Adrenergic mechanisms in the bullfrog and turtle’

TAKEHIKO AZUMA,2 ALBERT0 BINIA,3 AND MAURICE B. VISSCHER Dabartment of Phvsiolo~v. Uniuersitv of Minnesota. Minneubolis. Minnesota

AZUMA, TAKEHIKO, ALBERTO BINIA, AND MAURICE B. VISSCHER. Adrenergic mechanisms in the bullfrog and turtle. Am. J. Physiol. 2og(6): 1287-1294. rg65.-Epinephrine and nor- epinephrine contents of tissues and perfusates have been meas- ured by fluorimetric methods to ascertain which catecholamine is the sympathetic transmitter in bullfrogs and turtles. Except for adrenal and sympathetic chain, the predominant catechol- amine in bullfrogs is epinephrine. In snapping turtles, norepi- nephrine predominates+ During perfusion of bullfrog heart or liver without stimulation, only traces of catecholamine appear in perfusates, whereas during sympathetic nerve stimulation a large output of epinephrine occurs. In the bullfrog epinephrine rather than norepinephrine seems to be the sympathetic mediator. The situation may be the reverse in the turtle. Environmental temperature did not alter bullfrog tissue cate- cholamine. Cardiac sympathetic denervation did not decrease myocardial catecholamine within 6 weeks at low temperatures, but in animals maintained at 20 C survival was not achieved. Epinephrine levels in bullfrog ventricle were not lowered by 5 hr of contractions induced by electrical stimulation at 3o/min compared with controls in arrest. The fact that myocardial catecholamine stores are not depleted by contractile activity may result either from absence of utilization or from equiva- lence between breakdown and synthesis.

heart catecholamine; bullfrog organ catecholamine content; liver catecholamine content; brain catecholamine content; adrenal catecholamine content; heart work and catecholamine depletion

1~ IS GENERALLY ACCEPTED that in warm-blooded ani- mals, norepinephrine is the predominant catecholamine in most organs and tissues which have sympathetic innervation and that it is the chemical transmitter released by the postganglionic endings of the sympa- thetic system. In amphibia, however, only a few investi- gations have been carried out. In 1946 von Euler (14) found only epinephrine in extracts of the frog heart. A

Received for publication 26 April 1965. 1 This work was supported by Public Health Service Grants

HE-032 I 2 and FF-523. 2 Public Health Service International Postdoctoral Research

Fellow. Present address: Dept. of Physiology, University of Tokyo, Tokyo, Japan.

3 Rockefeller Foundation Postdoctoral Fellow. Present address : Dept. of Physiology, Cuyo University, Mendoza, Argentina.

few years later Gstlund (I I) reported that both epi- nephrine and norepinephrine exist in the frog heart, but that the predominant one is epinephrine. These latter observations have been confirmed recently by ‘Falck et al. (4) and Govyrin and Leontieva (5). Aside from the initial studies by Loewi (8) using a primitive fluorescence method, no reports on the character of the catechol- amine content of perfusates have appeared.

The question arises: which is the adrenergic trans- mitter in the frog heart, norepinephrine or epinephrine? Von Euler suggested that it is norepinephrine and he believed that epinephrine found in the heart was probably largely generated by chromafhne cells. However, Falck et al. demonstrated, in the frog heart, by means of a specific fluorescent staining method for the cellular detection of certain monoamines, that epinephrine is located in the intracardiac nerves and that there is no evidence for the existence of chromaffine tissue in the heart. They suggested that epinephrine may be the adrenergic transmitter in this animal.

The purpose of the present study is to gather further evidence relating to these two contradictory opinions, as well as to investigate the effect of changes in the physio- logical environment on the catecholamine content of the frog heart.

METHODS

Bullfrogs (Ranu cutesbeiuna), weighing from I oo to 300 g, were used mainly as experimental animals. They were anesthetized by intracisternal injection of 0.2-0.3 ml of 20% urethane. The heart and other organs were quickly removed and blotted on gauze to remove blood. Samples ranging from 0.5 to 3.0 g in weight, measured to milligram values with a torsion balance, were minced and kept in I o % trichloroacetic acid in deep freeze until homogenization. In the case of the hearts the whole organ was employed, except when atria and ventricle were analyzed separately, and ordinarily two or more hearts were pooled for analysis. In some experiments the snapping turtle (Chelydra serpentina) was employed.

Perfusion and sympathetic stimulution of the heart. After anesthetization both vagosympathetic trunks were dissected and cut. The heart was exposed in situ, the right arterial trunk, left carotid and pulmocutaneous arteries, and both superior caval veins were ligated,

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1288 AZUMA, BINIA, AND VISSCHER

TABLE I. Analyses of pookd samph of who&? heart (R. catesbeiana)

After reaching pH 8.4 the stirring was continued for 5-6 min, with occasional adjustment of the pH, and

Sa$fle . No. of Hearts

Epinephrine, pg/g Wet Tissue

Norepinephrine, pg/g Wet Tissue

then the alumina was allowed to settle for 3-5 min. After decanting the supernatant fluid, the aluminum

I

2

2 I -65 2 I .84

oxide was transferred quantitatively with distilled water to a glass column and washed with distilled water until

3 2 I l Zl 0 4 2 0.98 0

5 2 1 l 49 0

6 2 I .g8 0.05

the of the effluent was below ph 7. pressure Positive was occasionally applied to the top of the column to accel- erate the flow. For elution 2 X 2.5 ml of 0.25 N acetic acid were used, and the eluate was collected in a conical

6 2 I .02 0.04 8 2 2.22 0 9 3 I .65 0

IO 3 I l 20 0

AvET I .52 <o.or

centrifuge tube. The supernatant fluid previously de- canted, which usually contained light particles of alumi- num oxide in suspension, was centrifuged and the alumina at the bottom of the tube, after being washed with distilled water and centrifuged to remove the water, was submitted to elution with I ml of 0.25 N

and the left aorta and inferior caval vein were cannu- acetic acid. After being stirred with a glass rod for I-Z

lated. The heart was perfused from the venous side at a min, the mixture was centrifuged and the supernatant constant pressure with Ringer solution (NaCl I I I, fluid (I ml) was added to the eluate (5 ml) obtained KC1 2.82, CaClg 1.70, NaH2P04 1.25, NazHP04 3.75 from the column. The eluates were stored in the re- mM/liter) adjusted to pH 7. One milliliter of o. I % frigerator until the assay. The aluminum oxide was atropine sulfate was applied on the surface of the heart prepared according to Crout (3). Double-distilled water at least 30 min prior to the nerve stimulation. Bilateral and reagents of the highest purity were used throughout peripheral vagosympathetic stimulation was performed the procedure as well as in the fluorometric assay. with electric pulses of 30 cycles/set, 2-msec duration, Fluorometric assay. The trihydroxyindole method as and supraliminal strength. Five to six periods of stimula- described by von Euler and Lishajko (20) was used as tion with a duration of 10-15 min each were performed follows : in every experiment followed by “resting” intervals I) To o. 1-0.6 ml of eluate, previously centrifuged, during which the heart rate returned to the level before were added 2 ml of I M acetate buffer, pH 6.5, and the stimulation. The perfusate flowing from the right aorta contents mixed in a IO-ml graduated cylinder. before and during stimulation was collected in two 2) One milliliter of a 0.025 % solution of potassium separate graduated cylinders containing IO ml of 50 % ferricyanide was added, mixed thoroughly, and allowed trichloroacetic acid until a total volume of IOO ml in to stand for exactly 3 min. each of them was completed. The preparation of the 3) One milliliter of a mixture of 5 N NaOH plus eluates from these samples was made immediately after ascorbic acid 2 % (g and I ml, respectively), prepared completion of the experiment. immediately before use, was added and the contents

Preparation of samples. The perfusates collected in tri- thoroughly mixed. chloroacetic acid as described above were mixed, 4) The volume was made to 5 ml with distilled water, centrifuged at 3,400 rpm for IO min to remove small amounts of protein, and immediately processed. The tissue sample was thawed at room temperature and homogenized in a glass tissue grinder submerged in an ice-water bath. The homogenate was centrifuged at 3,400 rpm for 10 min, the supernatant fluid was sepa- rated, and the precipitate was washed with a few milli- liters of IO % trichloroacetic acid and centrifuged. The combined supernatant fluids were immediately proc- essed.

Preparation of eluates. The procedure was essentially the same for tissues and perfusates. To the clear superna- tant solutions were added z ml of I M sodium acetate, 0.2 M disodium ethylenediaminetetraacetic acid (3 ml to the tissue supernatant fluids, 4 ml to perfusate superna- tant fluids), and 0.5 g of aluminum oxide. The mixture, with constant stirring on a magnetic stirrer, was brought to pH 5 by dropwise addition of 2 N NHdOH and then to pH 8.4 by further dropwise addition of 0.5 N NHdOH. This step took in all cases 10-15 min, since faster neu- tralization produced low recoveries.

mixed and allowed to stand for 15-20 min before read- ing the fluorescence.

Standards. Standard solutions of epinephrine and norepinephrine were freshly prepared by diluting with 0.01 N HCl a more concentrated solution stored in the refrigerator and were treated simultaneously in the same way as the eluate. The quantities used in each experiment depended on the expected value of the eluate. At least two standards embracing the expected range were employed.

Blanks. Reagent and sample blanks were run simul- taneously with the eluate and the standard solutions. The reagent blanks were prepared with 0.01 N HCl instead of standard solutions of epinephrine or norepi- nephrine. The sample blanks were prepared by adding all the reagents in the mentioned order and an aliquot of the eluate after the addition of the NaOH-ascorbic acid mixture. Some authors (I, 13, 18) did not add potassium ferricyanide to the sample blank. This seems improper because it gives a lower reading than the

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ADRENERGIC MECHANISMS IN BULLFROG AND TURTLE 128~

reagent blank, as also occurs when the NaOH-ascorbic TABLE 2. Analyses of heart perfusates before and during acid is added before the ferricyanide (20). sympathetic nerue stimulation - (R. catesbeiana)

Instrumentation. The Aminco Bowman spectrophoto- fluorometer w-as used. The series of tubes was read at tw-o different activation wavelengths: first at 400 rnp w-ith fluorescence wavelength at 500 rnp, and then at 435 rnp with fluorescence wavelength at 540 rnp. At both sets of wavelengths the instrument was adjusted so that the galvanometer reading was go scale divisions with the higher of the two standards of epinephrine, which was usually o. I pg. The fluorescence ratio epi- nephrine/norepinephrine is I .5/1 in the first case and 6/1 in the second case.

Method of calculation . From the above fluorescence

- 1

Aw Increase in Heart Rate, %

29 30 33 23 27 21

18

38

Exp. No. Epinephrine,

pg/roo ml

0.02 0.52

ratios the quantities of epinephrine and norepinephrine were calculated follow-ing Crout (3).

Recovery exjOeriments. Recoveries of epinephrine and hand, existence of norepinephrine could not be detected except in two in which very small amounts of norepi- nephrine may have been present, although 0.05 pg/g is the limit of accuracy for the method employed.

Figure I, A and B, illustrates emission spectra at two activating wavelengths of a frog heart eluate, epineph- rine and norepinephrine standards and blanks. Curves were obtained with a spectrophotofluorometer by scanning the emitted light from 300 to 700 rnp with the activation source constant at 400 rnp (Fig. IA), and 435 rnp (Fig. IB). The fluorescence peaks of the heart eluate in both parts of the figure correspond exactly in position and contour to those of the epinephrine stand- ard.

A few estimations were made in order to compare catecholamine contents of the ventricle with those of the atrium in R. catesbeiana, but no significant difference was detected between these two parts of the heart.

Characterization of substance released by sympathetic stimula- tion. Perfusions of the heart with Ringer solution were carried out on eight hearts for identifying the chemical transmitter released by cardiac sympathetic stimula- tions. The perfusate flowing from an aorta was collected separately before and during the stimulations and assayed. Table 2 presents the results of these experiments. Significant release of epinephrine into the perfusate was brought about by the sympathetic stimulation in all cases, whereas very small amounts of norepinephrine were detected in only two perfusates obtained during the stimulation. In one of them the same amount of norepinephrine was also found in the perfusate obtained before the stimulation. It is doubtful that these small values of norepinephrine are significant, because they are within the lower limit of the sensitivity of the fluori- metric method used in the present investigation. At any rate it is obvious that the main substance liberated in the perfusate by the sympathetic stimulation is not norepinephrine but epinephrine. This fact suggests that in the frog, sympathetic control of the heart is mediated by epinephrine.

Figure 2, A and B, illustrates emission spectra of an eluate of the perfusate obtained during the nerve stimu- lation together with those of standards and blanks at

norepinephrine, when added separately or together tissues and perfu sates n amounts rangin g from 0.5 to

to

pug, varied from 75 to I oo 76. The values reported here are not corrected for the percentage recovery.

RESULTS

Catecholamines in the frog heart. Table I shows the results of IO independent estimations. In order to minimize a possible variation in catecholamine contents of the heart which might be brought about by changes in environ- mental temperature, frogs were kept at room tempera- ture (18-22 C) for 24 hr before removal of their hearts. As shown in the table, the quantitatively predominant - catecholamine in the frog heart was epinephrine. The amounts of epinephrine were between I a .nd z ,ug/g of wet tissue in g cases out of IO, and this average was 1.27 kg/g. In the maioritv

J d of the cases, on the other

1

Frog Heart

Activation 435 MM : 0.003 s:41

Frog Heart

Activation 400 MM: 0.003 s: 41

FIG. I. A and II.- emission spectra of: (I) epinephrine 0.05 pug, (2) norepinephrine 0.05 pg, (3) reagent blank, (4) a frog heart eIuate, (5) sample blank. Activation wavelength: 400 rnp (A) and 435 mP W)-

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1290

600 400 i -

Perfusion Experiment

Activation 400

Yi7o-oo3 .

-

Perfusion Experiment

Activation 435 MM:0.003 s:41

FIG. 2. A and B: emission spectra of: (I) epinephrine 0.05 lug (I;) norepinephrine 0.05 pg, (3) reagent blank, (4) eluate of a perfusate of the heart obtained during sympathetic stimulation, (5) sample blank. Activation wavelength: 400 rnp (A) and 435 mE.c @I.

two activation wavelengths as in Fig. I. The fluorescent peaks of the eluate correspond exactly to those of the epinephrine standard.

In these experiments, effectiveness of the nerve stimu- lation was checked by the induced positive chronotropic effects. Increases in the heart rate caused by the stimu- lation were between 18 and 38 70. As may be seen in Table 2, there was no correlation between the degree of the positive chronotropic action and the amount of the epinephrine released.

Efect of environmental temperature. In order to observe the effect of environmental temperature on catechol- amine contents of the heart, estimations were made on the following four groups of frogs. In groups 1 and 2 frogs were kept for I day at room temperature (18-22 C) and at a cold temperature (0.4 C), respectively. In groups 7 and 4 they were kept for I week at room and c cold temperatures, respectively. Ten independent sam- ples were taken from each of groups I and 2 (Fig. 3A), and eight samples from each of groups 3 and 4 (Fig. 3B). Values of the epinephrine contents obtained from nine samples were between I and 2 ,ug/g in group 2 as well as in group I. The averages were I .52 and I .2 7 lug/g. The difference between the two was not statistically signifi- cant at the 5 76 level of significance. Though the values of the epinephrine contents in groups 3 and 4 showed slightly wider variations, the majority of them were also within or very close to the 1-2 ,ug/g range. The averages were 1.39 and I .30 pg/g, and no statistically significant difference was found between the two.

Every sample in each group contained only traces of or no norepinephrine. The averages were less than o. #OI

pg/g in group I, 0.0 1 pg/g in group 2, 0.0 1 lug/g in group

AZUMA, BINIA, AND VTSSCHER

3, and 0.02 ,ug/g in group 4. As mentioned previously, however, these values are within the error of the method.

Effect of cardiac actizdy. The effect of cardiac activity on the catecholamine contents of the ventricle was studied with the following experimental arrangement. Ventricles were isolated and perfused with Ringer solution, and driven with electric pulses of 3o/min and supraliminal strength for 5 hr through a pair of elec- trodes which were attached to the inside and outside of the ventricle. Perfusion pressure was set so as to make the cardiac output about I ml/min. Ventricles in spon- taneous arrest in Ringer solution for the same period of time were used as controls.

Six pairs of independent estimations were made and the results are shown in Fig. 4. The averages of epi- nephrine contents of driven and still ventricles were I .a5 and I. I I ,ug,ig, respectively. The difference was not statistically significant at the 5 76 level of signifi- cance. Norepinephrine was not detected except in one pair in which a very small amount (0.01 pg/g) of nor- epinephrine was found. The differences between the epinephrine contents of the normal heart and those of these two groups of ventricles were not statistically significant.

Catecholamine contents of other organs, the liver, spleen, brain, skeletal muscle, sympathetic chain, and adrenal of the bull frog, were also estimated. As shown in Table 3, norepinephrine in reliably significant quanti- ties appeared only in brain, sympathetic chain, and adrenal gland tissue. The adrenal is especially interesting because in remarkable contrast with the same organ of warm-blooded animals, the greater part of catechol- amine was norepinephrine and the ratio of the amount of epinephrine to that of norepinephrine was about I : 2.

The reverse has been found with the catecholamine composition of the adrenal of warm-blooded animals. This reversal of the ratio of these catecholamines in the frog adrenal is considered, in connection with the predominance of epinephrine in the other organs, an interesting problem requiring further study.

Analyses have also been made of the catecholamine content of organs in the snapping turtle (C. serjuxtina).

Table 4 presents these data. It will be apparent that in this species a situation very different from that in the bullfrog prevails. Figure 5, A and B, presents the emission spectra of a turtle heart eluate, in which it is apparent that the predominant catecholamine is norepinephrine in contrast to the situation found with eluates from bullfrog hearts.

DISCUSSION

In all warm-blooded animals the predominant cate- cholamine in most organs and tissues is norepinephrine, and epinephrine is always a small fraction of the total catecholamine content. On the contrary, the main catecholamine found in the frog heart was epinephrine, and the amount of norepinephrine was nil or a trace. This finding is essentially consistent with the observa-

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ADRENERGIC MECHANISMS IN BULLFROG AND TURTLE

3.0

I------ 0 A 24 Hours

18-22 C

CT \ i.9 l

CT 3.

8 Q

I .o

1

: l a

l

0'

FIG. 3. Effect of environmental temperature on epinephrine content of bullfrog heart.

tions previously published. Von Euler (14) found 3-5 pg/g of epinephrine and no,norepinephrine in the frog heart by a biological assay. Ostlund (I I) found, using a similar method, that about 70 7c of the total catechol- amine in the heart of R. temporaria is epinephrine. With the same species of frog Falck et al. (4) reported that the greater part of catecholamines is epinephrine in the ventricle as well as in the atrium and that the amount of norepinephrine is very small. Their figures (1.5 pg/g of epinephrine and 0.01 rug/g of norepinephrine in the atrium and 1 .g and 0.01 ,ug/g of each in the atrium) are quite similar to those obtained in the present experi- ments. Concerning the amount of norepinephrine, exceptionally high values were reported by Govyrin and Leontieva (5), who found appreciable amounts of norepinephrine (7.16 & I. I o and 4.28 & I .26 pg/g) in the atrium and the ventricle of R. temporaria. But the predominant catecholamine was also epinephrine (9.43 & 1.57 pg/g in the atrium and 6.80 zt 1.44 ,ug/g in the ventricle). Their figures were expressed per gram of dried tissue, I g of which corresponded to between 6.7 and IO g of wet tissue. Although they employed the method of von Euler and Floding (18) their findings were contradictory and unexplained.

It is concluded that, opposite to the case in warm- blooded animals and in the turtle, the main catechol- amine in frog’s organs (except the adrenal, brain, and sympathetic chain) is epinephrine. This conclusion might suggest the existence of a biosynthetic pathway of catecholamine in the frog different from that in warm- blooded animals.

As noted above, no investigation has hitherto been carried out on the nature of the sympathetic transmitter of the frog except the classical work of Loewi (8) in which he reported that the cardiac fluid contents in the frog heart during several repeated cardiac sympathetic stimulations showed a green fluorescence which was comparable to that shown by epinephrine solution. He did not mention anything concerning the existence of norepinephrine in the liquid. Separate quantitations of

these two substances were impossible by the method he used. As far as this problem is concerned, the present work seems to be the first which demonstrates that the sympathetic stimulation releases epinephrine from the frog heart with at most traces of norepinephrine. The same finding was obtained with four perfusion experi- ments with the frog liver. The perfusate flowing from the hepatic vein before and during the splanchnic nerve stimulation was collected in two separate containers, and was processed and assayed in the same manner as the perfusate of the heart. A small but significant amount of epinephrine was found in the perfusate obtained during the stimulation. No norepinephrine was detected in the perfusate.

The idea that the sympathetic postganglionic trans- mitter in warm-blooded animals is norepinephrine has been developed on the basis of the following facts (I 5). I) Norepinephrine is the predominant catecholamine in most organs and tissues which have sympathetic

nervation. 2) Chronic sympathectomy of organs pro- duces a sharp decrease in their norepinephrine con- tents, whereas the decrease in epinephrine brought about by the procedure does not seem to be so clear. 3) Regeneration of adrenergic fibers produces the reap- pearance of the initial levels of norepinephrine. 4) Stimulation of adrenergic nerves releases much more norepinephrine into the blood stream than epinephrine. 5) The effects of such stimulation show the characteristics of norepinephrine action directly on the stimulated organ.

ks far as these criteria for determining the nature of the sympathetic chemical transmitter are concerned, the findings obtained in the present investigation seem to support the idea that epinephrine may be the adren- ergic transmitter in the specific case of the frog. Epi- nephrine is the predominant catecholamine in most organs. Stimulation of the cardiac sympathetic nerves releases mainly (or perhaps only) epinephrine. The effect of such stimulation produces an increase in the heart

3.0

0

Control (Whole Heart)

Still Ventricte

Driven Ventricle

FIG. 4. Ef!fect of bullfrog ventricle.

contractile activity on epinephrine content of

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1292 AZUMA BINIA, AND VISSCHER

TABLE 3. Catecho~amines in various organs of R. catesbeiana

Brain

Liver

Spleen

Sympathetic chain

Adrenal

Skeletal muscle

Epinephrine, pg/g Wet Tissue

Norepinephrine, pg/g Wet Tissue

I .I5 0.53 0.84 0.38

0.02 0

0.21 0

0.11 0

0.08 0

0.21 0

0.11 0

0.21 0

0.17 0

0.06 0

2.94 IO.09

845 1,680 1,360 1,980

0.02 0

0.03 0

Turtle Heart

Activation 400 MM ; 0.003 s:37

Turtle Heart

Activation 435 MM : 0.003 s:41

rate and the contractile force. This increase, of course, is not specific for either epinephrine or norepinephrine since both catecholamines have the same effect on the isolated perfused heart. But, at least in the present experiment, this action seemed likely to have been mediated by epinephrine since epinephrine was the main (or the only) one of the two that was detected in the perfusate obtained during the stimulation.

(2)

FIG. 5. A and B: emission spectra of: (I) epinephrine 0.05 pg, norepinephrine 0.05 pg, (3) reagent blank, (4) a turtle heart

eluate, (5) sample blank. Activation wavelength: 400 rnp (A) and 435 m/J (9. s ee Fig. I for contrast with bullfrog.

epinephrine contained in it could not be considered in connection with the nervous mechanism. One of the present authors, however, has found evidence which

As mentioned before, von Euler had postulated nor- epinephrine as the chemical transmitter in the frog heart (15, 16, I 7), despite the fact that he found from 3 to 5 pg/g of epinephrine in the frog heart (I 4). These high figures and wide range of variation, however, led him to conclude that epinephrine cannot be the adren- ergic transmitter, since if epinephrine were bound to the nerves the contents would be smaller and more constant. He thought it likely that the epinephrine found in the frog heart is the product of chromaffine cells (15, 17) which, as noted before, Falck et al. (4) could not confirm. But &lund (I I), Falck et al. (4), and the present authors found the contents of epineph- rinc to bc smaller and more constant than the figures given by von Euler. (Gstlund: 0.76-1 .o pg/g; Falck et al. and the present result: I .o-2.0 pg/g).

Von Euler also gave weight to the report of &tlund (I I) that the spleen which has abunda.nt sympathetic supply contains more norepinephrine than epinephrine. The present result, however, indicates that the spleen of the bullfrog contains mainly epinephrine. This differ- ence in results cannot be due to a difference in the species of the frog used, because Auton and Sayre ( I ) also reported a predominance of epinephrine in the spleen of R. temporaria, which was used by ostlund. It might be attributable to the difference in the methods of estimation, bioassay being utilized by Gstlund.

A third objection has been that the existence of sym- pathetic innervation in the frog ventricle is not verified (16). I f there is no sympathetic supply in the ventricle,

indicates the existence of such innervation (T. Azuma and H. Hayashi, unpublished observations). The evi- dence was obtained by the following experiment. The frog heart, excised with its nerve supply and cut open along the midline to expose the endocardial surface, was put in a Ringer solution bath with epicardium underneath and driven electrically. Membrane action potentials were recorded from a ventricular fiber with a glass-pipette microelectrode and the effect of the sympa- thetic stimulation was observed on the shape of the potentials. In order to avoid the possible influence of the transmitter liberated in the sinus and atrium by the stimulation, the Ringer solution bath in which the preparation was placed was divided into two parts by a partition with a semicircular notch. The partition was set in such a manner that the atrioventricular ring of the preparation lay across the notch. The space between the preparation and the partition was compactly plugged with Vaseline-soaked cotton. The Ringer solution in the ventricular side of the bath was thus completely separated from that in the atria1 side, so that the trans- mitter released in the atrium or sinus could no longer diffuse to the ventricular surface through the environ- mental saline solution. That it could not do so was confirmed by the fact that no appreciable changes in ventricular membrane potential were produced even several minutes after the introduction of epinephrine at a high concentration into the atria1 side of the bath. The stimulation of the sympathetic nerves of the prepa- ration produced the characteristic changes (6) in the shape of the action potentials recorded from an apical

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ADRENERGIC MECHANISMS IN BULLFROG AND TURTLE -93

TABLE 4. Analyses of jmled samfdes of organs of turtle (C. serpent ina)

Atrium

Ventricle

Pancreas

Spleen

Epinephrine, pg/g Wet Tissue

Norepinephrine, pg/g Wet Tissue

0.04 o*34 0.03 0.41

0.02 0.46 0 0.50 0.03 0.3’

0 0.24 0 0.27

0.01 o-35 0 0.29

0 o-33

ventricular fiber within a few minutes. This fact indi- cates the existence of a sympathetic nerve supply to the frog ventricle.

These considerations do not necessarily lead to the conclusion that the sympathetic transmitter in the frog is epinephrine, though the experimental facts lend support to the idea. For example, the hypothesis pro- posed by Burn (2), that the sympathetic postganglionic transmitter is acetylcholine and that the ace tylcholine secondarily releases catecholamines from their storage site, must be considered. At any rate, it seems clear that epinephrine plays the most important role in the final action of the sympathetic system in the frog, and that epinephrine may well be the sympathetic trans- mitter in this species.

The effect of chronic sympathectomy on the cate- cholamine contents of the heart was also studied. A simple operative method was devised which enables one to cut vagosympathetic trunks of the frog anes- thetized with ether practically without bleeding. Vago- sympathectomized frogs kept at a cold temperature (o-4 C) did not show any decrease in the catecholamine contents of their hearts even after 8 weeks or more. Those kept at room temperature, on the other hand, did not survive more than 4 days. Autopsy revealed an extremely dilated gastrointestinal tract with enormous gas retention. This could not be due to an infection because control frogs which were treated in the same

REFERENCES

I.

2.

3*

4.

6.

AUTON, A. II., ANI) D. F. SAYM. A study of the factors aflcct- ing the aluminum oxide trihydroxindole procedure for the analysis of catecholamines. J. Pharmacol. Exptl. Therap. 138:

360-375, 1962. BURN, J. H. The Autonomic Nervous System. Oxford: Blackwell,

I 963. CROUT, J. R. Catecholamines in urine. In: Standard Methods of Clinical Chemistry. New York: Academic, I 96 I, vol. 3, p. 62-80. FALCK, B., J. HAGGENDAL, AND C. H. OWMAN. The localiza- tion of adrenaline in adrenergic nerves in the frog. Quart. J. Exptl. Physiol. 48 : 253-257, 1963. GOVYRIN, V. A., AND G. R. LEONTIEVA. Effect of sympathetic denervation on myocardial catecholamine content in the frog. FisioZ. Zh. SSSR 4g : 556-569, 1963. HAYASHI, H., AND T. AZUMA. Effect of sympathomimetic

way except for cutting of the nerves could survive for a much longer period of time and never showed such gastrointestinal change. The effect of chronic denerva- tion of the frog heart deserves further study.

Results of the experiment on the effect of environ- mental temperature indicate that the epinephrine con- tent of the bullfrog heart is not affected by a drastic decrease in environmental temperature at which the animals have been kept for a day or a week. The only investigation involving a similar experiment is that carried out by Govyrin and Leontieva (5). They re- ported that the epinephrine and norepinephrine con- tents of the atrium and epinephrine contents of the ventricle are lower in the frog which has been kept in the cold temperature (4-6 C). They did not mention the duration of the period of cold exposure. The reduc- tion in the catecholamine contents was, according to their observation, found to be partly restored 30 min after bringing the animal into room temperature. The extreme discrepancy between these two results is not explicable at the present. It may be pointed out, how- ever, that Musacchia et al. (IO) observed that in cold torpor for about 2 weeks, catecholamine contents of the heart of ground squirrel, as well as those of the heart, kidney, and liver of turtle, show no change.

It is also interesting that an isolated ventricle, after pumping the perfusing fluid of 3 liters in volume, shows no decrease in its catecholamine level. Whatever the role the catecholamines may play in the contraction of an isolated heart driven electrically, it is evident either that they are not consumed in the process or that they are utilized and immediately resynthesized. No investiga- tion has previously been made on the relation between the cardiac activity and catecholamine contents of frog heart muscle. Lee and Shideman (7) found that after depletion of catecholamines by reserpine the papillary muscle of the cat showed depressed contractile force. Maxwell et al. (g), on the other hand, found that a drastic reduction in catecholamine content of the left ventricle of the rabbit did not necessarily result in severe impairment of its contractility. No previous report could be found on the question of whether or not the catecholamine content of the heart is affected bY the cardiac work.

7.

8.

90

I 0.

amincs on the transrnernbrane action potential of toad heart. Japan. J. Physiol. 12 : 2 10-224, 1962.

LEE, Mr. C., AND F. E. SHIDEMAN. Role of myocardial cate-

cholamines in cardiac contractility. Science I 2g : 967-968, I 959.

LOEWI, 0. Quantitative und qualitative Untersuchungen

fiber den Sympathicusstoff. Arch. Ges. Physiol. 237 : 504-5 14,

1936s MAXWELL, R. A., J. J. KELLEY, JR., AND E. T. ECKHARDT. Effect of catecholamine depletion on an index of myocardial

contractility in isolated rabbit hearts. Proc. Sot. ExptZ. Biol.

Med. 116: 672-674, 1964.

MUSACCHIA, X. J., M. JELLINEK, AND T. COOPER. Effect of hibernation and cold torpor on tissue catecholamine content. Proc. SOL Exptl. Biol. Med. I I o : 856-857, 1962.

by 10.220.33.2 on April 13, 2017

http://ajplegacy.physiology.org/D

ownloaded from

I’94 AZUMA, BINLA, AND VISSCHER

I I. &LUND, E. The distribution of catecholamines in lower ani- mals and their effect on the heart. AC&Z PIzysiol. &and. 31 : Suppl. x I 2, p. 1-67, 1954.

I 2. PEART, W. S. The nature of splenic sympathin. J, physz’ol., London 108: 491-501, 1949.

13. SCHAEPDRYVER, A. F. Differential fluorimetric estimation of adrenaline and noradrenaline in urine. Arch. Intern. Pharmaco- dyn. 115 : 233-245, 1958.

14. VoN EULER, U. S. A specific sympathomimetic ergone in adrenergic nerve fibers (sympathin) and its relation to ad- renaline and noradrenalin. Acta Fhysiol. Stand. I 2 : 73-97,

19469 15~ VON EULER, U. S. Noradrenaline. Springfield, Ill. : Thomas,

I956*

16. VON EULER, U. S. Neurotransmission in the adrenergic nervous system. Harvey Lectures Ser. 55 : 43-65, 1961.

I;r. VON EULER, U. S. Autonomic neuroeffector transmission. Handbook of Physiology. Neurofihysiology. Washington, D. C. : Am. Physiol. Sot., 1959, sect. I, vol. I, p. 215-237.

18. VON EULER, U. S., AND I. FLODLNG. A fluorimetric micro- method for differential estimation of adrenaline and nor- adrenalin. Acta Physiol. &and. 33 : Sup@. I I 8, p. 45-62, 1965.

Ig. VON EULER, U. S., AND S. HELLNER-BJ~RKMAN. Effect ofin- creased adrenergic nerve activity on the content of nor- adrenaline and adrenaline in cat organs. Acta Physiol. &and. 33 : Suppl. 118, p. 17-20, 1955.

20. VON EULER, U. S., AND F. LISHAJKO. The estimation of cate- cholamines in urine. Acta Physiol. Stand. 45 : I 22-I 32, 1959.

by 10.220.33.2 on April 13, 2017

http://ajplegacy.physiology.org/D

ownloaded from