histamine release and intracellular potentials during...

Histamine Release and Intracellular Potentials DuringAnaphylaxis in the Isolated Heart

By GEORGE A. FEIGEN, P H D . , E. M. VAUGHAN WILLIAMS, D.M., B.Sc,

JANICE K. PETERSON, A.B., AND C. B. NIELSEN, M.S.

ALTHOUGH cardiovascular collapse hasbeen recognized as a concomitant of

gross anaphylaxis in many species of animal,it lias been the general opinion that the circu-latory irregularities associated with fatalanaphylactic shock are secondary manifesta-tions, usually resulting from the asphyxiacaused by bronchospasm. While this view, firstproposed by Auer and Lewis in 1910,1 is per-fectly plausible in the case of the guinea pig,it cannot be held with equal strength in thecase of the rabbit2 and the dog,8'4 neither ofwhich dies of bronchoconstriction.

The first direct proof of the involvement ofthe heart itself as a target organ in anaphy-laxis is due to Cesaris-Demel.5 He showed,with excellently designed experiments, thatisolated hearts of immunized rabbits andguinea pigs would react specifically to suchantigens as beef serum, horse serum, cow'smilk, and egg white, used previously to sensi-tize the intact animal. The work of Cesaris-Demel is to be distinguished from the studiesof his successors in 2 important respects: first,he clearly recognized the nonspecific effectsof the crude antigens he employed and there-fore was very sedulous in challenging his prep-arations with doses below those producingnonspecific reactions; second, he reehallengedthem with the same dose to show that the or-gan had been desensitized.

Because of the ease with which tissue ana-phylaxis could be demonstrated in the guineapig gut0-7 and uterus,8 it is not surprising

From the Department of Physiology, StanfordUniversity School of Medicine, Stanford, Calif.

Supported by a researcli grant H-3693, from theAllergy and Immunology Division, U.S. Public HealthService, and by grants-in-aid from the Sun .FranciscoHeart Association, the Sonoma County Heart Associa-tion, and the Santa Clara County Heart Association.

Beceived for publication January 11, 1960.

that the technically more difficult technic ofcardiac anaphylaxis should have been neg-lected. Most of the studies made between 1911and 1938 are difficult to analyze immunologi-cally because of the use of impure—and some-times bizarre—antigens which, it is realizedtoday, could produce nonspecific reactions."

Studies upon the course of anaphylaxis inthe isolated heart, made in guinea pigs,5'10"'-rabbits;"'-11-15 cats,12'10 and rats,17 show thatthe severe disorganization of the heart beat,succeeding an effective dose of antigen,varies qualitatively with the species testedand quantitatively with the degree of immu-nization of the host. According to the com-parative studies of Wilcox and Andrus,11 thepattern of the cardiac reaction to an effectiveclose of antigen, as well as to histamine, inthe case of the guinea pig and the rabbit, ischaracterized by an increase in the rate andamplitude of contraction, a delay in atrioven-tricular conduction, abnormalities of originand spread of excitation in the ventricles,and an acute reduction of the coronary flow.In the cat, on the other hand, Andrus andWilcox12 found the coronary flow to be in-creased without a significant alteration of theheart rate. The parallelism between theeffects of histamine and anaphylactic shock12

has been shown in many animals includingthe rat;17 in this species both the amplitudeof cardiac contractions and coronary floware abruptly reduced after an effective doseof either of these agents.

While the previous studies had demon-strated that cardiac anaphylaxis could beengendered by antigen-antibody reactionstaking place in the heart itself, they had notestablished that any physiologically activematerial was elaborated during the processthat could give rise to the characteristic

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714 FEIGEN, VAUGHAN WILLIAMS, PETERSON, NIELSEN

changes observed.* Furthermore, in spite ofthe likelihood that the electrocardiographiechanges were consequent upon the reactionof the coronary bed to histamine and toanaphylaxis, the independent participationof the atria was not clearly excluded.

The experiments, forming the substance ofthe present report, show that a physiologicallyactive material, identified pharmacologicallyand chemically as histamine, is liberatedduring cardiac anaphylaxis and that evenpreparations of isolated atria will both liber-ate histamine and exhibit characteristicirregularities.

MethodsAll experiments were made on male guinea pigs,

ranging in weight between 450 and 600 Gm. Theanimals were sensitized by 2 intraperitoneal in-jections of 10 nig. of 4-times crystallized ovalbuiningiven on consecutive days; they were used for theperfusion experiments on the fourteenth to theninetieth day following the second injection. Theelectrophysiologic experiments were performed onguinea pigs which had been injected 14 to 24 daysprior to the experiment.

The balanced salt medium, used for both theelectrophysiologic and the perfusion experiments,was Chenoweth's modification of Tyrode's solu-tion ;18 all salts used were of analytical reagentgrade, made up in water redistilled over alkalinepermanganate. The solutions were gassed with 5per cent carbon dioxide in oxygen, at 37.5 C.

Perfusion ExperimentsGuinea pigs, primed with an intraperitoneal in-

jection of 3 mg. of Na heparin in 0.9 per centNaCI solution, were sacrificed 15 minutes later bya blow to the base of the skull. The thoracic cagewas opened, the ascending aorta dissected free, anda cannula inserted and secured into the vessel. Thevena cava was transected, and the heart fedwanned Chenoweth's solution until the muscle andthe chambers had been cleared of blood. The can-nulated heart was next removed from the carcassand appropriately affixed to the stnndpipe of anAnderson Heart Perfusion apparatus.t A nylonthread was passed through the apex of the heartso that one strand was connected to an ink-writinglever while the other bore a 1.5 Gm. load.

All test solutions were introduced through a

*Tlie immunologic release of histamine-like ma-terials from resting slices of various guinea pig tis-sues, including the heart, has been reported by H.O. Schilcl, J. Pliysiol. 95: 393, 1939.

•(•Metro Industries, Long Island City, N.Y.

short length of rubber tubing forming the unionbetween the side-arm and a constant temperaturereservoir. The solutions from either the principalstandpipe or the side-arm were fed at the sameconditions of temperature and pressure, i.e., 37.5C. and 70 cm. H2O. The ovalbuinin and histaminetest solutions were made up in concentrations per-mitting the use of effective doses in volumes notexceeding 0.25 ml. Since the side-arm was madeof capillary tubing and had a capacity of only 2ml., no turbulence, hence no serious dilution of thetest "slug," occurred as the dose was washed intothe aorta.

The heart was perfused for about 30 minutesfrom the main standpipe while several controlestimates were made of the rate, amplitude, andminute perfusion volume. The organ was then per-fused from the side-arm for 15 minutes and simi-lar measurements were taken. When the 2 sets ofmeasurements were in sufficient agreement, theheart was challenged with 0.1 ml. of a 1 per centsolution of ovalbuinin in Chenoweth's solution.When the kymograph exhibited a definite changein rate or amplitude, a beaker was placed underthe system to collect the effluent, the period of col-lection being determined by the duration of theeffect. The volume of the sample and the durationwere noted, and an aliquot portion was stored inthe deep-freeze for subsequent bioassay. When theeffect of the first challenge had worn off, the flowfrom the side-arm was discontinued and the per-fusion from the main standpipe resumed. Twentyminutes later the side-arm flow was re-establishedand the organ rechallenged with ovalbuinin in themanner described above. Finally, the heart wasthen removed, the atria trimmed away, the cham-bers opened and blotted, and the organ weighedin a closed weighing bottle.

Electrophysiologic MethodsThe apparatus employed to obtain simultaneous

records of contractions and intracellular potentialswas, in essence, a copy of that already describedby Vaughan Williams.10' 20 Guinea pig atria weresuspended horizontally in a 15 nil. bath maintainedat 37.0 ± 0.1 C. Contractions were recorded withan RCA 5734 transducer and were displayed on 1beam of a Tektronix 502 oscilloscope. Intracellularpotentials were displayed on the other beam. Themicroelectrodes had resistances of 10 to 60 MQand tip potentials less than 10 mV. The cathodefollower was an electrometer (ME 1400), carriedon the micromanipulator. The latter permittedmovements of 0.5/* to be made smoothly. The atrinwere driven electrically through Ag-AgCl elec-trodes by a stimulator which also triggered thesweep. The x-plate voltage of the oscilloscope wasfed into an electronic circuit which, at the end ofeach sweep, enabled a pulse to be delivered to a

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ANAPHYLAXIS IN ISOLATED HEART 715

Grass oscilloscope camera, modified to shift oneframe in response to each pulse. Photographs werethus obtained on stationary film during consecutiveheart beats, and the film was shifted automaticallyone frame between the beats.Characterization

Quantitative and qualitative analyses were madeby bioassay and paper chrornatography, respec-tively.Bioassay

The perfusates were assayed for potency, incomparison with known concentrations of hista-inine, upon ileal strips obtained from normal maleguinea pigs. The solutions21 and methods22 usedhave been described in other reports from thislaboratory. Further details will be found under"Results."

Paper ChromatographyPerfusates were dried from the frozen state, re-

dissolved in water amounting to one-tenth of theoriginal volume, and extracted with M-butanol ina liquid extractor. The butanol was removed byvacuum distillation, the residue was taken up inwater and spot-tested on filter paper with sulfanilicacid reagent. The samples were prepared by beingeitlier first concentrated in a lyophil apparatus ordirectly extracted from Chenoweth's medium withbutanol according to the method of Mclntire, Roth,and Shaw.23

ResultsCharacterization and Bioassay of the Active Prin-ciple Released during Cardiac Anaphylaxis

Presumptive evidence for the belief thathistamine was liberated during the anaphy-laetic reaction was furnished by the resultsof preliminary pharmacologic and chemicalexperiments on the perfusates. Pharmacologicstudies showed that the perfusates obtainedduring the height of the anaphylactic re-sponse to ovalbumin (fig. 1) : (a) reproducedthe characteristic effects when tested uponhearts of normal guinea pigs and (b) causedcontraction of the guinea pig ileum but notof the rat gut (fig. 2 top) ; (c) their stimulat-ing action on the guinea pig gut was blockedby pyribenzamine (fig. 2 bottom) but not byatropine, and (d) the destruction of theactive material by tissue enzymes was pre-vented by the presence of semicarbazide inthe incubation mixture. An analysis of thisevidence shows that the cardiac effects couldhave been produced either by histamine or

EFFECTS OF OVALBUMIN AND HISTAMINE ON SENSITIZED HEART

Figure 1Effects of ovalbumin and histamine on the heartof a guinea pig, actively sensitized with ovalbumin.The figure shoivs first a normal pattern, then a re-action of the heart to 0.10 mg. of ovalbumin. Re-challenging the heart with ovalbumin is withouteffect, showing that the heart is desensitized. Theimmunologic response is to be compared xvith thenonspecific reaction to 0.1 ml. of 1O'S M histamine.Arrows mark time in seconds after a given event.The 30-second time-base is given under "normal."

serotonin, but not by acetylcholine. Sero-tonin and acetylcholine are excluded in (b)since both agents affect the rat gut. Thepresumption of histamine is strengthened in(c) by the fact that the response of theguinea pig ileum to the perfusate and tohistamine, as shown in figure 2 bottom, isblocked by pretreating the muscle for 60seconds with 0.5 jug./ml. of pyribenzamine.The presumption of histamine is finallystrengthened by the fact that the destructionof the active principle, as well as of histamine,by the monoamine oxidase system of the gutis inhibited by semicarbazide.Identification of Histamine in Perfusates

Although the active material could not beidentified by isolation and crystallization,sufficient evidence was obtained by paperchromatography to suggest that the activematerial was, in fact, histamine. The datadisplayed in table 1 make it quite evidentthat the amount of histamine released by asingle heart at the height of its reaction toantigen would be below the limits of identifi-

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716 PEIGBN, VAUGHAN WILLIAMS, PETERSON, NIELSEN

Guineo Pig Gut Rat Gut

Iaii 10'*0.1 > »•*O.I i IO"*H.

O.Sal O.T a l <L«i|O'*M. O.UHO"*a. O.SilO"9M.R I * I M. H. N.

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Rl- Challanga with Ovalbumin

R2- RKhall ingt with Ovalbumin

J J

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O.I • 100.2i IO"*

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0.5 ml.

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2.0 ml.

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il. 0.15110* U.

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0.2H0* M.

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0.5*

for

PBZ

6 0 *

4.0 ml.

Rl

4.0 ml.

Rl

H O.li 10

0.2l 10"*

O.UIO*4M.

6 .0 B

R 2

Figure 2Pharmacologic behavior of perfusates collected during cardiac ana phyla xis. Top. Effectsof histamine and "active" perfusate (Rl), on guinea pig ileum in contrast to their effectson rat gut. Neither histamine nor perfusate has a significant effect on rat muscle com-pared to acetylcholine (Ach). Bottom. Assay of perfusates obtained during initial chal-lenge (Rl) and rechallenge (R2) of sensitized heart exhibited in figure 1. No activityis exhibited by R2 in conformtmce with desensitization. Both Rl and histamine areblocked by pyribenzamine.

cation. In order to obtain active material atan adequate concentration, the perfusatesobtained from 10 experiments were pooled,deproteinized with ZnSO4) and the supernatetreated with KaPO4-Na2SO4 reagent.24 Theaqueous solution was shaken with an equalvolume of «-butauol, the butanol extractdried with anhydrous Na2S04, and evapo-rated under reduced pressure over a boilingwater bath. The residue was taken up in 1ml. of water and applied to filter paper and

dried. Several applications were necessaryto build up a sufficient concentration toproduce a definite pink color with Pauly'ssulfanilic acid reagent. The results of paperelectrophoresis, performed with the aid of aKarler-Misco apparatus, showed a definitepink streak, after development with sulfa-nilic acid, in the same region as that foundfor known solutions of histamine made upin Chenoweth's medium. Chromatographicanalysis of active perfusates, enriched with

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Table 1Effects of Ovalbumin and Histamine on the Sensitized Guinea Pig Heart

Control S.E. Challenge S.E.Per centchange

Reactions to ovalbumin*Heart rate (beats/min.)Amplitude (mm.)Coronary flow (ml./Gm./min.)

Heart rate (beats/min.)Amplitude (mm.)Coronary flow (ml./Gm./min.)

206.826.46.48

±13.19± 2.10± 0.72

Reactions to histaminot184.515.62.50

±13.69± 2.58± 0.41

256.656.33.36

215.834.31.92

±16.10± 5.03± 0.48

±18.43± 4.44± 0.42

+ 24.1+113.3- 48.1

+ 17.0+120.0- 23.2

'Reaction to 1 mg. of ovalbumin.•(•Reaction to 0.25 ml. of 10'3M histamine.

solutions of crystalline histamine diphosphate,also showed a single streak when tested withthe sulfanilic acid reagent.Bioassay

Owing to the limited quantities of physio-logically active material yielded during theanaphylactie reaction, it was necessary toselect those sections of gninea pig ileum mostsensitive to histamine for use as test objectsfor the bioassay method. Furthermore, itwas necessary to establish the form of thefunction that existed between the concentra-tion of histamine and the response of thegut, in order to evaluate differences inregional sensitivity and also to provide abasis for extrapolation of outlying values,if that became necessary.

The preliminary experiments were madeby removing the entire small intestine andtesting 5-em. portions for response to variousdoses of histamine ranging in final concentra-tion from 2.5 X 10'7 to 1 X 10"5M. It wasfound that the sigrnoidal dose-response curveto histamine could be described successfullyby the logistic relationship of von Krogh25

"'(£)"*in which x is the concentration of histaminerelated to a given degree of response, k is aconstant, y is the proportion of the totalcontraction in the presence of x molar hista-mine, and 1/n the slope of the line relatinglog x to log y/l-y. Although no fundamental


significance need be imputed to the relation-ship, i.e., adsorption, it is noteworthy that thesame relationship may be derived on proba-bilistic grounds, as has been done by Winderet al.,28 using the probit transformation ofBliss.27

Dose-response values obtained for the in-dividual strips were fitted to the von Kroghequation. A preliminary inspection of the in-dividual lines showed that they would fall into3 natural classes according to the magnitudeof their k values. The y/l-y quotients obtain-ed at each histamine concentration were thenaveraged for all strips comprising a particu-lar class, the standard errors calculated, andlines fitted visually to the array obtained forthat category. The results displayed infigure 3 show that the guinea pig intestineemployed in Experiment 78 could be dividedinto at least 3 major portions, of about 50cm. each, according to their sensitivity tohistamine. A comparison among the groupson the basis of k values shows that the gra-dient of sensitivity of the guinea pig ileumto histamine progressively increases in theaboral direction, the median effective con-centrations of histamine being 6.4 X 10~7,1.46 X KH, and 2.7 X 10-°M for groups A(0.50 cm. from the eecum), B (50-100 cm.),and C (100-160 cm.), respectively. The varia-tion in sensitivity among 3 guinea pigs,assayed in the same manner as that of Experi-ment 78, is given in table 2.



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718 FEIGEN, VAUGHAN WILLIAMS, PETERSON, NIELSEN

10''

Ragionol Sensitivity of Guinio Pig lltum To

A -Itripi I-to 10-SOcm}B- i l r ip i M-20 (50-l00em.lC - strips 20-32 1100-ISOcm)y • • of motimal contraction

Table 2Regional Sensitivity of Guinea Pig Ilcum toHistamine

r/i-r

Figure 3Regional sensitivity of guinea pig ileum to hista-mine. Relationship "between dose and degree of re-sponse as fitted by the logistic transformation ofvon Krogh (see text). Horizontal bars are thestandard errors.

Reaction of the Sensitized Heart to Perfusion withAntigen

The response of the sensitized guinea pigheart to an effective dose of antigen (oval-bumin) is characterized by 3 distinct events:an acceleration in heart rate, an increase inthe amplitude of contraction, and a decreasein the coronary flow. The violence of thereaction depends upon the antibody contentof the tissue and upon the challenging doseof antigen employed in the experiment. Dur-ing the more intense reactions, a characteris-tic A-V block is observed which may wax andwane for several minutes following a singledose of antigen, as exhibited in figure 1.Usually, a second challenge with antigen isineffective, indicating desensitization; occa-sionally, with smaller initial challengingdoses, an effect of A'ariable intensity may beobserved when the organ is rechallenged.

The results of experiments performed onthe hearts of 18 sensitized guinea pigs, givenin table 1, show that the percentile changesin rate, amplitude, and coronary flow,* ex-hibited during anaphylaxis, are quantitativelyvery similar to those shown after an effectivedose of histamine, although it is quite ap-

*The term ' ' coronary flow'' as used here is merelythe Volume of perfusato per unit time. This is com-pounded of true coronary flow and "leakage flow,"as described by Wilcox and Andrus.11

Expt.

77

78

79

Group*

ABC

ABC

ABC

k

3.0 X 10"'M4.5 X 10"'8.6 X 10"'

6.4 X 10"'1.5 X 10"G

2.7 X 10"6

5.2 X 1(T7

6.6 X 10"'8.8 X 10-'

1/n

0.600.360.16

0.410.550.55

0.550.250.24

*JDistance from cecal end: A=0 to 5 cm.; B=50 to10 cm.; C=100 to 160 cm.

parent that the absolute performance of theheart is never entirely restored to that of theinitial control period after it has reacted tothe antigen.

Bioassays conducted on the perfusates ob-tained during the height of response showeda net output of physiologically active materialequivalent to 1.1 X 10"8 ± 2.1 X 10"° moles ofhistamine per gram of heart per minute. Onthe basis that the recovery of a known amountof histamine is onljr a small portion of thatinstilled into the preparation, it can safelybe assumed that the total amount of histaminereleased per unit time during anaphylaxismust be several times greater than the netvalue found in these experiments.Electrophysiologic Changes in Sensitized AtriaTreated with Antigen or Histamine

Wilcox and Andrus11 considered that theelectrocardiographic changes recorded direct-ly from the isolated organ undergoing ana-phylaxis were caused by the effects of coro-nary constriction on the electrical activity ofthe heart, and that the cardiac irregularitiesobserved might have resulted from myocardialanoxia. Because in certain instances cardiacirregularities could be observed in the absenceof a reduced coronary flow, it was suspectedthat these phenomena might equally welloriginate from the atrium. The purpose ofthese experiments was to determine whethersensitized atria could independently react to

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ANAPHYLAXIS IN ISOLATED HEART 7.1.9

ant igen and, if they could, to establish

whether the acceleration in rate and the

ar rhy thmias could be accounted for by altera-

tions in the electrical activity of individual

cells of a kind that could have been produced

by locally released histamine.

Electrical Records

Simultaneous measurements of contractionsand intracellular potentials were made in thepresence of a wide range of concentrations ofhistamine with both rabbit atria at 31 C. andguinea pig atria at 37 C. Records were ob-tained from 7 pairs of atria removed fromguinea pigs sensitized with ovalbumin, 14 to24 days previously. When control recordshad been obtained, the atria were challengedby addition of ovalbumin directly to theorgan bath. A reaction was observed in everyexperiment, though in 1 preparation thereaction was small; this pair of atria sub-sequently proved to be extremely insensitiveto histamine. In all other experiments, thereaction was sufficiently strong for the atria to"break away" from the stimulus and beatspontaneously, in spite of the fact that thestimulation rate chosen was at least 50 percent higher than the frequency of the naturalpacemaker. In 2 experiments the reactionwas violent enough to precipitate fibrillation.In concentrations of histamine up to 10"°M,the atria continued to follow the stimulus.There were, however, marked changes in theshape of the intracellular action potential:(a) the resting potential was reduced, (b)the overshoot was reduced, (c) the rate ofrise of the action potential was greatly re-duced, and (d) the tail of the repolarizationphase was prolonged. There was always,also, an increase in the amplitude of thecontraction which may be associated with theprolonged repolarization time.28 At higherconcentrations of histamine, the atria beganto beat spontaneously, and at still higherconcentrations they fibrillated.

The effects of the antigen-antibody reactionwere precisely similar, and in each experimentan attempt was made to match them with anappropriate concentration of histamine. In


Figure 4Effects of specific, antigen upon intracellular po-tentials of atrial fibers. A and B. Control recordsat slow and fast siveep speeds. C and D. Recordstaken shortly after challenge with ovalbumin. K.The 2 horizontal traces are 100 mV apart and girothe Y-axis calibration for all records in figures Iand 5. The X-axis calibrations apply to the stoicand fast sweep speeds in figures 4A., 4.B, 4C, and:in figures 5E and 5F. F. Depolarization phase ofaction potential after exposure to histamine. Sweepspeed as in D.

some experiments the atria were challengedwith ovalbumin before being exposed tohistamine; in others the histamine was givenfirst, but the order made no difference in theresults. In each experiment, when the anti-gen-antibody reaction was complete and theatria had been washed for half an hour withthe control solution, the atria were rechalleng-ed with more ovalbumin. The second chal-lenge had no effect. At the height of thereaction to the challenging dose of ovalbumin(fig. 4C) an aliquot sample of 2 ml. was with-drawn from the bath for bioassay accordingto the method described in a prior section ofthis report. The titration of the materialreleased by the atrium is displayed in figure5.

Examples of records taken from 1 experi-ment are presented in figures 4 and 6. Infigure iA the lower trace represents the con-traction of the atria in control solution; thetraces above are successive, superimposedrecords of the intracellular action potentialat slow and fast sweep speeds. The appro-



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720 FBIGBN, VAUGHAN WILLIAMS, PETERSON, NIELSEN

r

0.8 ml H 0.09 ml H 0.08 ml O.< ml H 0.09 ml H 0. OS m I

I.O*ICr°H. * I C.SilO~*N. S.O«IO"8M.

3.0»l0"*M.

Tigure 5Bioassay of material released by the shocked atria.Al is a sample of the aliquot of material with-drawn from the bath folloiving challenge withovalbumin.

priate calibrations are given in figure iE.Figure 4B depicts the depolarization phaseonly of the action potential at a fast sweepspeed, in order that the rate of rise of theaction potential can be accurately measured.After the control records had been taken, theatria were challenged with ovalbumin, and thephotographs shown in figures 4C and 42) weretaken. In 4C, the contraction is depicted onthe upper trace. In 4D, the atria had brokenaway from the stimulus and were beatingspontaneously, making it difficult to "catch"a rising phase of the action potential. Thus,the record 4D does not contain the whole ofthe depolarization phase, but it is obviousthat it is much slower, especially since thesweep speed is about twice as slow as in 4B.Figure 4F shows the rising phase of an actionpotential, at the same sweep as in 42), photo-graphed during exposure of the atria to hista-mine later on in the experiment.

In this experiment, about 90 seconds afterchallenging them with a dose of 20 mg. ofovalbumin, the atria began to fibrillate.Regular trains of action potentials wererecorded as in figure GA, each associated withan a trial contraction. These rapid contrac-tions sometimes caused the recording leverof the transducer to vibrate at its naturalfrequency, introducing an artifact into thecontraction record. Occasionally, short burstsof 3 or 4 action potentials would follow oneanother in very rapid succession, causing thecontractions to diminish28 as shown in figure6B. The records 6C and 67) were taken during

the subsequent exposure of the same pair ofatria to histamine. The similarity betweenthe records is sufficiently obvious to requireno comment.

It has been suggested (see "Discussion")that the effects of an antigen-antibody re-action on the intracellular potentials ofsmooth muscle could have been due to therelease of acetylcholine. .In the atria, at anyrate, as shown in figures 62? and 6.F, the effectsof acetylcholine are in complete contrast tothose of ovalbumin and histamine, since, afteracetylcholine, (a) the contractions diminish,(b) the resting potential is increased, (c) therate of rise is not reduced but is often in-creased, and (d) the repolarization phase isnot prolonged but shortened. Although therate of rise in figure 6F is a little slower thanthat of the control at the beginning of theexperiment (fig. 42?), it was no slower thanthe control taken after recovery from thehistamine.

DiscussionThe dose-response curve of a pharmacologic

system generally has been considered to bethe outward expression of some fundamentalphysical process by which a population ofcells or a tissue adsorbs a given drug andreacts to it. Although it is evident, a priori,that the intensity of the reaction must depend,in some way, on the amount of active materialbound, information about the character of thephysical adsorption in usually lacking, andthe precise relationship between the bindingisotherm and the reaction curve is unknown.Recent experiments in this laboratory20 showthat the reaction curve to antigen and hista-mine differs quantitatively, at least, from theexperimentally determined physical adsorp-tion isotherm describing the binding of anti-body to tissue. This discrepancy arises, inpart, because many cells may be capable ofadsorbing more of the agent than the amountnecessary to produce the maximal effect (cf.Bliss), and also because there is a distributionof sensitiveness among cells to a given doseof the stimulating agent; furthermore, anti-bodies may be bound to tissues which arephysiologically "inert."




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Most developments of the dose-responseproblem by toxicologie statisticians have quiteproperly regarded responses as being quanta]because they have dealt with a clear end-point, death. The fact that the dose-responsecurve of muscle and the dose-mortality curvesof unicellular organisms or insects can belinearized by plotting in each case the log-arithm of the probit against the logarithm ofthe dose does not entitle us to consider thereaction of smooth muscle cells to be quanta],as has been done by Chen et al.30

Since the probit and the logistic transfor-mations fit the present data equally well, wehave selected the latter transformation toexpress our results because it does not makeany assumption about the mechanism of drug-tissue interaction.

Katsh and Marshall111 showed that theSchultz-Dale reaction of the sensitized guineapig's uterus was accompanied by an increasein the frequency of the spike potentials, andby increments in the duration and amplitudeof the contractions. Because similar changeswere noted when these uteri were treatedwith acetylcholiue, the authors suggested thatacetylcholine might be liberated during ana-phylaxis. In the absence of any supportingchemical or pharmacologic experiments, themere similarity between the electrophysiologiereactions in the 2 situations hardly constitutespresumptive evidence for this view, partic-ularly since other agents, such as histamine,were not ruled out.

Owing to the great difference, shown in thepresent study, between the effects of acetyl-choline and those of anaphylaxis upon thecharacter of the atrial action potentials, it ispossible to rule out acetylcholine as a causalagent entirely on electrophysiologie grounds.The effects observed in the present study areiu complete contrast with those of aeetyl-choline, which increases the resting potentialand the overshoot20 and usually also increasesthe rate of rise of the action potential, at thesame time diminishing the contractions.

Strong anaphylactic reactions and largedoses of histamine precipitated fibrillation,


Figure 6Effects of specific antigen, Mstamine, and acetyl-choline on the intracellular potentials of atrialfibers. A and B. Fibrillation precipitated by oval-bumin. C and D. Fibrillation precipitated by his-tamine. The X-axis calibration in B applies to all4 records. E and F. Effects of acetylcholine onthe same pair of atria. Calibrations as in figure 4E.

confirming the previous observations of Rij-lant15 and the more recent studies of Pennaet al..,32 both of whom noted a similar effectin the case of shocked rabbit atria. Burstsof action potentials would occur in which themembrane appeared to be depolarized againas soon as it had repolarized to a sufficientextent to be re-excitable. All these effects ofhistamine and of the anaphylactic reactionwould be consistent with the hypothesis thatthe membrane had become excessively perme-able to sodium. The diminution of restingpotential, overshoot, and rate of rise could allbe consequences of an accumulation of intra-cellular sodium.

The demonstration that histamine is re-leased during atrial anaphylaxis by no meansdefines this tissue as its single source, nordoes it imply that the cardiac irregularitiesare, iu all cases, of atrial origin, but it doessuggest that the sinus tachycardia, observedby Wilcox and Andrus,11 could have beenstrictly local in origin and not a sequel ofcoronary constriction in the ventricles.

SummaryThe response of the sensitized guinea pig

heart to perfusion, with an effective dose of



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722 FEIGBN, VAUGHAN WILLIAMS, PETERSON, NIELSEN

ovalbumin, was found to consist of an ac-celeration of the rate, an increase in theamplitude of contraction, and a decrease incoronary flow, confirming the earlier observa-tions of Wilcox and Andrus. The characteris-tic mechanical reaction of the isolated atriawas an increase in amplitude and frequencyof contraction, the more intense effects result-ing in fibrillation. Electrophysiologically,atrial activity was demonstrable by bursts ofaction potentials, recurring as soon as themembrane had repolarized sufficiently to bere-excited. The resting potential was reduced,and the overshoot and the rate of rise of theaction potential were diminished.

All of the mechanical and electrical eventsnoted in the Langendorff heart and the a trialpreparations during anaphylaxis could be re-produced precisely by an appropriate dose ofliistamine. Evidence for the release of hista-mine by both the perfused heart and theisolated atria was first obtained by pharma-cologic methods and then confirmed by paperchromatography of butanolic extracts.* Hista-lnine was quantitatively estimated by a bioas-say method which is discussed in detail inthis report.

Acknowledgment'We are indebted to Mrs. D. W. Peterson and Mr.

Harold Moore for their assistance during certainphases of tliese investigations.

Summario in InterlinguaIn confirmation del observationes de Wilcox o

Andrus, il esseva constatate que le responsa del sen-sibilisato corde del porco de India al perfusion conuii dose effieaco de ovalbumina. consiste in un aug-niento del frequentia e del amplitude del contractione in un reduction del fluxo coronari. Le characteristicreaction mechanic del isolate atrios esseva un aug-nionto del amplitude e del frequentia del contractiones.Le plus intense effectos resultava, in fibrillation. Elec-trophysiologicamente, activitate atrial esseva demon-strable per auginentos explosive in le potentiates deaction que rocurreva si tosto quo le membranas essevasufficienteniente repolarisate pro que lor re-excitation

"Dr. W. E. Brocklehurst has found in 3 experi-ments that the sensitized guinea pig heart liberatesSBS-A as well as liistamine. (Brocklehurst, W. E.:Occurrence of SBS-A. ,T. Physiol. 150: .I960, inpress.)

esseva possibile. Le potential de reposo esseva re-ducite, e le resalto e le rapiditate del augmento delpotential de action esseva reducite.

Onine le evenimentos mechanic e electric notate inlo corde de Langendorff e in le preparato atrialdurante un anaphylaxe poteva esser reproducite pre-cisemente per un dose appropriate de histamina. In-dicios pro le liberation de histamina per lo cordeperfusionate si ben que per le atrios isolate essevaprimo obtenite per methodos pharniacologic o con-firmate subsequentemente per cliromatographia a pa-piro de extractos butanolic. Le histamina esseva es-timate quantitativemente per un niethodo de bio-essayage que es discutite in detalio in le presentereporto.

References1. AUER, J., AND LEWIS, P. A.: Physiology of the

immediate reaction of anaphylaxis in theguinea pig. J. Exper. Med. 12: 151, 1910.

2. —: Lethal cardiac anaphylaxis in the rabbit.J. Exper. Med. 14: 476, 1911.

8. MANWARING, W.: Anaphylaxis in the dog. Bull.Johns Hopkins Hosp. 21: 275, 1910.

4. VOEGTLIN, C, AND BERNHEIM, B. M.: Liver inits relation to anaphylactic shock. J. Pharma-col. & Exper. Thorap. 2: 507, 1911.

5. CESARIS-DEMEL, A.: Recherches sur 1'anaphylaxie.Sur le mode do se comporter du coeur isoled'aniinaux sensibilises. Arch. ital. biol. 54:141, 1910-1911.

0. SCHUI.TZ, W. H.: Physiological studies in anaphy-laxis. Reaction of smooth muscle of the guineapig sensitized with horse serum. J. Pharmacol.& Exper. Therap. 1: 549, 1910.

7. —: Physiological studies in anaphylaxis. I I .Reaction of smooth muscle from guinea pigsrendered tolerant to large doses of serum. J.Pharmacol. & Exper. Therap. 2: 221, 1910.

8. DALE, H. H.: Anaphylaetie reaction of plainmuscle in the guinea pig. J. Pharmacol. &Exper. Therap. 4: 167, 1913.

9. LAUNOY, L.: Contribution a 1'etude do 1'actiondu serum du boeuf et du serum de cheval surle coeur isole du cobaye: I. Action du serumdu cheval et du serum du boeuf sur le coeurisole d'aniinaux normaux. Ann. Inst. Pasteur25: 561, 1911.

10. MENDELEEFF, P., HANNEVART, G., AND PLATOIT-NOKF, M. C.: Anaphylaxie humorale et. cellu-laire. Arch, intermit, physiol. 24: 145, 1924-1925.

.11. WILCOX, H. B., AND ANDRUS, E. C : Anaphylaxisin the isolated heart. J. Exper. Med. 67: ]69,1938.

12. ANDRUS, E. C, AND WILCOX, H. B.: Effects ofanaphylaxis upon the coronary arteries in theisolated heart. J. Exper. Med. 69: 545, 1939.

Circulation Research, Volume VIII, July i960



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13. LEYTON, A. S., LEYTON, H. G., AND SOWTON, S.

C. M.: Anaphylactic effects shown in perfusionexperiments on the excised heart. J. Physiol. 23.50: 265, 1916.

14. DEMOOR, J.: Action de l'cxtrait thyroidien dochien sur le coeur isole du lapin normal on du 24.lapin sensibilise. Arch, intcrnat. physiol. 18:369, 1921.

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diaque spontanees ot reactionelles au cours do 25.1'anaphylaxie locallc. Automatismes Secon-dairos. Arch, intcrnat. physiol. 23: 61, 1924. 20.

16. Sciiur.TZ, W. H.: Physiological studies in ana-phylaxis. IV. Eeaction of the cat toward horseserum. J. Pharmacol. & Bxpor. Therap. 3: 299,1912.

17. MCEI.ROV, W. T.: General studies on anaphylnxis 27.and rat heart antiserum. Stanford University,Ph.D. dissertation, 1956. 28.

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21. FEIGEN, G. A., AND CAMPBELL, D. H.: Eel.itive 31.sensitivity of the niucosal and peritoneal sur-faces of guiiie.1 pig ileum to histamine, acetyl-eholine, and specific antigens. Am. J. Physiol.145: 676, 19'46. 32.

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VAUGHAN WILLIAMS, E. M.: Study of intraeellu-lar potentials and contractions in atria, includ-ing evidence for an after-potential. J. Physiol.149: 78, 1959.

NIELSEN, C. B., TERRES, G., AND FEIGEN, G. A.:Adsorption of antibody in vitro and magnitudeof the Sehultz-Dale reaction of guinea pigileum. Science 130: 41, 1959.

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KATSH, S., AND MARSHALL, J.: Electrical andmechanical responses of uterine smooth muscleduring anaphylaxis in vitro. Am. J. Physiol.196: 39, 1959.

PENNA, M., ILLANES, A., UBILLA, M., AND MU-.TIOA, S.: Effect of histamine and of the ana-phylactic reaction on isolated guinea pig atria.Circulation Besoarch 7: 521. 1959.




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NIELSENGEORGE A. FEIGEN, E. M. VAUGHAN WILLIAMS, JANICE K. PETERSON and C. B.

Histamine Release and Intracellular Potentials During Anaphylaxis in the Isolated Heart

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1960 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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