 3 ). In 3 5 exp e rim en ts w ith fu lly sens itiz ed str ip s

h alf-m ax im a l con tractio n w as ob tain ed a t ep ineph rin e concen tratio ns in the

ran ge 0 .5 to 5 X 10-8 (m ean : 3 .4 X 10-8 ± 0 .3 X 10-8 (SEM )). M ax im al co n-

trac tion (1 5 to 3 0 pe r cen t sho rten ing ) is gene rally ach iev ed a t a co ncen tra tion

of 10 -i, and occasion ally a t a con cen tra tion o f 1 0-i.

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13 4 ROB ERT F . FURCHGOTT AND SUCH IN BHADRAKOM

T he ac tua l co ncen tra tion -ac tiv ity cu rves ob ta in ed w ith fu lly sen sitized str ip s

g en era lly dev ia te co nsid erab ly from curves p red icted b y C lark ’s equ atio n (1 937 ),

CM SC = K + (E q ua tion 1 )

wh ere C is the con trac tile re spo nse a t d rug co ncen tra tion 8 , CM is th e m ax im al

con trac tile respon se (a t ve ry h igh d ru g con cen tra tion ) , and K is the ap pa ren t

d issoc ia tion con stan t fo r the d ru g-recep to r com plex .5 Th is dev ia tion re su lts

from rela tive ly sm a ller re spo nses o f th e arte ria l s tr ip s a t in te rm ed ia te h igh

con cen tra tions than w ou ld be pred ic ted from th e eq ua tion w hen K va lues a re

in tro duced b ased o n the resp on ses to ep ineph rine co ncen tra tio ns p rodu c ing le ss

th an on e-ha lf m ax im a l re sp onse (f ig s . 3A and 3B ).

If an a rte r ial s tr ip , a fte r b ein g su b jec ted to o ne ep inephrine con cen tra tion

serie s , is w ashed and a llow ed to re lax fu lly (ov e r a pe riod of 1 to 1 .5 ho urs),

an d is th en sub jected to a seco nd co ncen tra tion serie s , the re is gene ra lly no

sign ifican t d ifference b etw een the abso lu te h eig h ts o f m ax im a l con tractio n in the

tw o se rie s (0 ± 5 pe r cen t). H ow ev e r, the sensitiv ity o f a strip tow ard ep i-

nephrine in a secon d se ries is alw ays redu ced be low its s en sitiv ity in a firs t

se rie s . T h is reduc tion is m ost m arked a t con trac tion leve ls b elow ha lf-m ax im al

co n trac tion , w ith anyw here from tw o to fiv e tim es m ore ep ineph rine be ing

requ ired in the seco nd serie s th an in the firs t to p ro duce eq u iva len t respon ses .

A t h ig he r lev els o f con tractio n the d iffe ren ce in sensitiv ity becom es le ss m arked .

A s a re su lt o f th is d ifferen tia l change in sensitiv ity , cu rves from second con cen tra -

tion ser ie s m uch m o re c losely app rox im a te theo retica l cu rves p red icted by

C lark ’s eq ua tion than d o curves from firs t con cen tra tion se ries (f ig s . 3C and

3D ) .

T he reduced sensitiv ity o f a s trip d urin g a secon d con cen tra tion se ries w ith

ep ineph rin e is the re su lt o f changes b roug h t ab ou t du ring ex po su re to h ig h

co ncen tra tion s o f ep in ep hrine du rin g the firs t concen tratio n se rie s . T h is is

ind ica ted by th e find ing tha t a 5 to 10 m inu te exp osu re o f a fu lly sensitiz ed

strip to a s ing le h ig h d ose o f ep in ephrine , capab le o f p ro duc ing m ax im al con-

trac tion , reduces sen sitiv ity ju st a s e ffec tive ly as does exp osu re to g raded doses

o f ep in ep hrine in a concen tra tion se ries (tab le 2 ). Th e d ec rease in sen sitiv ity is

no t lim ited to ep in ephrine . A fte r exp osu re to a h igh co ncen tra tion of th is d rug ,

the re is a s im ila r d ec rease in sensitiv ity to norep ineph rin e, acety lch o line , an d

h istamine.

T he equ atio n a s g iven is s im p ly an algeb raic rear ran gem en t of C la rk’s equ atio n ,

10 0

K X = -

w here X is the d rug co ncen tra tion , K is the apparen t asso cia tion co nstan t be tw een drug andrecep to r, and V is the respon se in per cen t o f the m ax im al resp onse . Th e fo rm u la in the

tex t is p re fe rred becau se o f the comm on use o f an ana log ous fo rm ula in enzym e chemistry

and becau se it ha s been fou nd m ore conven ien t in a fu rthe r d ev elopm ent o f the theo ry of

d rug-recep to r in te rac tion (F urchg o tt, to b e pub lished).

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REACT ION S OF AORT IC ST R IPS T O DRUG S 13 5

T he redu ctio n in sen sitiv ity to low do ses o f stim u la ting dru gs fo llow ing ex-

p osu re to a do se o f a d rug causing essen tia lly m ax im a l con trac tion has been

reported p rev io usly fo r sh eep ca ro tid a rte ry (W ilk ie , 192 8), gu in ea p ig ileum

(C an ton i and Eastm an , 194 6) and gu inea p ig u te rus (D an ie l, 1 952 ). A cco rd in g

to C an ton i and Eastm an , inc rea sin g the K + concen tratio n of the ir m ed ium

seve ra l fo ld cou n te rac ted th is reduc tion in sensitiv ity in th e case o f g u inea p ig

ileum . In ou r ow n expe rim en ts w ith rab b it ao rta in creas in g the K + concen tra -

tio n of the regu la r K reb s so lu tion u p to 2 .5 tim es w as ine ffec tive in th is rega rd .

Equ ally ine ffectiv e w ere a num ber o f o the r a ltera tions o f th e reg u la r K rebs so lu -

tio n . The on ly m anne r in w hich th e d ecrease in sen sitiv ity fo llow ing exposu re

to h igh ep ineph rin e co ncen tra tion s cou ld be pa rtly rev ersed w as to a llow su ff i-

TABLE 1

C om pa riso n o f e ffe c ts o f ep inep hrine a nd o th er drugs o n str ips o f rabb it aor ta

CONCENTRATION REQUIRED FOR RELATIVE

DRUG RESPON SE HEIGHT OF

M in irn :1 d etec tab le Max ima l response RESPONSE

1 -ep inephr ine C 1-2 0 X 10 -10 1-10 X 10 ’ 10 0

l-no rep ineph rine C 1-2 0 X 100 1-10 X 10 ’ 10 0

h istam ine C 2 -20 X 10 ‘-40 75-97

ac ety lcho lin e C 1-5 0 X 10 1-10 X 10 10 -45

iso pro py lartere no l R5 1 -10 X 10 ’ 1-10 X 10 5-1 0

C 10-6 ‘5 x I0 72-80

In th is ta b le C deno tes con tra ctio n arid R re lax ation . T he concen tration req u ired for

m in im a l d ete cta b le resp on se w as d eterm ined on fu lly sen sitized str ips an d repre sen ts tha t

con cen tra tion g iv in g a 1 to 2 mm . d isp lacem ent o f th e lever po in t. A ll concen tra tio ns are

co ncen tra tion s of the salts , no t o f the free b ases. (S ee sec tion on METHODS fo r sa lts used .)

* Relaxat ion w ith iso propy la rte reno l w as de te rm ined on s trip s b roug h t to a s tate o f

m oderate ton e by prio r ad d itio n of one o f the o ther d ru gs lis ted in tab le . R e lative he ig h to f m ax im al response in case of re laxa tio n is based on com pariso n of abso lu te inc rease in

leng th of s trip on re laxa tion w ith abso lu te decrease in leng th on m axim al co n traction w ith

ep inephrine.

c ien t tim e (2 to 3 h ours) to elap se afte r th e w ash ou t o f th e large d e -sens itiz in g

d ose o f tha t d ru g (tab le 2 ) .

Norep in ep hrin e an d Iso pro py larle reno l. In six expe rim en ts ep ineph rin e b ita r-

tra te and m onohydrated no rep in ep hrine b ita rtra te w ere te sted on the sam e

a rter ia l s trip a t a s im ila r low con cen tra tion (1 0 to 1 0-8 ). T he av erage ra tio

o f the re spo nse to n orep inephrine re la tive to th at o f ep inep hrin e w as 1 .18 ± .05

(SEM ). E xcep t fo r th is som ew ha t g rea ter sens itiv ity a t low concen tra tion s,

the g ene ra l b eh av io r o f th e str ip s tow ard n orep in ephrine is the sam e as th at

tow ard ep inephrine (tab le 1 ) . I f m ax im al con tractio n is p ro duced by th e add itio n

o f on e o f these d ru gs at th e neces sary h igh concen tratio n , add itio n of the o th e r

d ru g a t the sam e concen tra tion , and in the p re sen ce of th e firs t d rug , p rod uces

n o add itio na l co n trac tion .

In con tra st to ep in ep hrine and no rep in ep hrine , isop ropy la rte reno l g iv es no

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13 6 ROB ERT F . FURCHGOPT AND SUCH IN BHADRAKOM

con trac tio ns in th e con cen tra tion range b etw een 10-s and 10-v . O n ly w h en a

concen tratio n of the o rd er o f 10_ s is reached does co n trac tion firs t occu r (f ig .

4A ). F urth er inc rea se in co ncen tra tion lead s to g rea ter co n trac tion , w ith the

m ax im al respon se be ing a tta ined at abou t 5 X 10 Th is m ax im a l con trac tile

resp on se w as foun d in five ex pe rim en ts to be abou t 75 pe r cen t o f tha t ob tain ed

w ith ep in ep hrine . B o th th e ra te o f sho rten in g on add ition o f a stim u latin g do se

TABLE 2

Sen8 i tiv i ty to low con cen tra tio ns o f e p in eph rin e befo re an d a fter ex po sure to a co nc en tra tion

causing max ima l con tract ion

#{163}XP. NO . NIPS. CONCN .

CONTRACTION B l IGHT

EP I

B efore

10 ’ Epineph .

A fter W ashou t of 10 ‘Epinephr ine

30 4 0 6-70 ’ 1 20’ 180 ’

mm . mm . mm . mm . mm .

1 3 X 10-’ 2 7 4* 8 12 -

2 3 X 10-’ 3 .2 - 1.5 2. 5 -

10-8 17 - 9 13 -

3 3 X 10-’ 17 - 5 6 8

4 3 X 10’ 7 .5 2 * 4 5 .5 6

S tr ip s in a ll exp er im en ts w ere approx im ate ly 3 cm . long w hen fu lly s tre tch ed und er

tens ion of lev er . C on tractio n he igh t is in mm. ris e o f leve r poin t on kym ograp h record .

* Strip had no t ye t com ple ted re laxa tion fo llow ing w ash ou t of 1 0-’ ep inephrine.

i i’ _ __

(A )

F IG . 4 . Responses to isopro py la rte ren o l hyd roch lo ride . A . C on trac tion of un treated

ar te ria l s trip w ith h igh concen tra tio ns o f isop ropy la rte reno l (I) , and re laxa tion afte r

w ashou t (W ). B . and C . R elaxa tio n w ith low con cen tra tions o f iso p ropy lar te reno l o f s trip s

in itia lly b rou gh t to a s ta te o f m odera te tone by the prev iou s add ition of ep inep hrine (E )

and ace ty lch o lin e (A C ).

of iso p ropy lar te reno l and the ra te o f relaxa tio n on w ashou t a re slow er than in the

case o f ep inephrine (com pare f ig s. 4A and 2A ).

In o rde r to show c lea rly the re lax ing e ffect o f isop rop y la rte ren o l o n aorticstrips, it is f irs t necessa ry to b ring the str ip s to a sta te o f low to m odera te to ne

w ith a stim u la ting d ru g . T he stim u la ting drug m ay b e ace ty lcho lin e, h is tam in e ,

o r even o th e r sym path om im etic s such as ep in ep hrine and norep ineph rine . S ub-

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REACT ION S OF AORT IC STR IPS T O DRUGS 13 7

sequen t add itions o f isop rop y la rte ren o l ove r th e low concen tratio n rang e of

10-’ to 10-v now p ro duce sig n if ican t re lax atio n (f ig s . 4B and 4C ). The m ax im a l

re lax ing effec t o ccu rs b etw een 10 -i an d 10-6 . W hen the con cen tra tion is in -

c rea sed fu rthe r, re laxa tion g ives w ay to con trac tion . Ind eed , the co ncen tra tion

o f isop rop y la rte ren o l cau sing m ax im a l re laxa tion freq uen tly has a b ip hasic

e ffec t, w ith the in itia l re laxa tion be ing fo llow ed by a sm a ll, s low ly d ev e loped

con tractio n . In term s of ab so lu te chan ges in leng th the m ax im al re lax ing e ffec t

o f iso p ropy lar te reno l is u sua lly b etw een 7 and 15 pe r cen t o f its m ax im a l con-

trac ting e ffect.

I f s trip s a re b rou gh t in itia lly to a sta te o f m ax im a l, ra the r th an low to m od-

e rate , tone w ith ep ineph rin e o r no rep in ep hrine , iso p ro py la r tereno l can n o lon ge r

cau se re laxa tion . In exp erim en ts w ith m ax im a l to ne due to h is tam ine , o u r

re su lts have no t b een con sisten t. In th ree such ex pe rim en ts , iso p ropy lar te reno l

w as ab le to p rod uce a sm a ll deg ree o f relax atio n , b u t in th ree o the rs it p rodu ced

no d etec tab le relaxa tion .

R e laxa tion w ith isop rop y la rte ren o l is v ery sim ila r bo th qu an tita tiv e ly and

qua lita tive ly to th a t p rod uced by ep in ep hrine on a rte r ial s tr ip s p rev io usly

trea ted w ith D ib en am ine (F urchgo tt, 1 952 a,b ) . H ow ev er , ep in ep hrine , u n like

isop rop y la rte ren o l, n eve r g ives re laxa tion of un trea ted str ip s f irs t b rou gh t to a

sta te o f m ode ra te ton e b y the ad d ition o f o the r stim u la ting drugs. T h is is the

case no t on ly w ith con cen tra tion s o f ep in ep hrine w hich alo ne g ive co n trac tion

b u t a lso w ith m uch low er co ncen tra tion s (10 -’ to 1 0 ’# {1 76} ).

Ace ty li.h o line a nd H istam ine . C erta in cha rac te ris tics o f acety lch o line and

h istam ine as s t imulan ts of ao rtic strip s a re lis ted in tab le 1 . The usu al m in im a l

effec tive co ncen tra tio n of acety lch o line is ab ou t 5 X 10-i (a lthou gh it va r ies

m arked ly in d iffe ren t strip s) , an d th a t o f h istam ine abou t 5 X 1O_8 . The quan ti-

ta tive re la tion of sen sitiv itie s to ep inephrine , to h istam ine , an d to ace ty lcho line

v a rie s g reatly in d iffe ren t str ip s . W ith co ncen tratio ns o f ace ty icho line up to

ab ou t five tim es th e m in im al e ffec tiv e lev e l, resp on se is app ro x im a te ly p ro por-

tio na l to con cen tra tion . H ow eve r , in the case o f h istam ine th ere is u sua lly

an ino rd in ate ly large respon se as th e co ncen tra tion is ra ised 2 to 5 tim es ab ove

th e m in im a l e ffec tiv e leve l (f ig . 5A and B ).

Th e tim e cha rac te ris tic s o f re sp onse fo r ace ty icho lin e and frequ en tly fo r

h istam ine are ve ry sim ila r to tho se a lready d escribed fo r ep inephrine . H ow eve r,

in abo u t ha lf o f th e expe rim en ts w ith low to m od era te con cen tra tions o f h is-

tam in e a gradua l rise in tone occurred fo r a s lon g as 10 m in u te s fo llow ing th e

in itial rap id con trac tion (fig . 5B ). In con tras t to th is , the re som e tim es occu rred

(4 ou t o f 10 ex pe rim en ts) a g radu a l lo ss o f ton e of strip s fo llow ing a tta inm en t

o f m ax im a l con trac tion w ith h igh concen tratio ns o f h istam ine .

R e lax atio n w as neve r ob ta ined eith er w ith ace ty lcho lin e o r h istam ine (ove r

th e ran ge 10b0 to 10 -i), rega rd les s o f w he the r the str ip be ing ’te sted w as in itia llya t re st o r in a s tate o f m od era te to h igh ton e d ue to the p rev io us add ition of

an o the r stim u la ting drug .

So d ium N itr ite an d S pon taneou s Ton e. W hen N aNO2 is ad ded a t a con cen tra -

tio n of 1 0-i to strip s o f rabb it ao rta in a sta te o f e leva ted tone d ue to th e p rey -

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13 8 ROB ERT F . FURCHGOT T AND SUCH IN BHADRAKOM

ous add itio n of a stim u latin g dru g , re laxa tion a lw ays occurs (fig . 6A ). T he d eg ree

o f re laxa tion d oes n o t d ep end on the pa rticu la r stim u la tin g d ru g bu t on the

in itial tonu s leve l. A t low to m od era te lev els (co n trac tion he igh ts up to abo u t

30 p er cen t o f the m ax im um p ro duced by ep inephrine ) , the re laxa tion on ad d i-

tio n of NaNO2 is com ple te . Indeed , in m any ex pe rim en ts , the strip s ac tua lly

(A ) 5Xt0

-

.01 3 M

FIG . 5. T yp ica l resp onses to ac ety ich o lin e b rom id e (AC ), h istam ine d ip hosph ate (H ),

an d K C I. A rrow ind ica tes ad d itio n of d rug an d W ind ica tes change of flu id . C oncen tra tion

of K C1 is in excess o f th at in regu la r K rebs so lu tion .

(A

( -

1 -I

S.

NQP4O2 . )O 3 J-

(C ’)

0

(D ) Nc*N01

F IG . 6 . R espo nses to sod ium nitr ite . R eco rd A w as ob ta in ed w ith s trip ap prox im ate ly

3 cm . in leng th (under ten sion of lev er ), w h ile a ll o th er records w ere o b tained w ith strips

app rox im ate ly 6 cm . in leng th . V ertica l ba r in record B is 1 cm . on o rig in al reco rd . A .

Relax ation by N aN O 2 o f strip in itia lly b rou gh t to a s tate o f m od erate tone w ith ep inephrine .

B . E ffec t o f N aNO 2 on fu lly stre tched s tr ip s. E x tra e long ation afte r N aN O 2 in low er trac ing

w as g reates t ob ta in ed in presen t exp er im en ts . C . E ffec t o f N aN O 2 added to s tr ip u nder-

g o ing g radua l e long a tion f iv e m inu tes a fte r a ttachm ent to w eigh ted lev er. Tw o low er curves

a re from sam e record o ne hour and tw o hou rs la te r. D . E ffec t o f N aNO 2 added a t beg in n ing

o f g radua l phase of re laxa tion fo llow ing in itia l rap id ph ase of re lax a tion on w ash ou t o f

stim ulatin g d ose of ep inep hrine.

re lax to slig h tly g reate r len g ths than they h ad be fo re the eleva tion o f ton e w ith

the stim u latin g d ru g . R e lax a tion beg ins with in a few second s o f the add ition of

N aNO2 and is in itia lly even m ore rap id than relaxa tion resu lting from th e

rem ova l o f the stim u latin g drug by rap id change o f so lu tion (com pare f ig s. 6A

and 6D ). H ow eve r, a s in the case o f the rem ov al o f such drug by w ash in g , the

fast in itial p hase of re lax a tion afte r NaNO 2 is fo llow ed by a slow ph ase , w ith

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Li i

a-

REACT IO NS O F AORT IC ST RIPS TO DRUG S 13 9

the leve r ap pro ach ing the b ase line o f com ple te relax atio n alo ng an ex pon en tial

cu rve w ith a ha lf- tim e o f ab ou t 1 0 to 15 minu tes .

T he fin d ing tha t a strip in itia lly in a sta te o f low to m odera te ton e du e tothe p rev io us add itio n of a stim u la ting drug o ften re laxes on add ition o f 10-i

N aNO 2 to a leng th slig h tly g reate r than it h ad p rio r to th e add ition of th e

s t imula t ing drug su ggested tha t m any aortic str ip s p ossess a sligh t sp on tan eo us

tone w h ich can be su ppre ssed b y n itr ite . T h is w as sh ow n to b e th e case in a

la rge num ber o f ex pe rim en ts w ith strip s w h ich h ad been a llow ed to a ttain e s-

sen tia lly cons tan t len g th in ab sence of stim u la ting drug s. S uch str ip s o n the

add itio n of 10 -i N aNO 2 usu ally und e rw en t a s ligh t e long a tion (fig . 6B ). T he

e long atio n , appa ren tly re f lec ting the su pp re ssion o f spo n taneous to ne , am oun ted

a t m o st to on ly a frac tion of a per cen t o f the in itia l leng th . It w as no t inc rea sed

- LOG EP I CONCEN TR AT IO N

F IG . 7 . E ffect o f p resence of v ar io us concen tra tions of N aN O 2 on resp onse to prog ress iv e

inc reases in concen tra tion o f l-ep inephrine b ita rtrate . T he strips u sed w ere a ll from the

sam e aorta . T he leve l o f m ax im al respon se at 10-i ep in ep hrine w ith e ach con cen tra tio n ofN aNO 2 is based on the ef fec t o f the add ition of each con cen tration of N aN O 2 to the co n tro l

s trip in the presence of 10 -i ep in ep hrine . N aN O 2 concen tra tions w ere as fo llow s: no ne 0

(con tro l), 10 -’ X , 10’L 10 # {149}.

by ra ising th e n itra te co ncen tra tion to 1 0 T he fin a l re laxed leng th o f an in -

d iv idua l s trip trea ted w ith N aNO 2 w as the sam e w he the r th e str ip h ad b een

in an un stim u lated state o r in a sta te o f low drug -ind uced tone prio r to trea t-

men t .

T he sm a ll am oun t o f sp on taneou s to ne w hich can be su ppre ssed w ith n itrite

persis ts desp ite n um erous changes of the so lu tion b ath ing ao rtic strips . It a lso

persis ts in th e p re sence o f D iben am ine , a trop ine o r d iph enhydram in e , so tha t it

seem s un like ly th at it is du e to a co n tinu ous relea se o f ep ineph rin e, no rep in ep h-

rin e, acety lch o line o r h istam ine w ith in the s trip s . O n the o the r hand , in terfe r ing

w ith the en ergy m e tabo lism of a rte r ial s tr ip s b y sh if ting from ae rob ic to anae ro b ic

co nd itio ns in th e absen ce o f g lucose d oes supp re ss spon tan eo us ton e just a s

e ffec tive ly as n itr ite (F urch go tt, to b e p ub lished ) .

T he re su lts o f a typ ica l qu an titativ e expe rim en t o n N aNO2 -ep in ep hrine

an tag on ism are p lo tted in f ig . 7 . A t an y g iv en ep in ep hrine con cen tra tion th e

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14 0 ROB ERT F . FURCIIG OT T AND SUCH IN BHADRAKOM

d eg ree o f dep res sion in creases a s the N aNO 2 con cen tra tion in crease s from

10-6 to 1 0 A t any g iven n itr ite con cen tra tion the degree o f dep ress ion de -

c rea se s as the ep ineph rin e con cen tra tion inc rea se s. T hus, in the ex pe rim en t o f

f ig . 7 , 1 0 -i N aNO2 is ab le to sup pre ss com ple te ly th e re sp onse to ep inep hrin e

up to a con cen tra tion approach ing 10-8 , ye t it low ers the re sp onse to 1 0 ep i-

nephrine on ly 6 pe r cen t. T h is ab ility o f ep in ep hrine in h igh con cen tra tion to

“b reak th ro ugh ” n itr ite inh ib ition ind ica tes tha t the in h ib itio n do es n o t depend

on a d irec t in terfe ren ce w ith the con tractile m ech an ism of th e vascu la r sm oo th

musc le .

P otas sium C hlo ride . If th e K concen tratio n (0 .005 8 N ) of the K rebs-b i-

ca rbon ate so lu tio n ba th ing an arte ria l s trip is inc rea sed 3 to 4 tim es by the

add itio n of ex tra K C 1 , th e str ip con tracts . A s show n in fig . 5C , b o th the ra te

of shor ten ing on add itio n o f KC I an d th e ra te o f relaxa tion on w ashou t a re

ve ry m uch slow er than in th e case o f ep ineph rin e. A dd itio ns o f K C I w h ich no

m o re than doub le the K + con cen tra tion o f the K reb s so lu tion a re u sua lly in -

capab le o f caus ing v is ib le con trac tion o f a r ter ia l s trip s . H ow eve r , such sub -th re sho ld add itions d e fin ite ly p o ten tia te the con trac tile re sp onse p ro duced by

low concen tra tio ns o f ep inephrine . If s trip s a re b rou gh t to a sta te o f m od erate

to ne by the add itio n of KC 1 , th ey a re u sua lly m ore resis tan t to the re lax ing

effec ts o f isop rop y la rte ren o l and N aNO2 than str ip s b rou gh t to a com parab le

state o f ton e w ith ep in ep hrine , acety lch o line o r h is tam ine .

D I sCU s s I oN . The use o f sp ira lly cu t strip s o f ar te rie s fo r in vitro s tud ie s w as

firs t de sc rib ed by L ew is an d K oessle r (1 927 ), w ho used p ig ca ro tid a r te r ies as a

sou rce . In th e case o f a rte r ies o f ab ou t 2 to 3 mm . d iam e ter , such as the rab b it

ao rta , sp irally cu t str ip s a re p refe rab le to r ing s o r transec ted rings (such as

used freq uen tly w ith la rg e r a rte rie s) , because they g iv e adequa te kym og raph

reco rds w ith on ly m oderate leve r am plif ica tion . W e have been unab le to find

any prev ious rep orts o n the use o f th e rabb it ao rta as a te st ob jec t fo r the re -

ac tion s of a rteria l sm ooth m usc le , ye t it app ea rs to h av e certa in d is tinc t ad -

van tag es. Its sou rce is a comm on laborato ry an im a l. P repa ring sp ira lly cu t

str ip s from it is n o t d iff icu lt. S uch strip s resp ond w e ll to d rug s an d a re th in

enough to pe rm it re la tive ly rap id d iffus ion of d ru gs from the su rro und ing m ed ium

in to th e ir deepe r laye rs . A lso w hen such str ip s a re susp en ded in w e ll ox ygena ted

so lu tion , the re is n o dange r o f anae ro b ic con d ition s p reva iling in d eep er lay ers

a s th ere m igh t b e w ith ring s or s trips from m uch th icker a rte ries o f th e type

frequen tly ob ta ined from slaug h terhouse an im als.

A no the r ad van tage of s trip s o f rabb it ao rta un de r o ur ex pe rim en tal con d ition s

is tha t they possess a lm o st neg lig ib le spon taneous tone an d n ev e r ex h ib it rh y th -

m ic con trac tion . T h is m akes them espec ially w ell su ited fo r q uan tita tive stud ies

on drugs cau sing con trac tio n , s ince th e va riab le in flu en ce of these fac to rs o n th e

response to such dru gs does n o t h av e to be ev a lua ted . In th is reg ard they d iffe r

from ce rta in p rep ara tions o f a r ter ie s from la rg er an im a ls w h ich m ay deve lop

bo th cons ide rab le spon tan eo us to ne and rh y thm ic co n trac tion du ring the course

o f an expe rim en t (e .g. , R oth lin , 192 0 ; W eiss , 19 20 ; D ucret, 193 1).

Th e re sp onses o f strip s o f rab b it ao rta to ep inep hrin e, h is tam in e, ace ty l-

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REACT ION S OF AORT IC STR IPS T O DRUGS 14 1

ch o line , and N aNO2 are sim ila r in m any resp ec ts to the re spo nses o f r ing s o r

str ip s o f v a riou s p rin c ipa l d is tribu ting arte rie s o f slaugh terho use an im als , such

as stud ied by num erou s ea rlier w orke rs . (Fo r om prehensive rev iew s of lite ra tu resee p ap e rs by R o th lin (1 920 ) an d M onn ier (194 3)). A s fo r iso p ropy lar te reno l,

w e have been un ab le to f ind an y prev iou s rep orts a s to its effec ts o n iso la ted

p repa ratio ns o f a rte rie s . A m ong the d rug s w h ich w e h av e so far investig ated it

is un ique in tha t it c an cause re laxa tion a t low concen tra tion s and con tractio ns a t

h igh concen tratio ns. T h is f ind in g g ives d irec t su ppo rt to the conc lusio n o f

A hlq u ist (19 49) , ba sed on b loo d p re ssu re expe rim en ts w ith rabb its , tha t iso -

p rop y la rte ren o l can cau se vasoco nstr ictio n as w ell a s vasod ila tio n .

R etu rn ing to the m ore gene ra l p rop ertie s o f s trip s o f rab b it ao rta , it sho u ld

b e no ted th at the g rad ua l in crease s in leng th and sensitiv ity o f su ch strip s

a fte r they a re m oun ted in m u scle cham bers a re sim ila r to inc rea se s o bse rv ed

b y o the rs w ith p rep ara tions o f iso lated a rter ie s from la rge r an im asi (e .g. , Ro th -

u n , 1 920 , and D ucre t, 193 1) . In o ur ow n exp erim en ts th e g radua l in crease in

leng th w as ap prox im ate ly an expon en tial fun ctio n of tim e . A ve ry s im ila r ph ase

o f g radua l e long atio n w as fo und to fo llow the in itia l rap id ph ase o f relaxa tio n

of rab b it ao rtic strip s a f ter the w asho u t o f stim u latin g d ru gs. A ltho ugh w e a re

iio t p repa red to sp ecu la te a s to w ha t e lem en ts in the strip s a re g radua lly leng th -

en ing u nde r su ch c ircum stances, ou r exp erim en ts w ith N aNO2 appear to ru le

ou t the pos sib ility tha t th e ph en om enon o f g radua l e lon ga tion is d ue to a g rad ua l

lo ss o f spo n taneous ton e.

T he g radua l in crease in sens itiv ity o f fre sh ly m oun ted strip s to low do ses o f

s tim ula ting drugs m ay represen t a g radua l reco very from a s ta te o f re la tive ly

poo r sensitiv ity b rou gh t on b y the traum a and stim u la tio n to w hich th e strip s

are sub jec ted du ring th eir p repa ratio n . T h is is con son an t w ith the fin d ing th at

w hen strips w h ich have becom e fu lly sen sitized are s tim u la ted to m ax im al con -

trac tion by exposu re to a h igh concen tra tion of ep inephrine , they rev er t to a

s tate o f re la tive ly p oor sen sitiv ity to low concen tration s o f s tim ulating d rugs .

Fo llow in g such an exposu re sensitiv ity aga in inc rea se s a s a func tio n of tim e .

Th e red uc tion in sen sitiv ity re su ltin g from expo su re to h igh con cen tra tions o f

ep inephrine acco un ts fo r the f ind in g tha t con cen tra tion -activ ity cu rv es o b-

ta ined in second ep in ephrine con cen tra tion se rie s dev iate le ss from theore tica l

cu rves than do concen tra tio n -ac tiv ity cu rves ob ta ined in firs t se rie s . If it is

a ssum ed tha t ch an ges in th e sta te o f the sm oo th m usc le w hich lead to reduced

sen sitiv ity deve lop a t h ig h leve ls o f con trac tion during a firs t s er ie s , then it

w ou ld be exp ec ted th at the ac tua l co ncen tratio n -ac tiv ity cu rve from such a

se rie s w ou ld fo llow a theo retica l cu rve up to m od era te lev els o f con tractio n bu t

fa ll be low it a t h igh lev e ls o f con tractio n . In te rm s of Equa tion 1 th is effec t

m igh t b e rep re sen ted by a prog re ssiv e inc rea se in the m agn itud e of K at h igh

leve ls o f con trac tion .6 Furthe rm ore , if the changes lead in g to reduced sen sitiv ity

‘O n the b asis of C lark’s orig ina l fo rm u lat ion , th e K in eq uation 1 sh ou ld be the d is-

sociatio n con sta n t fo r th e dru g-recep to r com p lex . H ow ev er, recen t w ork in th is labo rato ry

ind icates that K is n o t the ac tua l d issociatio n con sta n t, b u t p rob ab ly that co nstan t tim es

ano ther fun ction w h ich is dep en den t on th e n um ber of to ta l recep to rs, th e sta te o f m etab o-

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14 2 ROB ERT F . FL JRCHGOTF AND SUCH IN BHADRAKOM

are large ly unreve rsed by the tim e a secon d con cen tra tion se ries is run , any

fu rth e r ch an ges d ev elo p ing a t h ig h lev els o f co n trac tion du rin g the seco nd se rie s

a re like ly to be le ss m arked . T he iefo re , the re spo nses ob ta ined a t a ll lev els o f

co n trac tion s in a second se rie s w ou ld be expec ted to fa ll fa ir ly c lose to a theo -

re tical co ncen tra tio n -ac tiv ity cu rve . In te rm s o f E qu atio n 1 , K a t the beg inn in g

of a secon d serie s w ou ld be h igh er th an at th e beg in n ing o f a f irs t se rie s , bu t

w ou ld rem a in m o re n ea rly con stan t at a ll co n trac tion leve ls du ring th e secon d

series .

S UMMAR Y

1 . T he gen era l p rope rtie s o f sp ira lly cu t str ip s o f rab b it ao rta a s pha rm a-

co log ica l te st ob jec ts h av e been desc ribed . S uch strip s po sse ss o n ly a v ery sm a ll

am oun t o f spo n taneou s ton e and n ev er exh ib it rhy thm ic con trac tio ns. W hen

fre sh ly m oun ted in m usc le cham bers they unde rgo a grad ua l elo nga tion and

gradua l inc rea se in sensitiv ity to stim u latin g d ru gs ove r a pe rio d of abo u t tw o

to th ree hou rs . If a fu lly sens itiz ed str ip is caused to con tract m ax im a lly by

tem po ra ry exp osu re to a h igh concen tratio n o f ep inephrine , it rev erts to a sta te

o f low sen sitiv ity .

2 . D rug s w hich w ere fou nd to g ive on ly con tractio n o f ao rtic strip s fa ll in

the fo llow in g orde r a s to po tency : n o rep inephrine an d ep ineph rin e > h is tam ine

> a ce ty ic ho li ne .

3 . S od ium nitr ite cau ses com p le te re laxa tion of str ip s in itially p laced in a

sta te o f low to m od era te tone by the p rev iou s ad d ition o f a s tim u la ting d rug . I t

also ap pears to b e ab le to com p lete ly sup pres s spo n taneous to ne of ao rtic str ip s .

H ow eve r, in the face o f m ax im al to ne d ue to h igh con cen tra tions o f ep ineph rin e,

it c au ses on ly sligh t re lax atio n .

4 . Isop rop y la rte ren o l causes re laxa tion a t low con cen tra tion s. H ow eve r, a t

h igh co ncen tratio ns it causes con trac tion .

5 . Th e tim e-ch arac te r is tic s o f con trac tion on add ition , and o f re laxa tiona fter w asho u t, o f ep ineph rin e and ce rtain o the r d ru gs have been desc ribed .

6 . C oncen tratio n -ac tiv ity cu rv es fo r ep in ep hrine have been presen ted . T he

re latio n of the ob se rv ed cu rv es to theo re tical cu rv es p red ic ted by C la rk ’s equa -

tio n has been d iscussed .

R E FERENCES

AHLQUIST , R . P .: A m . J. P hysio l., 153 : 586 , 1948 .

CANTON! , G . L ., AND E A S TMAN , G .: THIS JOURNAL , 87 : 39 2 , 1 946 .

CLARK , A . J .: in H effte r’s H andbuch d er Expe rem en tellen Pha rm aco log ie, vo l. 4 , J. Spr in -

g er , B erlin , 1947 .

DANIEL , E . E .: Fed . P roc ., 11 : 3 35 , 1 952 .

DUCRET , S .: A rch . f. d . ges. P hys io l., 227 : 7 53 , 1931 .

FURCHGOr r , R . F .: TH IS JOU RN AL , 99 : 1 , 1950 .

lism of the m usc le , e tc . A s w ill b e sh ow n in a la ter p ub lica tion , con cen tra tion -a ctiv ity cu rve s

of the type pred ic ted by C la rk ’s eq ua tio n can on ly be expec ted w hen th is func tio n rem ain s

con sta n t th rou ghout an ex per im ent.

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REACT ION S OF AORT IC ST RIPS TO DRUG S 14 3

FURCHGOT T , R . F .: F ed . P roc ., 11 : 217 , 1952a.

F IJRCHGOT ’F , R . F .: TH IS JOURNAL , 106 : 387 , 1 952b .

I t REBS , H . A ., A ND H EN SE LE IT , K .: Z . f. phys io l. C h em ., 210 : 33 , 1932 .

LEWIS , J . H ., AN D K OE SSL ER , K . K .: A rch . In t. M ed ., 39 : 1 82 , 1927 .

MEYER , 0 . B .: Z . f . B io l., 4 8 : 352 , 1 905 .

MONN IE R , M .: H e lv . P hys io l. A c ta , 2 : 2 79 , 1 943 .

ROTHL IN , E .: B io ch em . Z ., 11 1 : 219 , 1 920 .

WEiss , S .: A rch . f. d . g es . P hys io l., 1 81 : 2 13 , 19 20 .

\V! ! J uE , D .: TH IS JO URNAL , 34 : 1 , 1928 .