- Email: [email protected]
The effect of drugs on the alimentary canal of the African migratory locust Locusta migratoria
Comp. Biochem. Physiol., 1966, Vol. 17, pp. 755 to 764. Pergamon Press Ltd. Printed in Great Britain
THE EFFECT OF DRUGS ON THE ALIMENTARY CANAL OF THE AFRICAN MIGRATORY LOCUST LOCUSTA MIGRATORIA M. A. FREEMAN Department of Physiology and Pharmacology, Chelsea College of Science and Technology, London, S.W.3 (Received 24 August 1965)
Abstract--1. The effect of various compounds on the isolated fore gut and hind gut of Locusta migratoria and hind gut of Periplaneta americana is described. 2. The hind gut of locust was sensitive to 5-hydroxytryptamine, dopamine and tyramine but showed no response to acetylcholine, adrenaline, DOPA and tryptamine. 3. The fore gut of locust contracted in the presence of 5-hydroxytryptamine, dopamine, adrenaline and noradrenaline. It did not respond to acetylcholine, tryptamine and DOPA. 4. The response of locust intestine to 5-hydroxytryptamine was inhibited by bromolysergic acid diethylamide. 5. The hind gut of Periplaneta was sensitive to tryptamine and 5-hydroxytryptamine. The response to 5-HT was inhibited by lysergic acid diethylamide. 6. It is suggested that the intestine of locust would be of use in the analysis of possible chemical mediators in insects.
INTRODUCTION PREVIOUS work on the pharmacology of insect gut has been confined mainly to the isolated gut of Periplaneta americana. Kooistra (1950) found that the whole gut was sensitive to acetylcholine and that the response to this drug was potentiated by anticholinesterases. According to Davey (1962) and C o l h o u n ( 1 9 6 3 ) , 5-hydroxytryptamine, 5,6-dihydroxytryptamine and tryptamine produce contractions of the isolated hind gut. Cameron (1953) found a compound in the corpus cardiacum of Periplaneta which he tentatively identified as an o-diphenol. This compound excited the isolated hind gut and heart. Various pharmacologically active compounds have since been extracted from insect tissues and in many cases these have been tested on the isolated gut and heart (Unger, 1957; Gersch et al., 1960). However, little information is available as to the sensitivity and selectivity of these tissues to drugs. T h e present paper describes the effect of various naturally occurring compounds on the isolated fore gut and hind gut of Locusta migratoria. T h e results are compared with those from the hind gut of Periplaneta americana. 755
756
M.A. FREEMAN
METHODS Mature adult Locusta migratoria of both sexes were used. In the fore-gut preparation, ligatures were placed around the oesophagus and the junction of the gizzard and mid gut. The hind-gut preparation consisted of the ileum, colon and rectum. Tissues were suspended in a 5 ml gut bath in an aerated solution of insect saline solution (NaC1, 8.2g; CaC12, 0.22g; MgC12, 0.19g; KHCO3, 0-4 g; and KH~PO4, 0.82 g/1. water; Hoyle, 1953). The preparation was attached to a Sanborn force transducer FTA-100-1 and subjected to a load of 0.1-0.2 g. Contractions were recorded using a Sanborn carrier preamplifier and pen recorder. The chart speed was 0.25 mm/sec and the sensitivity was set at 0.2 g/cm. All drugs were prepared in insect saline solution. RESULTS (a) The isolated hind gut of Locusta (1) Acetylcholine (ACh). No responses to ACh in concentrations of 0.250/zg/ml were observed. (2) 5-Hydroxytryptamine (5-HT). Fig. 1 shows the effect of three concentrations of 5 - H T on the hind gut. In each case there was a delay of approximately 1 rain before the onset of the response which consisted of a series of rapid contractions. The amplitude of the contractions varied with the concentration of drug. The hind gut was more sensitive to 5 - H T than to any other compound tested; it responded to concentrations of 5-HT as low as 20-40 ng/ml. (3) Tryptamine. The hind gut was insensitive to tryptamine in concentrations of 0.1-50/xg/ml. (4) 3,4-Dihydroxyphenylalanine(DOPA). No consistent results were obtained with DOPA. Occasionally the hind gut gave a single contraction after 5-10 min with concentrations of the drug above 10 ~g/ml. However, there was no relation between responses and concentrations of drug. (5) 3-Hydroxytyramine (dopamine). The hind gut was sensitive to dopamine in concentrations greater than 1/~g/ml. Fig. 2 shows the response to 10/xg dopamine (2 ttg/ml). The duration of the individual contractions produced by dopamine was generally longer than those seen with 5-HT, although the amplitude of the contractions to dopamine was never as great as those produced by 5-HT. (6) Tyramine. Tyramine in concentrations of 10 gg/ml produced contractions similar to those seen with dopamine but of smaller amplitude. (7) Adrenaline and noradrenaline. The preparation was insensitive to these compounds in concentrations of 0-5-50/xg/ml. (b) The isolatedfore gut of Locusta When first set up in the bath the fore gut was quiescent but after about 1 hr contractions at a frequency of about 2-3/min were seen and these continued throughout the experimental period (4-5 hr). The fore gut and mid gut of Locusta contain a brown fluid which is extruded from the mouth when the animals are handled. Addition of this fluid to the bath inhibited the spontaneous contractions of the isolated fore gut (Fig. 3).
EFFECT
OF DRUGS
ON
ALIMENTARY
CANAL
OF
LOCUSTA MIGRATOR1A
757
O'1 /zg 5 H T .--
!~!
:::
ii ;i
illlll
0'2/~g
5HT
!illiiiii~ !~!!!! !I!
:~: !~i ::::
I~i~::: ,:= iiii I~::
;i
~i =:
FtG. 1. Response of the isolated hind gut of Locusta migrator~a to increasing concentrations of 5 - H T (20, 40 and 80 ng/ml). There was a delay of about 1 m i n before the onset of the response.
~T!!!!
lOp.g
!!!!!
!!! ::::
Dopomine i]
rrJ!Irlrlfr!!llrlllflillIl! llIHill!!!
!Jrrrf
'
FxG. 2. Response of the isolated hind gut of Locusta migratoria to 2 / z g / m l dopamine.
758
M.A.
FPY_~MAN
The response of the isolated fore gut to drugs varied according to whether the preparation was contracting or quiescent.
FIG. 3.
I n h i b i t i o n of the s p o n t a n e o u s contractions of t h e isolated fore g u t of
Locusts b y g u t fluid; 50/zl of fluid w e r e a d d e d to t h e b a t h at arrow.
(1) 5-Hydroxytryptamine. The fore gut was sensitive to 5-HT in concentrations above 0"1/zg/ml. Fig. 4 shows the effect of 5-HT on a quiescent preparation. There was a prolonged increase in tone of the tissue; small contractions appeared
~z~
iiiiiiiill ~lili:I::::I!!i!I:,i!iI]~!liiiiI[]~li!!! ~
::::iIi::it ::::liiI ~:l::i:i]::!::i
,::::: :::i ....:: i!::
ilii
iiii !iiiiiii!ilii!i!!iiii!i!ii~i
Ui!ili!i i iii!lil!i:il lli/iiili iiil i iiiill!:iiillil
F I o . 4. R e s p o n s e of the isolated fore g u t of Locusta migratoria to increasing c o n centrations of 5 - H T (0"2, 0"4 a n d 0"8 ftg/ml). T h i s t y p e of r e s p o n s e is seen b e f o r e t h e onset of s p o n t a n e o u s contractions.
as the concentration of the drug was increased. The response of a fore gut showing spontaneous activity was more pronounced (Fig. 5). (2) Doparnine. Dopamine had no effect on a quiescent preparation. However, when dopamine was added to a preparation showing spontaneous activity two types of responses were seen according to the concentration of the drug. For example, 10/~g dopamine (2 pg/ml) decreased the amplitude and frequency of contractions while 20/~g dopamine (4/~g/ml) produced small contractions of longer duration (Fig. 6). (3) Adrenal~ and noradrenaline. These compounds produced no response on quiescent preparations. When added to a fore gut showing spontaneous activity
EFFECT
OF DRUGS
ON ALIMI~NTARY
CANAL
OF
LOCUSTA M1GRATORIA
~.
HH ii{{
FxG. 5. The effect of 5 fig 5 - H T (1 pg/ml) on an isolated fore gut showing slight spontaneous activity.
FIG. 6. T h e effect of dopamine on an isolated fore gut showing spontaneous activity; 10 pg dopamine (2 pg/rnl) inhibited the contractions of the locust fore gut while in the presence of 20 pg dopamine (4 pg/nd) contractions of longer duration and low amplitude are seen
759
760
M . A . FREEMAN
i iiii IIii iii~
iJ
iiii FIo. 7. Response of the isolated fore gut of Locusta to 10/zg adrenaline (2/zg/ml). adrenaline (Fig. 7) and noradrenaline produced an increase in the amplitude and frequency of contractions. (c) The effect of bromolysergic acid diethylamide (BOL) on the fore and hind gut T h e responses of both fore and hind gut of Locusta to 5 - H T were inhibited by BOL. Fig. 8 shows that the contractions of hind gut produced by 0.2/zg/ml 5 - H T were completely inhibited by 40 ng/ml BOL. Increased concentrations of
::i::iiii::ii:.-'.-~i'.5~ iiff ~ ~ -l~i ~-..::.... ......... !:: ::igliii::ggii~il iili i! !iiil]iiil iii[i~2: ................ •: : : : : : : : ::::: : : : : : : : : : : : :
...... ~iiiiil,i :::: : : ~:::1:: |
~.,... :.:.!! ,.... :/?::?:i;I~ igiiigiiigli:.I~ ~ii iii ~I~... s!sli!::f!i!si~:.li~ iil:: !ii ii ::=~ = =~:=":* :F: t
i ,~.r
ii::iiil iiiiiiiilliiiliii iiiil Ti
i~!~l~l~l~r..li~ ~:2~I~ :-t lliilil~iliii!li~ii lliilililiiiiliiii lliilililiiiili!ii lllililillii~l!!i!
i!iii
til't '
ill
l~::.--l~.,2-!iV!i!li~ilii~qii~i
FIG. 8. Inhibition of the response of hind gut of Locusta to 1/xg 5-HT by 0"2/zg bromolysergie acid diethylamide. The antagonist was added 2 rain before the next dose of 5-HT. B O L did not produce contractions of the hind gut. T h e concentration of B O L giving complete inhibition of the response to 5 - H T also inhibited the responses of hind gut to dopamine and tyramine. T h e response of the fore gut to 5 - H T could be partially inhibited by low concentrations of B O L (0.2-2/~g/ml). However, increased concentrations of the
EFFECT OF DRUGS ON ALIMENTARY CANAL OF L O C U S T A M I G R A T O R I A
761
antagonist produced an increase in the amplitude and frequency of spontaneous contractions. This effect appeared to be irreversible. (d) The isolated hind gut of Periplaneta Previous work on the pharmacology of the hind gut of Periplaneta involved the use of isotonic levers; Kooistra (1950), however, used an optical method in studying the whole gut. To allow a direct comparison of the hind gut of the American cockroach with that of the locust, recordings were made using the force transducer system described above. The hind gut of cockroach did not respond to acetylcholine, adrenaline or noradrenaline in concentrations of 0.1-50/zg/ml. 5-HT in concentrations greater than 1/zg/ml produced a prolonged increase in tone, but there was little difference in the amplitude of the contraction with increasing concentrations of drug. A
FIO. 9. Response of the isolated hind gut of Periplanetato 10/xg and 20/xg 5-HT and inhibition of the response to 10/zg 5-HT by 20/zg lysergic acid diethylamide. similar response was obtained with tryptamine, the minimum effective concentration being approximately 10/zg/ml. The response to 5-HT could be completely inhibited by lysergic acid diethylamide (LSD). Fig. 9 shows the effect of two concentrations of 5-HT on the isolated fore gut and the inhibition of the response by LSD. No excitation of the gut by higher concentrations of LSD was observed. DISCUSSION The present results have shown that the hind gut of Locusta differs in many important respects from the fore gut. The hind gut showed little or no spontaneous activity and reacted to drugs such as 5-HT and dopamine by large contractions corresponding to an increase in tension of up to 1 g. In contrast, semitivity of the fore gut to drugs other than 5-HT was seen only in preparations showing spontaneous contractions. The period of quiescence of the fore gut lasted for about 1 hr during which the bath was emptied and refilled several times.
762
M.A. FREEMAN
This could indicate that the fore gut contained substances which inhibited the action of certain drugs and which were graduaUy washed from the tissue, the onset of spontaneous activity and sensitivity to drugs such as noradrenaline and dopamine being coincident. There is some evidence to support this view. The addition of the fluid content of a fresh fore gut to an isolated fore gut showing spontaneous activity produced a prolonged period of quiescence. It is to be expected that some of this fluid would be present in the isolated preparation. The inhibitory effect of gut fluid on the action of drugs on the fore gut is being investigated. In addition to the differences in the behaviour of isolated fore and hind gut in the presence and absence of drugs, there were marked differences in the sensitivity of these tissues to drugs. For example, adrenaline and noradrenaline which were inactive on the hind gut produced an increase in both amplitude and frequency of fore-gut contractions. These differences are summarized in Table 1. TABLE 1 Minimum effective dose
5-Hydroxytryptamine Tyramine Dopamine Noradrenaline Adrenaline Aeetylcholine DOPA Tryptamine
Hind gut
Fore gut
20 ng/ml 10/~g/ml 1 p.g/ml * * * * *
0.2 pg/ml Not tested 0"5 Izg/ml 1 Izg/ml 1 I~g/ml * * *
* No response to drug in concentrations of 0.5 to 50/~g/ml The results obtained with the isolated fore gut of Locusts are of particular interest as no information as to the pharmacology of insect fore gut has previously been available. This may be related to the delicate nature of the fore gut of many insects. For example, Davey (1964) describes the fore gut of Periplaneta as unsuitable for use with conventional mechanographic techniques. A study of the pharmacology of insect tissues may give some indication as to the type of compounds which could act as chemical mediators. Several of the compounds tested have been found in insect tissues. Acetylcholine. In contrast to the results of Kooistra (1950) on Periplaneta and of Gersh (1955) on larvae of Chaoborus, the gut of Locusta was found to be insensitive to ACh. It has been suggested that ACh may be important in the control of contractions of insect gut (Davey, 1964). These results indicate that there may be generic variations in the response of tissues and that generalizations as to the
EFFECT OF DRUGS O N ALIMENTARY CANAL OF L O C U S T A M I G R A T O R I A
763
role of ACh in insects may not be valid at this stage. However, it should be noted that in this series of experiments no response to ACh was obtained with the hind gut of Periplaneta. This could be explained either by differences in the method of recording or by the fact that contractions to ACh recorded by Kooistra (1950) with whole gut were produced by the fore or mid gut. 5-Hydroxytryptamine. Both hind gut and fore gut of Locusta responded to low concentrations of 5-HT, greater sensitivity being shown by the hind gut. Davey (1962) reports that the hind gut of Periplaneta contains a compound showing certain properties of a tryptamine derivative and suggests that this compound may be released from the hind gut in the presence of homogenates of corpus cardiacum. The sensitivity of the hind gut of locust to 5-HT might suggest that an indolalkylamine is involved in the control of the gut in this species. Preliminary investigations into the compounds present in locust gut have failed to show the presence of 5-HT. The gut, however, contains a compound which when tested on mammalian preparations has similar properties to those of noradrenaline and adrenaline. Adrenaline and noradrenaline. The excitation of the fore gut by adrenaline and noradrenaline is of interest since many invertebrate tissues are relatively insensitive to these compounds. The presence of these amines in insect tissues has been reported by Ostlund (1954), and von Euler (1961) and Barton Browne et al. (1961) found an adrenaline-like compound in the corpus cardiacum of Periplaneta. The differences in the pattern of the responses to drugs shown by the fore gut and hind gut of Locusta and in the sensitivity to drugs suggest that these tissues would be of use in the analysis of pharmacologically active compounds in extracts of insect tissues. Acknowledgements--I am indebted to Professor S. E. Dicker for reading and discussing this paper and to the Anti-Locust Research Centre for the supply of animals. REFERENCES BARTON BROWNE L., DOBSON L. F., HODGSON E. S. 8: KmALY J. K. (1961) Adrenergic properties of the cockroach corpus cardiacum. Gen. Comp. Endocrinol. 1, 232-236. CAMERON M. L. (1953) Secretion of an o-diphenol in the corpus cardiacum of insects. Nature, Lond. 192, 349-350. COLI-IOLrN E. H. (1963) Synthesis of 5-hydroxytryptamine in the American cockroach. Experientia 19, 9-10. DAVEY K. G. (1962) T h e mode of action of the corpus cardiacum on the hind gut in Periplaneta americana. J. exp. Biol. 39, 319-324. DAVEY K. G. (1964) T h e control of visceral muscle in insects. In Advances in Insect Physiology, Vol. 2. Academic Press, N.Y. VON EULER U. S. (1961) Occurrence of catechol amines in the Acrania and invertebrates. Nature, Lond. 190, 170-171. GERSH M. (1955) Untersuchungen tiber Auslosung und Steuerung der Darmbewegungen bei der Larve yon Ckaoborus. Biol. Zbl. 74, 601-628. GERSCH M., FiscrmR F., UNGIm H. & KOCH H. (1960) Die Isollerung neurohormonaler Factoren aus dem Nervensystem der Ktichenschabe Periplaneta americana. Z. Naturf. 15, 319-322. HOYLE G. (1953) Potassium ions and insect nerve muscle. J . exp. Biol. 30, 121-135.
764
M . A . FREEMAN
KOOIST~ G. (1950) Contribution to the knowledge of the action of acetylcholine on the intestine of Periplaneta americana L. Physiol. Comp. 2, 75-80. OSTLtrtqD E. (1954) T h e distribution of catechol amines in lower animals and their effect on the heart. Acta Physiol. Scand. 31 (Suppl. 112), 1-65. U N G ~ H. (1957) Untersuchungen zur neurohormonalen Beeinflussung der Herzttitigkeit bei Schaben (P. orientalis, P. americana, PhyUodroma germanica). Biol. Zbl. 76, 439--442.
THE EFFECT OF DRUGS ON THE ALIMENTARY CANAL OF THE AFRICAN MIGRATORY LOCUST LOCUSTA MIGRATORIA M. A. FREEMAN Department of Physiology and Pharmacology, Chelsea College of Science and Technology, London, S.W.3 (Received 24 August 1965)
Abstract--1. The effect of various compounds on the isolated fore gut and hind gut of Locusta migratoria and hind gut of Periplaneta americana is described. 2. The hind gut of locust was sensitive to 5-hydroxytryptamine, dopamine and tyramine but showed no response to acetylcholine, adrenaline, DOPA and tryptamine. 3. The fore gut of locust contracted in the presence of 5-hydroxytryptamine, dopamine, adrenaline and noradrenaline. It did not respond to acetylcholine, tryptamine and DOPA. 4. The response of locust intestine to 5-hydroxytryptamine was inhibited by bromolysergic acid diethylamide. 5. The hind gut of Periplaneta was sensitive to tryptamine and 5-hydroxytryptamine. The response to 5-HT was inhibited by lysergic acid diethylamide. 6. It is suggested that the intestine of locust would be of use in the analysis of possible chemical mediators in insects.
INTRODUCTION PREVIOUS work on the pharmacology of insect gut has been confined mainly to the isolated gut of Periplaneta americana. Kooistra (1950) found that the whole gut was sensitive to acetylcholine and that the response to this drug was potentiated by anticholinesterases. According to Davey (1962) and C o l h o u n ( 1 9 6 3 ) , 5-hydroxytryptamine, 5,6-dihydroxytryptamine and tryptamine produce contractions of the isolated hind gut. Cameron (1953) found a compound in the corpus cardiacum of Periplaneta which he tentatively identified as an o-diphenol. This compound excited the isolated hind gut and heart. Various pharmacologically active compounds have since been extracted from insect tissues and in many cases these have been tested on the isolated gut and heart (Unger, 1957; Gersch et al., 1960). However, little information is available as to the sensitivity and selectivity of these tissues to drugs. T h e present paper describes the effect of various naturally occurring compounds on the isolated fore gut and hind gut of Locusta migratoria. T h e results are compared with those from the hind gut of Periplaneta americana. 755
756
M.A. FREEMAN
METHODS Mature adult Locusta migratoria of both sexes were used. In the fore-gut preparation, ligatures were placed around the oesophagus and the junction of the gizzard and mid gut. The hind-gut preparation consisted of the ileum, colon and rectum. Tissues were suspended in a 5 ml gut bath in an aerated solution of insect saline solution (NaC1, 8.2g; CaC12, 0.22g; MgC12, 0.19g; KHCO3, 0-4 g; and KH~PO4, 0.82 g/1. water; Hoyle, 1953). The preparation was attached to a Sanborn force transducer FTA-100-1 and subjected to a load of 0.1-0.2 g. Contractions were recorded using a Sanborn carrier preamplifier and pen recorder. The chart speed was 0.25 mm/sec and the sensitivity was set at 0.2 g/cm. All drugs were prepared in insect saline solution. RESULTS (a) The isolated hind gut of Locusta (1) Acetylcholine (ACh). No responses to ACh in concentrations of 0.250/zg/ml were observed. (2) 5-Hydroxytryptamine (5-HT). Fig. 1 shows the effect of three concentrations of 5 - H T on the hind gut. In each case there was a delay of approximately 1 rain before the onset of the response which consisted of a series of rapid contractions. The amplitude of the contractions varied with the concentration of drug. The hind gut was more sensitive to 5 - H T than to any other compound tested; it responded to concentrations of 5-HT as low as 20-40 ng/ml. (3) Tryptamine. The hind gut was insensitive to tryptamine in concentrations of 0.1-50/xg/ml. (4) 3,4-Dihydroxyphenylalanine(DOPA). No consistent results were obtained with DOPA. Occasionally the hind gut gave a single contraction after 5-10 min with concentrations of the drug above 10 ~g/ml. However, there was no relation between responses and concentrations of drug. (5) 3-Hydroxytyramine (dopamine). The hind gut was sensitive to dopamine in concentrations greater than 1/~g/ml. Fig. 2 shows the response to 10/xg dopamine (2 ttg/ml). The duration of the individual contractions produced by dopamine was generally longer than those seen with 5-HT, although the amplitude of the contractions to dopamine was never as great as those produced by 5-HT. (6) Tyramine. Tyramine in concentrations of 10 gg/ml produced contractions similar to those seen with dopamine but of smaller amplitude. (7) Adrenaline and noradrenaline. The preparation was insensitive to these compounds in concentrations of 0-5-50/xg/ml. (b) The isolatedfore gut of Locusta When first set up in the bath the fore gut was quiescent but after about 1 hr contractions at a frequency of about 2-3/min were seen and these continued throughout the experimental period (4-5 hr). The fore gut and mid gut of Locusta contain a brown fluid which is extruded from the mouth when the animals are handled. Addition of this fluid to the bath inhibited the spontaneous contractions of the isolated fore gut (Fig. 3).
EFFECT
OF DRUGS
ON
ALIMENTARY
CANAL
OF
LOCUSTA MIGRATOR1A
757
O'1 /zg 5 H T .--
!~!
:::
ii ;i
illlll
0'2/~g
5HT
!illiiiii~ !~!!!! !I!
:~: !~i ::::
I~i~::: ,:= iiii I~::
;i
~i =:
FtG. 1. Response of the isolated hind gut of Locusta migrator~a to increasing concentrations of 5 - H T (20, 40 and 80 ng/ml). There was a delay of about 1 m i n before the onset of the response.
~T!!!!
lOp.g
!!!!!
!!! ::::
Dopomine i]
rrJ!Irlrlfr!!llrlllflillIl! llIHill!!!
!Jrrrf
'
FxG. 2. Response of the isolated hind gut of Locusta migratoria to 2 / z g / m l dopamine.
758
M.A.
FPY_~MAN
The response of the isolated fore gut to drugs varied according to whether the preparation was contracting or quiescent.
FIG. 3.
I n h i b i t i o n of the s p o n t a n e o u s contractions of t h e isolated fore g u t of
Locusts b y g u t fluid; 50/zl of fluid w e r e a d d e d to t h e b a t h at arrow.
(1) 5-Hydroxytryptamine. The fore gut was sensitive to 5-HT in concentrations above 0"1/zg/ml. Fig. 4 shows the effect of 5-HT on a quiescent preparation. There was a prolonged increase in tone of the tissue; small contractions appeared
~z~
iiiiiiiill ~lili:I::::I!!i!I:,i!iI]~!liiiiI[]~li!!! ~
::::iIi::it ::::liiI ~:l::i:i]::!::i
,::::: :::i ....:: i!::
ilii
iiii !iiiiiii!ilii!i!!iiii!i!ii~i
Ui!ili!i i iii!lil!i:il lli/iiili iiil i iiiill!:iiillil
F I o . 4. R e s p o n s e of the isolated fore g u t of Locusta migratoria to increasing c o n centrations of 5 - H T (0"2, 0"4 a n d 0"8 ftg/ml). T h i s t y p e of r e s p o n s e is seen b e f o r e t h e onset of s p o n t a n e o u s contractions.
as the concentration of the drug was increased. The response of a fore gut showing spontaneous activity was more pronounced (Fig. 5). (2) Doparnine. Dopamine had no effect on a quiescent preparation. However, when dopamine was added to a preparation showing spontaneous activity two types of responses were seen according to the concentration of the drug. For example, 10/~g dopamine (2 pg/ml) decreased the amplitude and frequency of contractions while 20/~g dopamine (4/~g/ml) produced small contractions of longer duration (Fig. 6). (3) Adrenal~ and noradrenaline. These compounds produced no response on quiescent preparations. When added to a fore gut showing spontaneous activity
EFFECT
OF DRUGS
ON ALIMI~NTARY
CANAL
OF
LOCUSTA M1GRATORIA
~.
HH ii{{
FxG. 5. The effect of 5 fig 5 - H T (1 pg/ml) on an isolated fore gut showing slight spontaneous activity.
FIG. 6. T h e effect of dopamine on an isolated fore gut showing spontaneous activity; 10 pg dopamine (2 pg/rnl) inhibited the contractions of the locust fore gut while in the presence of 20 pg dopamine (4 pg/nd) contractions of longer duration and low amplitude are seen
759
760
M . A . FREEMAN
i iiii IIii iii~
iJ
iiii FIo. 7. Response of the isolated fore gut of Locusta to 10/zg adrenaline (2/zg/ml). adrenaline (Fig. 7) and noradrenaline produced an increase in the amplitude and frequency of contractions. (c) The effect of bromolysergic acid diethylamide (BOL) on the fore and hind gut T h e responses of both fore and hind gut of Locusta to 5 - H T were inhibited by BOL. Fig. 8 shows that the contractions of hind gut produced by 0.2/zg/ml 5 - H T were completely inhibited by 40 ng/ml BOL. Increased concentrations of
::i::iiii::ii:.-'.-~i'.5~ iiff ~ ~ -l~i ~-..::.... ......... !:: ::igliii::ggii~il iili i! !iiil]iiil iii[i~2: ................ •: : : : : : : : ::::: : : : : : : : : : : : :
...... ~iiiiil,i :::: : : ~:::1:: |
~.,... :.:.!! ,.... :/?::?:i;I~ igiiigiiigli:.I~ ~ii iii ~I~... s!sli!::f!i!si~:.li~ iil:: !ii ii ::=~ = =~:=":* :F: t
i ,~.r
ii::iiil iiiiiiiilliiiliii iiiil Ti
i~!~l~l~l~r..li~ ~:2~I~ :-t lliilil~iliii!li~ii lliilililiiiiliiii lliilililiiiili!ii lllililillii~l!!i!
i!iii
til't '
ill
l~::.--l~.,2-!iV!i!li~ilii~qii~i
FIG. 8. Inhibition of the response of hind gut of Locusta to 1/xg 5-HT by 0"2/zg bromolysergie acid diethylamide. The antagonist was added 2 rain before the next dose of 5-HT. B O L did not produce contractions of the hind gut. T h e concentration of B O L giving complete inhibition of the response to 5 - H T also inhibited the responses of hind gut to dopamine and tyramine. T h e response of the fore gut to 5 - H T could be partially inhibited by low concentrations of B O L (0.2-2/~g/ml). However, increased concentrations of the
EFFECT OF DRUGS ON ALIMENTARY CANAL OF L O C U S T A M I G R A T O R I A
761
antagonist produced an increase in the amplitude and frequency of spontaneous contractions. This effect appeared to be irreversible. (d) The isolated hind gut of Periplaneta Previous work on the pharmacology of the hind gut of Periplaneta involved the use of isotonic levers; Kooistra (1950), however, used an optical method in studying the whole gut. To allow a direct comparison of the hind gut of the American cockroach with that of the locust, recordings were made using the force transducer system described above. The hind gut of cockroach did not respond to acetylcholine, adrenaline or noradrenaline in concentrations of 0.1-50/zg/ml. 5-HT in concentrations greater than 1/zg/ml produced a prolonged increase in tone, but there was little difference in the amplitude of the contraction with increasing concentrations of drug. A
FIO. 9. Response of the isolated hind gut of Periplanetato 10/xg and 20/xg 5-HT and inhibition of the response to 10/zg 5-HT by 20/zg lysergic acid diethylamide. similar response was obtained with tryptamine, the minimum effective concentration being approximately 10/zg/ml. The response to 5-HT could be completely inhibited by lysergic acid diethylamide (LSD). Fig. 9 shows the effect of two concentrations of 5-HT on the isolated fore gut and the inhibition of the response by LSD. No excitation of the gut by higher concentrations of LSD was observed. DISCUSSION The present results have shown that the hind gut of Locusta differs in many important respects from the fore gut. The hind gut showed little or no spontaneous activity and reacted to drugs such as 5-HT and dopamine by large contractions corresponding to an increase in tension of up to 1 g. In contrast, semitivity of the fore gut to drugs other than 5-HT was seen only in preparations showing spontaneous contractions. The period of quiescence of the fore gut lasted for about 1 hr during which the bath was emptied and refilled several times.
762
M.A. FREEMAN
This could indicate that the fore gut contained substances which inhibited the action of certain drugs and which were graduaUy washed from the tissue, the onset of spontaneous activity and sensitivity to drugs such as noradrenaline and dopamine being coincident. There is some evidence to support this view. The addition of the fluid content of a fresh fore gut to an isolated fore gut showing spontaneous activity produced a prolonged period of quiescence. It is to be expected that some of this fluid would be present in the isolated preparation. The inhibitory effect of gut fluid on the action of drugs on the fore gut is being investigated. In addition to the differences in the behaviour of isolated fore and hind gut in the presence and absence of drugs, there were marked differences in the sensitivity of these tissues to drugs. For example, adrenaline and noradrenaline which were inactive on the hind gut produced an increase in both amplitude and frequency of fore-gut contractions. These differences are summarized in Table 1. TABLE 1 Minimum effective dose
5-Hydroxytryptamine Tyramine Dopamine Noradrenaline Adrenaline Aeetylcholine DOPA Tryptamine
Hind gut
Fore gut
20 ng/ml 10/~g/ml 1 p.g/ml * * * * *
0.2 pg/ml Not tested 0"5 Izg/ml 1 Izg/ml 1 I~g/ml * * *
* No response to drug in concentrations of 0.5 to 50/~g/ml The results obtained with the isolated fore gut of Locusts are of particular interest as no information as to the pharmacology of insect fore gut has previously been available. This may be related to the delicate nature of the fore gut of many insects. For example, Davey (1964) describes the fore gut of Periplaneta as unsuitable for use with conventional mechanographic techniques. A study of the pharmacology of insect tissues may give some indication as to the type of compounds which could act as chemical mediators. Several of the compounds tested have been found in insect tissues. Acetylcholine. In contrast to the results of Kooistra (1950) on Periplaneta and of Gersh (1955) on larvae of Chaoborus, the gut of Locusta was found to be insensitive to ACh. It has been suggested that ACh may be important in the control of contractions of insect gut (Davey, 1964). These results indicate that there may be generic variations in the response of tissues and that generalizations as to the
EFFECT OF DRUGS O N ALIMENTARY CANAL OF L O C U S T A M I G R A T O R I A
763
role of ACh in insects may not be valid at this stage. However, it should be noted that in this series of experiments no response to ACh was obtained with the hind gut of Periplaneta. This could be explained either by differences in the method of recording or by the fact that contractions to ACh recorded by Kooistra (1950) with whole gut were produced by the fore or mid gut. 5-Hydroxytryptamine. Both hind gut and fore gut of Locusta responded to low concentrations of 5-HT, greater sensitivity being shown by the hind gut. Davey (1962) reports that the hind gut of Periplaneta contains a compound showing certain properties of a tryptamine derivative and suggests that this compound may be released from the hind gut in the presence of homogenates of corpus cardiacum. The sensitivity of the hind gut of locust to 5-HT might suggest that an indolalkylamine is involved in the control of the gut in this species. Preliminary investigations into the compounds present in locust gut have failed to show the presence of 5-HT. The gut, however, contains a compound which when tested on mammalian preparations has similar properties to those of noradrenaline and adrenaline. Adrenaline and noradrenaline. The excitation of the fore gut by adrenaline and noradrenaline is of interest since many invertebrate tissues are relatively insensitive to these compounds. The presence of these amines in insect tissues has been reported by Ostlund (1954), and von Euler (1961) and Barton Browne et al. (1961) found an adrenaline-like compound in the corpus cardiacum of Periplaneta. The differences in the pattern of the responses to drugs shown by the fore gut and hind gut of Locusta and in the sensitivity to drugs suggest that these tissues would be of use in the analysis of pharmacologically active compounds in extracts of insect tissues. Acknowledgements--I am indebted to Professor S. E. Dicker for reading and discussing this paper and to the Anti-Locust Research Centre for the supply of animals. REFERENCES BARTON BROWNE L., DOBSON L. F., HODGSON E. S. 8: KmALY J. K. (1961) Adrenergic properties of the cockroach corpus cardiacum. Gen. Comp. Endocrinol. 1, 232-236. CAMERON M. L. (1953) Secretion of an o-diphenol in the corpus cardiacum of insects. Nature, Lond. 192, 349-350. COLI-IOLrN E. H. (1963) Synthesis of 5-hydroxytryptamine in the American cockroach. Experientia 19, 9-10. DAVEY K. G. (1962) T h e mode of action of the corpus cardiacum on the hind gut in Periplaneta americana. J. exp. Biol. 39, 319-324. DAVEY K. G. (1964) T h e control of visceral muscle in insects. In Advances in Insect Physiology, Vol. 2. Academic Press, N.Y. VON EULER U. S. (1961) Occurrence of catechol amines in the Acrania and invertebrates. Nature, Lond. 190, 170-171. GERSH M. (1955) Untersuchungen tiber Auslosung und Steuerung der Darmbewegungen bei der Larve yon Ckaoborus. Biol. Zbl. 74, 601-628. GERSCH M., FiscrmR F., UNGIm H. & KOCH H. (1960) Die Isollerung neurohormonaler Factoren aus dem Nervensystem der Ktichenschabe Periplaneta americana. Z. Naturf. 15, 319-322. HOYLE G. (1953) Potassium ions and insect nerve muscle. J . exp. Biol. 30, 121-135.
764
M . A . FREEMAN
KOOIST~ G. (1950) Contribution to the knowledge of the action of acetylcholine on the intestine of Periplaneta americana L. Physiol. Comp. 2, 75-80. OSTLtrtqD E. (1954) T h e distribution of catechol amines in lower animals and their effect on the heart. Acta Physiol. Scand. 31 (Suppl. 112), 1-65. U N G ~ H. (1957) Untersuchungen zur neurohormonalen Beeinflussung der Herzttitigkeit bei Schaben (P. orientalis, P. americana, PhyUodroma germanica). Biol. Zbl. 76, 439--442.