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Spider cardiac physiology, III. responses of a tarantula heart to certain catecholamines, amino-acids, and 5-hydroxytryptamine
Comp. gen. Pharmac., x97 o, x, I 8 5 - t 9 5
I8 5
SPIDER CARDIAC PHYSIOLOGY, III. RESPONSES OF A TARANTULA HEART TO CERTAIN CATECHOLAMINES, AMINO-ACIDS, AND 5-HYDROXYTRYPTAMINE* ROBERT
G. S H E R M A N ~
AND R A L P H
A. P A X
Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A.
(Recdved x2 Dec., x969) ABSTRACT x. Epinephrine and norepinephrine at concentrations greater than x × to -6 M produce an increase in heart-rate and heart-beat amplitude by their actions on the cardiac ganglion. 2. Gamma aminobutyric acid at concentrations greater than x × xo-5 M produces a decrease in heart-rate and a dual phased heart-beat amplitude response (decrease, then an increase) by its actions on the cardiac ganglion. 3. D-Glutamate at concentrations greater than x × xo - ' M produces an increase in heart-rate and a decrease in heart-beat amplitude by its actions on the cardiac ganglion. 4. L-Glutamate at concentrations greater than x × xo-4 M has little effect on heart-rate, but produces an increase in heart-beat amplitude by a direct action on the myocardium. 5. 5-Hydr°xytryp tamine at concentrations greater than x x xo-' M evokes a decrease in heart-rate and heart-beat amplitude by its actions on the cardiac ganglion. 6. v-Aspartate, L-aspartate, and glycine, in concentrations up to x × xo-* M, have no appreciable effects on heart-rate and heart-beat amplitude. IN t h e p r e c e d i n g p a p e r it was shown t h a t acetylcholine p r o d u c e s a n increase in the r a t e a n d a m p l i t u d e o f t h e h e a r t - b e a t o f the spider Eurypelma marxi S i m o n ( S h e r m a n a n d Pax, x97o ). I n t h e e x p e r i m e n t s p r e s e n t e d here e p i n e p h r i n e a n d n o r e p i n e p h r i n e , selected a m i n o - a c i d s , a n d 5 - h y d r o x y t r y p t a m i n e have b e e n e x a m i n e d for t h e i r effects o n isolated h e a r t p r e p a r a t i o n s o f the spider E. marxi. T h e p r i n c i p a l a i m o f this s t u d y was to determ i n e i f a n y o f these c o m p o u n d s m i g h t b e considered as possible n e u r o t r a n s m i t t e r s in s p i d e r hearts. A t the s a m e time it was h o p e d t h a t sufficient i n f o r m a t i o n w o u l d be o b t a i n e d a b o u t the p h a r m a c o l o g y o f this h e a r t to permit comparisons with other neurogenic hearts. * Please address all correspondence to Dr. Ralph A. Pax, Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A. t Present address: Department of Zoology, University of Toronto, 25 Harbord Street, Toronto 5, Ontario, Canada.
M A T E R I A L S AND M E T H O D S The materials and methods used in this study are the same as those described previously (Sherman and Pax, I97o ). RESULTS
CATECHOLAMINES The catecholamines, epinephrine hydrochloride and norepinephrine hydrochloride, e a c h were a p p l i e d to 5 different h e a r t s a t c o n c e n t r a t i o n s r a n g i n g f r o m I x 10 -6 M to x × to--4 M . B o t h p r o d u c e d a n increase in heart-rate and heart-beat amplitude, and at c o n c e n t r a t i o n s g r e a t e r t h a n I × IO-5 M a t r a n s i e n t increase in m y o c a r d i a l tonns (Fig. x). T h e responses to these drugs a r e i m m e d i a t e , b u t the m a x i m u m effects o c c u r a b o u t 45 seconds after d r u g a p p l i c a t i o n . O n c e t h e m a x i m u m increase is seen, t h e r a t e a n d a m p l i t u d e o f t h e c o n t r a c t i o n s r e m a i n quite constant for the d u r a t i o n o f t h e t r e a t m e n t . U p o n d r u g r e m o v a l t h e y b e g i n to decrease g r a d u a l l y u n t i l p r e t r e a t m e n t levels a r e seen from IO to 15 m i n u t e s later.
I86
Comp. gen. Pharmac.
SHERMAN AND PAX
A d o s e - r e s p o n s e curve for the effect o f e p i n e p h r i n e on t h e h e a r t - r a t e is shown in Fig. ~. T h e d o s e - r e s p o n s e curve for n o r e p i n e p h r i n e is essentially t h e same. T h r e s h o l d c o n c e n t r a t i o n for the effect o f b o t h drugs on
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the h e a r t - r a t e effects o f these amplitude and Table I. T h e e p i n e p h r i n e is
is a b o u t I X I o - 6 M . The c a t e c h o l a m i n e s on h e a r t - b e a t m y o c a r d i a l tonus are listed in t h r e s h o l d c o n c e n t r a t i o n for a b o u t 5 x I o -6 M , w h i l e t h a t
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:FIG. i.--Response of the heart to epinephrine. The response to norepinephrine is essentially the same. Continuous record from top left to bottom right. Saline removed at first arrow and 5 x I o - s 5 I epinephrine applied at the second arrow. Time marker equals I second; downward excursion of time marker represents a heart-beat amplitude of 50 mg.
I97O, x
CATEGHOLAMINESAND SPIDER HEART
for norcpinephrinc is approximately z X z0 -5 M . In general, norepinephrine does not have quite as great an effectas does epinephrine. The site of action of epinephrine and norepinephrinc was investigated by applying each of them to dcganglionatcd hearts while monitoring changes in myocardial tonns and by recording the cardiac ganglion electrical
I87
the heart-beat (Fig. 4). T h e maximum inhibitory effect occurs within zo seconds of administration. Several seconds after the ma~im~tm effects of GABA are seen, the heart-rate begins to return to the prctreatment rate, but it does not completely recover as long as GABA is on the heart. Concurrently with the recovery in rate, and even
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Fro. 2.~Dme--response curve for the effect of epinephrine on the heart-rate. Each point represents the mean response to 7 replicatiom made on at least 4 different hearts. Vertical lines represent one standard deviation.
activity of intact hearts while these drugs wcrc on the heart. Both catecholamlncs, in concentrations as great as x X xo "4 M , produced no rccordable changes in the myocardial tonus of deganglionated hearts, but both increased the frequency of ganglionic bursts as well as the amount of activltyin each burst when applied to intact hearts (Fig. 3 A,
S). G A M M A AMINOBUTYRIG ACID Gamma aminobutyric acid ( G A B A ) at concentrations greater than I × xo -5 M produccs a decrease in the rate and amplitude of
more striking, the amplitude of the heart-beat begins to increase and soon exceeds the amplitude seen before the treatment was
begun. Upon removal of GABA a small further increase in heart-beat amplitude occurs, but this increase is only a transient one. In all cases both the rate and amplitude returned to prctreatrncnt levels within 6 minutes of the end of the treatment. A dose-response curve for the effect of G A B A on the heart-rate is shown in Fig. 5. T h e threshold concentration required to produce a decrease in the heart-rate is
188
SHERMAN AND PAX
Comp. gen. Pharraac.
J Fla. 3.--Effects of epinephrine and GABA on cardiac ganglion electrical activity. A, Burst frequency in saline. B, xo seconds after administration of 5 × Io-~ M epinephrine. C, Burst frequency in saline. D, xo seconds after application of 5 × io-~ M GABA. t:, Same as D, except ao seconds later. F, Io seconds after GABA had been replaced by saline.
I970 , I
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CATECHOLAMINES AND SPIDER HEART
change in myocardial tonus, but when applied to intact hearts it causes sporadic firing of the cardiac ganglion along with occasional bursts of prolonged duration and reduced amplitude (Fig. 3D). After about 3o seconds in G A B A , co-ordinated bursts at
5 × I 0 -s M. The effect of various concentrations of G A B A on the amplitude of the heart-beat is shown in Table IL Each number represents the m c a n change in heart-beat amplitude from the control. This was determined by averaging the amplitudes of the
Table L ~ E F F E C T S OF EPINEPHRINE AND NOREPINEPHRINE ON HEART-BEAT AMPLITUDE
AND
MYOCARDIAL TONUB DRUG CONCENTRATION
(M)
CHANGE IN I-IEART-BEAT AMPLITUDE*
INCREASE IN Tom~s
N o . oF
(mg.)
INCREASES
(rag.)
ToNE
N o . oF REPLICATIONS
Epinephrine I XIO -6
io
(-Io-25)
5 X 10 -e I × I O -5
3° 55 65 85
(m-55) (3°-I35) (3o-I I5) (3o--175)
5 X IO -5 I X IO - i
0
xo (o-4o)
40 (o--4o) 8o (0--220) IO0 (30--200)
Norepinephrine
*
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I × IO - s 5 × xo-S I X I o -5
IO ( - - IO--35 )
5 x i o -s
30 (o-95)
io (o--3o) 45 (o--80)
I XIO -t
45 (15-95)
9° (4(>-xoo)
0 0
I5 (o-55)
Mean and range.
Table II.--EFFECT OF GAMMA AMINOBUTYRIC Acre ON HEART-BEAT AMPLITUDE CONCENTRATION
(M)
~tEAN CHANGE IN AMPLITUDEFROM CONTROL (rag.)
First Minute I XIO -5
5
Second Minute
O
×Io-5 i Xio -4
--5
5 ×1o-4
--3 °
--20
first IO beats that occurred in each of the 3 minutes that GABA was on the heart and comparing these values with the m e a n heart-beat amplitude for the io beats that occurred just prior to GABA application. T h e threshold dose required to produce a change in heart-beat amplitude is about 5 × to -s M. T h e dual phased nature of the amplitude response is readily apparent from the table. When applied to deganglionated hearts, GABA does not produce any recordable
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an increased rate appear (Fig. 3E) and a few seconds after removal of G A B A , the burst amplitude exceeds that seen before the G A B A treatment was begun (Fig. 3F). Since these responses arc comparable to those seen in the mechanical recordings (Fig. 4), it is clear that the various effectsof G A B A arc duc to its actions on the cardiac ganglion. P m R O T O X ~ AND G A B A Picrotoxin blocks the effects of G A B A on the ncurogenlc hearts of decapod crustaceans
I9o
SHERMAN A N D P A X
(Florey, i957) , b u t n o t on t h e n e u r o g e n i c
Liraulus polypheraus h e a r t ( P a x a n d S a n b o r n , I967a). F r o m a c o m p a r a t i v e s t a n d p o i n t it w o u l d b e o f interest to k n o w i f p i c r o t o x i n blocks the effects o f G A B A on t h e n e u r o g e n i c E. marxi heart. F o u r h e a r t s w e r e t r e a t e d for u p to 3 ° m i n u t e s w i t h 5 × t o - 4 M p i c r o t o x i n a n d t h e n w i t h 5 × I o-~ M p i c r o t o x i n plus G A B A in c o n c e n t r a t i o n s r a n g i n g f r o m
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Comp. gen. Pharmac.
5 × t 0 -6 M to 5 × 10 -4 M . P i c r o t o x i n a l o n e h a d no effect on t h e heart. F u r t h e r m o r e , in no case d i d p i c r o t o x i n r e d u c e t h e effectiveness o f G A B A . OTHER AMINO-ACIDS D - G l u t a m i c a c i d a t c o n c e n t r a t i o n s u p to I × xo-4 M h a d little effect on the heart. A t 5 × I ° - * M it p r o d u c e d a n a v e r a g e increase
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197o, x
CATECHOLAMINESA N D
in heart-rate of 12 beats per minute (3 hearts) and a decrease in the amplitude of the contractions of about 3o m g . At I X I O - s M these effects were even more pronounced, often resulting in such weak contractions that the heart-rate could not be measured accurately. L-Glutamic acid had no appreciable effect on the heart at concentrations up to
0 n
191
in a few instances, but only at concentrations of I X 1 0 - 4 M . O f the amino-acids used, D-glutamate was the only one other than GABA that had an appreciable effect on the electrical activity of the cardiac ganglion. The response of the ganglion to D-glutamate consisted of a decrease in the burst amplitude and a general appearance of asynchronous firing.
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mean increase in heart-beat amplitude of 3 o m g • (3 hearts); this effect was even greater at I x xo-a M. Only rate increases less than 5 beats per minute occurred at any of the concentrations used. D- and L-Aspartic acid and glycine usually produced only slight increases in heart-rate and heart-beat amplitude at concentrations ranging from I X I O - 5 M t o I X I O - 8 M ( 3 hearts). Increases in heart-rate greater than 5 beats per minute and increases in heartbeat amplitude greater than I o nag. were seen
When applied to deganglionated hearts, none of the amino-acids except L-glutamate produced any recordable change in myocardial tonus at concentrations up to I X I O -s M. L-Glutamate caused a sustained contraction of the deganglionated myocardium at concentrations of I X IO-4 M to I × I o ~ M ( F i g . 6). Each contraction lasted for the duration of the drug treatment. U p o n removal of the L-glutamate the contraction gradually subsided over a period of several minutes until the control myocardial tonus was reached.
x92
SHERMAN AND PAX
A number of small oscillatory contractions occur in the record of the response to L-glutamate. Such contractions were seen in a n u m b e r of hearts (but not all) about I5-3o minutes after removal of the cardiac ganglion, a procedure which causes an immediate cessation of the heart-beat (Bursey and Sherman, x97o). These contractions took the form of small twitches which occurred in localized areas of the heart. The significance of these uncoordinated contractions is not
Comp. gen. Pharmac.
5 - H T at 5 × IO-s M produced a decrease in the amplitude of the ganglionic bursts and a slight decrease in burst frequency when administered to intact hearts. At the same concentration it h a d no recordable effect on the myocardial tonus of deganglionated hearts. DISCUSSION The catecholamines, epinephrine and norepinephrine, are the most effective compounds
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FIO. 6.--Effect of L-glutamate on the myocardial tonus of a deganglionated heart. Saline removed at first arrow and 5 x to -t M L-glutamate'applied at second arrow. L-Glutamate removed at third arrow and saline applied at fourth arrow. Time marker equals x second; downward excursion of time marker represents a change in tension of ~5 mg. clear, but they perhaps are analogous to the contractions reported by Carlson (I9O4, 1907) to occur in the deganglionated heart of the horseshoe crab (Limulus polypt~mus) when it is placed in an isotonic sodium chloride solution. 5-HYDROXYTRYPTAMINE
5-Hydroxytryptamine (5-HT) was applied to 4 hearts at concentrations ranging from x × lO -6 M to 5 × I°-3 M. Only occasional small changes in the rate and amplitude of the heart-beat were seen at concentrations up to I × I o -s M. At 5 × 10 -8 M the amplitude of the heart-beat was reduced to the point where it was barely recordable (Fig. 7). At the same concentration the heart-rate was reduced by about 13 beats per minute. These effects were constant for the duration of the treatment. Within a few seconds after removal o f 5 - H T a striking increase in heartbeat amplitude occurred which resulted in an amplitude nearly twice that of the control. This increase was only transitory and within 5 minutes after the end of the treatment the amplitude as well as the heart-rate had returned to pretreatment levels.
of those tested in producing an increase in the rate or amplitude of the E. marxi heart-beat. These effects are comparable to those of ACh on this same heart (Sherman and Pax, 197o ) . However, the time course of the response to these catecholamines is quite different from that of ACh. Unlike ACh, once the m a x i m u m response to epinephrine and norepinephrine is seen, there is little change in the response for as long as the drugs are on the heart, and upon their removal the response only gradually diminishes. Such a time course could be explained by the assumption that a rapid enzymatic process for the termination of the actions of epinephrine and norepinephrine is either absent or else is not involved to any great extent in this heart. The lack of involvement of such a process for terminating the actions of catecholamines has been reported for a number of other animals (Koelle, I965). L-Glutamic acid has been proposed as the excitatory transmitter at crustacean (Takeuchi and Takeuchi, x964) and insect (Ber~-mk and Miller, I968 ) neuromuscular junctions. Since L-glutamate was the only compound used in this study to produce
I970 , X
CATECHOLAMINES
recordable contractions of deganglionated hearts, it seems possible that it might be involved in neuromuscular transmission in spider hearts. GABA is the most effective compound of those tested in producing a decrease in the heart-rate and heart-beat amplitude. Perhaps the most striking effect of GABA is the
AND
SPIDER
HEART
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dual phased heart-beat amplitude response. The initial reduction in amplitude appears to be due to a decrease in the synchronization of the cardiac ganglion impulses, as suggested by a prolongation of the bursts and reduced burst amplitudes. As apparent resynchronization occurs, as suggested by the appearance of bursts having shorter durations
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FIG. 7.--Response of the heart to 5-HT. Continuous record from top left to bottom right. Saline removed at first arrow and 5 x IO-a M 5-HT applied at second arrow. 5-HT removed at third arrow and saline applied at fourth arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5° mg.
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Comp. gen. Pharmae.
SHERMAN AND PAX
and increased amplitudes, the heart-beat amplitude is seen to be larger. The occurrence of the dual phased amplitude response and the partial recovery of the reduced heart-rate during GABA treatment indicate that the heart may become desensitized to GABA. An alternate explanation is that GABA may be metabolized by the cardiac ganglion, thereby reducing its inhibitory effects on the heart. Either of these possibilities could lead to a re-synchronization of the cardiac ganglion impulses, but additional information is required before definitive statements can be made in this regard. The results presented here and in the preceding paper (Sherman and Pax, 197o ) provide a basis for comparing the pharmacology of a spider heart with that of other neurogenic hearts. The pharmacology of only a few neurogenic hearts is sufficiently well characterized to permit meaningful comparisons. These include the hearts of decapod crustaceans, certain scorpions, and Limulus polyphemus. The E. marxi heart appears similar to these hearts with respect to its responses to catecholamines and to GABA. Catecholamines are excitatory in all of the above hearts; GABA is known to be inhibitory in all, except in scorpions where it has yet to be tested (GmTey, i922 ; Bain, I929; Welsh, i939b ; Kanungo, 1957 ; Florey, I957 ; Maynard, 196I; Pax and Sanborn, x967a; Abbott, Lang, and Parnas, 1969). The effects of 5 - H T on neurogenic hearts are not so uniform. It is not particularly effective on the E. marxi heart, has marked inhibitory effects on the heart ofL. polyphemus, and has pronounced excitatory effects on the hearts of deeapod crustaceans (Florey and Florey, I954; Maynard and Welsh, I959; Kerkut and Price, I964; Belamarich and Terwilligcr, I966; Cooke, I966; Pax and Sanborn, I967b ). Acetylcholine also has different effects on neurogenic hearts. It is excitatory on the hearts of decapod crustaceans, L. polyphemus, and E. marxi, but it is inhibitory on the hearts of scorpions. Eserine potentiates the effects of ACh on the hearts of decapod crustaceans, L. polyphemus, E. marxi, and one scorpion, but it apparently
does not potentiate the effects of ACh on another scorpion. Atropine blocks the actions of ACh on the hearts of decapod crustaceans and E. marxi, but not on the hearts of L. polyphemus and scorpions (Carlson, x9o6; Welsh, x939a,b , t942; Davenport, x94I, 1942 ; Garrey, 1942; Kanungo, 1957; Zwicky, x968). From a consideration of the above, there seems to be no set pattern to the pharmacological responses ofneurogenic hearts. The responses of hearts to applied acetylcholine often have been used as a means of determining if a particular heart is neurogenic or myogenic, acetylcholine being excitatory on neurogenic hearts and inhibitory on myogenic hearts (Prosser, x96I ). As discussed above, an excitatory response to acetylcholine does not always occur in neurogenic hearts, and conversely it appears that an inhibitory response to acetylcholine does not always occur in myogenic hearts (Miller and Metcalf, x968 ). For this reason, the effect of ACh as the sole means of distinguishing between neurogenie and myogenic hearts should be discontinued. ACKNOWLEDGEMENTS
This investigation was supported by a U.S.P.H.S. predoctoral fellowship from the Institute of General Medical Sciences to R. G. Sherman and by a U.S.P.H.S. research grant from the National Heart Institute to R. A. Pax. Critical reading of the manuscript by Dr. H. L. Atwood is gratefully acknowledged. REFERENCES
ABBOTT, B. C., LANG, F., and PARNAS, I. (1969), 'Physiological properties of the heart and cardiac ganglion of Lirauluspolyphemus ', Comp. Bioc~,m. Physiol., a~ 149--158. B~N, W. A. (x929), ' T h e action of adrenalin and of certain drugs upon the isolated crustacean heart ', Q.oT. exp. Biol., xg, ~97-3o8. BELAmAmCH, F. A., and TERW'ILLIOER,R. C. (I966), ' Isolation and identification of cardioexcitor hormone from the pericardial organs of Cancer borealis', Am. Zool., 6, Ioi-Io6. BERAa~-ZK, R., and MILLER, P. L. (1968), ' The
action of iontophoretically applied glutamate on insect muscle fibres ', o7. exp. Biol., 49, 83-93. BURSSY, C. R., and SHEgMAN, R. G. (I97o), 'Spider cardiac physiology, I. Structure and function of the cardiac ganglion ', Comp. gen. Pharmac., x, i6o-17o.
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CATEGHOLAMINES
CARLSON,A. J. (i9o4) , ' Further evidence of the nervous origin of the heartbeat in Limulus ', Am. 07. Physiol., x~, 471-498. - - - - (I9o6), ' On the point of action of drugs on the heart with special reference to the heart of Limulus ', Ibid., x7~ I77-2Io. -- - (x9o7) , ' The relation of the normal heart rhythm to the artificial rhythm produced by sodium chloride ', Ibid., xT, 478-486. COOKE, I. M. (1966), ' The sites of action of pericardial organ extract and 5-hydroxytryptamine in the decapod crustacean heart ', Am. Zool., 6, IO7-I2I. DAVENPORT, D. (I941), ' T h e effects of acetylcholine, atropine and nicotine on the isolated heart of the commercial crab, Cancer magister D a n a ' , Physiol. Zool., t 4 , 178-. I85. - - (1942), ' F u r t h e r studies in the pharmacology of the heart of Canzer magisWr Dana ', Biol. Bull. mar. biol. Lab., Woods Hole, 82, 255o60.
FLOREY, E. (1957) , ' F u r t h e r evidence for the transmitter function of factor I ', Naturwissenschaflen, 44, 404-405. - - - - and FLOREY,E. (x954) , ' I~ber die m6gliche Bedeutung yon Enteramin (5-oxy-Tryptamin) als nervo~er Aktiom substanz bei Cephalopoden und dekapoden Crustacean ', Z. aVaturforsch., 9~ 58--68. GARREY, W. A. (x9o0), ' T h e relation of respiration to rhythm in the cardiac ganglion of Limulus polyphemus ', 07. gen. Physiol., 4~ I49-156. m _ _ (x942), ' A n analysis of the action of acetylcholine on the cardiac ganglion of Limulus polyphemus ', Am. 07. Physiol., x36, t82 t93.
KAmmoo, M. S. (1957), ' Cardiac physiology of the scorpion Palartmaeus bengalensis Koch ', Biol. Bull. mar. biol. Lab., Woods Hole, xx3, I35I4O. K~Rmrr, G. A., and Pmce, M. A. (i964) , ' Chromatographic separation of cardioaccelerators (6-HT and a mucopeptide) from Cardnus heart ', Comp. Biochem. Physiol., xI, 45-
5 o• KOeLLe, G. B. (I965), 'Neurohumoral transmission and the autonomic nervous system ', in The Pharmacological Basis of Therapeutics (ed. Goovu~a% L. S., and GILMAN,A.), pp. 399-440. New York: Macmillan.
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HEART
I95
MAYNARD, D. M. (I96I), ' C a r d i a c inhibition in decapod Crustacea ', in .Nervous Inhibition (ed. FLom~v, E.), pp. I44-I78. New York: Pergamon Press. - - and WELSH,J. H. (x959), ' Neurohormones of the pericardial organs of brachyuran Crustacea ', 07. Physiol., Lord., t49, 2t5-227. MILLER, T., and METCALS, R. L. (I968), ' Site of action of pharmacologically active compounds on the heart of Periplaneta americana L. ', o7. Insect Physiol., x,t, 383-394. PAX, R. A., and SANBORN,R. C. (I967a), ' Cardioregulation in Limulus, II. G a m m a aminobutyric acid, antagonists and inhibitor nerves ', Biol. Bull. mar. biol. Lab., Woods Hole, x3a~ 38t-39 L (i967b), ' Cardioregulation in Limulus, I I I . Inhibition by 5-hydroxytryptamine and antagonism by bromolysergic acid diethylamide and picrotoxin ', Ibid., x32, 392--4o 3. PROSSZR, C. L. (x96I), 'Circulation of body fluids ', in Comparative Animal Physiolog~ (ed. PRosseR, C. L., and BROWN, F. A.), 2nd ed., pp. 386-416. Philadelphia: Saunders. SHERMAN, R. G., and PAX, R. A. (197o), ' Spider cardiac physiology, II. Responses of a tarantula heart to cholinergic compounds ', Comp. gen. Pharmac., x, i 7 I - I 8 4 . TAKEUCHI, A., and TAI~UCHI, N. (t964), ' T h e effect on crayfish muscle of iontophoretically applied glutamate ', o7. Physiol., Lond., x7o, 096317 • WELSH, J. H. (1939a), ' C h e m i c a l mediation in crustaceans, I. The occurrence of acetylcholine in nervous tissues and its action on the decapod heart ', 07. exp. Biol., x6, 198-2t 9. (i939b), 'Chemical mediation in crustaceans, II. The action of acetylcholine and adrenalin on the isolated heart of Panulirus argus ', Physiol. Zool., xo~ 231-237 . (1942), 'Chemical mediation in crustaceans, IV. The action of acetyleholine on isolated hearts of Homarus and Carcinides ', 07. cell. comp. Physiol., x9~ 27t-279. ZWlCKV, K. T. (1968), ' Innervation and pharmacology of the heart of Urodacus, a scorpion ', Comp. Biochem. Physiol., a4, 799-808.
Key WordIndex: Spider heart, spider cardiac ganglion, tarantula, Eurypelma marxi, epinephrine, norepinephrine, GABA, picrotoxin, D-glutamate, L-glutamate, 5-hydroxytryptamine.
I8 5
SPIDER CARDIAC PHYSIOLOGY, III. RESPONSES OF A TARANTULA HEART TO CERTAIN CATECHOLAMINES, AMINO-ACIDS, AND 5-HYDROXYTRYPTAMINE* ROBERT
G. S H E R M A N ~
AND R A L P H
A. P A X
Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A.
(Recdved x2 Dec., x969) ABSTRACT x. Epinephrine and norepinephrine at concentrations greater than x × to -6 M produce an increase in heart-rate and heart-beat amplitude by their actions on the cardiac ganglion. 2. Gamma aminobutyric acid at concentrations greater than x × xo-5 M produces a decrease in heart-rate and a dual phased heart-beat amplitude response (decrease, then an increase) by its actions on the cardiac ganglion. 3. D-Glutamate at concentrations greater than x × xo - ' M produces an increase in heart-rate and a decrease in heart-beat amplitude by its actions on the cardiac ganglion. 4. L-Glutamate at concentrations greater than x × xo-4 M has little effect on heart-rate, but produces an increase in heart-beat amplitude by a direct action on the myocardium. 5. 5-Hydr°xytryp tamine at concentrations greater than x x xo-' M evokes a decrease in heart-rate and heart-beat amplitude by its actions on the cardiac ganglion. 6. v-Aspartate, L-aspartate, and glycine, in concentrations up to x × xo-* M, have no appreciable effects on heart-rate and heart-beat amplitude. IN t h e p r e c e d i n g p a p e r it was shown t h a t acetylcholine p r o d u c e s a n increase in the r a t e a n d a m p l i t u d e o f t h e h e a r t - b e a t o f the spider Eurypelma marxi S i m o n ( S h e r m a n a n d Pax, x97o ). I n t h e e x p e r i m e n t s p r e s e n t e d here e p i n e p h r i n e a n d n o r e p i n e p h r i n e , selected a m i n o - a c i d s , a n d 5 - h y d r o x y t r y p t a m i n e have b e e n e x a m i n e d for t h e i r effects o n isolated h e a r t p r e p a r a t i o n s o f the spider E. marxi. T h e p r i n c i p a l a i m o f this s t u d y was to determ i n e i f a n y o f these c o m p o u n d s m i g h t b e considered as possible n e u r o t r a n s m i t t e r s in s p i d e r hearts. A t the s a m e time it was h o p e d t h a t sufficient i n f o r m a t i o n w o u l d be o b t a i n e d a b o u t the p h a r m a c o l o g y o f this h e a r t to permit comparisons with other neurogenic hearts. * Please address all correspondence to Dr. Ralph A. Pax, Department of Zoology, Michigan State University, East Lansing, Michigan 48823, U.S.A. t Present address: Department of Zoology, University of Toronto, 25 Harbord Street, Toronto 5, Ontario, Canada.
M A T E R I A L S AND M E T H O D S The materials and methods used in this study are the same as those described previously (Sherman and Pax, I97o ). RESULTS
CATECHOLAMINES The catecholamines, epinephrine hydrochloride and norepinephrine hydrochloride, e a c h were a p p l i e d to 5 different h e a r t s a t c o n c e n t r a t i o n s r a n g i n g f r o m I x 10 -6 M to x × to--4 M . B o t h p r o d u c e d a n increase in heart-rate and heart-beat amplitude, and at c o n c e n t r a t i o n s g r e a t e r t h a n I × IO-5 M a t r a n s i e n t increase in m y o c a r d i a l tonns (Fig. x). T h e responses to these drugs a r e i m m e d i a t e , b u t the m a x i m u m effects o c c u r a b o u t 45 seconds after d r u g a p p l i c a t i o n . O n c e t h e m a x i m u m increase is seen, t h e r a t e a n d a m p l i t u d e o f t h e c o n t r a c t i o n s r e m a i n quite constant for the d u r a t i o n o f t h e t r e a t m e n t . U p o n d r u g r e m o v a l t h e y b e g i n to decrease g r a d u a l l y u n t i l p r e t r e a t m e n t levels a r e seen from IO to 15 m i n u t e s later.
I86
Comp. gen. Pharmac.
SHERMAN AND PAX
A d o s e - r e s p o n s e curve for the effect o f e p i n e p h r i n e on t h e h e a r t - r a t e is shown in Fig. ~. T h e d o s e - r e s p o n s e curve for n o r e p i n e p h r i n e is essentially t h e same. T h r e s h o l d c o n c e n t r a t i o n for the effect o f b o t h drugs on
t'
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the h e a r t - r a t e effects o f these amplitude and Table I. T h e e p i n e p h r i n e is
is a b o u t I X I o - 6 M . The c a t e c h o l a m i n e s on h e a r t - b e a t m y o c a r d i a l tonus are listed in t h r e s h o l d c o n c e n t r a t i o n for a b o u t 5 x I o -6 M , w h i l e t h a t
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:FIG. i.--Response of the heart to epinephrine. The response to norepinephrine is essentially the same. Continuous record from top left to bottom right. Saline removed at first arrow and 5 x I o - s 5 I epinephrine applied at the second arrow. Time marker equals I second; downward excursion of time marker represents a heart-beat amplitude of 50 mg.
I97O, x
CATEGHOLAMINESAND SPIDER HEART
for norcpinephrinc is approximately z X z0 -5 M . In general, norepinephrine does not have quite as great an effectas does epinephrine. The site of action of epinephrine and norepinephrinc was investigated by applying each of them to dcganglionatcd hearts while monitoring changes in myocardial tonns and by recording the cardiac ganglion electrical
I87
the heart-beat (Fig. 4). T h e maximum inhibitory effect occurs within zo seconds of administration. Several seconds after the ma~im~tm effects of GABA are seen, the heart-rate begins to return to the prctreatment rate, but it does not completely recover as long as GABA is on the heart. Concurrently with the recovery in rate, and even
60--
50--
40
i
m 30
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I
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CONC[NTRATION(W
Fro. 2.~Dme--response curve for the effect of epinephrine on the heart-rate. Each point represents the mean response to 7 replicatiom made on at least 4 different hearts. Vertical lines represent one standard deviation.
activity of intact hearts while these drugs wcrc on the heart. Both catecholamlncs, in concentrations as great as x X xo "4 M , produced no rccordable changes in the myocardial tonus of deganglionated hearts, but both increased the frequency of ganglionic bursts as well as the amount of activltyin each burst when applied to intact hearts (Fig. 3 A,
S). G A M M A AMINOBUTYRIG ACID Gamma aminobutyric acid ( G A B A ) at concentrations greater than I × xo -5 M produccs a decrease in the rate and amplitude of
more striking, the amplitude of the heart-beat begins to increase and soon exceeds the amplitude seen before the treatment was
begun. Upon removal of GABA a small further increase in heart-beat amplitude occurs, but this increase is only a transient one. In all cases both the rate and amplitude returned to prctreatrncnt levels within 6 minutes of the end of the treatment. A dose-response curve for the effect of G A B A on the heart-rate is shown in Fig. 5. T h e threshold concentration required to produce a decrease in the heart-rate is
188
SHERMAN AND PAX
Comp. gen. Pharraac.
J Fla. 3.--Effects of epinephrine and GABA on cardiac ganglion electrical activity. A, Burst frequency in saline. B, xo seconds after administration of 5 × Io-~ M epinephrine. C, Burst frequency in saline. D, xo seconds after application of 5 × io-~ M GABA. t:, Same as D, except ao seconds later. F, Io seconds after GABA had been replaced by saline.
I970 , I
I89
CATECHOLAMINES AND SPIDER HEART
change in myocardial tonus, but when applied to intact hearts it causes sporadic firing of the cardiac ganglion along with occasional bursts of prolonged duration and reduced amplitude (Fig. 3D). After about 3o seconds in G A B A , co-ordinated bursts at
5 × I 0 -s M. The effect of various concentrations of G A B A on the amplitude of the heart-beat is shown in Table IL Each number represents the m c a n change in heart-beat amplitude from the control. This was determined by averaging the amplitudes of the
Table L ~ E F F E C T S OF EPINEPHRINE AND NOREPINEPHRINE ON HEART-BEAT AMPLITUDE
AND
MYOCARDIAL TONUB DRUG CONCENTRATION
(M)
CHANGE IN I-IEART-BEAT AMPLITUDE*
INCREASE IN Tom~s
N o . oF
(mg.)
INCREASES
(rag.)
ToNE
N o . oF REPLICATIONS
Epinephrine I XIO -6
io
(-Io-25)
5 X 10 -e I × I O -5
3° 55 65 85
(m-55) (3°-I35) (3o-I I5) (3o--175)
5 X IO -5 I X IO - i
0
xo (o-4o)
40 (o--4o) 8o (0--220) IO0 (30--200)
Norepinephrine
*
o (-xo-xo)
I × IO - s 5 × xo-S I X I o -5
IO ( - - IO--35 )
5 x i o -s
30 (o-95)
io (o--3o) 45 (o--80)
I XIO -t
45 (15-95)
9° (4(>-xoo)
0 0
I5 (o-55)
Mean and range.
Table II.--EFFECT OF GAMMA AMINOBUTYRIC Acre ON HEART-BEAT AMPLITUDE CONCENTRATION
(M)
~tEAN CHANGE IN AMPLITUDEFROM CONTROL (rag.)
First Minute I XIO -5
5
Second Minute
O
×Io-5 i Xio -4
--5
5 ×1o-4
--3 °
--20
first IO beats that occurred in each of the 3 minutes that GABA was on the heart and comparing these values with the m e a n heart-beat amplitude for the io beats that occurred just prior to GABA application. T h e threshold dose required to produce a change in heart-beat amplitude is about 5 × to -s M. T h e dual phased nature of the amplitude response is readily apparent from the table. When applied to deganglionated hearts, GABA does not produce any recordable
O
+io
+25 +15
Third Minute O
+t5 +35 +45
an increased rate appear (Fig. 3E) and a few seconds after removal of G A B A , the burst amplitude exceeds that seen before the G A B A treatment was begun (Fig. 3F). Since these responses arc comparable to those seen in the mechanical recordings (Fig. 4), it is clear that the various effectsof G A B A arc duc to its actions on the cardiac ganglion. P m R O T O X ~ AND G A B A Picrotoxin blocks the effects of G A B A on the ncurogenlc hearts of decapod crustaceans
I9o
SHERMAN A N D P A X
(Florey, i957) , b u t n o t on t h e n e u r o g e n i c
Liraulus polypheraus h e a r t ( P a x a n d S a n b o r n , I967a). F r o m a c o m p a r a t i v e s t a n d p o i n t it w o u l d b e o f interest to k n o w i f p i c r o t o x i n blocks the effects o f G A B A on t h e n e u r o g e n i c E. marxi heart. F o u r h e a r t s w e r e t r e a t e d for u p to 3 ° m i n u t e s w i t h 5 × t o - 4 M p i c r o t o x i n a n d t h e n w i t h 5 × I o-~ M p i c r o t o x i n plus G A B A in c o n c e n t r a t i o n s r a n g i n g f r o m
tnlnllJh~,Jl
Comp. gen. Pharmac.
5 × t 0 -6 M to 5 × 10 -4 M . P i c r o t o x i n a l o n e h a d no effect on t h e heart. F u r t h e r m o r e , in no case d i d p i c r o t o x i n r e d u c e t h e effectiveness o f G A B A . OTHER AMINO-ACIDS D - G l u t a m i c a c i d a t c o n c e n t r a t i o n s u p to I × xo-4 M h a d little effect on the heart. A t 5 × I ° - * M it p r o d u c e d a n a v e r a g e increase
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197o, x
CATECHOLAMINESA N D
in heart-rate of 12 beats per minute (3 hearts) and a decrease in the amplitude of the contractions of about 3o m g . At I X I O - s M these effects were even more pronounced, often resulting in such weak contractions that the heart-rate could not be measured accurately. L-Glutamic acid had no appreciable effect on the heart at concentrations up to
0 n
191
in a few instances, but only at concentrations of I X 1 0 - 4 M . O f the amino-acids used, D-glutamate was the only one other than GABA that had an appreciable effect on the electrical activity of the cardiac ganglion. The response of the ganglion to D-glutamate consisted of a decrease in the burst amplitude and a general appearance of asynchronous firing.
¢
tt
-10 n
"~ -20
SPIDER H E A R T
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-30 - -
I
I
5xlO"~
10"
I ,
I
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5x10-'
Fro. 5.--Dose-response curve for the effect of GABA on the heart-rate. Each point represents the mean response to at least 5 replications made on at least 4 different hearts. Vertical lines represent one standard deviation. I X IO -4 M.
At
5 × io -4 M
it
produced
a
mean increase in heart-beat amplitude of 3 o m g • (3 hearts); this effect was even greater at I x xo-a M. Only rate increases less than 5 beats per minute occurred at any of the concentrations used. D- and L-Aspartic acid and glycine usually produced only slight increases in heart-rate and heart-beat amplitude at concentrations ranging from I X I O - 5 M t o I X I O - 8 M ( 3 hearts). Increases in heart-rate greater than 5 beats per minute and increases in heartbeat amplitude greater than I o nag. were seen
When applied to deganglionated hearts, none of the amino-acids except L-glutamate produced any recordable change in myocardial tonus at concentrations up to I X I O -s M. L-Glutamate caused a sustained contraction of the deganglionated myocardium at concentrations of I X IO-4 M to I × I o ~ M ( F i g . 6). Each contraction lasted for the duration of the drug treatment. U p o n removal of the L-glutamate the contraction gradually subsided over a period of several minutes until the control myocardial tonus was reached.
x92
SHERMAN AND PAX
A number of small oscillatory contractions occur in the record of the response to L-glutamate. Such contractions were seen in a n u m b e r of hearts (but not all) about I5-3o minutes after removal of the cardiac ganglion, a procedure which causes an immediate cessation of the heart-beat (Bursey and Sherman, x97o). These contractions took the form of small twitches which occurred in localized areas of the heart. The significance of these uncoordinated contractions is not
Comp. gen. Pharmac.
5 - H T at 5 × IO-s M produced a decrease in the amplitude of the ganglionic bursts and a slight decrease in burst frequency when administered to intact hearts. At the same concentration it h a d no recordable effect on the myocardial tonus of deganglionated hearts. DISCUSSION The catecholamines, epinephrine and norepinephrine, are the most effective compounds
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FIO. 6.--Effect of L-glutamate on the myocardial tonus of a deganglionated heart. Saline removed at first arrow and 5 x to -t M L-glutamate'applied at second arrow. L-Glutamate removed at third arrow and saline applied at fourth arrow. Time marker equals x second; downward excursion of time marker represents a change in tension of ~5 mg. clear, but they perhaps are analogous to the contractions reported by Carlson (I9O4, 1907) to occur in the deganglionated heart of the horseshoe crab (Limulus polypt~mus) when it is placed in an isotonic sodium chloride solution. 5-HYDROXYTRYPTAMINE
5-Hydroxytryptamine (5-HT) was applied to 4 hearts at concentrations ranging from x × lO -6 M to 5 × I°-3 M. Only occasional small changes in the rate and amplitude of the heart-beat were seen at concentrations up to I × I o -s M. At 5 × 10 -8 M the amplitude of the heart-beat was reduced to the point where it was barely recordable (Fig. 7). At the same concentration the heart-rate was reduced by about 13 beats per minute. These effects were constant for the duration of the treatment. Within a few seconds after removal o f 5 - H T a striking increase in heartbeat amplitude occurred which resulted in an amplitude nearly twice that of the control. This increase was only transitory and within 5 minutes after the end of the treatment the amplitude as well as the heart-rate had returned to pretreatment levels.
of those tested in producing an increase in the rate or amplitude of the E. marxi heart-beat. These effects are comparable to those of ACh on this same heart (Sherman and Pax, 197o ) . However, the time course of the response to these catecholamines is quite different from that of ACh. Unlike ACh, once the m a x i m u m response to epinephrine and norepinephrine is seen, there is little change in the response for as long as the drugs are on the heart, and upon their removal the response only gradually diminishes. Such a time course could be explained by the assumption that a rapid enzymatic process for the termination of the actions of epinephrine and norepinephrine is either absent or else is not involved to any great extent in this heart. The lack of involvement of such a process for terminating the actions of catecholamines has been reported for a number of other animals (Koelle, I965). L-Glutamic acid has been proposed as the excitatory transmitter at crustacean (Takeuchi and Takeuchi, x964) and insect (Ber~-mk and Miller, I968 ) neuromuscular junctions. Since L-glutamate was the only compound used in this study to produce
I970 , X
CATECHOLAMINES
recordable contractions of deganglionated hearts, it seems possible that it might be involved in neuromuscular transmission in spider hearts. GABA is the most effective compound of those tested in producing a decrease in the heart-rate and heart-beat amplitude. Perhaps the most striking effect of GABA is the
AND
SPIDER
HEART
I93
dual phased heart-beat amplitude response. The initial reduction in amplitude appears to be due to a decrease in the synchronization of the cardiac ganglion impulses, as suggested by a prolongation of the bursts and reduced burst amplitudes. As apparent resynchronization occurs, as suggested by the appearance of bursts having shorter durations
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FIG. 7.--Response of the heart to 5-HT. Continuous record from top left to bottom right. Saline removed at first arrow and 5 x IO-a M 5-HT applied at second arrow. 5-HT removed at third arrow and saline applied at fourth arrow. Time marker equals i second; downward excursion of time marker represents a heart-beat amplitude of 5° mg.
I94
Comp. gen. Pharmae.
SHERMAN AND PAX
and increased amplitudes, the heart-beat amplitude is seen to be larger. The occurrence of the dual phased amplitude response and the partial recovery of the reduced heart-rate during GABA treatment indicate that the heart may become desensitized to GABA. An alternate explanation is that GABA may be metabolized by the cardiac ganglion, thereby reducing its inhibitory effects on the heart. Either of these possibilities could lead to a re-synchronization of the cardiac ganglion impulses, but additional information is required before definitive statements can be made in this regard. The results presented here and in the preceding paper (Sherman and Pax, 197o ) provide a basis for comparing the pharmacology of a spider heart with that of other neurogenic hearts. The pharmacology of only a few neurogenic hearts is sufficiently well characterized to permit meaningful comparisons. These include the hearts of decapod crustaceans, certain scorpions, and Limulus polyphemus. The E. marxi heart appears similar to these hearts with respect to its responses to catecholamines and to GABA. Catecholamines are excitatory in all of the above hearts; GABA is known to be inhibitory in all, except in scorpions where it has yet to be tested (GmTey, i922 ; Bain, I929; Welsh, i939b ; Kanungo, 1957 ; Florey, I957 ; Maynard, 196I; Pax and Sanborn, x967a; Abbott, Lang, and Parnas, 1969). The effects of 5 - H T on neurogenic hearts are not so uniform. It is not particularly effective on the E. marxi heart, has marked inhibitory effects on the heart ofL. polyphemus, and has pronounced excitatory effects on the hearts of deeapod crustaceans (Florey and Florey, I954; Maynard and Welsh, I959; Kerkut and Price, I964; Belamarich and Terwilligcr, I966; Cooke, I966; Pax and Sanborn, I967b ). Acetylcholine also has different effects on neurogenic hearts. It is excitatory on the hearts of decapod crustaceans, L. polyphemus, and E. marxi, but it is inhibitory on the hearts of scorpions. Eserine potentiates the effects of ACh on the hearts of decapod crustaceans, L. polyphemus, E. marxi, and one scorpion, but it apparently
does not potentiate the effects of ACh on another scorpion. Atropine blocks the actions of ACh on the hearts of decapod crustaceans and E. marxi, but not on the hearts of L. polyphemus and scorpions (Carlson, x9o6; Welsh, x939a,b , t942; Davenport, x94I, 1942 ; Garrey, 1942; Kanungo, 1957; Zwicky, x968). From a consideration of the above, there seems to be no set pattern to the pharmacological responses ofneurogenic hearts. The responses of hearts to applied acetylcholine often have been used as a means of determining if a particular heart is neurogenic or myogenic, acetylcholine being excitatory on neurogenic hearts and inhibitory on myogenic hearts (Prosser, x96I ). As discussed above, an excitatory response to acetylcholine does not always occur in neurogenic hearts, and conversely it appears that an inhibitory response to acetylcholine does not always occur in myogenic hearts (Miller and Metcalf, x968 ). For this reason, the effect of ACh as the sole means of distinguishing between neurogenie and myogenic hearts should be discontinued. ACKNOWLEDGEMENTS
This investigation was supported by a U.S.P.H.S. predoctoral fellowship from the Institute of General Medical Sciences to R. G. Sherman and by a U.S.P.H.S. research grant from the National Heart Institute to R. A. Pax. Critical reading of the manuscript by Dr. H. L. Atwood is gratefully acknowledged. REFERENCES
ABBOTT, B. C., LANG, F., and PARNAS, I. (1969), 'Physiological properties of the heart and cardiac ganglion of Lirauluspolyphemus ', Comp. Bioc~,m. Physiol., a~ 149--158. B~N, W. A. (x929), ' T h e action of adrenalin and of certain drugs upon the isolated crustacean heart ', Q.oT. exp. Biol., xg, ~97-3o8. BELAmAmCH, F. A., and TERW'ILLIOER,R. C. (I966), ' Isolation and identification of cardioexcitor hormone from the pericardial organs of Cancer borealis', Am. Zool., 6, Ioi-Io6. BERAa~-ZK, R., and MILLER, P. L. (1968), ' The
action of iontophoretically applied glutamate on insect muscle fibres ', o7. exp. Biol., 49, 83-93. BURSSY, C. R., and SHEgMAN, R. G. (I97o), 'Spider cardiac physiology, I. Structure and function of the cardiac ganglion ', Comp. gen. Pharmac., x, i6o-17o.
197o , t
CATEGHOLAMINES
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FLOREY, E. (1957) , ' F u r t h e r evidence for the transmitter function of factor I ', Naturwissenschaflen, 44, 404-405. - - - - and FLOREY,E. (x954) , ' I~ber die m6gliche Bedeutung yon Enteramin (5-oxy-Tryptamin) als nervo~er Aktiom substanz bei Cephalopoden und dekapoden Crustacean ', Z. aVaturforsch., 9~ 58--68. GARREY, W. A. (x9o0), ' T h e relation of respiration to rhythm in the cardiac ganglion of Limulus polyphemus ', 07. gen. Physiol., 4~ I49-156. m _ _ (x942), ' A n analysis of the action of acetylcholine on the cardiac ganglion of Limulus polyphemus ', Am. 07. Physiol., x36, t82 t93.
KAmmoo, M. S. (1957), ' Cardiac physiology of the scorpion Palartmaeus bengalensis Koch ', Biol. Bull. mar. biol. Lab., Woods Hole, xx3, I35I4O. K~Rmrr, G. A., and Pmce, M. A. (i964) , ' Chromatographic separation of cardioaccelerators (6-HT and a mucopeptide) from Cardnus heart ', Comp. Biochem. Physiol., xI, 45-
5 o• KOeLLe, G. B. (I965), 'Neurohumoral transmission and the autonomic nervous system ', in The Pharmacological Basis of Therapeutics (ed. Goovu~a% L. S., and GILMAN,A.), pp. 399-440. New York: Macmillan.
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MAYNARD, D. M. (I96I), ' C a r d i a c inhibition in decapod Crustacea ', in .Nervous Inhibition (ed. FLom~v, E.), pp. I44-I78. New York: Pergamon Press. - - and WELSH,J. H. (x959), ' Neurohormones of the pericardial organs of brachyuran Crustacea ', 07. Physiol., Lord., t49, 2t5-227. MILLER, T., and METCALS, R. L. (I968), ' Site of action of pharmacologically active compounds on the heart of Periplaneta americana L. ', o7. Insect Physiol., x,t, 383-394. PAX, R. A., and SANBORN,R. C. (I967a), ' Cardioregulation in Limulus, II. G a m m a aminobutyric acid, antagonists and inhibitor nerves ', Biol. Bull. mar. biol. Lab., Woods Hole, x3a~ 38t-39 L (i967b), ' Cardioregulation in Limulus, I I I . Inhibition by 5-hydroxytryptamine and antagonism by bromolysergic acid diethylamide and picrotoxin ', Ibid., x32, 392--4o 3. PROSSZR, C. L. (x96I), 'Circulation of body fluids ', in Comparative Animal Physiolog~ (ed. PRosseR, C. L., and BROWN, F. A.), 2nd ed., pp. 386-416. Philadelphia: Saunders. SHERMAN, R. G., and PAX, R. A. (197o), ' Spider cardiac physiology, II. Responses of a tarantula heart to cholinergic compounds ', Comp. gen. Pharmac., x, i 7 I - I 8 4 . TAKEUCHI, A., and TAI~UCHI, N. (t964), ' T h e effect on crayfish muscle of iontophoretically applied glutamate ', o7. Physiol., Lond., x7o, 096317 • WELSH, J. H. (1939a), ' C h e m i c a l mediation in crustaceans, I. The occurrence of acetylcholine in nervous tissues and its action on the decapod heart ', 07. exp. Biol., x6, 198-2t 9. (i939b), 'Chemical mediation in crustaceans, II. The action of acetylcholine and adrenalin on the isolated heart of Panulirus argus ', Physiol. Zool., xo~ 231-237 . (1942), 'Chemical mediation in crustaceans, IV. The action of acetyleholine on isolated hearts of Homarus and Carcinides ', 07. cell. comp. Physiol., x9~ 27t-279. ZWlCKV, K. T. (1968), ' Innervation and pharmacology of the heart of Urodacus, a scorpion ', Comp. Biochem. Physiol., a4, 799-808.
Key WordIndex: Spider heart, spider cardiac ganglion, tarantula, Eurypelma marxi, epinephrine, norepinephrine, GABA, picrotoxin, D-glutamate, L-glutamate, 5-hydroxytryptamine.