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Spider cardiac physiology, II. responses of a tarantula heart to cholinergic compounds
Comp. gen. Pharmac., i97o , x, I 7 z - I 8 4
I7X
SPIDER CARDIAC PHYSIOLOGY, II. RESPONSES OF A TARANTULA HEART TO CHOLINERGIC COMPOUNDS* ROBERT
G. S H E R M A N t
AND R A L P H
A. P A X
Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A. (Rtg~ilgd 12
Dec., I969)
ABSTRACT x. Acetylcholine produces a marked increase in the rate and amplitude of the tarantula heart-beat by its actions on the cardiac ganglion. These effects are greatly potentiated by eserine and are considerably reduced by atropine, hexamethonium, and D-tubocurarine. 2. Methacholine produces a moderate increase in heart-rate and a pronounced increase in the amplitude of the contractions. Nicotine produces a marked increase in beart-rate, hut either no change or else a reduction in heart-beat amplitude. 3. The effects of methacholine are blocked completely by atropine, hut are unaffected by hexamethonium. The effects of nicotine are reduced considerably by hexamethonium, but not by atropine. 4. It is suggested that both nicotinic and muscarinic receptor sites for acetylcboline may be present in the cardiac ganglion.
THE effectsof certain chemicals, particularly acetylcholine (ACh), are often used as one means of distinguishing between ncurogcnic and myogenic hearts (Prosscr, z96I ). I f A C h causes a decrease in heart-rate,the heart-beat is thought to bc myogenic. If it produces an incrcasc in heart-rate, the heart-beat is thought to bc ncurogcnic. Kadzlcla and Kokocifiski (1966) reported that A C h produccd a decrease in the ratc and amplitude of the heart-beat o f Tegenaria atrica which suggests t h a t the heart-beat o f this spider is myogenic. However, conclusive evidence as to the origin of the heart-beat in T. attica has not been obtained. I n the previous paper the heart-beat o f the tarantula Eurypelma marxi Simon was shown to be neurogenie (Bursey a n d Sherman, z97o ). I t would be o f interest to determine if the heart o f this spider is also slowed b y A C h . Experiments were conducted to determine the effects o f A C h on the E. marxi heart as * Please address all correspondence to Dr. Ralph A. Pax, Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A. I" Present address: Department of Zoology, University of Toronto, 25 Harbord Street, Toronto 5, Ontario, Canada.
w e U as to determine what effects certain chollnergic blocking agents and an anticholincsterasc had on the rcspomc to A C h . Other experiments involving nicotine and methacholinc were carried out to obtain information about the cholinoccptivc sitesin this heart. MATERIALS AND M E T H O D S The method for isolating the heart and the recording apparatus used were described in the first paper of this series (Bursey and Sherman, I97o). For nearly all experiments the preparation consisted of an intact isolated heart pinned dorsal side up in a recording dish which had been filled partially with paraffin, Insect pins (size ooo) were placed at both ends of the heart and along the side opposite to that where the transducer was attached. This served to anchor the heart in place. In certain instances it was advantageous to modify the preparation described above. When the effects of drugs on cardiac ganglion electrical activity were to be studied, the heart was split ventrally by making a longitudinal incision along the entire length of the heart. The heart was pinned out flat, dorsal side up, in the recording dish with pins placed at both ends and sidesof the heart. This held ittightlyto the paraffi and very littlemovement occurred with each contraction. Hearts prepared in this way did not pull away from the recording electrode when solutions bathing the heart were changed.
x 72
SHERMAN AND
A third preparation consisted of a heart from which the cardiac ganglion had been removed, but which was otherwise left intact. The ganglion was easily dissected by simply teasing it free from the external heart surface with forceps. Deganglionated hearts were used to study the effects of drugs on the myocardium, free from any influence of the cardiac ganglion. Each of the drugs was dissolved in spider physiological solution (saline) as shortly before use as possible. Drugs were administered by replacing the saline bathing the heart with the drug solution by means of a syringe. Unless otherwise noted, hearts were exposed to each drug for a period of 3 minutes. After exposure, the rate and amplitude of the heart-beat were allowed to return to the pretreatment level before the next treatment. During this recovery period, which lasted from 5 to 15 minutes, at least 3 and as many as 7 saline washes were made. Only i drug was used on a particular heart unless the interactions of 2 or more drugs were to be investigated. In no case were drugs tested on a heart that had been in isolation for more than 3 hours. Hearts used had heart-rates between 49 and 20 beats per minute during the course of an experiment. If, upon isolation, the initial heartrate was greater than 49 beats per minute, the heart was allowed to beat in saline until the rate had dropped to less than 5° beats per minute. This was done because a greater amount of variability in rates was observed for hearts beating faster than 5° beats per minute than for those with lower rates. For all drugs except ACh heart-rates were measured for the minute just preceding each treatment and for each minute thereafter. In the case of ACh the minute intervals were subdivided into 6 equal intervals of xo seconds each. This was done because ACh has its maximum effect immediately after application and the effect is often a transient one. The amplitude of the heart contractions was measured in terms of rag. ofpuU on the transducer. The heart-beat amplitude was determined for the io beats just prior to the beginning of a drug treatment and for the xo beats immediately after application of a drug. In addition, to consecutive beats were also measured at minute intervals during the drug treatment. RESULTS CONTROL HEARTS T o d e t e r m i n e the a m o u n t o f v a r i a b i l i t y in isolated h e a r t - r a t e s , 5 h e a r t s were a l l o w e d to b e a t in isolation for 3 hours while b a t h e d only i n saline. T h e m e a n h e a r t - r a t e seen i n i t i a l l y was 45 beats p e r m i n u t e a n d t h e r a t e g r a d u a l l y d e c l i n e d to a m e a n r a t e a t the e n d o f 2 4 beats p e r m i n u t e . T h e greatest c h a n g e
PAX
Comp. gen. Pkarmac.
in r a t e f r o m one m i n u t e to the n e x t for a n y h e a r t was -4-2 beats p e r m i n u t e ; usually there was e i t h e r no c h a n g e or a difference o f o n l y -4-i b e a t p e r m i n u t e . M e a s u r e m e n t s o f the v a r i a b i l i t y in h e a r t b e a t a m p l i t u d e were also m a d e for t h e 5 control hearts. Since the a m o u n t o f pull e x e r t e d b y the h e a r t on t h e t r a n s d u c e r is a function o f t h e a m o u n t o f tension p l a c e d on t h e heart, this tension was a d j u s t e d so t h a t the a m p l i t u d e o f the h e a r t - b e a t r e c o r d e d was b e t w e e n 4 ° a n d 6o nag. a t the start o f e a c h c o n t r o l period. A m p l i t u d e m e a s u r e m e n t s were m a d e o f e a c h o f xo consecutive h e a r t b e a t s a t xo - m i n u t e intervals t h r o u g h o u t t h e 3-hour control period. T h e r e was v e r y little v a r i a t i o n b e t w e e n t h e m e a n for one i o - b e a t i n t e r v a l a n d the next. U s u a l l y t h e r e was n o c h a n g e in t h e m e a n a m p l i t u d e . T h e greatest changes ever seen were 4- i o rag. T o d e t e c t w h a t effect t h e p e r i o d i c saline washes m i g h t h a v e on the h e a r t - r a t e , rates were m e a s u r e d for t h e m i n u t e j u s t p r i o r to a n d i m m e d i a t e l y after the c o m p l e t i o n o f e a c h wash t h r o u g h o u t t h e control r e c o r d i n g period. T h e m e a n c h a n g e in r a t e for the 5 h e a r t s was + x b e a t p e r m i n u t e ; t h e largest changes ever seen were - - 2 a n d + 3 beats p e r minute. T h e effect o f the saline washes on h e a r t b e a t a m p l i t u d e was d e t e r m i n e d b y m e a s u r i n g t h e xo beats j u s t p r i o r to a n d i m m e d i a t e l y following e a c h wash. T h e r e was n o m e a n difference b e t w e e n t h e h e a r t - b e a t a m p l i t u d e seen before a n d after t h e washings. T h e greatest a m p l i t u d e changes seen were - - 1 5 a n d + I o rag.
ACETYLCHOLINE T h e response o f t h e h e a r t to x x to -4 M acetylcholine (ACh) consists initially o f a sustained c o n t r a c t i o n w h i c h after a few seconds gives w a y to i n d i v i d u a l b e a t s t h a t occur a t a g r e a t l y increased f r e q u e n c y (Fig. I A). As t h e i n c r e a s e d m y o c a r d i a l tonus declines, t h e a m p l i t u d e o f t h e h e a r t - b e a t increases a n d soon reaches a m a x i m u m . A f t e r the m a x i m u m effects a r e seen, t h e response begins to decline c o n s i d e r a b l y ; however, i n c r e a s e d r a t e a n d a m p l i t u d e a r e seen t h r o u g h out the drug treatment. Upon the removal of
I970, •
CHOLINERGIC
COMPOUNDS
A C h h e a r t activity r e t u r n s to p r e t r e a t m e n t levels w i t h i n 2 - 5 m i n u t e s , d e p e n d i n g directly o n the dose applied. A l t h o u g h the response to A C h declines d u r i n g the course of a single t r e a t m e n t , there is n o decrease i n the
AND
SPIDER
I73
HEART
response to several successive t r e a t m e n t s w h e n the h e a r t is washed with saline for 5 - i 5 m i n u t e s b e t w e e n each one. A dose--response curve for the effect of A C h o n the heart-rate is shown i n Fig. 2. F r o m
A
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B
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iiiiiiiiii,iii,iiiiiiiiiiliiiiiiiiiiiiiiiiiii i lliiiiii!iiiiiiiiiiiiiiiiiiiiliiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii Fxo. i.--Response of the heart t o ACh and eserine. A, Saline removed at first arrow and I × I o - ' M ACh applied at second arrow. B, Saline removed at first arrow and x x xo-s M ACh applied at second arrow. C, Saline removed at first arrow and x x xo-s M eserine applied at second arrow. D, Eserine removed at first arrow and x × xo-5 M ACh applied at second arrow. Time marker equals x second; downward excursion of time marker represents a heart-beat amplitude of 5o mg.
174
Corap. gen. Pharmac.
SHERMAN AND PAX
this figure it appears that concentrations as low as 5 × xo -e M are effective. The effect of A C h on heart-beat amplitude and myocardial tonus is shown in Table L At concentrations of I × I o -5 M and lower, the response, if any, is an increase in heart-beat
response is readily apparent. After ACh and eserine treatments, the time required for the heart-rate and heart-beat amplitude to return to pretreatment levels is nearly three times longer than after ACh alone,
50
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Fzo. 2.--Dose-response curve for the rate effect of ACh before and after treatment with eserine. Each point represents the mean response to at least 5 replications made on at least 5 different hearts. O, Before eserine; O, After eserine. Vertical lines represent one standard deviation. amplitude. Higher concentrations produce both an increase in beat amplitude and a transient increase in myocardial tonns. ANT~CHOLmmTE~ T o determine if an enzyme might be present which terminates the action of ACh, 8 hearts were treated with ~ × x0 -5 M eserine (physostigmine) for 5 minutes prior to the administration of ACh. Eserine alone produced a mean increase in heart-rate of I I beats per minute and a mean increase in heart-beat amplitude of 40 rag. (Fig. I C). The effect of eserine treatment on the response of the heart to I × xo-~ M ACh is shown in Fig. xB, D. The potentiation of the
A dose-response curve for the effect of ACh on the heart-rate after eserine treatment is shown in Fig. ':,. ACh, in concentrations of 5 × x ° - t M and IXXO- S M , gave rate increases from 3 to 4 times those seen before treatment with eserine. Concentrations which gave no rate increases before eserine, such as x × 10-6 M, gave rate increases after eserine. Concentrations greater than x × io -5 M A C h produced a prolonged tetany of the heart after eserine and were not included in this figure. Eserine also potentiated the effects of ACh on heart-beat amplitude and myocardial tonus (Table I). A greater increase in amplitude probably would have been seen after
I97o, •
t75
CHOLINERGIG COMPOUNDS AND SPIDER HEART
blocked the increased heart-beat amplitude produced by ACh. Three hearts were treated with 5 × 10-4 M C6 for 15 minutes and then with 5 × 10-4 M C6 pins 5 × 10-4 M ACh. C6 alone had little immediate effect on the heart-rate, but in the fifteenth minute after the beginning of the treatment the rate had decreased an average of 8 beats per minute. No change in the
A C h and eserine treatment if the tremendous increase in myocardial tonus had not occurred. CHOLINERGIC BLOCKING AGENTS
Three different compounds which block the actions of A C h in a variety of animals wcrc tested on the tarantula heart. These three blocking agents were atropine sulphate,
Table/.--EFFECTS OF ACETYLCHOLINE AND ESERINE ON H E A R T - B E A T AMPLITUDE AND MYOC, ARDL~L TONUS
DRUG CONCENTRATION
(M)
C H A N G E IN HEART-BEAT AMPLITUDE* (mg.)
INCREASEIN TONUS* (mg.)
No. oF TONE INCREASES
No. OF REPLICATIONS
Acetylcholine I × I 0 -s
5 X IO -6 I X I 0 -5
5 X I O -5 I × I 0 -~' 5 × IO-~
o io 25 40 45 7°
(-io-io) (-5-I5) (IO-55) (IS-IOO) (2o--85) (35-I2O)
0 0 0
5 6
(0-30) 60 (2o-~o5) i~,o (2o-I8O) ~o
IO IO
8 5
Eserine I XIO -6
40 (I5--85)
Acetylcholine after eserine I
X I O -7
5 × i o -7 I X I 0 -6 5 × IO-6 I × I O -5
15 (o--25) I5 (5-25) 25 ( - IO--5o) 2o ( - 15-45) 35 ( - xo--8o)
O
o (o-io) 30 (IO-7O)
50 (io-x2o) 2io (5o-34o)
* Mean and range. hexamethonium chloride (C6), and D-tubocurarine chloride (DTc). Five hearts were treated with I × xo -4 M atropine for 5 minutes and then with I × 10-4 M atropine plus x × 10-4 M ACh. Atropine alone had no immediate effect on the heart-rate, but after 5 minutes a mean decrease in the rate of 5 beats per minute was seen. At this time there was also a mean increase in heart-beat amplitude of 2o nag. After treatment with atropine, the response to ACh was considerably reduced, but not completely abolished. The rate increase due to ACh before atropine averaged 24 beats per minute, while after atropine it was only 6 beats per minute (Fig. 3)- Atropine also
amplitude of the contractions occurred. The effectiveness of A C h in producing an increase in rate was reduced from a mean of 30 beats per minute to i8 beats per minute by treatment with C6 (Fig. 4). The amplitude response to A C h was not affected. v T c at I × 10-4 M was applied to 3 hearts for 3° minutes and then I × t 0 -4 M DTc plus x × IO-4 M A C h were administered. DTc alone produced an initial mean increase in heart-rate of 4 beats per minute, but after 3° minutes the heart-rate had decreased an average of 9 beats per minute. No change in heart-beat amplitude was seen. v T c reduced the rate response to A G h in 2 hearts from 3 ° to 12 beats per minute; in the third heart the
x76
SHERMAN AND PAX
r a t e response was r e d u c e d from 30 to t8 beats per m i n u t e . T h e increased h e a r t - b e a t a m p l i t u d e p r o d u c e d b y A C h was n o t affected. T h e records shown i n Fig. 4 for C6 are essentially the same as those o b t a i n e d i n the experim e n t s done with DTc.
Comp. gen. Pharmac.
SXTS o r A C ~ ACTION T h e r e are p r i n c i p a l l y two locations where A C h could be a c t i n g to p r o d u c e changes i n h e a r t activity; the c a r d i a c g a n g l i o n a n d the h e a r t m u s c u l a t u r e . T o d e t e r m i n e if A C h has a direct effect o n t h e m y o c a r d i u m , it was
A
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iiiiiiiiiiiii,,i,iiiiii,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiijitiiiiiiiiiifii,iiifiiiif iiiiiiiiiiiiiiiiiiiJiiiJij Fzo. 3.--Effect of atropine on the response to ACh. A, Saline removed at first arrow and x × Io -4 M AC'h applied at second arrow. B, Saline removed at first arrow and I x zo -* M atropine applied at second arrow. C, Atropine removed at first arrow after a 5-minute treatment and ~ x I o - ' M ACh plus z x xo -* M atropine applied at second arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5° ms.
I97o, x
CHOLINEROIG COMPOUNDS AND SPIDER HEART
applied to the heart after removal of the cardiac ganglion. A C h did not evoke a recordable change in the myocardial tonus of deganglionated hearts. Thus the response of the heart to A C h appears to be due to its actions on the cardiac ganglion. T o demonstrate more clearly that the site of action is the cardiac ganglion, the electrical activity of the ganglion was monitored while
I77
A C h was administered to intact hearts that had been treated with eserine. The increase in ganglionic activity produced by such a treatment is readily observable (Fig. 5A, B, C). It is apparently the continuous firing of the ganglion which produces the sustained contractions seen irt the mechanical recordings of eserine and A C h treated hearts
(Fig. ID).
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¢¢ iiiiiiiii•iiiii•iiiiiiiiiiiiiii•••iiiimmiiiiiiiiiii••••iiiiiiiiiiiiiiiiiiiiiiiii•i••iii•iiiii••i••iiiii••iiiiii•iiiiiiiiiiiii•iiiiiiiiii FIG. 4.--Effect of C6 on the response to ACh. A, Saline removed at first arrow and 5 × Io-' M A C h applied at second arrow. B, Saline removed at first arrow and 5 × I o - ' M C6 applied at second arrow. C, C6 removed at first arrow after a I5-minute treatment and 5 × xo-4 M ACh plus 5 × I o - ' M C6 applied at second arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5o nag.
17 8
Comp.gen. Pharmac.
SHERMAN AND ]PAX
1
FIo. 5.--Effects of cholinergic compounds on cardiac ganglion electrical activity. A, Burst frequency in saline. B, Io seconds after application of I × ro-s M A C h . C, ro seconds after application of I × ro-s M A C h after treatment with eserlne. D, ro seconds after application of x × IO-5 M A C h plus i × ro-~ M atropine after a 5-mlnute atropine treatment of the same heart as in C. E, Burst frequency in saline (different heart). F, IO seconds after application of 5 x ro-5 M methacholine. G, Burst frequency in saline (different heart). H, io seconds after application of 5 × x°-5 M nicotine. Vertical llne equals 3oo ~tV. ; horizontal line equals 2 seconds.
I970, •
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C H O L I N E R G I C COMPOUNDS AND SPIDER H E A R T
T o d e m o n s t r a t e t h a t the action o f the blocking agents also occurs in t h e c a r d i a c ganglion, t h e response o f the g a n g l i o n to A C h
was m o n i t o r e d before a n d after t r e a t m e n t w i t h at r o p i n e. As shown in Fig. 5 C, D, treatment with atropine blocked the
A
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+ + iiiiiiiiiiiiiiiiiiilliiiiiiiiiitiiiiiiiiit~iiiiiiiiiiiiiiiiiiiiiiiiiii11tll]iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiill Fzo. 6.--Response of the heart to methacholine and atropine. A, Saline removed at first arrow and I x io-4 Mmethacholine applied at second arrow. B, Saline removed at first arrow and 5 x zo-5 Mmethacholine applied at second arrow. C, Saline removed at first arrow gnd z × IO-4 M atropine applied at second arrow. D, Atropine removed after a 5-minute treatment at first arrow and 5 x IO-6 M methacholine plus I x Io -4 M atropine applied at second arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5o rag.
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SHERMAN AND PAX
response of the eserinized cardiac ganglion to ACh. METHACHOLINE
Methacholine (acetyl-beta-methylcholine) chloride, a mnscarinic cholinomimetic, at concentrations from t × 10-6 M to t × i o -* M produced an increase in the rate and amplitude of the heart-beat, as well as a transient increase in myocardial tonus (Fig. 6A). In contrast to ACh, the response to methacholine did not diminish during the course of a single treatment. After a treatment, the rate and amplitude of the heart-beat returned to control levels within 5 minutes. The effect of methacholine on heart-beat amplitude was more pronounced than the effect on the heart-rate. For example, i × io -4 m methacholine produced a mean rate increase of 12 beats per minute and a mean increase in heart-beat amplitude of 5° rag. (6 replications, 2 on each of 3 hearts). A C h at the same concentration gave a mean increase in heart-rate of 3° beats per minute and a mean increase in heart-beat amplitude of 45 nag. Thus, the effect of methacholine on heart-beat amplitude is comparable to that of ACh, while the effect on the heart-rate is considerably less. The site of methacholine action was investigated in the same manner as was ACh. Methacholine, like ACh, did not produce any recordable change in myocardial tonns when applied to deganglionated hearts. The effect of methacholine on cardiac ganglion electrical activity is shown in Fig. 5 E, F. Both an increase in the rate of bursting and an increase in the amount of activity in each burst were seen upon application of methacholine. T h e p o t e n t i a t i o n of the ACh effect by eserine indicates that a cholinesterase is present in the cardiac ganglion. Since methacholine differs in structure from ACh only by the presence of a methyl group at the beta position, it was of interest to know if eserine would also potentiate the action of methacholine. Treatment of a heart with i × lO-5 M eserine for I o minutes did not appreciably enhance the effects of methacholine on the heart. The rate increase produced by 5 × I o - S M methacholine before
Corap. gen. Pharma¢.
eserine was 13 beats per minute, while afterward it was 15 beats per minute. There was no increase in the amplitude response to methacholine after treatment with eserine. Atropine and C6 were also tested to see if they would block the effects of methacholine on the heart. Treatments with these compounds were identical with those in the experiments involving ACh. Atropine effectively blocked both the rate and amplitude response produced by methacholine. The mean increase in rate evoked by 5 × I o - s M methacholine before atropine was i2 beats per minute, while after atropine it was only i beat per minute (4 hearts) (Fig. 6). No increase in heart-beat amplitude occurred after atropine treatment. C6, on the other hand, did not alter the response of the heart to methacholine. The mean rate increase before C6 was 14 beats per minute, while afterwards it was I6 beats per minute (3 hearts). Also, there was no change in the amplitude response. NICOTINE
Nicotine produced an immediate increase in heart-rate when applied in concentrations from I × I O - e M to 5 × I o - 5 M (5 hearts). The effect of nicotine on heart-beat amplitude was variable. In a few instances nicotine produced a slight increase in amplitude (about IO mg.), but in other instances either there was no change or else a decrease. The latter case was particularly true for concentrations higher than I X lO-5 M (Fig. 7D). The response of one heart to I × 10-5 M nicotine is shown in Fig. 7A, where an increase in the rate of 2 7 beats per minute occurred, with little change in heart-beat amplitude. The heart apparently becomes desensitized to nicotine, for soon after its administration the response starts to decline and when low doses are applied (i.e., i × io -e M) the rate often returns to pretreatment levels during the course of the treatment. Furthermore, upon repeated treatments with the same concentration of nicotine the response becomes progressively less. Nicotine did not produce any recordable change in myocardial tonus when applied to
I970 , I
18I
C H O L I N E R O I C COMPOUNDS AND SPIDER H E A R T
d e g a n g l i o n a t e d hearts. W h e n 5 × I O -6 M nicotine was a p p l i e d to i n t a c t hearts, a n increase i n cardiac g a n g l i o n electrical activity was seen, b u t the activity consisted o f small a n d irregular bursts (Fig. 5G, I-I).
T h u s , the site of action of nicotine is also the cardiac ganglion. A t r o p i n e a n d C6 were also tested for their ability to block the effect of nicotine o n the heart. T r e a t m e n t s with these c o m p o u n d s
A
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Fro. 7.--Response of the heart to nicotine and C6. A, Saline removed at first arrow and x × to -5 M nicotine applied at second arrow. B, Saline removed at first arrow and z x Io -~ M 0_,6applied at second arrow. C, C6 removed after a x5-minute treatment at first arrow and z × lo -6 M nicotine plus z × Io -4 M C6 applied at second arrow. D, Saline removed at first arrow and 5 x io -s M nicotine applied at second arrow (different heart). Time marker equals I second; downward excursion of time marker represents a heart-beat amplitude of 5° mg.
I82
SHERMAN AND PAX
w e r e the same as for A C h . C6 r e d u c e d the r a t e response p r o d u c e d by i X IO-5 M nicotine f r o m a m e a n increase o f 19 beats p e r m i n u t e to 8 beats p e r m i n u t e (3 hearts) (Fig. 7).
Comp. gen. Pharmac.
A t r o p i n e d i d n o t alter the response to nicotine in 3 o f 5 hearts. I n the o t h er 2, the m e a n rate increase p r o d u c e d by nicotine was r e d u c e d f r o m x5 to 7 beats per m i n u t e .
A
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FIO. 8.--Additive effects of nicotine and methacholine on the heart. A, Saline removed at first arrow and 5 x i o - ' M nicotine applied at second arrow. B, Saline removed at first arrow and 5 x i o - ' M methacholine applied at second arrow. C, Saline removed at first arrow and 5 x Io -6 M nicotine plus 5 x to -6 M methacholine applied at second arrow. D, Atropine removed after a 5-minute treatment at first arrow and 5 × Io-6 M nicotine plus 5 x I o - ' M methacholine plus i x t o - ' M atropine applied at second arrow. Time marker equals x second; downward excursion of time marker represents a heartbeat amplitude of5o mg.
x97o , x
C H O L I N E R G I C COMPOUNDS A N D SPIDER H E A R T
I83
Although there appears to be a partial blocking of the nicotine effect by atropine in these 2 cases, a greater than usual densensitization of the heart m a y have occurred.
that atropine selectively blocked the action of methacholine while leaving that of nicotine unaffected.
NICOTINE AND ~ETHACHOLINE T h e principal effects of nicotine and methachoLine on the heart are quite different and it m a y be that these drugs are acting at different sites in the cardiac ganglion. I f this is the case, the individual effects of these cholinomimetics should sum when both are applied to the heart at the same time. T o test this hypothesis, a heart was treated first with nicotine, then methacholine, and then with both of t h e m together. Fig. 8 shows the results of this experiment. Nicotine alone in a concentration of 5 × IO-e M produced an increase in the rate of 2o beats per minute. Little change in heart-beat amplitude occurred, but there was a small transient increase in tonus (Fig. 8A). Methacholine alone, at 5 x I o -5 M, produced an increase of 6 beats per minute in the rate and a pronounced increase in the amplitude of the heart-beat (about 5o rag.) (Fig. 8B). When both compounds were applied together, both a large increase in rate (2 x beats per minute) and in beat amplitude (4o rag.) were seen (Fig. 8C). The rate increase was greater than 3 times that seen for methacholine alone (2 x in comparison with 6 beats per minute). T o further investigate the additive effects of methacholine and nicotine, this same heart was treated with I x IO-4 M atropine for 5 minutes. T h e same doses of nicotine and methacholine as were used above were then applied together with I × I O-4 M atropine. T h e results of these treatments are shown in Fig. 8D. Before atropine, nicotine and methacholine, when applied together, produced a rate increase of 21 beats per minute and an increase in both heart-beat amplitude and myocardial tonns. After atropine, the simultaneous treatment with nicotine and methacholine produced a rate increase of 15 beats per minute, with no increase in either heart-beat amplitude or myocardial tonus. Since methacholine alone initially produced a rate increase of 6 beats per minute and an increase in heart-beat amplitude, it appears
DISCUSSION The observation that the response to A C h declines during the course of a treatment indicates that the heart either metabolizes A C h or becomes desensitized to it. Since successive treatments with A C h are equally effective, the decline in response is not due to desensitization. When treatment with eserine, a potent antichoLinesterase, precedes treatment with ACh, no decline in response occurs; in fact, the effects of A C h are greatly enhanced. These results indicate that a cholinesterase which metabolizes ACh is present in the cardiac ganglion. The specificity of the cholinesterasc was investigated by the experiment involving eserine and methachoUne. T r e a t m e n t with eserine did not appreciably increase the effectiveness of methacholine. Thus it appears that the cholinesterase present is quite specific for ACh. T h e results of the experiments involving cholinomimetics and cholinergic blocking agents indicate that the action of A C h on the heart is not a simple one and that A C h is probably acting on more than one kind of receptor site in the cardiac ganglion. Nicotine mimics the rate-changing effects of ACh, but not the heart-beat amplitude effects. Methacholine mimics the heart-beat amplitude effects of ACh, but only partially mimics the rate effects. T h e effects of nicotine are reduced considerably by C6, but not by atropine; the effects of methacholine are blocked by atropine, but not by C6. W h e n nicotine and methachoLine are applied simultaneously to the heart a response occurs which is greater than that seen to either of t h e m alone. Thus it appears that nicotine and methacholine are acting at different sites in the cardiac ganglion and that both nicotinic and muscarinic receptor sites for A C h are present. I n a previous paper it was shown that different nerve-cell types can be distinguished on a morphological basis within the cardiac ganglion of E. marxi (Bursey and Sherman,
I84
SHERMAN AND PAX
I97o ) . I f these morphological cell types are the abdomen is quite small, a drug injection indicative of a separation of the cells into made anywhere in the abdomen might propacemaker and follower cells as is the case for duce a considerable increase in internal the lobster cardiac ganglion (Maynard, x955; pressure. Such a pressure increase could lead Hartline, I967) then, according to the to a decrease in heart-rate as well as a results obtained in this study, the nicotinic decrease in heart-beat amplitude. Although receptor sites would be located on the pace- the authors state that ' test injections did not maker cells and the muscarinic sites would be produce any change in the action of the located for the most part on the follower cells. heart ', the upward shift in the baseline of However, since methacholine produces a their records during the time that the heartmoderate increase in heart-rate and since rate was reduced indicates that a change in atropine reduces the rate response produced pressure m a y have occurred as a result of the by ACh, some of the muscariuic sites m a y injection. also be located on pacemaker cells. An examination of the effects of ACh on I n the only other pharmacological study of isolated heart preparations of Tegenaria atrica spider hearts it was reported by Kadziela and is necessary to determine whether the differKokocifiski (i966) that A C h produced a ences in results reported are due to species decrease in the rate and amplitude of the differences or to differences in experimental heart-beat of Tegenaria atrica. These results methods. are exactly opposite to those obtained in this study for the tarantula Eurypelma marxi. ACKNOWLEDGEMENTS While it is possible that the differences in This investigation was supported by a results are due to species differences, it seems U.S.P.H.S. predoctoral fellowship from the just as possible that the differences are due to Institute of General Medical Sciences to R. G. and by a U.S.P.H.S. research grant from the totally different manner in which the two Sherman the National Heart Institute to R. A. Pax. studies were made. In this regard, a number Critical reading of the manuscript by Dr. H. L. of problems seem apparent in the methods Atwood is gratefully acknowledged. used by Kadziela and Kokocifiski (1966). By using an in vivo heart preparation, the conREFERENCES centration of drug acting on the heart is not BURSEY, C. R., and SHm~r~AN, R. G. (I97O), 'Spider cardiac physiology, I. Structure and the same as that injected, since some dilution function of the cardiac ganglion ', Comp. gen. must occur due to the blood present. Since Pharmac., x, I6o--x7o. they supposedly only recorded electrical HAnTLm'E, D. K. (x967) , ' Impulse identification events and not mechanical events, there is and axon mapping of the nine neurons in the cardiac ganglion of the lobster Homarus araericonsiderable doubt about the validity of canus ', o7. exp. Biol., 47, 327-34 °. their statements concerning heart-beat amplitudes. However, their recordings of 'the KA~zm~, W., and KoKocn~srJ, W. (i966), ' The effect of some neurohormones on the heart-rate action potentials of the heart ' m a y in fact be of spiders ', Experientia, ~ , 45-46. only movement artefacts, since their record- MAYNARD, D. M. (x955) , ' Activity in a crustacean ganglion, II. Pattern and interaction in burst ings, made by inserting metal electrodes into formation ', Biol. Bull. mar. biol. Lab., Woods the abdomen, are quite unlike the electrical Hole, xog, 40o-436. recordings reported for other spider hearts PROSSER, C. L. (x961), ' Circulation of body (Sherman and Pax, I968; Bursey and fluids ', in Comparative Animal Physiology (ed. Paos~mR, C. L., and BROWN,F. A.), and ed., pp. Sherman, x97o ). 386-4x6. Philadelphia: Saunders. Another serious problem in using an in vivo R. G., and PAX, R. A. (x968), ' T h e heart preparation in spiders is that by inject- SHEn~mN, heart-beat of the spider, Geolycosa missouriensis ', ing solutions into the abdomen the volume Comp. Biochem. Physiol., 26~ 529--536. injected alone could produce a change in Key Word Index: Spider heart, spider cardiac heart-rate and heart-beat amplitude. Since ganglion, tarantula, Eut.ypelma marxi, acetylthe abdomen is essentially a closed system choline, eserine, atropine, hexamethonium, nand since it would seem that the volume of tubocurarine, methacholine, nicotine.
I7X
SPIDER CARDIAC PHYSIOLOGY, II. RESPONSES OF A TARANTULA HEART TO CHOLINERGIC COMPOUNDS* ROBERT
G. S H E R M A N t
AND R A L P H
A. P A X
Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A. (Rtg~ilgd 12
Dec., I969)
ABSTRACT x. Acetylcholine produces a marked increase in the rate and amplitude of the tarantula heart-beat by its actions on the cardiac ganglion. These effects are greatly potentiated by eserine and are considerably reduced by atropine, hexamethonium, and D-tubocurarine. 2. Methacholine produces a moderate increase in heart-rate and a pronounced increase in the amplitude of the contractions. Nicotine produces a marked increase in beart-rate, hut either no change or else a reduction in heart-beat amplitude. 3. The effects of methacholine are blocked completely by atropine, hut are unaffected by hexamethonium. The effects of nicotine are reduced considerably by hexamethonium, but not by atropine. 4. It is suggested that both nicotinic and muscarinic receptor sites for acetylcboline may be present in the cardiac ganglion.
THE effectsof certain chemicals, particularly acetylcholine (ACh), are often used as one means of distinguishing between ncurogcnic and myogenic hearts (Prosscr, z96I ). I f A C h causes a decrease in heart-rate,the heart-beat is thought to bc myogenic. If it produces an incrcasc in heart-rate, the heart-beat is thought to bc ncurogcnic. Kadzlcla and Kokocifiski (1966) reported that A C h produccd a decrease in the ratc and amplitude of the heart-beat o f Tegenaria atrica which suggests t h a t the heart-beat o f this spider is myogenic. However, conclusive evidence as to the origin of the heart-beat in T. attica has not been obtained. I n the previous paper the heart-beat o f the tarantula Eurypelma marxi Simon was shown to be neurogenie (Bursey a n d Sherman, z97o ). I t would be o f interest to determine if the heart o f this spider is also slowed b y A C h . Experiments were conducted to determine the effects o f A C h on the E. marxi heart as * Please address all correspondence to Dr. Ralph A. Pax, Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A. I" Present address: Department of Zoology, University of Toronto, 25 Harbord Street, Toronto 5, Ontario, Canada.
w e U as to determine what effects certain chollnergic blocking agents and an anticholincsterasc had on the rcspomc to A C h . Other experiments involving nicotine and methacholinc were carried out to obtain information about the cholinoccptivc sitesin this heart. MATERIALS AND M E T H O D S The method for isolating the heart and the recording apparatus used were described in the first paper of this series (Bursey and Sherman, I97o). For nearly all experiments the preparation consisted of an intact isolated heart pinned dorsal side up in a recording dish which had been filled partially with paraffin, Insect pins (size ooo) were placed at both ends of the heart and along the side opposite to that where the transducer was attached. This served to anchor the heart in place. In certain instances it was advantageous to modify the preparation described above. When the effects of drugs on cardiac ganglion electrical activity were to be studied, the heart was split ventrally by making a longitudinal incision along the entire length of the heart. The heart was pinned out flat, dorsal side up, in the recording dish with pins placed at both ends and sidesof the heart. This held ittightlyto the paraffi and very littlemovement occurred with each contraction. Hearts prepared in this way did not pull away from the recording electrode when solutions bathing the heart were changed.
x 72
SHERMAN AND
A third preparation consisted of a heart from which the cardiac ganglion had been removed, but which was otherwise left intact. The ganglion was easily dissected by simply teasing it free from the external heart surface with forceps. Deganglionated hearts were used to study the effects of drugs on the myocardium, free from any influence of the cardiac ganglion. Each of the drugs was dissolved in spider physiological solution (saline) as shortly before use as possible. Drugs were administered by replacing the saline bathing the heart with the drug solution by means of a syringe. Unless otherwise noted, hearts were exposed to each drug for a period of 3 minutes. After exposure, the rate and amplitude of the heart-beat were allowed to return to the pretreatment level before the next treatment. During this recovery period, which lasted from 5 to 15 minutes, at least 3 and as many as 7 saline washes were made. Only i drug was used on a particular heart unless the interactions of 2 or more drugs were to be investigated. In no case were drugs tested on a heart that had been in isolation for more than 3 hours. Hearts used had heart-rates between 49 and 20 beats per minute during the course of an experiment. If, upon isolation, the initial heartrate was greater than 49 beats per minute, the heart was allowed to beat in saline until the rate had dropped to less than 5° beats per minute. This was done because a greater amount of variability in rates was observed for hearts beating faster than 5° beats per minute than for those with lower rates. For all drugs except ACh heart-rates were measured for the minute just preceding each treatment and for each minute thereafter. In the case of ACh the minute intervals were subdivided into 6 equal intervals of xo seconds each. This was done because ACh has its maximum effect immediately after application and the effect is often a transient one. The amplitude of the heart contractions was measured in terms of rag. ofpuU on the transducer. The heart-beat amplitude was determined for the io beats just prior to the beginning of a drug treatment and for the xo beats immediately after application of a drug. In addition, to consecutive beats were also measured at minute intervals during the drug treatment. RESULTS CONTROL HEARTS T o d e t e r m i n e the a m o u n t o f v a r i a b i l i t y in isolated h e a r t - r a t e s , 5 h e a r t s were a l l o w e d to b e a t in isolation for 3 hours while b a t h e d only i n saline. T h e m e a n h e a r t - r a t e seen i n i t i a l l y was 45 beats p e r m i n u t e a n d t h e r a t e g r a d u a l l y d e c l i n e d to a m e a n r a t e a t the e n d o f 2 4 beats p e r m i n u t e . T h e greatest c h a n g e
PAX
Comp. gen. Pkarmac.
in r a t e f r o m one m i n u t e to the n e x t for a n y h e a r t was -4-2 beats p e r m i n u t e ; usually there was e i t h e r no c h a n g e or a difference o f o n l y -4-i b e a t p e r m i n u t e . M e a s u r e m e n t s o f the v a r i a b i l i t y in h e a r t b e a t a m p l i t u d e were also m a d e for t h e 5 control hearts. Since the a m o u n t o f pull e x e r t e d b y the h e a r t on t h e t r a n s d u c e r is a function o f t h e a m o u n t o f tension p l a c e d on t h e heart, this tension was a d j u s t e d so t h a t the a m p l i t u d e o f the h e a r t - b e a t r e c o r d e d was b e t w e e n 4 ° a n d 6o nag. a t the start o f e a c h c o n t r o l period. A m p l i t u d e m e a s u r e m e n t s were m a d e o f e a c h o f xo consecutive h e a r t b e a t s a t xo - m i n u t e intervals t h r o u g h o u t t h e 3-hour control period. T h e r e was v e r y little v a r i a t i o n b e t w e e n t h e m e a n for one i o - b e a t i n t e r v a l a n d the next. U s u a l l y t h e r e was n o c h a n g e in t h e m e a n a m p l i t u d e . T h e greatest changes ever seen were 4- i o rag. T o d e t e c t w h a t effect t h e p e r i o d i c saline washes m i g h t h a v e on the h e a r t - r a t e , rates were m e a s u r e d for t h e m i n u t e j u s t p r i o r to a n d i m m e d i a t e l y after the c o m p l e t i o n o f e a c h wash t h r o u g h o u t t h e control r e c o r d i n g period. T h e m e a n c h a n g e in r a t e for the 5 h e a r t s was + x b e a t p e r m i n u t e ; t h e largest changes ever seen were - - 2 a n d + 3 beats p e r minute. T h e effect o f the saline washes on h e a r t b e a t a m p l i t u d e was d e t e r m i n e d b y m e a s u r i n g t h e xo beats j u s t p r i o r to a n d i m m e d i a t e l y following e a c h wash. T h e r e was n o m e a n difference b e t w e e n t h e h e a r t - b e a t a m p l i t u d e seen before a n d after t h e washings. T h e greatest a m p l i t u d e changes seen were - - 1 5 a n d + I o rag.
ACETYLCHOLINE T h e response o f t h e h e a r t to x x to -4 M acetylcholine (ACh) consists initially o f a sustained c o n t r a c t i o n w h i c h after a few seconds gives w a y to i n d i v i d u a l b e a t s t h a t occur a t a g r e a t l y increased f r e q u e n c y (Fig. I A). As t h e i n c r e a s e d m y o c a r d i a l tonus declines, t h e a m p l i t u d e o f t h e h e a r t - b e a t increases a n d soon reaches a m a x i m u m . A f t e r the m a x i m u m effects a r e seen, t h e response begins to decline c o n s i d e r a b l y ; however, i n c r e a s e d r a t e a n d a m p l i t u d e a r e seen t h r o u g h out the drug treatment. Upon the removal of
I970, •
CHOLINERGIC
COMPOUNDS
A C h h e a r t activity r e t u r n s to p r e t r e a t m e n t levels w i t h i n 2 - 5 m i n u t e s , d e p e n d i n g directly o n the dose applied. A l t h o u g h the response to A C h declines d u r i n g the course of a single t r e a t m e n t , there is n o decrease i n the
AND
SPIDER
I73
HEART
response to several successive t r e a t m e n t s w h e n the h e a r t is washed with saline for 5 - i 5 m i n u t e s b e t w e e n each one. A dose--response curve for the effect of A C h o n the heart-rate is shown i n Fig. 2. F r o m
A
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iiiiiiiiii,iii,iiiiiiiiiiliiiiiiiiiiiiiiiiiii i lliiiiii!iiiiiiiiiiiiiiiiiiiiliiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii Fxo. i.--Response of the heart t o ACh and eserine. A, Saline removed at first arrow and I × I o - ' M ACh applied at second arrow. B, Saline removed at first arrow and x x xo-s M ACh applied at second arrow. C, Saline removed at first arrow and x x xo-s M eserine applied at second arrow. D, Eserine removed at first arrow and x × xo-5 M ACh applied at second arrow. Time marker equals x second; downward excursion of time marker represents a heart-beat amplitude of 5o mg.
174
Corap. gen. Pharmac.
SHERMAN AND PAX
this figure it appears that concentrations as low as 5 × xo -e M are effective. The effect of A C h on heart-beat amplitude and myocardial tonus is shown in Table L At concentrations of I × I o -5 M and lower, the response, if any, is an increase in heart-beat
response is readily apparent. After ACh and eserine treatments, the time required for the heart-rate and heart-beat amplitude to return to pretreatment levels is nearly three times longer than after ACh alone,
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Fzo. 2.--Dose-response curve for the rate effect of ACh before and after treatment with eserine. Each point represents the mean response to at least 5 replications made on at least 5 different hearts. O, Before eserine; O, After eserine. Vertical lines represent one standard deviation. amplitude. Higher concentrations produce both an increase in beat amplitude and a transient increase in myocardial tonns. ANT~CHOLmmTE~ T o determine if an enzyme might be present which terminates the action of ACh, 8 hearts were treated with ~ × x0 -5 M eserine (physostigmine) for 5 minutes prior to the administration of ACh. Eserine alone produced a mean increase in heart-rate of I I beats per minute and a mean increase in heart-beat amplitude of 40 rag. (Fig. I C). The effect of eserine treatment on the response of the heart to I × xo-~ M ACh is shown in Fig. xB, D. The potentiation of the
A dose-response curve for the effect of ACh on the heart-rate after eserine treatment is shown in Fig. ':,. ACh, in concentrations of 5 × x ° - t M and IXXO- S M , gave rate increases from 3 to 4 times those seen before treatment with eserine. Concentrations which gave no rate increases before eserine, such as x × 10-6 M, gave rate increases after eserine. Concentrations greater than x × io -5 M A C h produced a prolonged tetany of the heart after eserine and were not included in this figure. Eserine also potentiated the effects of ACh on heart-beat amplitude and myocardial tonus (Table I). A greater increase in amplitude probably would have been seen after
I97o, •
t75
CHOLINERGIG COMPOUNDS AND SPIDER HEART
blocked the increased heart-beat amplitude produced by ACh. Three hearts were treated with 5 × 10-4 M C6 for 15 minutes and then with 5 × 10-4 M C6 pins 5 × 10-4 M ACh. C6 alone had little immediate effect on the heart-rate, but in the fifteenth minute after the beginning of the treatment the rate had decreased an average of 8 beats per minute. No change in the
A C h and eserine treatment if the tremendous increase in myocardial tonus had not occurred. CHOLINERGIC BLOCKING AGENTS
Three different compounds which block the actions of A C h in a variety of animals wcrc tested on the tarantula heart. These three blocking agents were atropine sulphate,
Table/.--EFFECTS OF ACETYLCHOLINE AND ESERINE ON H E A R T - B E A T AMPLITUDE AND MYOC, ARDL~L TONUS
DRUG CONCENTRATION
(M)
C H A N G E IN HEART-BEAT AMPLITUDE* (mg.)
INCREASEIN TONUS* (mg.)
No. oF TONE INCREASES
No. OF REPLICATIONS
Acetylcholine I × I 0 -s
5 X IO -6 I X I 0 -5
5 X I O -5 I × I 0 -~' 5 × IO-~
o io 25 40 45 7°
(-io-io) (-5-I5) (IO-55) (IS-IOO) (2o--85) (35-I2O)
0 0 0
5 6
(0-30) 60 (2o-~o5) i~,o (2o-I8O) ~o
IO IO
8 5
Eserine I XIO -6
40 (I5--85)
Acetylcholine after eserine I
X I O -7
5 × i o -7 I X I 0 -6 5 × IO-6 I × I O -5
15 (o--25) I5 (5-25) 25 ( - IO--5o) 2o ( - 15-45) 35 ( - xo--8o)
O
o (o-io) 30 (IO-7O)
50 (io-x2o) 2io (5o-34o)
* Mean and range. hexamethonium chloride (C6), and D-tubocurarine chloride (DTc). Five hearts were treated with I × xo -4 M atropine for 5 minutes and then with I × 10-4 M atropine plus x × 10-4 M ACh. Atropine alone had no immediate effect on the heart-rate, but after 5 minutes a mean decrease in the rate of 5 beats per minute was seen. At this time there was also a mean increase in heart-beat amplitude of 2o nag. After treatment with atropine, the response to ACh was considerably reduced, but not completely abolished. The rate increase due to ACh before atropine averaged 24 beats per minute, while after atropine it was only 6 beats per minute (Fig. 3)- Atropine also
amplitude of the contractions occurred. The effectiveness of A C h in producing an increase in rate was reduced from a mean of 30 beats per minute to i8 beats per minute by treatment with C6 (Fig. 4). The amplitude response to A C h was not affected. v T c at I × 10-4 M was applied to 3 hearts for 3° minutes and then I × t 0 -4 M DTc plus x × IO-4 M A C h were administered. DTc alone produced an initial mean increase in heart-rate of 4 beats per minute, but after 3° minutes the heart-rate had decreased an average of 9 beats per minute. No change in heart-beat amplitude was seen. v T c reduced the rate response to A G h in 2 hearts from 3 ° to 12 beats per minute; in the third heart the
x76
SHERMAN AND PAX
r a t e response was r e d u c e d from 30 to t8 beats per m i n u t e . T h e increased h e a r t - b e a t a m p l i t u d e p r o d u c e d b y A C h was n o t affected. T h e records shown i n Fig. 4 for C6 are essentially the same as those o b t a i n e d i n the experim e n t s done with DTc.
Comp. gen. Pharmac.
SXTS o r A C ~ ACTION T h e r e are p r i n c i p a l l y two locations where A C h could be a c t i n g to p r o d u c e changes i n h e a r t activity; the c a r d i a c g a n g l i o n a n d the h e a r t m u s c u l a t u r e . T o d e t e r m i n e if A C h has a direct effect o n t h e m y o c a r d i u m , it was
A
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iiiiiiiiiiiii,,i,iiiiii,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiijitiiiiiiiiiifii,iiifiiiif iiiiiiiiiiiiiiiiiiiJiiiJij Fzo. 3.--Effect of atropine on the response to ACh. A, Saline removed at first arrow and x × Io -4 M AC'h applied at second arrow. B, Saline removed at first arrow and I x zo -* M atropine applied at second arrow. C, Atropine removed at first arrow after a 5-minute treatment and ~ x I o - ' M ACh plus z x xo -* M atropine applied at second arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5° ms.
I97o, x
CHOLINEROIG COMPOUNDS AND SPIDER HEART
applied to the heart after removal of the cardiac ganglion. A C h did not evoke a recordable change in the myocardial tonus of deganglionated hearts. Thus the response of the heart to A C h appears to be due to its actions on the cardiac ganglion. T o demonstrate more clearly that the site of action is the cardiac ganglion, the electrical activity of the ganglion was monitored while
I77
A C h was administered to intact hearts that had been treated with eserine. The increase in ganglionic activity produced by such a treatment is readily observable (Fig. 5A, B, C). It is apparently the continuous firing of the ganglion which produces the sustained contractions seen irt the mechanical recordings of eserine and A C h treated hearts
(Fig. ID).
A
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tt •iii••••iiiiiiiiiiiiiiiiiiiii•••i•••iiiiiii•••i•i•iiiiiiii•iiiii•iiiiiiiiiii•••ii•iiiiiiii•••••iiiii•iii••iiiiiiiiii•ii•iiiiiiiiii•i•ii•i••• C
¢¢ iiiiiiiii•iiiii•iiiiiiiiiiiiiii•••iiiimmiiiiiiiiiii••••iiiiiiiiiiiiiiiiiiiiiiiii•i••iii•iiiii••i••iiiii••iiiiii•iiiiiiiiiiiii•iiiiiiiiii FIG. 4.--Effect of C6 on the response to ACh. A, Saline removed at first arrow and 5 × Io-' M A C h applied at second arrow. B, Saline removed at first arrow and 5 × I o - ' M C6 applied at second arrow. C, C6 removed at first arrow after a I5-minute treatment and 5 × xo-4 M ACh plus 5 × I o - ' M C6 applied at second arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5o nag.
17 8
Comp.gen. Pharmac.
SHERMAN AND ]PAX
1
FIo. 5.--Effects of cholinergic compounds on cardiac ganglion electrical activity. A, Burst frequency in saline. B, Io seconds after application of I × ro-s M A C h . C, ro seconds after application of I × ro-s M A C h after treatment with eserlne. D, ro seconds after application of x × IO-5 M A C h plus i × ro-~ M atropine after a 5-mlnute atropine treatment of the same heart as in C. E, Burst frequency in saline (different heart). F, IO seconds after application of 5 x ro-5 M methacholine. G, Burst frequency in saline (different heart). H, io seconds after application of 5 × x°-5 M nicotine. Vertical llne equals 3oo ~tV. ; horizontal line equals 2 seconds.
I970, •
z79
C H O L I N E R G I C COMPOUNDS AND SPIDER H E A R T
T o d e m o n s t r a t e t h a t the action o f the blocking agents also occurs in t h e c a r d i a c ganglion, t h e response o f the g a n g l i o n to A C h
was m o n i t o r e d before a n d after t r e a t m e n t w i t h at r o p i n e. As shown in Fig. 5 C, D, treatment with atropine blocked the
A
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+ + iiiiiiiiiiiiiiiiiiilliiiiiiiiiitiiiiiiiiit~iiiiiiiiiiiiiiiiiiiiiiiiiii11tll]iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiill Fzo. 6.--Response of the heart to methacholine and atropine. A, Saline removed at first arrow and I x io-4 Mmethacholine applied at second arrow. B, Saline removed at first arrow and 5 x zo-5 Mmethacholine applied at second arrow. C, Saline removed at first arrow gnd z × IO-4 M atropine applied at second arrow. D, Atropine removed after a 5-minute treatment at first arrow and 5 x IO-6 M methacholine plus I x Io -4 M atropine applied at second arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5o rag.
180
SHERMAN AND PAX
response of the eserinized cardiac ganglion to ACh. METHACHOLINE
Methacholine (acetyl-beta-methylcholine) chloride, a mnscarinic cholinomimetic, at concentrations from t × 10-6 M to t × i o -* M produced an increase in the rate and amplitude of the heart-beat, as well as a transient increase in myocardial tonus (Fig. 6A). In contrast to ACh, the response to methacholine did not diminish during the course of a single treatment. After a treatment, the rate and amplitude of the heart-beat returned to control levels within 5 minutes. The effect of methacholine on heart-beat amplitude was more pronounced than the effect on the heart-rate. For example, i × io -4 m methacholine produced a mean rate increase of 12 beats per minute and a mean increase in heart-beat amplitude of 5° rag. (6 replications, 2 on each of 3 hearts). A C h at the same concentration gave a mean increase in heart-rate of 3° beats per minute and a mean increase in heart-beat amplitude of 45 nag. Thus, the effect of methacholine on heart-beat amplitude is comparable to that of ACh, while the effect on the heart-rate is considerably less. The site of methacholine action was investigated in the same manner as was ACh. Methacholine, like ACh, did not produce any recordable change in myocardial tonns when applied to deganglionated hearts. The effect of methacholine on cardiac ganglion electrical activity is shown in Fig. 5 E, F. Both an increase in the rate of bursting and an increase in the amount of activity in each burst were seen upon application of methacholine. T h e p o t e n t i a t i o n of the ACh effect by eserine indicates that a cholinesterase is present in the cardiac ganglion. Since methacholine differs in structure from ACh only by the presence of a methyl group at the beta position, it was of interest to know if eserine would also potentiate the action of methacholine. Treatment of a heart with i × lO-5 M eserine for I o minutes did not appreciably enhance the effects of methacholine on the heart. The rate increase produced by 5 × I o - S M methacholine before
Corap. gen. Pharma¢.
eserine was 13 beats per minute, while afterward it was 15 beats per minute. There was no increase in the amplitude response to methacholine after treatment with eserine. Atropine and C6 were also tested to see if they would block the effects of methacholine on the heart. Treatments with these compounds were identical with those in the experiments involving ACh. Atropine effectively blocked both the rate and amplitude response produced by methacholine. The mean increase in rate evoked by 5 × I o - s M methacholine before atropine was i2 beats per minute, while after atropine it was only i beat per minute (4 hearts) (Fig. 6). No increase in heart-beat amplitude occurred after atropine treatment. C6, on the other hand, did not alter the response of the heart to methacholine. The mean rate increase before C6 was 14 beats per minute, while afterwards it was I6 beats per minute (3 hearts). Also, there was no change in the amplitude response. NICOTINE
Nicotine produced an immediate increase in heart-rate when applied in concentrations from I × I O - e M to 5 × I o - 5 M (5 hearts). The effect of nicotine on heart-beat amplitude was variable. In a few instances nicotine produced a slight increase in amplitude (about IO mg.), but in other instances either there was no change or else a decrease. The latter case was particularly true for concentrations higher than I X lO-5 M (Fig. 7D). The response of one heart to I × 10-5 M nicotine is shown in Fig. 7A, where an increase in the rate of 2 7 beats per minute occurred, with little change in heart-beat amplitude. The heart apparently becomes desensitized to nicotine, for soon after its administration the response starts to decline and when low doses are applied (i.e., i × io -e M) the rate often returns to pretreatment levels during the course of the treatment. Furthermore, upon repeated treatments with the same concentration of nicotine the response becomes progressively less. Nicotine did not produce any recordable change in myocardial tonus when applied to
I970 , I
18I
C H O L I N E R O I C COMPOUNDS AND SPIDER H E A R T
d e g a n g l i o n a t e d hearts. W h e n 5 × I O -6 M nicotine was a p p l i e d to i n t a c t hearts, a n increase i n cardiac g a n g l i o n electrical activity was seen, b u t the activity consisted o f small a n d irregular bursts (Fig. 5G, I-I).
T h u s , the site of action of nicotine is also the cardiac ganglion. A t r o p i n e a n d C6 were also tested for their ability to block the effect of nicotine o n the heart. T r e a t m e n t s with these c o m p o u n d s
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Fro. 7.--Response of the heart to nicotine and C6. A, Saline removed at first arrow and x × to -5 M nicotine applied at second arrow. B, Saline removed at first arrow and z x Io -~ M 0_,6applied at second arrow. C, C6 removed after a x5-minute treatment at first arrow and z × lo -6 M nicotine plus z × Io -4 M C6 applied at second arrow. D, Saline removed at first arrow and 5 x io -s M nicotine applied at second arrow (different heart). Time marker equals I second; downward excursion of time marker represents a heart-beat amplitude of 5° mg.
I82
SHERMAN AND PAX
w e r e the same as for A C h . C6 r e d u c e d the r a t e response p r o d u c e d by i X IO-5 M nicotine f r o m a m e a n increase o f 19 beats p e r m i n u t e to 8 beats p e r m i n u t e (3 hearts) (Fig. 7).
Comp. gen. Pharmac.
A t r o p i n e d i d n o t alter the response to nicotine in 3 o f 5 hearts. I n the o t h er 2, the m e a n rate increase p r o d u c e d by nicotine was r e d u c e d f r o m x5 to 7 beats per m i n u t e .
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FIO. 8.--Additive effects of nicotine and methacholine on the heart. A, Saline removed at first arrow and 5 x i o - ' M nicotine applied at second arrow. B, Saline removed at first arrow and 5 x i o - ' M methacholine applied at second arrow. C, Saline removed at first arrow and 5 x Io -6 M nicotine plus 5 x to -6 M methacholine applied at second arrow. D, Atropine removed after a 5-minute treatment at first arrow and 5 × Io-6 M nicotine plus 5 x I o - ' M methacholine plus i x t o - ' M atropine applied at second arrow. Time marker equals x second; downward excursion of time marker represents a heartbeat amplitude of5o mg.
x97o , x
C H O L I N E R G I C COMPOUNDS A N D SPIDER H E A R T
I83
Although there appears to be a partial blocking of the nicotine effect by atropine in these 2 cases, a greater than usual densensitization of the heart m a y have occurred.
that atropine selectively blocked the action of methacholine while leaving that of nicotine unaffected.
NICOTINE AND ~ETHACHOLINE T h e principal effects of nicotine and methachoLine on the heart are quite different and it m a y be that these drugs are acting at different sites in the cardiac ganglion. I f this is the case, the individual effects of these cholinomimetics should sum when both are applied to the heart at the same time. T o test this hypothesis, a heart was treated first with nicotine, then methacholine, and then with both of t h e m together. Fig. 8 shows the results of this experiment. Nicotine alone in a concentration of 5 × IO-e M produced an increase in the rate of 2o beats per minute. Little change in heart-beat amplitude occurred, but there was a small transient increase in tonus (Fig. 8A). Methacholine alone, at 5 x I o -5 M, produced an increase of 6 beats per minute in the rate and a pronounced increase in the amplitude of the heart-beat (about 5o rag.) (Fig. 8B). When both compounds were applied together, both a large increase in rate (2 x beats per minute) and in beat amplitude (4o rag.) were seen (Fig. 8C). The rate increase was greater than 3 times that seen for methacholine alone (2 x in comparison with 6 beats per minute). T o further investigate the additive effects of methacholine and nicotine, this same heart was treated with I x IO-4 M atropine for 5 minutes. T h e same doses of nicotine and methacholine as were used above were then applied together with I × I O-4 M atropine. T h e results of these treatments are shown in Fig. 8D. Before atropine, nicotine and methacholine, when applied together, produced a rate increase of 21 beats per minute and an increase in both heart-beat amplitude and myocardial tonns. After atropine, the simultaneous treatment with nicotine and methacholine produced a rate increase of 15 beats per minute, with no increase in either heart-beat amplitude or myocardial tonus. Since methacholine alone initially produced a rate increase of 6 beats per minute and an increase in heart-beat amplitude, it appears
DISCUSSION The observation that the response to A C h declines during the course of a treatment indicates that the heart either metabolizes A C h or becomes desensitized to it. Since successive treatments with A C h are equally effective, the decline in response is not due to desensitization. When treatment with eserine, a potent antichoLinesterase, precedes treatment with ACh, no decline in response occurs; in fact, the effects of A C h are greatly enhanced. These results indicate that a cholinesterase which metabolizes ACh is present in the cardiac ganglion. The specificity of the cholinesterasc was investigated by the experiment involving eserine and methachoUne. T r e a t m e n t with eserine did not appreciably increase the effectiveness of methacholine. Thus it appears that the cholinesterase present is quite specific for ACh. T h e results of the experiments involving cholinomimetics and cholinergic blocking agents indicate that the action of A C h on the heart is not a simple one and that A C h is probably acting on more than one kind of receptor site in the cardiac ganglion. Nicotine mimics the rate-changing effects of ACh, but not the heart-beat amplitude effects. Methacholine mimics the heart-beat amplitude effects of ACh, but only partially mimics the rate effects. T h e effects of nicotine are reduced considerably by C6, but not by atropine; the effects of methacholine are blocked by atropine, but not by C6. W h e n nicotine and methachoLine are applied simultaneously to the heart a response occurs which is greater than that seen to either of t h e m alone. Thus it appears that nicotine and methacholine are acting at different sites in the cardiac ganglion and that both nicotinic and muscarinic receptor sites for A C h are present. I n a previous paper it was shown that different nerve-cell types can be distinguished on a morphological basis within the cardiac ganglion of E. marxi (Bursey and Sherman,
I84
SHERMAN AND PAX
I97o ) . I f these morphological cell types are the abdomen is quite small, a drug injection indicative of a separation of the cells into made anywhere in the abdomen might propacemaker and follower cells as is the case for duce a considerable increase in internal the lobster cardiac ganglion (Maynard, x955; pressure. Such a pressure increase could lead Hartline, I967) then, according to the to a decrease in heart-rate as well as a results obtained in this study, the nicotinic decrease in heart-beat amplitude. Although receptor sites would be located on the pace- the authors state that ' test injections did not maker cells and the muscarinic sites would be produce any change in the action of the located for the most part on the follower cells. heart ', the upward shift in the baseline of However, since methacholine produces a their records during the time that the heartmoderate increase in heart-rate and since rate was reduced indicates that a change in atropine reduces the rate response produced pressure m a y have occurred as a result of the by ACh, some of the muscariuic sites m a y injection. also be located on pacemaker cells. An examination of the effects of ACh on I n the only other pharmacological study of isolated heart preparations of Tegenaria atrica spider hearts it was reported by Kadziela and is necessary to determine whether the differKokocifiski (i966) that A C h produced a ences in results reported are due to species decrease in the rate and amplitude of the differences or to differences in experimental heart-beat of Tegenaria atrica. These results methods. are exactly opposite to those obtained in this study for the tarantula Eurypelma marxi. ACKNOWLEDGEMENTS While it is possible that the differences in This investigation was supported by a results are due to species differences, it seems U.S.P.H.S. predoctoral fellowship from the just as possible that the differences are due to Institute of General Medical Sciences to R. G. and by a U.S.P.H.S. research grant from the totally different manner in which the two Sherman the National Heart Institute to R. A. Pax. studies were made. In this regard, a number Critical reading of the manuscript by Dr. H. L. of problems seem apparent in the methods Atwood is gratefully acknowledged. used by Kadziela and Kokocifiski (1966). By using an in vivo heart preparation, the conREFERENCES centration of drug acting on the heart is not BURSEY, C. R., and SHm~r~AN, R. G. (I97O), 'Spider cardiac physiology, I. Structure and the same as that injected, since some dilution function of the cardiac ganglion ', Comp. gen. must occur due to the blood present. Since Pharmac., x, I6o--x7o. they supposedly only recorded electrical HAnTLm'E, D. K. (x967) , ' Impulse identification events and not mechanical events, there is and axon mapping of the nine neurons in the cardiac ganglion of the lobster Homarus araericonsiderable doubt about the validity of canus ', o7. exp. Biol., 47, 327-34 °. their statements concerning heart-beat amplitudes. However, their recordings of 'the KA~zm~, W., and KoKocn~srJ, W. (i966), ' The effect of some neurohormones on the heart-rate action potentials of the heart ' m a y in fact be of spiders ', Experientia, ~ , 45-46. only movement artefacts, since their record- MAYNARD, D. M. (x955) , ' Activity in a crustacean ganglion, II. Pattern and interaction in burst ings, made by inserting metal electrodes into formation ', Biol. Bull. mar. biol. Lab., Woods the abdomen, are quite unlike the electrical Hole, xog, 40o-436. recordings reported for other spider hearts PROSSER, C. L. (x961), ' Circulation of body (Sherman and Pax, I968; Bursey and fluids ', in Comparative Animal Physiology (ed. Paos~mR, C. L., and BROWN,F. A.), and ed., pp. Sherman, x97o ). 386-4x6. Philadelphia: Saunders. Another serious problem in using an in vivo R. G., and PAX, R. A. (x968), ' T h e heart preparation in spiders is that by inject- SHEn~mN, heart-beat of the spider, Geolycosa missouriensis ', ing solutions into the abdomen the volume Comp. Biochem. Physiol., 26~ 529--536. injected alone could produce a change in Key Word Index: Spider heart, spider cardiac heart-rate and heart-beat amplitude. Since ganglion, tarantula, Eut.ypelma marxi, acetylthe abdomen is essentially a closed system choline, eserine, atropine, hexamethonium, nand since it would seem that the volume of tubocurarine, methacholine, nicotine.