- Email: [email protected]
Biochemical and physiological effects of octopamine and selected octopamine agonists on the oviducts of Locusta migratoria
J. Insecr Physiol. Vol. 39, No. 5, pp. 393400, Printed in Great Britain. All rights reserved
1993 Copyright 0
OO22-1910/93 $6.00 + 0.00 1993 Pergamon Press L.td
Biochemical and Physiological Effects of Octopamine and Selected Octopamine Agonists on the Oviducts of Locusta migratoria ANGELA
B. LANGE,*
PETER K. C. TSANG*
Received 22 September 1992; revised 9 November 1992
The effects of octopamine and some selected octopamine agonists on neurally-evoked contractions and cyclic AMP levels of the lateral oviducts of the locust, Locusta migratoriu, have been examined. Octopamine caused reversible, dosedependent decreases in both the basal tonus and amplitude of neurally-evoked contractions of the lateral oviducts, and inhibited myogenic contractions. The 2-aminooxazoline, AC6 [2-+chloro-o-toluidino)-2_oxazoline], and the substituted phenyliminoimidazolidines (PIIs), NC5 (2,~diethyl-PII) and NC7 (2-methyl-&chloro-PII), were each capable of eliciting similar responses to octopamine on neurally-evoked contractions. The vertebrate a-adrenergic receptor antagonist phentolamine blocked the physiological effects of all agonists tested. The effect of these agonists on cyclic AMP levels was also examined. Octopamine and the three agonists were able to increase the cyclic AMP content of the lateral oviducts in a dose-dependent manner. The increases in cyclic AMP were inhibited in the presence of various vertebrate receptor antagonists. The results of this study indicate that AC6, NC5, and NC7 all act as agonists to the octopamiq-like receptors present on locust oviduct and confirm previous studies for the agonistic properties of these agents. Octopamine
agonists
Aminooxazoline
Phenyliminoimidazolidine
INTRODUCTION
One approach in the development of safer and more selective insecticides is to select targets which are either specific to or preferentially-located in insects thereby minimizing toxicity to non-target organisms (Evans et al., 1989). In the search for novel insecticides, much attention has been directed at the monophenolic amine, octopamine [ 1-b -hydroxyphenyl)-2-aminoethanol] (Jennings et al., 1987). Octopamine is widely distributed in insects and is one of the most abundant biogenic amines in the insect nervous system (Evans, 1985) where it may act as a neurotransmitter, neurohormone or neuromodulator (Evans, 1980; Orchard, 1982). The use of octopamine by the vertebrate nervous system is less clear and it has been suggested that octopamine receptors linked to adenylate cyclase may be unique to invertebrates (Evans, 1980; Jennings et al., 1988). Targeting the octopamine receptor has promised to be a useful approach since the discovery of the pesticidal properties of formamidines, which were subsequently shown to be octopaminergic agonists (Hollingworth and Murdock, 1980). In the search for other innovative types of insecticides, a computerized database was used to examine all com-
*Department of Zoology, University of Toronto, Erindale College, 3359 Mississauga Road, Mississauga, ON, Canada L5L IC6.
Octopamine
Oviducts
Locust
pounds with activity against the two-spotted spider mite, Tetranychus urticae, and with chemical structures similar to those of octopamine and the formamidines (Jennings et al., 1988). These compounds were then screened for agonist activity using the octopamine receptors linked to adenylate cyclase in Periplaneta americana (Jennings et al., 1988). In this manner, the 2-aminooxazolines were identified and one member of this class, AC6 [2-(4-chloro-o-toluidino)-2-oxazoline] (see Fig. l), was found to be a potent octopaminergic agonist. Its physiological, biochemical and behavioural effects on a variety of tissues known to contain octopamine receptors have been described (Jennings et al., 1988; Lange and Orchard, 1990). In earlier studies, the light organ of Photinus was used in an effort to develop potent octopamine agonists and it was found that some substituted 2-(phenylimino)imidazolidines (PIIs) were extremely potent and selective agonists of octopamine receptors (Nathanson, 1985a, b). Structure-activity studies were performed to create compounds with even greater potency. Two members of the PIIs, NC5 (2,6-diethyl-PII) and NC7 (2-methyl-4chloro-PII) (see Fig. l), were discovered to be particularly potent and their physiological, biochemical and behavioural effects on a variety of preparations have been described (Nathanson, 1985a, b; Evans, 1987; Lange and Orchard, 1990; Orr et al., 1991). 393
394
ANGELA B. LANGE and PETER K. C. TSANG NH-
CH2 0 >fZlV~
OH
Octopamine
photoperiod and were fed fresh wheat seedlings, supplemented with bran. Dissections
Oviducts from mature adult female locusts were dissected out via a mid-ventral incision under physiological saline (composition in mM: NaCl 150; KC1 10; CaCl, 4; MgCl, 2; NaHCO, 4; HEPES 5, pH 7.2; sucrose 90; trehalose 5). Neurally-evoked contractions
NC5
CH2CH3
FIGURE 1. Chemical structures of octopamine (OA), AC6 [2-(4chloro-o-toluidino)-2-oxazoline], NC7 (2-methyl-4-chloro-phenyliminoimidazolidine) and NC5 (2,6-diethyl-phenyliminoimidazolidine). Note the similarities in structure of octopamine and the three agonists. Adapted from Lange and Orchard (1990).
In the present study, the effects of octopamine, AC6, NC7 and NC5 on the oviducts of the locust, Locusta migratoria, were examined. Gctopamine plays a physiological role in the control of locust oviducts (Lange and Orchard, 1984a, b) which receive innervation from two octopaminergic dorsal unpaired median neurones located in the seventh abdominal ganglion (Orchard and Lange, 1985). These neurones project to the lower lateral and upper common oviduct (Lange and Orchard, 1984b). Octopamine reversibly reduces the amplitude of neurally-evoked contractions in a dose-dependent manner and causes a relaxation in basal tonus (Lange and Orchard, 1984a; Orchard and Lange, 1985). The effects of octopamine appear to be mediated by the second messenger cyclic AMP (Lange and Orchard, 1986). Thus, the locust oviduct is a well described, model system for the study of octopaminergic actions. The physiological and biochemical effects of octopamine on locust oviduct are compared to those of the aminooxazoline, AC6 and the phenyliminoimidazolidines, NC5 and NC7. The ability of several vertebrate cl-adrenergic receptor antagonists to block these biochemical effects has also been examined in order to determine the receptor specificity for these octopamine agonists. MATERIALS
AND METHODS
Animals
Mature adult female locusts of Locusta migratoria were used throughout this study. They were reared under crowded conditions at 30°C on a 12 h light: 12 h dark
The innervated region of the oviducts (lateral oviducts, posterior to the attachment of the ovaries, and the common oviduct) along with the oviducal nerves were dissected from adult female locusts, 10-20 days after fledgling. Both of the free ends of the lateral oviducts were attached to the wax base of a perspex chamber using minuten pins and the posterior end of the common oviduct was attached to an AE 875 miniature force transducer (Aksjeselskapet Mikro-elektronikk, Oslo, Norway) via a fine thread, allowing for the detection of isotonic mechanical events. Muscular contractions were recorded using a Linear 1200 Chart Recorder. The preparations were held at an angle of approximately 45” to the horizontal during all recordings and maintained in 1 ml of saline. One oviducal nerve was stimulated via a suction electrode which used electrical pulses delivered by a Grass S88 Stimulator. The pulses delivered were of 1 ms duration delivered at 30 Hz for 2 s every 10 s (Lange and Orchard, 1984a). The preparations were stimulated in this manner for approximately 10 min prior to experimentation in order to equilibrate the muscles to the tension caused by its attachments to the dish and to the force transducer. The saline in the dish was replaced at regular intervals. Test chemicals were applied by first removing 500 ~1 of saline from the dish and then replacing it with 500 ~1 of test chemical at twice the desired final bath concentration. This procedure took approximately 5 s. Controls were performed in which the 500 ~1 of saline was removed and replaced by another 500~1 of saline not containing any test chemicals. In this manner, it was found that there were no physiological effects induced solely by the exchange of saline. Octopamine (Sigma Chemical Co., St Louis, MO, U.S.A.) was prepared as a stock solution at 10e3M in saline and then diluted with saline to the desired concentration prior to use. Phentolamine HCl (Ciba-Geigy) was prepared as a stock solution at 10W2Min saline and also diluted with saline prior to use. AC6 (a gift from American Cyanamid, Princeton, NJ, U.S.A.) and NC7 and NC5 (both gifts from J. A. Nathanson, Department of Neurology, Harvard Medical School, Boston, MA, U.S.A.) were made up as lo-‘M solutions in dimethyl sulfoxide (DMSO) (Sigma Chemical) and diluted with saline prior to use. The highest percentage of DMSO in the bath solution (0.1%) did not alter muscular contractions of the preparation.
OCTOPAMINE
AGONISTS ON OVIDUCTS
Cyclic AMP determinations
The effects of octopamine, AC6, NC7, NC5 and various antagonists upon the cyclic AMP content of the oviduct muscles of adult female locusts, 10-20 days post-fledgling, were examined. The innervated regions of the lateral oviducts were dissected under physiological saline. The lateral oviducts (two from each locust) were incubated individually for 10 min in 100 ,ul saline containing the desired concentration of drugs (see Lange and Orchard, 1986; for time-course). The phos3-isobutyl- 1-methylxanthine phodiesterase inhibitor, (IBMX) (Sigma Chemical Co.), at a concentration of 5 x 10-4M, was used in all incubations. In the trials with the three octopamine agonists, the controls contained DMSO at 0.001% which was the highest concentration of DMSO used in the test solutions. Experiments
(4
10-6M OA
395
were terminated by the addition of 400~1 of boiling Tris/EDTA buffer (O.O5M, pH 7.5, containing 4mM EDTA). The mixture was boiled for 5 min and then sonicated. The resultant suspension was centrifuged at 88OOgin an Eppendorf 5413 Centrifuge for 10 min. The pellet was dissolved in 50~1 of 0.5M NaOH and then heated in a water bath for 2 h at 55°C for subsequent protein determination using the Bio-Rad Protein Assay with human y-globulin as standard (see Bradford, 1976). For this assay, the standard tubes also contained 0.5M NaOH (1.0%) to control for the NaOH present in the sample tubes. Cyclic AMP levels were determined in a suitable aliquot of the supernatant by radioimmunoassay using a commercially available protein binding assay kit (Amersham Corporation, Arlington Heights, IL, U.S.A.). The results are based upon at least 4 samples.
(W
10-6M OA + 10-5M PA
(El
lo-*M
Ii
‘I
II A
(B)
10-5M OA
OA + 10-5M PA
I
I . ‘-
lo-‘M
PA
FIGURE 2. The effects of addition of octopamine and the a-adrenergic antagonist, phentolamine, upon neurally-evoked contractions of the locust oviduct. The oviducal nerve was stimulated at 30 Hz for 2 s every 10 s. Arrows indicate the addition of octopamine (OA) and/or phentolamine (PA). Solid bars represent washes with physiological saline. Note the decrease in the basal tonus, the amplitude of neurally-evoked contractions and in myogenic contractions after addition of octopamine, and the ability of phentolamine to block the effects of octopamine. Horizontal scale represents 1min.
ANGELA B. LANGE
396
Values for these experiments were expressed as pmol cyclic AMP per mg of protein. The following chemicals were prepared as lo-‘M stock solutions, usinR either water or ethanol as solvent, and then diluted with saline prior to use: phenoxybetuamine HCl (Smith, Kline and French) and phentolamine HCl (Ciba-Geigy); all other chemicals were obtained from Sigma Chemical Company.
(A)
and PETER K. C. TSANG RESULTS
Eflects of octopamine, AC6, NC7 and NC5 on neurallyevoked contractions
The effects of D,L-octopamine, AC6, NC7 and NC5 upon neurally-evoked contractions of the innervated region of the locust oviducts are shown in Figs 2-4. As found previously by Orchard and Lange (1985)
AC6
__I 50 mg
(B)
NC1
NC5
I
I
60 mg
FIGURE 3. The effects of addition of AC6, NC7 and NC5 upon neurally-evoked contractions of the locust oviduct. Arrows indicate the addition of 10-6M of either (A) AC6, (B) NC7 or (C) NC5. Note the decrease in the amplitude of neurally-evoked contractions and in myogenic contraction after addition of each agonist. Exchange of solution produced an artefact spike in (B) and (C). Horizontal scale represents I min.
OCTOPAMINE
octopamine resulted in a dose-dependent decrease in basal tonus and amplitude of neurally-evoked contractions (Figs 2 and 4). In addition, the myogenic contractions observed between the neurally-evoked contractions were abolished in the presence of octopamine. The effects of octopamine were reversible after washing with saline [Fig. 2(A) and (B)]. The a-adrenergic antagonist, phentolamine (IO-‘M), caused a transient increase in basal tonus and a slight increase in the amplitude of neurally-evoked contractions [Fig. 2(C)] and was also an effective blocker of the effects of 10p6M and 10m5M octopamine [Fig. 2(D) and (E)]. The effects of AC6, NC7 or NC5 upon neurallyevoked contractions are shown in Fig. 3(A-C). The addition of each of the three agonists led to a decrease in the amplitude of neurally-evoked contractions and an inhibition of myogenic contractions which are evident between each neurally-evoked contraction (Fig. 3). The effect upon basal tonus however was not as pronounced as that observed with octopamine. The effects of all three agonists were reversible after washing with saline and, as with octopamine, phentolamine (lo-‘M) was able to block the effects of all three agonists 100% (not shown). As can be seen in Fig. 4, octopamine, AC6, NC5 and NC7 lead to apparent dose-dependent decreases in the amplitude of neurally-evoked contractions. The threshold for this response was about 10-8M for octopamine with maximum decrease (52%) occurring at 10e4M. NC5 and NC7 were the next most effective agonists with thresholds between 10-8M and lo-‘M for NC5 and between 10m9 and IO-‘M for NC7, and decreases in amplitude of neurally-evoked
120
397
AGONISTS ON OVIDUCTS
r
TABLE 1. The effects of octopamine, AC6, NC7 and NC5 on the amplitude of neurally-evoked contractions of the innervated region of the oviducts Drug
Threshold (M)
Octopamine AC6 NC7 NC5
IO-8-10-7 lo-*-lo-’ 10-9-10~8 IO-8-10-7
Maximum (M)
;z$
10-d IO-6 IO-& IO-5
7.8 x IO-’ 4.9 X IO-” 1.7 X IO-’ 2.5 x IO-’
*The concentration which results in 50% of the maximal inhibition of amplitude of contraction.
contraction of 26 and 24% respectively. AC6 was a fairly poor agonist, decreasing the amplitude of neurallyevoked contractions by only about 11% at 10-6M. Table 1 compares the threshold, maxima and ED,, of octopamine, AC6, NC5 and NC7. Phentolamine (lo-‘M) was capable of decreasing the effectiveness of either octopamine or any of the three agonists, AC6, NC7 and NC5 and resulted in the dose-response curves being shifted to the right in a manner similar to that shown before for octopamine (Orchard and Lange, 1985).
500
r
400
-
(A)
AC-~ T
T
l Octopamine
z ‘Z z
500 400
(B)
NC-J
-
V
NC-J
l Octopamine
500 r
Concentration
(c) NC-5
[M] log scale
FIGURE 4. Dose-response curves showing the effect of octopamine, AC6, NC7 and NC5 upon the amplitude of neurally-evoked contractions. The response is expressed as the percentage of the amplitude of contraction during drug application to that immediately prior to addition. Responses are statistical at IO-‘M or greater for octopamine and NCS, at 10e6M or greater for NC7 and at 10m5Mor greater for AC6 (Students r-test, P ~0.05). The symbols represent mean f SE, ” 34.
Concentration
0
NC-5
l
Octopamine
[M] log scale
FIGURE 5. Dose-response curves showing the effect of octopamine and (A) AC6, (B) NC7 and (C) NC5 on cyclic AMPcontent of the lateral oviducts. The values (mean + SE, n 2 4) were determined after IO min of incubation. The basal level is 26.5 f 15.2 pmol cAMP/mg protein.
ANGELA B. LANGE and PETER K. C. TSANG
398
TABLE 2. The effects of octopamine, AC6, NC7 and NC5 on cyclic AMP content of the lateral oviducts Drug
Threshold (M)
Maximum (M)
Octopamine AC6 NC7 NC5
lo-s-10-7 10-7-10-6 lo-r-lo-’ 10-E-10-’
10-d 10-d 10-e 10-5
1.5 x 1.7 x 2.6 x 6.8 x
10-e 10-e lo-’ 10-7
*The concentration at which cyclic AMP levels reach 50% of their maximal increase.
EfSects of octopamine and its agonists on cyclic AMP content D, L-Octopamine, AC6, NC7 and NC5 elevated cyclic AMP content of lateral oviducts in a dosedependent manner (Fig. 5). As can be seen from Fig. 5, octopamine increased the cyclic AMP content of the oviduct by approximately 16-fold at 10m4M. NC7 and NC5 were almost as effective as octopamine, whereas
AC6 only increased the cyclic AMP content by about 6fold at 10e4M. Table 2 compares the threshold, maxima and ED,, of octopamine, AC6, NC7 and NC5. Eflects of antagonists on agonist-induced increases in cyclic AMP levels The effectiveness of a wide range of aminergic antagonists in blocking the increases in cyclic AMP content of the lateral oviducts caused by D, L-octopamine, AC6, NC7 and NC5 was examined (Fig. 6). At lo-‘M, mianserin, metoclopramide and phentolamine were each capable of inhibiting the effects of 10-6M of either octopamine, AC6, NC7 or NC5. Phenoxybenzamine, propranolol and chlorpromazine were much less effective antagonists, resulting in only a small inhibition or, in some cases, actually a small potentiation, of the effects of octopamine, AC6, NC7 and NC5.
FIGURE 6. The ability of selected aminergic antagonists (at lo-‘M) to inhibit octopamine-stimulated and agonist-stimulated increases in cyclic AMP levels in the lateral oviducts. Values (mean + SE, n > 4) are expressed as the percentage decrease in cyclic AMP levels during a 10 min incubation in 10m6Mof either (A) octopamine, (B) AC6, (C) NC7 or (D) NC5 in the presence of 5 x 10e4M IBMX and antagonist. Resting level of 26.5 f 15.2 pmol cyclic AMP/mg protein represents the amount of cyclic AMP accumulated during 10min in IBMX alone and was subtracted from the experimental values. The resting levels (for AC6 = 18.4 f 7.0 pmol/mg protein; for NC7 = 24.2 f 6.0 pmol/mg protein; and for NC5 = 4.4 f 0.8 pmol/mg protein) represent 10 min in IBMX and DMSO (0.001%) and were subtracted from the experimental values. *Indicates that the effects of the agonist were potentiated by the antagonist. Note that phenoxybenzamine was dissolved in ethanol and therefore ethanol (0.2%) was added to determine resting levels.
OCTOPAMINE AGONISTS ON OVIDUCTS
DISCUSSION
It has previously been shown that neurones containing octopamine project to the locust oviduct, and that this neurochemical has potent modulatory actions upon contraction of this visceral muscle (Lange and Orchard, 1984b; Orchard and Lange, 1985). Octopamine exerts many of its actions through receptors associated with the activation of adenylate cyclase (Lange and Orchard, 1986). Therefore it seems that locust oviducts may provide a model system for studying the pharmacological properties of octopamine receptors by using both physiological and biochemical parameters. The results of the present investigation extend our knowledge of the pharmacology of oviduct octopamine receptors. Thus, addition of octopamine to the locust oviducts led to a dose-dependent decrease in basal tonus, amplitude of neurally-evoked contractions and myogenic contractions. The aminooxazoline, AC6, and the phenyliminoimidazolidines, NC5 and NC7, were also capable of inhibiting myogenic contractions and decreasing the amplitude of neurally-evoked contractions, although they were less effective than octopamine. The thresholds for the effects of octopamine and the three octopamine agonists for reducing the amplitude of neurally-evoked contractions lay between IO-’ and 10-7M with the threshold for NC7 actually below 10p8M. The order of potency for the effect on neurally-evoked contractions as judged by EDso was AC6 > NC7 > NC5 > octopamine but when viewed as maximal effect the rank order was octopamine > NC5 > NC7 > AC6. Indeed, NC5, NC7 and AC6 were only partial agonists of octopamine when examining this physiological parameter. The addition of phentolamine, an antagonist known to block octopamine receptors, led to a slight increase in the basal tonus of the muscle and a slight increase (approximately 5-10%) in the amplitude of neurally-evoked contractions. The ability of phentolamine to enhance neurallyevoked contractions indicates that octopamine was being released during electrical stimulation of the oviducal nerve, and so the neurally-evoked contractions were already partially inhibited (Lange and Orchard, 1984a). Application of phentolamine antagonizes this amine and would allow the neurally-evoked contractions to be fully expressed. Phentolamine (IO-‘M) was able to antagonize all three agonists, suggesting that they are indeed acting upon the octopamine receptor. AC6, NC5 and NC7 have been demonstrated to exert physiological effects on tissues which have been shown to be innervated to octopaminergic neurones. AC6 activates the firefly light organ in terms of both light intensity and duration of glow, (Jennings et al., 1988). Nathanson (1985b) found that both NC5 and NC7 stimulate light emission in the isolated firefly light organ in a dose-dependent manner and in a similar manner to octopamine. These physiological effects, as with those induced by octopamine, could be blocked by phentolamine (Nathanson, 1985a, b). Evans (1987) found the same trends in studying the effects of NC5 and NC7
399
on the extensor tibialis muscle of locusts. Octopamine, NC5 and NC7 were found to increase the amplitude and relaxation rate of SETi-induced twitch tension and decrease the frequency of a myogenic rhythm of contraction (Evans, 1987). Once again the effects of octopamine, NC5 and NC7 could be blocked by the phentolamine (Evans, 1987). Octopamine, AC6, NC7 and NC5 elevate cyclic AMP content of lateral oviducts in a dose-dependent manner. Octopamine was capable of increasing the cyclic AMP content of the oviducts by approximately 16-fold. Both NC7 and NC5 were nearly as effective as octopamine whereas AC6 was able to increase cyclic AMP content by only 6-fold. The rank order with respect to maximal effect was octopamine > NC7 > NC5 > AC6. The ability of NC5, NC7 and AC6 to increase cyclic AMP content is totally antagonised by phentolamine, metoclopramide and mianserin, three antagonists which also block octopamine-stimulated increases in cyclic AMP. This lends further credence to the belief that NC5, NC7 and AC6 are octopamine receptor agonists. The effects of these drugs on cyclic AMP levels in locust oviducts are comparable to those reported for the lantern of the firefly Photinus pyralis (Nathanson, 1985a, b), the extensor tibialis muscle of the locust Schistocerca gregaria (Evans, 1987) the nerve cord of the cockroach Periplaneta americana (Jennings et al., 1988), the fat body of Locusta (Lange and Orchard, 1990) and in membrane preparations of cockroach brain and nerve cords and in cockroach haemocytes (Orr et al., 1991). Both NC5 and NC7 are effective agonists of the locust oviduct octopamine receptor having similar thresholds and maximal effects to octopamine and this was also found on locust fat body although NC7 was less potent (Lange and Orchard, 1990). Nathanson (1985b) found NC5 to be a full and potent agonist of octopamine receptors in firefly light organ, cockroach and Munducu nerve cord with potencies ranging from 8 to 19 times that of octopamine. On the other hand NC7 was only a partial agonist in Manduca nerve cord, a full agonist in cockroach nerve cord and intermediate in firefly light organ. In extensor-tibiae muscle of Schistocerca gregaria, Evans (1987) found that the maximum increase in levels of cyclic AMP in response to both NC7 and NC5 were much less than that of octopamine although the maximum physiological effects were quite similar for all three drugs. The reverse was found for locust oviduct with the maximum increases in cyclic AMP induced by NC5 and NC7 similar to octopamine, but effect on neurallyevoked contractions much poorer than octopamine. This discrepancy reveals a more complicated relationship between octopamine receptors, cyclic AMP content and physiological effect than hitherto recognised. As an example of this, Evans (1987) discovered that the cyclic AMP response of the locust extensor-tibiae muscle to octopamine was made up of two components with NC5 and NC7 activating only the higher-affinity component. This was sufficient to cause the observed maximal physiological effects for NC5 and NC7, even though
ANGELA
400
B. LANGE
anId PETER K. C. TSANG
cyclic AMP levels never reach the maximal increase induced by octopamine. Both octopamine and AC6 were found to increase cyclic AMP levels in a dose-dependent manner in nerve cords of the cockroach Periplaneta americana (Jennings et al., 1988). The maximum effect of AC6 occurred at a concentration of 10p4M and at this concentration, AC6 was approximately 36% as effective as 10m4M octopamine in stimulating increases in cyclic AMP levels (Jennings et al., 1988). These results are similar to those found in this present study for AC6 on locust oviducts. The maximum effect of AC6 occurred at 10p4M and at this concentration, AC6 was 48% as effective as 10m4M octopamine in stimulating increases in cyclic AMP levels. In all of the above studies, the rank order of effectiveness of octopamine, NCS, NC7 and AC6 varied either between species or between different tissues within the same species (Nathanson, 1985a, b; Evans, 1987; Lange and Orchard, 1990). The differences in sensitivity to these three drugs may be due to either differences in their metabolism between species and tissue types or due to differing populations of octopaminergic receptor subtypes whose distribution varies between species and between tissue types (Nathanson, 1985b). However, the general trends in the results of these previous studies compares well with the findings in this present study, Thus NC5, NC7 an AC6 mimic the actions of octopamine at both the physiological and biochemical level, and these actions can be blocked by antagonists which are known to block octopamine receptors. Thus, the results support the suggestion that NC5, NC7 and AC6 are octopamine receptor agonists and further emphasize the usefulness of these agents in examining octopamine receptors. Furthermore, in view of the known deleterious effects of these agents on insects, further studies targeting the octopaminergic receptor for pesticidal purposes should prove fruitful. REFERENCES Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Evans P. D. (1980) Biogenic amines in the insect nervous system. Ado. Insect Physiol. 15, 317-473.
Evans P. D. (1985) Octopamine. In Comprehensive Insect Physiology and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 11, pp. 4999530. Pergamon Press, Oxford. Evans P. D. (1987) Phenyliminoimidazolidine derivatives activate both octopamine, and octopamine, receptor subtypes in locust skeletal muscle. J. exp. Biol. 129, 239-250. Evans P. D., Robb S. and Cuthbert B. A. (1989) Insect neuropeptides-Identification, establishment of functional roles and novel target sites for pesticides. Pestic. Sci. 25, 71-83. Hollingworth R. M., and Murdock L. L. (1980) Formamidine pesticides: octopamine-like actions in a firefly. Science 208, 7476. Jennings K. R., Kuhn D. G., Trotto S. H. and Whitney W. K. (1987) Monoamines as targets for insecticide discovery. In Trace Amines: Their Comparative Neurobiology and Clinical Significance (Eds Boulton A. R., Juorio A. V. and Downer R. G. H.), pp. 5343.
Humana Press, New Jersey. Jennings K. R., Kuhn D. G., Kukel C. F., Trotto S. H. and Whitney W. K. (1988) A biorationally synthesized octopaminergic insecticide: 2-(4-chloro-o-toluidino)-2-oxazoline. Pestic. Biochem. Physiol. 30, 190-197.
Lange A. B. and Orchard I. (1984a) Some pharmacological properties of neuromuscular transmission in the oviduct of the locust, Locusta migratoria. Archs Insect Biochem. Physiol. 1, 231-241. Lange A. B. and Orchard I. (1984b) Dorsal unpaired median neurons, and ventral bilaterally paired neurons, project to a viscera1 muscle in an insect. J. Neurobiol. 15, 441453. Lange A. B. and Orchard I. (1986) Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3’,5’-monophosphate (cyclic AMP). Brain Res. 363, 34&349. Lange A. B. and Orchard I. (1990) The action of phenyl-iminoimidazolidines and 2-aminooxazoline on octopamine receptors on locust fat body. Pesric. Biochem. Physiol. 37, 24-29. Nathanson J. A. (1985a) Characterization of octopamine-sensitive adenylate cyclase: Elucidation of a class of potent and selective octopamine, receptor agonists with toxic effects in insects. Proc. natn. Acad. Sci. U.S.A. 82, 599-603.
Nathanson J. A. (1985b) Phenyliminoimidazolidines. Characterization of a class of potent agonists of octopamine-sensitive adenylate cyclase and their use in understanding the pharmacology of octopamine receptors. Moiec. Pharmac. 28, 254-268. Orchard I. (1982) Octopamine in insects: neurotransmitter, neurohormone, and neuromodulator. Can. J. Zool. 60, 659669. Orchard I. and Lange A. B. (1985) Evidence for octopaminergic modulation of an insect viscera1 muscle. J. Neurobioi. 16, 171-181. Orr N., Orr G. L. and Hollingworth R. M. (1991) Characterization of a potent agonist of the insect octopamine-receptor-coupled adenylate cyclase. Insect Biochem. 21, 335-340.
Acknowledgements-This
work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We are grateful to Dr. James A. Nathanson for the gifts of NC5 and NC7 and to Dr Kent Jennings of American Cyanamid for arranging the gift of AC6 and to Dr Ian Orchard for helpful comments during the writing of this manuscript.
1993 Copyright 0
OO22-1910/93 $6.00 + 0.00 1993 Pergamon Press L.td
Biochemical and Physiological Effects of Octopamine and Selected Octopamine Agonists on the Oviducts of Locusta migratoria ANGELA
B. LANGE,*
PETER K. C. TSANG*
Received 22 September 1992; revised 9 November 1992
The effects of octopamine and some selected octopamine agonists on neurally-evoked contractions and cyclic AMP levels of the lateral oviducts of the locust, Locusta migratoriu, have been examined. Octopamine caused reversible, dosedependent decreases in both the basal tonus and amplitude of neurally-evoked contractions of the lateral oviducts, and inhibited myogenic contractions. The 2-aminooxazoline, AC6 [2-+chloro-o-toluidino)-2_oxazoline], and the substituted phenyliminoimidazolidines (PIIs), NC5 (2,~diethyl-PII) and NC7 (2-methyl-&chloro-PII), were each capable of eliciting similar responses to octopamine on neurally-evoked contractions. The vertebrate a-adrenergic receptor antagonist phentolamine blocked the physiological effects of all agonists tested. The effect of these agonists on cyclic AMP levels was also examined. Octopamine and the three agonists were able to increase the cyclic AMP content of the lateral oviducts in a dose-dependent manner. The increases in cyclic AMP were inhibited in the presence of various vertebrate receptor antagonists. The results of this study indicate that AC6, NC5, and NC7 all act as agonists to the octopamiq-like receptors present on locust oviduct and confirm previous studies for the agonistic properties of these agents. Octopamine
agonists
Aminooxazoline
Phenyliminoimidazolidine
INTRODUCTION
One approach in the development of safer and more selective insecticides is to select targets which are either specific to or preferentially-located in insects thereby minimizing toxicity to non-target organisms (Evans et al., 1989). In the search for novel insecticides, much attention has been directed at the monophenolic amine, octopamine [ 1-b -hydroxyphenyl)-2-aminoethanol] (Jennings et al., 1987). Octopamine is widely distributed in insects and is one of the most abundant biogenic amines in the insect nervous system (Evans, 1985) where it may act as a neurotransmitter, neurohormone or neuromodulator (Evans, 1980; Orchard, 1982). The use of octopamine by the vertebrate nervous system is less clear and it has been suggested that octopamine receptors linked to adenylate cyclase may be unique to invertebrates (Evans, 1980; Jennings et al., 1988). Targeting the octopamine receptor has promised to be a useful approach since the discovery of the pesticidal properties of formamidines, which were subsequently shown to be octopaminergic agonists (Hollingworth and Murdock, 1980). In the search for other innovative types of insecticides, a computerized database was used to examine all com-
*Department of Zoology, University of Toronto, Erindale College, 3359 Mississauga Road, Mississauga, ON, Canada L5L IC6.
Octopamine
Oviducts
Locust
pounds with activity against the two-spotted spider mite, Tetranychus urticae, and with chemical structures similar to those of octopamine and the formamidines (Jennings et al., 1988). These compounds were then screened for agonist activity using the octopamine receptors linked to adenylate cyclase in Periplaneta americana (Jennings et al., 1988). In this manner, the 2-aminooxazolines were identified and one member of this class, AC6 [2-(4-chloro-o-toluidino)-2-oxazoline] (see Fig. l), was found to be a potent octopaminergic agonist. Its physiological, biochemical and behavioural effects on a variety of tissues known to contain octopamine receptors have been described (Jennings et al., 1988; Lange and Orchard, 1990). In earlier studies, the light organ of Photinus was used in an effort to develop potent octopamine agonists and it was found that some substituted 2-(phenylimino)imidazolidines (PIIs) were extremely potent and selective agonists of octopamine receptors (Nathanson, 1985a, b). Structure-activity studies were performed to create compounds with even greater potency. Two members of the PIIs, NC5 (2,6-diethyl-PII) and NC7 (2-methyl-4chloro-PII) (see Fig. l), were discovered to be particularly potent and their physiological, biochemical and behavioural effects on a variety of preparations have been described (Nathanson, 1985a, b; Evans, 1987; Lange and Orchard, 1990; Orr et al., 1991). 393
394
ANGELA B. LANGE and PETER K. C. TSANG NH-
CH2 0 >fZlV~
OH
Octopamine
photoperiod and were fed fresh wheat seedlings, supplemented with bran. Dissections
Oviducts from mature adult female locusts were dissected out via a mid-ventral incision under physiological saline (composition in mM: NaCl 150; KC1 10; CaCl, 4; MgCl, 2; NaHCO, 4; HEPES 5, pH 7.2; sucrose 90; trehalose 5). Neurally-evoked contractions
NC5
CH2CH3
FIGURE 1. Chemical structures of octopamine (OA), AC6 [2-(4chloro-o-toluidino)-2-oxazoline], NC7 (2-methyl-4-chloro-phenyliminoimidazolidine) and NC5 (2,6-diethyl-phenyliminoimidazolidine). Note the similarities in structure of octopamine and the three agonists. Adapted from Lange and Orchard (1990).
In the present study, the effects of octopamine, AC6, NC7 and NC5 on the oviducts of the locust, Locusta migratoria, were examined. Gctopamine plays a physiological role in the control of locust oviducts (Lange and Orchard, 1984a, b) which receive innervation from two octopaminergic dorsal unpaired median neurones located in the seventh abdominal ganglion (Orchard and Lange, 1985). These neurones project to the lower lateral and upper common oviduct (Lange and Orchard, 1984b). Octopamine reversibly reduces the amplitude of neurally-evoked contractions in a dose-dependent manner and causes a relaxation in basal tonus (Lange and Orchard, 1984a; Orchard and Lange, 1985). The effects of octopamine appear to be mediated by the second messenger cyclic AMP (Lange and Orchard, 1986). Thus, the locust oviduct is a well described, model system for the study of octopaminergic actions. The physiological and biochemical effects of octopamine on locust oviduct are compared to those of the aminooxazoline, AC6 and the phenyliminoimidazolidines, NC5 and NC7. The ability of several vertebrate cl-adrenergic receptor antagonists to block these biochemical effects has also been examined in order to determine the receptor specificity for these octopamine agonists. MATERIALS
AND METHODS
Animals
Mature adult female locusts of Locusta migratoria were used throughout this study. They were reared under crowded conditions at 30°C on a 12 h light: 12 h dark
The innervated region of the oviducts (lateral oviducts, posterior to the attachment of the ovaries, and the common oviduct) along with the oviducal nerves were dissected from adult female locusts, 10-20 days after fledgling. Both of the free ends of the lateral oviducts were attached to the wax base of a perspex chamber using minuten pins and the posterior end of the common oviduct was attached to an AE 875 miniature force transducer (Aksjeselskapet Mikro-elektronikk, Oslo, Norway) via a fine thread, allowing for the detection of isotonic mechanical events. Muscular contractions were recorded using a Linear 1200 Chart Recorder. The preparations were held at an angle of approximately 45” to the horizontal during all recordings and maintained in 1 ml of saline. One oviducal nerve was stimulated via a suction electrode which used electrical pulses delivered by a Grass S88 Stimulator. The pulses delivered were of 1 ms duration delivered at 30 Hz for 2 s every 10 s (Lange and Orchard, 1984a). The preparations were stimulated in this manner for approximately 10 min prior to experimentation in order to equilibrate the muscles to the tension caused by its attachments to the dish and to the force transducer. The saline in the dish was replaced at regular intervals. Test chemicals were applied by first removing 500 ~1 of saline from the dish and then replacing it with 500 ~1 of test chemical at twice the desired final bath concentration. This procedure took approximately 5 s. Controls were performed in which the 500 ~1 of saline was removed and replaced by another 500~1 of saline not containing any test chemicals. In this manner, it was found that there were no physiological effects induced solely by the exchange of saline. Octopamine (Sigma Chemical Co., St Louis, MO, U.S.A.) was prepared as a stock solution at 10e3M in saline and then diluted with saline to the desired concentration prior to use. Phentolamine HCl (Ciba-Geigy) was prepared as a stock solution at 10W2Min saline and also diluted with saline prior to use. AC6 (a gift from American Cyanamid, Princeton, NJ, U.S.A.) and NC7 and NC5 (both gifts from J. A. Nathanson, Department of Neurology, Harvard Medical School, Boston, MA, U.S.A.) were made up as lo-‘M solutions in dimethyl sulfoxide (DMSO) (Sigma Chemical) and diluted with saline prior to use. The highest percentage of DMSO in the bath solution (0.1%) did not alter muscular contractions of the preparation.
OCTOPAMINE
AGONISTS ON OVIDUCTS
Cyclic AMP determinations
The effects of octopamine, AC6, NC7, NC5 and various antagonists upon the cyclic AMP content of the oviduct muscles of adult female locusts, 10-20 days post-fledgling, were examined. The innervated regions of the lateral oviducts were dissected under physiological saline. The lateral oviducts (two from each locust) were incubated individually for 10 min in 100 ,ul saline containing the desired concentration of drugs (see Lange and Orchard, 1986; for time-course). The phos3-isobutyl- 1-methylxanthine phodiesterase inhibitor, (IBMX) (Sigma Chemical Co.), at a concentration of 5 x 10-4M, was used in all incubations. In the trials with the three octopamine agonists, the controls contained DMSO at 0.001% which was the highest concentration of DMSO used in the test solutions. Experiments
(4
10-6M OA
395
were terminated by the addition of 400~1 of boiling Tris/EDTA buffer (O.O5M, pH 7.5, containing 4mM EDTA). The mixture was boiled for 5 min and then sonicated. The resultant suspension was centrifuged at 88OOgin an Eppendorf 5413 Centrifuge for 10 min. The pellet was dissolved in 50~1 of 0.5M NaOH and then heated in a water bath for 2 h at 55°C for subsequent protein determination using the Bio-Rad Protein Assay with human y-globulin as standard (see Bradford, 1976). For this assay, the standard tubes also contained 0.5M NaOH (1.0%) to control for the NaOH present in the sample tubes. Cyclic AMP levels were determined in a suitable aliquot of the supernatant by radioimmunoassay using a commercially available protein binding assay kit (Amersham Corporation, Arlington Heights, IL, U.S.A.). The results are based upon at least 4 samples.
(W
10-6M OA + 10-5M PA
(El
lo-*M
Ii
‘I
II A
(B)
10-5M OA
OA + 10-5M PA
I
I . ‘-
lo-‘M
PA
FIGURE 2. The effects of addition of octopamine and the a-adrenergic antagonist, phentolamine, upon neurally-evoked contractions of the locust oviduct. The oviducal nerve was stimulated at 30 Hz for 2 s every 10 s. Arrows indicate the addition of octopamine (OA) and/or phentolamine (PA). Solid bars represent washes with physiological saline. Note the decrease in the basal tonus, the amplitude of neurally-evoked contractions and in myogenic contractions after addition of octopamine, and the ability of phentolamine to block the effects of octopamine. Horizontal scale represents 1min.
ANGELA B. LANGE
396
Values for these experiments were expressed as pmol cyclic AMP per mg of protein. The following chemicals were prepared as lo-‘M stock solutions, usinR either water or ethanol as solvent, and then diluted with saline prior to use: phenoxybetuamine HCl (Smith, Kline and French) and phentolamine HCl (Ciba-Geigy); all other chemicals were obtained from Sigma Chemical Company.
(A)
and PETER K. C. TSANG RESULTS
Eflects of octopamine, AC6, NC7 and NC5 on neurallyevoked contractions
The effects of D,L-octopamine, AC6, NC7 and NC5 upon neurally-evoked contractions of the innervated region of the locust oviducts are shown in Figs 2-4. As found previously by Orchard and Lange (1985)
AC6
__I 50 mg
(B)
NC1
NC5
I
I
60 mg
FIGURE 3. The effects of addition of AC6, NC7 and NC5 upon neurally-evoked contractions of the locust oviduct. Arrows indicate the addition of 10-6M of either (A) AC6, (B) NC7 or (C) NC5. Note the decrease in the amplitude of neurally-evoked contractions and in myogenic contraction after addition of each agonist. Exchange of solution produced an artefact spike in (B) and (C). Horizontal scale represents I min.
OCTOPAMINE
octopamine resulted in a dose-dependent decrease in basal tonus and amplitude of neurally-evoked contractions (Figs 2 and 4). In addition, the myogenic contractions observed between the neurally-evoked contractions were abolished in the presence of octopamine. The effects of octopamine were reversible after washing with saline [Fig. 2(A) and (B)]. The a-adrenergic antagonist, phentolamine (IO-‘M), caused a transient increase in basal tonus and a slight increase in the amplitude of neurally-evoked contractions [Fig. 2(C)] and was also an effective blocker of the effects of 10p6M and 10m5M octopamine [Fig. 2(D) and (E)]. The effects of AC6, NC7 or NC5 upon neurallyevoked contractions are shown in Fig. 3(A-C). The addition of each of the three agonists led to a decrease in the amplitude of neurally-evoked contractions and an inhibition of myogenic contractions which are evident between each neurally-evoked contraction (Fig. 3). The effect upon basal tonus however was not as pronounced as that observed with octopamine. The effects of all three agonists were reversible after washing with saline and, as with octopamine, phentolamine (lo-‘M) was able to block the effects of all three agonists 100% (not shown). As can be seen in Fig. 4, octopamine, AC6, NC5 and NC7 lead to apparent dose-dependent decreases in the amplitude of neurally-evoked contractions. The threshold for this response was about 10-8M for octopamine with maximum decrease (52%) occurring at 10e4M. NC5 and NC7 were the next most effective agonists with thresholds between 10-8M and lo-‘M for NC5 and between 10m9 and IO-‘M for NC7, and decreases in amplitude of neurally-evoked
120
397
AGONISTS ON OVIDUCTS
r
TABLE 1. The effects of octopamine, AC6, NC7 and NC5 on the amplitude of neurally-evoked contractions of the innervated region of the oviducts Drug
Threshold (M)
Octopamine AC6 NC7 NC5
IO-8-10-7 lo-*-lo-’ 10-9-10~8 IO-8-10-7
Maximum (M)
;z$
10-d IO-6 IO-& IO-5
7.8 x IO-’ 4.9 X IO-” 1.7 X IO-’ 2.5 x IO-’
*The concentration which results in 50% of the maximal inhibition of amplitude of contraction.
contraction of 26 and 24% respectively. AC6 was a fairly poor agonist, decreasing the amplitude of neurallyevoked contractions by only about 11% at 10-6M. Table 1 compares the threshold, maxima and ED,, of octopamine, AC6, NC5 and NC7. Phentolamine (lo-‘M) was capable of decreasing the effectiveness of either octopamine or any of the three agonists, AC6, NC7 and NC5 and resulted in the dose-response curves being shifted to the right in a manner similar to that shown before for octopamine (Orchard and Lange, 1985).
500
r
400
-
(A)
AC-~ T
T
l Octopamine
z ‘Z z
500 400
(B)
NC-J
-
V
NC-J
l Octopamine
500 r
Concentration
(c) NC-5
[M] log scale
FIGURE 4. Dose-response curves showing the effect of octopamine, AC6, NC7 and NC5 upon the amplitude of neurally-evoked contractions. The response is expressed as the percentage of the amplitude of contraction during drug application to that immediately prior to addition. Responses are statistical at IO-‘M or greater for octopamine and NCS, at 10e6M or greater for NC7 and at 10m5Mor greater for AC6 (Students r-test, P ~0.05). The symbols represent mean f SE, ” 34.
Concentration
0
NC-5
l
Octopamine
[M] log scale
FIGURE 5. Dose-response curves showing the effect of octopamine and (A) AC6, (B) NC7 and (C) NC5 on cyclic AMPcontent of the lateral oviducts. The values (mean + SE, n 2 4) were determined after IO min of incubation. The basal level is 26.5 f 15.2 pmol cAMP/mg protein.
ANGELA B. LANGE and PETER K. C. TSANG
398
TABLE 2. The effects of octopamine, AC6, NC7 and NC5 on cyclic AMP content of the lateral oviducts Drug
Threshold (M)
Maximum (M)
Octopamine AC6 NC7 NC5
lo-s-10-7 10-7-10-6 lo-r-lo-’ 10-E-10-’
10-d 10-d 10-e 10-5
1.5 x 1.7 x 2.6 x 6.8 x
10-e 10-e lo-’ 10-7
*The concentration at which cyclic AMP levels reach 50% of their maximal increase.
EfSects of octopamine and its agonists on cyclic AMP content D, L-Octopamine, AC6, NC7 and NC5 elevated cyclic AMP content of lateral oviducts in a dosedependent manner (Fig. 5). As can be seen from Fig. 5, octopamine increased the cyclic AMP content of the oviduct by approximately 16-fold at 10m4M. NC7 and NC5 were almost as effective as octopamine, whereas
AC6 only increased the cyclic AMP content by about 6fold at 10e4M. Table 2 compares the threshold, maxima and ED,, of octopamine, AC6, NC7 and NC5. Eflects of antagonists on agonist-induced increases in cyclic AMP levels The effectiveness of a wide range of aminergic antagonists in blocking the increases in cyclic AMP content of the lateral oviducts caused by D, L-octopamine, AC6, NC7 and NC5 was examined (Fig. 6). At lo-‘M, mianserin, metoclopramide and phentolamine were each capable of inhibiting the effects of 10-6M of either octopamine, AC6, NC7 or NC5. Phenoxybenzamine, propranolol and chlorpromazine were much less effective antagonists, resulting in only a small inhibition or, in some cases, actually a small potentiation, of the effects of octopamine, AC6, NC7 and NC5.
FIGURE 6. The ability of selected aminergic antagonists (at lo-‘M) to inhibit octopamine-stimulated and agonist-stimulated increases in cyclic AMP levels in the lateral oviducts. Values (mean + SE, n > 4) are expressed as the percentage decrease in cyclic AMP levels during a 10 min incubation in 10m6Mof either (A) octopamine, (B) AC6, (C) NC7 or (D) NC5 in the presence of 5 x 10e4M IBMX and antagonist. Resting level of 26.5 f 15.2 pmol cyclic AMP/mg protein represents the amount of cyclic AMP accumulated during 10min in IBMX alone and was subtracted from the experimental values. The resting levels (for AC6 = 18.4 f 7.0 pmol/mg protein; for NC7 = 24.2 f 6.0 pmol/mg protein; and for NC5 = 4.4 f 0.8 pmol/mg protein) represent 10 min in IBMX and DMSO (0.001%) and were subtracted from the experimental values. *Indicates that the effects of the agonist were potentiated by the antagonist. Note that phenoxybenzamine was dissolved in ethanol and therefore ethanol (0.2%) was added to determine resting levels.
OCTOPAMINE AGONISTS ON OVIDUCTS
DISCUSSION
It has previously been shown that neurones containing octopamine project to the locust oviduct, and that this neurochemical has potent modulatory actions upon contraction of this visceral muscle (Lange and Orchard, 1984b; Orchard and Lange, 1985). Octopamine exerts many of its actions through receptors associated with the activation of adenylate cyclase (Lange and Orchard, 1986). Therefore it seems that locust oviducts may provide a model system for studying the pharmacological properties of octopamine receptors by using both physiological and biochemical parameters. The results of the present investigation extend our knowledge of the pharmacology of oviduct octopamine receptors. Thus, addition of octopamine to the locust oviducts led to a dose-dependent decrease in basal tonus, amplitude of neurally-evoked contractions and myogenic contractions. The aminooxazoline, AC6, and the phenyliminoimidazolidines, NC5 and NC7, were also capable of inhibiting myogenic contractions and decreasing the amplitude of neurally-evoked contractions, although they were less effective than octopamine. The thresholds for the effects of octopamine and the three octopamine agonists for reducing the amplitude of neurally-evoked contractions lay between IO-’ and 10-7M with the threshold for NC7 actually below 10p8M. The order of potency for the effect on neurally-evoked contractions as judged by EDso was AC6 > NC7 > NC5 > octopamine but when viewed as maximal effect the rank order was octopamine > NC5 > NC7 > AC6. Indeed, NC5, NC7 and AC6 were only partial agonists of octopamine when examining this physiological parameter. The addition of phentolamine, an antagonist known to block octopamine receptors, led to a slight increase in the basal tonus of the muscle and a slight increase (approximately 5-10%) in the amplitude of neurally-evoked contractions. The ability of phentolamine to enhance neurallyevoked contractions indicates that octopamine was being released during electrical stimulation of the oviducal nerve, and so the neurally-evoked contractions were already partially inhibited (Lange and Orchard, 1984a). Application of phentolamine antagonizes this amine and would allow the neurally-evoked contractions to be fully expressed. Phentolamine (IO-‘M) was able to antagonize all three agonists, suggesting that they are indeed acting upon the octopamine receptor. AC6, NC5 and NC7 have been demonstrated to exert physiological effects on tissues which have been shown to be innervated to octopaminergic neurones. AC6 activates the firefly light organ in terms of both light intensity and duration of glow, (Jennings et al., 1988). Nathanson (1985b) found that both NC5 and NC7 stimulate light emission in the isolated firefly light organ in a dose-dependent manner and in a similar manner to octopamine. These physiological effects, as with those induced by octopamine, could be blocked by phentolamine (Nathanson, 1985a, b). Evans (1987) found the same trends in studying the effects of NC5 and NC7
399
on the extensor tibialis muscle of locusts. Octopamine, NC5 and NC7 were found to increase the amplitude and relaxation rate of SETi-induced twitch tension and decrease the frequency of a myogenic rhythm of contraction (Evans, 1987). Once again the effects of octopamine, NC5 and NC7 could be blocked by the phentolamine (Evans, 1987). Octopamine, AC6, NC7 and NC5 elevate cyclic AMP content of lateral oviducts in a dose-dependent manner. Octopamine was capable of increasing the cyclic AMP content of the oviducts by approximately 16-fold. Both NC7 and NC5 were nearly as effective as octopamine whereas AC6 was able to increase cyclic AMP content by only 6-fold. The rank order with respect to maximal effect was octopamine > NC7 > NC5 > AC6. The ability of NC5, NC7 and AC6 to increase cyclic AMP content is totally antagonised by phentolamine, metoclopramide and mianserin, three antagonists which also block octopamine-stimulated increases in cyclic AMP. This lends further credence to the belief that NC5, NC7 and AC6 are octopamine receptor agonists. The effects of these drugs on cyclic AMP levels in locust oviducts are comparable to those reported for the lantern of the firefly Photinus pyralis (Nathanson, 1985a, b), the extensor tibialis muscle of the locust Schistocerca gregaria (Evans, 1987) the nerve cord of the cockroach Periplaneta americana (Jennings et al., 1988), the fat body of Locusta (Lange and Orchard, 1990) and in membrane preparations of cockroach brain and nerve cords and in cockroach haemocytes (Orr et al., 1991). Both NC5 and NC7 are effective agonists of the locust oviduct octopamine receptor having similar thresholds and maximal effects to octopamine and this was also found on locust fat body although NC7 was less potent (Lange and Orchard, 1990). Nathanson (1985b) found NC5 to be a full and potent agonist of octopamine receptors in firefly light organ, cockroach and Munducu nerve cord with potencies ranging from 8 to 19 times that of octopamine. On the other hand NC7 was only a partial agonist in Manduca nerve cord, a full agonist in cockroach nerve cord and intermediate in firefly light organ. In extensor-tibiae muscle of Schistocerca gregaria, Evans (1987) found that the maximum increase in levels of cyclic AMP in response to both NC7 and NC5 were much less than that of octopamine although the maximum physiological effects were quite similar for all three drugs. The reverse was found for locust oviduct with the maximum increases in cyclic AMP induced by NC5 and NC7 similar to octopamine, but effect on neurallyevoked contractions much poorer than octopamine. This discrepancy reveals a more complicated relationship between octopamine receptors, cyclic AMP content and physiological effect than hitherto recognised. As an example of this, Evans (1987) discovered that the cyclic AMP response of the locust extensor-tibiae muscle to octopamine was made up of two components with NC5 and NC7 activating only the higher-affinity component. This was sufficient to cause the observed maximal physiological effects for NC5 and NC7, even though
ANGELA
400
B. LANGE
anId PETER K. C. TSANG
cyclic AMP levels never reach the maximal increase induced by octopamine. Both octopamine and AC6 were found to increase cyclic AMP levels in a dose-dependent manner in nerve cords of the cockroach Periplaneta americana (Jennings et al., 1988). The maximum effect of AC6 occurred at a concentration of 10p4M and at this concentration, AC6 was approximately 36% as effective as 10m4M octopamine in stimulating increases in cyclic AMP levels (Jennings et al., 1988). These results are similar to those found in this present study for AC6 on locust oviducts. The maximum effect of AC6 occurred at 10p4M and at this concentration, AC6 was 48% as effective as 10m4M octopamine in stimulating increases in cyclic AMP levels. In all of the above studies, the rank order of effectiveness of octopamine, NCS, NC7 and AC6 varied either between species or between different tissues within the same species (Nathanson, 1985a, b; Evans, 1987; Lange and Orchard, 1990). The differences in sensitivity to these three drugs may be due to either differences in their metabolism between species and tissue types or due to differing populations of octopaminergic receptor subtypes whose distribution varies between species and between tissue types (Nathanson, 1985b). However, the general trends in the results of these previous studies compares well with the findings in this present study, Thus NC5, NC7 an AC6 mimic the actions of octopamine at both the physiological and biochemical level, and these actions can be blocked by antagonists which are known to block octopamine receptors. Thus, the results support the suggestion that NC5, NC7 and AC6 are octopamine receptor agonists and further emphasize the usefulness of these agents in examining octopamine receptors. Furthermore, in view of the known deleterious effects of these agents on insects, further studies targeting the octopaminergic receptor for pesticidal purposes should prove fruitful. REFERENCES Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Evans P. D. (1980) Biogenic amines in the insect nervous system. Ado. Insect Physiol. 15, 317-473.
Evans P. D. (1985) Octopamine. In Comprehensive Insect Physiology and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.), Vol. 11, pp. 4999530. Pergamon Press, Oxford. Evans P. D. (1987) Phenyliminoimidazolidine derivatives activate both octopamine, and octopamine, receptor subtypes in locust skeletal muscle. J. exp. Biol. 129, 239-250. Evans P. D., Robb S. and Cuthbert B. A. (1989) Insect neuropeptides-Identification, establishment of functional roles and novel target sites for pesticides. Pestic. Sci. 25, 71-83. Hollingworth R. M., and Murdock L. L. (1980) Formamidine pesticides: octopamine-like actions in a firefly. Science 208, 7476. Jennings K. R., Kuhn D. G., Trotto S. H. and Whitney W. K. (1987) Monoamines as targets for insecticide discovery. In Trace Amines: Their Comparative Neurobiology and Clinical Significance (Eds Boulton A. R., Juorio A. V. and Downer R. G. H.), pp. 5343.
Humana Press, New Jersey. Jennings K. R., Kuhn D. G., Kukel C. F., Trotto S. H. and Whitney W. K. (1988) A biorationally synthesized octopaminergic insecticide: 2-(4-chloro-o-toluidino)-2-oxazoline. Pestic. Biochem. Physiol. 30, 190-197.
Lange A. B. and Orchard I. (1984a) Some pharmacological properties of neuromuscular transmission in the oviduct of the locust, Locusta migratoria. Archs Insect Biochem. Physiol. 1, 231-241. Lange A. B. and Orchard I. (1984b) Dorsal unpaired median neurons, and ventral bilaterally paired neurons, project to a viscera1 muscle in an insect. J. Neurobiol. 15, 441453. Lange A. B. and Orchard I. (1986) Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3’,5’-monophosphate (cyclic AMP). Brain Res. 363, 34&349. Lange A. B. and Orchard I. (1990) The action of phenyl-iminoimidazolidines and 2-aminooxazoline on octopamine receptors on locust fat body. Pesric. Biochem. Physiol. 37, 24-29. Nathanson J. A. (1985a) Characterization of octopamine-sensitive adenylate cyclase: Elucidation of a class of potent and selective octopamine, receptor agonists with toxic effects in insects. Proc. natn. Acad. Sci. U.S.A. 82, 599-603.
Nathanson J. A. (1985b) Phenyliminoimidazolidines. Characterization of a class of potent agonists of octopamine-sensitive adenylate cyclase and their use in understanding the pharmacology of octopamine receptors. Moiec. Pharmac. 28, 254-268. Orchard I. (1982) Octopamine in insects: neurotransmitter, neurohormone, and neuromodulator. Can. J. Zool. 60, 659669. Orchard I. and Lange A. B. (1985) Evidence for octopaminergic modulation of an insect viscera1 muscle. J. Neurobioi. 16, 171-181. Orr N., Orr G. L. and Hollingworth R. M. (1991) Characterization of a potent agonist of the insect octopamine-receptor-coupled adenylate cyclase. Insect Biochem. 21, 335-340.
Acknowledgements-This
work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We are grateful to Dr. James A. Nathanson for the gifts of NC5 and NC7 and to Dr Kent Jennings of American Cyanamid for arranging the gift of AC6 and to Dr Ian Orchard for helpful comments during the writing of this manuscript.