- Email: [email protected]
Inhibition by acetylcholine of cockroach heart (Gromphadorhina portentosa)
Comp. gen. Pharmac.
4.°6
INHIBITION BY ACETYLCHOLINE OF COCKROACH HEART
(GROMPHADORHINAPORTENTOSA)
C A R L W. S C H A E F E R AND J A C Q U E L I N E
MILES
Systematics and Evolutionary Biology Section, Biological Sciences Group, University of Connecticut, Storrs o6268, U.S.A.
(Received 2I March, x97o) ABSTRACT i. The normal heart-rate (at 25° C.) of the Madagascar hissing cockroach is 66' 5 beats per minute. 2. There is no difference between sexes. 3. Io-~ M and Io -3 M acetylcholine slows the beat (to 56q and 63.i beats per minute respectively), whereas io -4 M ACh has no effect. 4. Glutamate (io -~ M) is also without effect, but 5-hydroxytryptamine (xo -2 M) is slightly stimulatory (68.8 beats per minute).
THE control of the insect h e a r t - b e a t is poorly understood, as is, indeed, the control of all insect visceral muscle. T h e beat appears i n m a n y insects to be of the i n n e r v a t e d myogenic type, with the i n n e r v a t i o n controlling a n intrinsically initiated a n d p r o m u l g a t e d contraction of the heart muscle. A l t h o u g h A C h is p r o b a b l y not a n e u r o m u s c u l a r transmitter i n insect skeletal muscle (Treherne, i966), it does have a n effect u p o n the contraction of insect h e a r t muscle. I t is not clear whether the effect is u p o n the muscle directly, or u p o n the n e u r o m u s c u l a r j u n c t i o n , or u p o n some intrinsic p a c e m a k e r ; a n d the effect varies, d e p e n d i n g often u p o n the insect group. I n the only cockroach (Dictyoptera) so far studied i n detail, Periplaneta americana L., A C h stimulated at low concentrations a n d m a y i n h i b i t at high (see references in Discussion). Little has been published of the effect of high concentrations, however, a n d we describe a case here where A C h intfibits at high c o n c e n t r a tions b u t appears to have no effect at a lower one. W e also include a few d a t a on the effect of glutamate and 5-HT.
METHODS AND MATERIALS Adults were used of the Madagascar hissing cockroach, Gromphadorhina portentosa Schaum, which has been kept in colony for some years at the University of Connecticut (on lab chow and water, at 3°° C. and 80 per cent R.H.). The cockroaches were immobilized by cooling, the head and thorax removed, and the abdominal dorsum bearing the heart isolated. The preparation was held at 25° C. in a water-bath and allowed to stand at least 15 minutes for temperature equilibration. Palm's (I946) Ringer's solution was used (formulation, in g. per litre distilled water: NaCI 9, KC1 o'4, CaC1, o'2, NaHCO~ o-x, glucose I). Drugs were dissolved in the Ringer's solution and used fresh (ACh as the chloride, 5-HT as the creatinine sulphate, glutamate as L-glutamic acid, all from Sigma Chemical Co., St. Louis, Mo.). In most experiments counts were made Io times per preparation of the number of contractions per minute, and these counts were averaged. All counts were taken at the fifth abdominal segment. Statistical significance was determined with Students' t-test. RESULTS T h e r e was no significant difference due to sex in the n u m b e r of contractions per m i n u t e of control hearts (Table I). Therefore, the
197o, x, 426-43o
427
I N H I B I T I O N OF H E A R T BY A C H
had no effect ( P > o . i ) , but the results with lO -3 M 5 - H T were almost significant at the 5 per cent level ( o . I > P > o . o 5 ) , and showed a slight stimulation (Table III).
results for males and females were combined, and the overall average (67"0 contractions per minute, W=28) was used as the control. High concentrations of A C h inhibit the heart (Table H). The statistical significance of these results, as compared to the control, is as follows : - IO-2 M ACh, P < o . o o I . Io -3 M ACh, P < o-ooi. IO-4 M ACh, o ' 6 < P < o - 7.
DISCUSSION Despite the wide experimental use of cockroaches, surprisingly little has been published on their normal heart contraction-rates. Only the rate of Periplaneta americana has been published, and different authors disagree on
Table / . - - C O N T R A C T I O N S
PER M I N U T E OF HEARTS OF M A L E AND FEMALE COCKROACHES CONSIDERED SEPARATELY
SEX
SPECIMEN
No. Female
I 2
6 5
3 4 5 6 7 8 9
IO
IO II I2
13 I4 I5 I6 I7 Male
N O . OF
6 IO IO 10 IO IO IO IO IO IO IO IO IO IO
I
5
2
I0
3 4 5 6 7 8 9
IO
IO II
N O . OF CONTRACTIONS PER M I N U T E
CONTRACTIONS
IO IO IO IO I0 I0 I0 IO
Average
T h e lowest concentration of ACh (lO -4 M) had no effect upon contractions of the heart. Because glutamate and 5 - H T have been implicated in the control of arthropod muscle, we tested their effects. Glutamate (lO -2 M)
(Average and Range) 6o.8 64-i 7o-2 6I'7 67'o 62-2 69.6 66. 7 65"5
(6o'I-6I'2) (63.1-65-2) (69"7-71-4) (6o'o-65"2) (65'9-68"I) (66"4-63'1) (68.6-7oq) (65.8-68.i) (64"9-67"5)
7I'2 (70"1-71"9)
67.1 6o. 3 7o'8 7I'9 7o'2 7o'5 69'o
(65.2-68.o) (58.8-61.2) (68"9-72.2) (68'9-74-o) (68'9-71.5) (67'4-72"2) (67'4-7o'5)
59'8 (57.8--61.8) 6I. 3 (6o.o-64.1) 62'6 (6o'o-64.2) 66-4 (65"9-67'3) 69.o (68.5-69.9) 68.I (67.2-69.1) 65"4 (64"6--66"4) 7I'4 (68"1-73-5) 68.6 (68.6--69.i) 65"4 (64"7-66"o) 66'8 (65'8-67.4) 67"o o"3 < P
it. The most frequently cited rate is that of Yeager and colleagues (Yeager and Hager, 1934; Yeager and Gahan, 1937) ; this rate ranges from 4 ° to 5 ° , and appears low, although temperatures were high (25-29 ° C.).
428
SCHAEFER
AND
I n at least one p a p e r the rate was b a s e d on b u t a few specimens (Yeager a n d H a g e r ,
Table//.--CONTRACTIONS P E R xo-2 M ACh Specimen No. I ,9
3 4 5 6 7 8 9 IO II I2
I3 14 I5 Average
Comp. gen. Pharmac.
MILES
tion) ( M c I n d o o , i939) ; 83, 28 ° C. (males) (Metcalf, W i n t o n , a n d F u k u t o , r964) ; 88
MINUTE
OF HEART
OF
G. portentosa*
I o -3 M ACh
No. of Contractions per Minute (Average and Range) 65-9 (63'`9-674) 580 (556-60'0) 5`9'6 (5i'7-55o) 47.i (46.5-48.o) 764 (73'2-8o-o) 457 (444-46"9) 61.6 (6o.6-63.2) 75'8 (75'9-76-9) 478 (46'9-49-2) 57"3 (55'6-594) 57'2 (55'6-58"2) 51.9 (51.7-52-6) 67. 3 (64.6-7o.6) 62.i (6o.o-64.5) 54"3 (526-55"6)
Specimen No. I `9
3 4 5 6 7 8 9 IO IX I2
I3
56. I
IO-4 M ACh
No. of Contractions per Minute (Average and Range) 62'I 6~' 5 6~'8 60.2 6I'7 64-o 62"3 63'6 64"6 63"7 64.`9 64'o 67' 3
(59'8-63"8) (59"4-63.4) (57.8-63.4) (58.4-61.5) (6o'o--62.4) (63.7-64.5) (6o'6-63'2) (6o'6-65'o) (6o'6-65"9) (6~'4-65"8) (6`9.3-66.7) (6o'6-66"7) (63"4-69.9)
Specimen No.
No. of Contractions per Minute (Average and Range) 66.6 65"9 66.2 67'4 66. 4 66'5 68.6 65.1 7o'o 66.8
I
2
3 4 5 6 7 8 9 IO
63. I
(63.8-68.2) (63"8-67-4) (65-2-68.2) (64'5-69"o) (63.8-7o.6) (64"5-69"o) (66.7-7o.6) (63.8-66.7) (68.2-71.4) (65.2-68.2)
66. 9
* Experiments were carried out in Palm' Ringer's solution with indicated amounts of ACh. In each case IO contractions were counted. Table III.--CONTRACTIONS PER MINUTE OF HEARTS OF G. portentosa*
Io -2 M GLUTAMATE Specimen No. I
2
3 4 5 6 7 8 9 IO
Average
No. of Contractions per Minute (Average and Range) 7o'7 64" 7 7o'6 68"4 69"o 66-6 66"I 66"7 68- 3 67"9
(67"4-75"o) (6I'8-67'4) (68"2-73'2) (65.2-7o.6) (65"9-71.4) (64'5-7o.6) (63-8-67.4) (64"5-69'8) (65.2-69.8) (65"2-7I'4)
67. 9
IO-~ M 5-HT Specimen No. I
9
3 4 5 6 7 8 9 IO
No. of Contractions per Minute (Average and Range) 64'6 67.x 67"4 67"3 67' 4 72"I 69" 7 7o"4 72"3 69.8
(67"8-66"7) (63.8-69.8) (63'8-69"8) (65"2-7o'6) (65-2-69.8) (69"8-74"1) (69.o-73.2) (66'7-74q) (69'o-74"I) (68-4-72.3)
68.8
* Experiments were carried out in Palm's Ringer's solution with glutamate or 5-HT. In each case io contractions were counted. I934). O t h e r s r e p o r t higher rates: 66-72 p e r m i n u t e , t e m p e r a t u r e u n r e p o r t e d (i p r e p a r a -
(females) a n d i o 5 (males), 25 ° C. ( O s m a n i a n d N a i d u , I96o); 8¢>-I2o, 3 °o C. (Krijgs-
197o, I
INHIBITION OF HEART BY ACH
m a n and Krijgsman-Berger, i952) ; 9o-I2O, 29 ° C. (Naidu, I955). Some of these wide ranges m a y reflect the sexual differences noted by O s m a n i and N a i d u (196o). T h e rate of Gromphadorhina portentosa (67.o per minute) is rather low a n d there is no sexual difference (Table I). This relatively low rate m a y be correlated with the animal's lethargy. N o one appears to have reported the effects of high A C h concentrations on cockroach hearts. O s m a n i a n d N a i d u (I96O) suggest that A C h at 1o -a (5"5 × IO-6 M ) partially inhibits, although N a i d u earlier (1955) h a d reported 1o -a to lO -5 A C h to be stimulatory. A C h o.I per cent (5"5× 1 o - 4 M ) partly inhibits the exposed Anopheles quadrimaculatus heart, b u t other concentrations were not tried (Jones, i956 ). T h e wax.moth heart (Galleria mellonella) is stimulated by low concentrations (2 × Io -~ to 7 × l°-a), partly inhibited by concentrations of 2 × IO-4 to 1.25 × IO-3 (about IO - 6 M to 7 × lO-6 M ) , a n d completely inhibited at concentrations > 2-5 × I o -3 (about 1.4 × Io -5 M ) (Folliott, 1957). This study with Galleria is the only one on insects directly comparable with our own, a n d there is a significant difference. Whereas low concentrations of A C h stimulate the Galleria heart, our lowest concentration (IO -4 M ) h a d no effect. It appears that the two p h e n o m e n a are different. I n the case of Galleria (and perhaps Periplaneta), inhibition m a y be due to a ' p o i s o n i n g ' of a tissue perhaps normally stimulated by ACh. I n the case of G. portentosa, A C h in high concentrations m a y normally inhibit the heart; or A C h m a y ordinarily have no effect, and high concentrations may here be poxsonous. T h e heart of G. portentosa is innervated. Methylene blue preparations show a pair of small ganglia asymmetrically a r r a n g e d within each segment, at least in the abdomen. These ganglia are the centres of a very loose and diffuse network of nerves which ramify over the surface of the heart.
429
It appears that the G. portentosa heart fits Prosser's ( 1 9 4 2 ) definition of an innervated myogenic muscle, b u t it is one relatively insensitive to ACh. A n o t h e r such muscle, more sensitive, m a y be that of the Periplaneta Malpighian tubules (Schaefer, 1968; more complete data to be published), although here the situation is complicated by the apparent absence of multiterminal innervation. Glutamate m a y well be the (or a) excitatory transmitter of insect skeletal muscle (Usherwood, Machili, and Leaf, 1968), b u t it appears to have no effect on the G. portentosa heart, at least at IO-2 m . 5-HT, which inlaibits insect skeletal muscle (Hill and Usherwood, I96i), stimulates the heart of P. americana (Davey, I961, I964), with a threshold of I o -8 M. T h e heart of G. portentosa is far less sensitive to 5 - H T , j u d g i n g from our results, which show a slight stimulatory effect at the high concentration of 10 - 2 m .
REFERENCES DAY,Y, K. G. (I96I), ' Substances controlling the rate of beating of the heart of Periplaneta ', Nature, Lond., 192, 284. - - - - (x964), ' The control of visceral muscles in insects ', Adv. Insect Physiol., 2, 2 x9-245. FOLUOTT, R. (1957), ' Action de l'ac~tylcholine sur le fonctionnement du vaisseau dorsal de la larve de Galleria mellonella L. ', Bull. Soc. Sci. Bretagne, 32, 12~-~24. HILL, R. B., and USHERWOOD, P. N. R. (I96I), ' The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerca gregaria ', J. Physiol., Lond., x57, 393-4ox. JONES, J. C. (I956), ' Effects of drugs on Anopheles heart rates ', J. exp. Zool., x33, 573-588. KRIJGSMAN, B. J., a n d KRIJGSMAN-BERGER, N. E. (I952), 'Physiological investigations into the heart function of arthropods. The heart of Periplaneta americana ', Bull. ent. Res., 42, 143-I55. MCINDoO, N. E. (I939) , ' Segmental blood vessels of the American cockroach (Periplaneta americana (L.)) ', ft. Morph., 65, 323-347. METCALF, R. L., WXNTON, M. Y., and FUKUTO, T. R. (I964) , ' T h e effects of cholinergic substances upon the isolated heart of Periplaneta americana ', J. Insect Physiol., zo, 353-361. NAIDU, M. B. (I955) , 'Physiological action of
drugs and insecticides on insects ', Bull. ent. Res., 4 6, 2o5-2~o.
43 °
S C H A E F E R A N D MILES
OSMANI, Z. H., a n d NAIDU, M. B. (196o), ' Effect of temperature on the frequency of the heartbeat of a cockroach ', Proc. zool. Soc. Calcutta, x3~ IO7xl 4. PALM, N. B. (I946), ' Studies on the peristalsis of the Malpighian tubes in insects ', Acta Univ. lund., 42, 1-39. PROSSER, C. L. (1942), ' A n analysis of the action of acetylcholine on hearts, particularly in arthropods ', Biol. Bull. mar. biol. Lab., Woods Hole, 83, I45-I64. SCHAEFER, C. W. (I968), ' I n h i b i t i o n by acetylcholine of Malpighian tubule muscle of the cockroach Periplaneta americana ', Ann. ent. Soc. Am., 6I, 226-227. TREHERNE, J. E. (x966), The Weurochemistry of Arthropods. Cambridge: University Press.
USHER'~,'OOD, P. N. R., MACHILI,P., and LEAF,G. (i968), ' L-Glutamate at insect excitatory nerve-muscle synapse ', Nature, Lond., 219, II69-I172. YEACEr~, J. F., a n d GAHAY, J. B. (1937), ' Effects of the alkaloid nicotine on the rhythmicity of isolated heart preparations from Periplaneta americana and Prodenia eridania ', .7. agric. Res., 55, I-I9" ----and HAOER, A. (I934), ' O n the rates of contraction of the isolated heart and Malpighian tube of the insect, Periplaneta orientalis: method ', Iowa St. Coll. J. Sci., 8, 391-395 .
Key Word Index: Insect visceral nmscle, insect heart, acetylcholine.
4.°6
INHIBITION BY ACETYLCHOLINE OF COCKROACH HEART
(GROMPHADORHINAPORTENTOSA)
C A R L W. S C H A E F E R AND J A C Q U E L I N E
MILES
Systematics and Evolutionary Biology Section, Biological Sciences Group, University of Connecticut, Storrs o6268, U.S.A.
(Received 2I March, x97o) ABSTRACT i. The normal heart-rate (at 25° C.) of the Madagascar hissing cockroach is 66' 5 beats per minute. 2. There is no difference between sexes. 3. Io-~ M and Io -3 M acetylcholine slows the beat (to 56q and 63.i beats per minute respectively), whereas io -4 M ACh has no effect. 4. Glutamate (io -~ M) is also without effect, but 5-hydroxytryptamine (xo -2 M) is slightly stimulatory (68.8 beats per minute).
THE control of the insect h e a r t - b e a t is poorly understood, as is, indeed, the control of all insect visceral muscle. T h e beat appears i n m a n y insects to be of the i n n e r v a t e d myogenic type, with the i n n e r v a t i o n controlling a n intrinsically initiated a n d p r o m u l g a t e d contraction of the heart muscle. A l t h o u g h A C h is p r o b a b l y not a n e u r o m u s c u l a r transmitter i n insect skeletal muscle (Treherne, i966), it does have a n effect u p o n the contraction of insect h e a r t muscle. I t is not clear whether the effect is u p o n the muscle directly, or u p o n the n e u r o m u s c u l a r j u n c t i o n , or u p o n some intrinsic p a c e m a k e r ; a n d the effect varies, d e p e n d i n g often u p o n the insect group. I n the only cockroach (Dictyoptera) so far studied i n detail, Periplaneta americana L., A C h stimulated at low concentrations a n d m a y i n h i b i t at high (see references in Discussion). Little has been published of the effect of high concentrations, however, a n d we describe a case here where A C h intfibits at high c o n c e n t r a tions b u t appears to have no effect at a lower one. W e also include a few d a t a on the effect of glutamate and 5-HT.
METHODS AND MATERIALS Adults were used of the Madagascar hissing cockroach, Gromphadorhina portentosa Schaum, which has been kept in colony for some years at the University of Connecticut (on lab chow and water, at 3°° C. and 80 per cent R.H.). The cockroaches were immobilized by cooling, the head and thorax removed, and the abdominal dorsum bearing the heart isolated. The preparation was held at 25° C. in a water-bath and allowed to stand at least 15 minutes for temperature equilibration. Palm's (I946) Ringer's solution was used (formulation, in g. per litre distilled water: NaCI 9, KC1 o'4, CaC1, o'2, NaHCO~ o-x, glucose I). Drugs were dissolved in the Ringer's solution and used fresh (ACh as the chloride, 5-HT as the creatinine sulphate, glutamate as L-glutamic acid, all from Sigma Chemical Co., St. Louis, Mo.). In most experiments counts were made Io times per preparation of the number of contractions per minute, and these counts were averaged. All counts were taken at the fifth abdominal segment. Statistical significance was determined with Students' t-test. RESULTS T h e r e was no significant difference due to sex in the n u m b e r of contractions per m i n u t e of control hearts (Table I). Therefore, the
197o, x, 426-43o
427
I N H I B I T I O N OF H E A R T BY A C H
had no effect ( P > o . i ) , but the results with lO -3 M 5 - H T were almost significant at the 5 per cent level ( o . I > P > o . o 5 ) , and showed a slight stimulation (Table III).
results for males and females were combined, and the overall average (67"0 contractions per minute, W=28) was used as the control. High concentrations of A C h inhibit the heart (Table H). The statistical significance of these results, as compared to the control, is as follows : - IO-2 M ACh, P < o . o o I . Io -3 M ACh, P < o-ooi. IO-4 M ACh, o ' 6 < P < o - 7.
DISCUSSION Despite the wide experimental use of cockroaches, surprisingly little has been published on their normal heart contraction-rates. Only the rate of Periplaneta americana has been published, and different authors disagree on
Table / . - - C O N T R A C T I O N S
PER M I N U T E OF HEARTS OF M A L E AND FEMALE COCKROACHES CONSIDERED SEPARATELY
SEX
SPECIMEN
No. Female
I 2
6 5
3 4 5 6 7 8 9
IO
IO II I2
13 I4 I5 I6 I7 Male
N O . OF
6 IO IO 10 IO IO IO IO IO IO IO IO IO IO
I
5
2
I0
3 4 5 6 7 8 9
IO
IO II
N O . OF CONTRACTIONS PER M I N U T E
CONTRACTIONS
IO IO IO IO I0 I0 I0 IO
Average
T h e lowest concentration of ACh (lO -4 M) had no effect upon contractions of the heart. Because glutamate and 5 - H T have been implicated in the control of arthropod muscle, we tested their effects. Glutamate (lO -2 M)
(Average and Range) 6o.8 64-i 7o-2 6I'7 67'o 62-2 69.6 66. 7 65"5
(6o'I-6I'2) (63.1-65-2) (69"7-71-4) (6o'o-65"2) (65'9-68"I) (66"4-63'1) (68.6-7oq) (65.8-68.i) (64"9-67"5)
7I'2 (70"1-71"9)
67.1 6o. 3 7o'8 7I'9 7o'2 7o'5 69'o
(65.2-68.o) (58.8-61.2) (68"9-72.2) (68'9-74-o) (68'9-71.5) (67'4-72"2) (67'4-7o'5)
59'8 (57.8--61.8) 6I. 3 (6o.o-64.1) 62'6 (6o'o-64.2) 66-4 (65"9-67'3) 69.o (68.5-69.9) 68.I (67.2-69.1) 65"4 (64"6--66"4) 7I'4 (68"1-73-5) 68.6 (68.6--69.i) 65"4 (64"7-66"o) 66'8 (65'8-67.4) 67"o o"3 < P
it. The most frequently cited rate is that of Yeager and colleagues (Yeager and Hager, 1934; Yeager and Gahan, 1937) ; this rate ranges from 4 ° to 5 ° , and appears low, although temperatures were high (25-29 ° C.).
428
SCHAEFER
AND
I n at least one p a p e r the rate was b a s e d on b u t a few specimens (Yeager a n d H a g e r ,
Table//.--CONTRACTIONS P E R xo-2 M ACh Specimen No. I ,9
3 4 5 6 7 8 9 IO II I2
I3 14 I5 Average
Comp. gen. Pharmac.
MILES
tion) ( M c I n d o o , i939) ; 83, 28 ° C. (males) (Metcalf, W i n t o n , a n d F u k u t o , r964) ; 88
MINUTE
OF HEART
OF
G. portentosa*
I o -3 M ACh
No. of Contractions per Minute (Average and Range) 65-9 (63'`9-674) 580 (556-60'0) 5`9'6 (5i'7-55o) 47.i (46.5-48.o) 764 (73'2-8o-o) 457 (444-46"9) 61.6 (6o.6-63.2) 75'8 (75'9-76-9) 478 (46'9-49-2) 57"3 (55'6-594) 57'2 (55'6-58"2) 51.9 (51.7-52-6) 67. 3 (64.6-7o.6) 62.i (6o.o-64.5) 54"3 (526-55"6)
Specimen No. I `9
3 4 5 6 7 8 9 IO IX I2
I3
56. I
IO-4 M ACh
No. of Contractions per Minute (Average and Range) 62'I 6~' 5 6~'8 60.2 6I'7 64-o 62"3 63'6 64"6 63"7 64.`9 64'o 67' 3
(59'8-63"8) (59"4-63.4) (57.8-63.4) (58.4-61.5) (6o'o--62.4) (63.7-64.5) (6o'6-63'2) (6o'6-65'o) (6o'6-65"9) (6~'4-65"8) (6`9.3-66.7) (6o'6-66"7) (63"4-69.9)
Specimen No.
No. of Contractions per Minute (Average and Range) 66.6 65"9 66.2 67'4 66. 4 66'5 68.6 65.1 7o'o 66.8
I
2
3 4 5 6 7 8 9 IO
63. I
(63.8-68.2) (63"8-67-4) (65-2-68.2) (64'5-69"o) (63.8-7o.6) (64"5-69"o) (66.7-7o.6) (63.8-66.7) (68.2-71.4) (65.2-68.2)
66. 9
* Experiments were carried out in Palm' Ringer's solution with indicated amounts of ACh. In each case IO contractions were counted. Table III.--CONTRACTIONS PER MINUTE OF HEARTS OF G. portentosa*
Io -2 M GLUTAMATE Specimen No. I
2
3 4 5 6 7 8 9 IO
Average
No. of Contractions per Minute (Average and Range) 7o'7 64" 7 7o'6 68"4 69"o 66-6 66"I 66"7 68- 3 67"9
(67"4-75"o) (6I'8-67'4) (68"2-73'2) (65.2-7o.6) (65"9-71.4) (64'5-7o.6) (63-8-67.4) (64"5-69'8) (65.2-69.8) (65"2-7I'4)
67. 9
IO-~ M 5-HT Specimen No. I
9
3 4 5 6 7 8 9 IO
No. of Contractions per Minute (Average and Range) 64'6 67.x 67"4 67"3 67' 4 72"I 69" 7 7o"4 72"3 69.8
(67"8-66"7) (63.8-69.8) (63'8-69"8) (65"2-7o'6) (65-2-69.8) (69"8-74"1) (69.o-73.2) (66'7-74q) (69'o-74"I) (68-4-72.3)
68.8
* Experiments were carried out in Palm's Ringer's solution with glutamate or 5-HT. In each case io contractions were counted. I934). O t h e r s r e p o r t higher rates: 66-72 p e r m i n u t e , t e m p e r a t u r e u n r e p o r t e d (i p r e p a r a -
(females) a n d i o 5 (males), 25 ° C. ( O s m a n i a n d N a i d u , I96o); 8¢>-I2o, 3 °o C. (Krijgs-
197o, I
INHIBITION OF HEART BY ACH
m a n and Krijgsman-Berger, i952) ; 9o-I2O, 29 ° C. (Naidu, I955). Some of these wide ranges m a y reflect the sexual differences noted by O s m a n i and N a i d u (196o). T h e rate of Gromphadorhina portentosa (67.o per minute) is rather low a n d there is no sexual difference (Table I). This relatively low rate m a y be correlated with the animal's lethargy. N o one appears to have reported the effects of high A C h concentrations on cockroach hearts. O s m a n i a n d N a i d u (I96O) suggest that A C h at 1o -a (5"5 × IO-6 M ) partially inhibits, although N a i d u earlier (1955) h a d reported 1o -a to lO -5 A C h to be stimulatory. A C h o.I per cent (5"5× 1 o - 4 M ) partly inhibits the exposed Anopheles quadrimaculatus heart, b u t other concentrations were not tried (Jones, i956 ). T h e wax.moth heart (Galleria mellonella) is stimulated by low concentrations (2 × Io -~ to 7 × l°-a), partly inhibited by concentrations of 2 × IO-4 to 1.25 × IO-3 (about IO - 6 M to 7 × lO-6 M ) , a n d completely inhibited at concentrations > 2-5 × I o -3 (about 1.4 × Io -5 M ) (Folliott, 1957). This study with Galleria is the only one on insects directly comparable with our own, a n d there is a significant difference. Whereas low concentrations of A C h stimulate the Galleria heart, our lowest concentration (IO -4 M ) h a d no effect. It appears that the two p h e n o m e n a are different. I n the case of Galleria (and perhaps Periplaneta), inhibition m a y be due to a ' p o i s o n i n g ' of a tissue perhaps normally stimulated by ACh. I n the case of G. portentosa, A C h in high concentrations m a y normally inhibit the heart; or A C h m a y ordinarily have no effect, and high concentrations may here be poxsonous. T h e heart of G. portentosa is innervated. Methylene blue preparations show a pair of small ganglia asymmetrically a r r a n g e d within each segment, at least in the abdomen. These ganglia are the centres of a very loose and diffuse network of nerves which ramify over the surface of the heart.
429
It appears that the G. portentosa heart fits Prosser's ( 1 9 4 2 ) definition of an innervated myogenic muscle, b u t it is one relatively insensitive to ACh. A n o t h e r such muscle, more sensitive, m a y be that of the Periplaneta Malpighian tubules (Schaefer, 1968; more complete data to be published), although here the situation is complicated by the apparent absence of multiterminal innervation. Glutamate m a y well be the (or a) excitatory transmitter of insect skeletal muscle (Usherwood, Machili, and Leaf, 1968), b u t it appears to have no effect on the G. portentosa heart, at least at IO-2 m . 5-HT, which inlaibits insect skeletal muscle (Hill and Usherwood, I96i), stimulates the heart of P. americana (Davey, I961, I964), with a threshold of I o -8 M. T h e heart of G. portentosa is far less sensitive to 5 - H T , j u d g i n g from our results, which show a slight stimulatory effect at the high concentration of 10 - 2 m .
REFERENCES DAY,Y, K. G. (I96I), ' Substances controlling the rate of beating of the heart of Periplaneta ', Nature, Lond., 192, 284. - - - - (x964), ' The control of visceral muscles in insects ', Adv. Insect Physiol., 2, 2 x9-245. FOLUOTT, R. (1957), ' Action de l'ac~tylcholine sur le fonctionnement du vaisseau dorsal de la larve de Galleria mellonella L. ', Bull. Soc. Sci. Bretagne, 32, 12~-~24. HILL, R. B., and USHERWOOD, P. N. R. (I96I), ' The action of 5-hydroxytryptamine and related compounds on neuromuscular transmission in the locust Schistocerca gregaria ', J. Physiol., Lond., x57, 393-4ox. JONES, J. C. (I956), ' Effects of drugs on Anopheles heart rates ', J. exp. Zool., x33, 573-588. KRIJGSMAN, B. J., a n d KRIJGSMAN-BERGER, N. E. (I952), 'Physiological investigations into the heart function of arthropods. The heart of Periplaneta americana ', Bull. ent. Res., 42, 143-I55. MCINDoO, N. E. (I939) , ' Segmental blood vessels of the American cockroach (Periplaneta americana (L.)) ', ft. Morph., 65, 323-347. METCALF, R. L., WXNTON, M. Y., and FUKUTO, T. R. (I964) , ' T h e effects of cholinergic substances upon the isolated heart of Periplaneta americana ', J. Insect Physiol., zo, 353-361. NAIDU, M. B. (I955) , 'Physiological action of
drugs and insecticides on insects ', Bull. ent. Res., 4 6, 2o5-2~o.
43 °
S C H A E F E R A N D MILES
OSMANI, Z. H., a n d NAIDU, M. B. (196o), ' Effect of temperature on the frequency of the heartbeat of a cockroach ', Proc. zool. Soc. Calcutta, x3~ IO7xl 4. PALM, N. B. (I946), ' Studies on the peristalsis of the Malpighian tubes in insects ', Acta Univ. lund., 42, 1-39. PROSSER, C. L. (1942), ' A n analysis of the action of acetylcholine on hearts, particularly in arthropods ', Biol. Bull. mar. biol. Lab., Woods Hole, 83, I45-I64. SCHAEFER, C. W. (I968), ' I n h i b i t i o n by acetylcholine of Malpighian tubule muscle of the cockroach Periplaneta americana ', Ann. ent. Soc. Am., 6I, 226-227. TREHERNE, J. E. (x966), The Weurochemistry of Arthropods. Cambridge: University Press.
USHER'~,'OOD, P. N. R., MACHILI,P., and LEAF,G. (i968), ' L-Glutamate at insect excitatory nerve-muscle synapse ', Nature, Lond., 219, II69-I172. YEACEr~, J. F., a n d GAHAY, J. B. (1937), ' Effects of the alkaloid nicotine on the rhythmicity of isolated heart preparations from Periplaneta americana and Prodenia eridania ', .7. agric. Res., 55, I-I9" ----and HAOER, A. (I934), ' O n the rates of contraction of the isolated heart and Malpighian tube of the insect, Periplaneta orientalis: method ', Iowa St. Coll. J. Sci., 8, 391-395 .
Key Word Index: Insect visceral nmscle, insect heart, acetylcholine.