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The pharmacology of the isolated foregut of the locust Schistocerca Gregaria—III. Identification of an octopamine2 receptor
Camp. Eiochem.
Physiol.
Vol. 92C, No. 2, pp. 333-336, 1989
0306-4492/89$3.00+ 0.00 0 1989PergamonPressplc
Printed in Great Britain
THE PHARMACOLOGY OF THE ISOLATED FOREGUT OF THE LOCUST SCHISTOCERCA GREGARIA-III. IDENTIFICATION OF AN OCTOPAMINE, RECEPTOR S. E. BANNER, R. H. OSBORNE,* M. J. WALKER and K. J. CATTELL Department of Science, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol BS16 lQY, UK. Telephone (0272) 656261 (Received 20 April 1988) Abetmet-1. Cktopamine (OA) (lO-7-lO-5 M) relaxed isolated foreguts. 2. The effects of OA were mimicked by synephrine. 3. Mianserin (IC, = 1 y M), cyproheptadine (rcso = 50 PM) and mepyramine (ICY= 50 PM) were non competitive antagonists of OA whereas yohimbine had no significant effect. 4. Caffeine-induced (1 PM) potentiation of OA-induced relaxation was antagonised by mianserin (1 PM). 5. It is concluded that OA relaxes the locust foregut by activating an adenylate cyclase-linked OA, receptor.
INTRODUCTION Octopamine (OA) is now considered to be an important biogenic amine in arthropods (for reviews see Axelrod and Saavedra, 1977; Hicks, 1977; and David and Coulon, 1985) where its functions seem to be comparable to those of noradrenaline and adrenaline in mammals. In insects, OA has been shown to act as a neurotransmitter, neurohormone and neuromodulator (Orchard, 1982; Evans, 1984a). The effects of OA on insect skeletal muscle preparations have been studied extensively (Evans and O’Shea, 1977, 1978). For example, two classes of OA receptors were described physiologically and pharmacologically by Evans (1981) using the extensor-tibiae preparation of the locust Schistocerca gregaria. These were designated OA class 1, which mediated the frequency of myogenic rhythm, and OA class 2 which modulated the rate of relaxation of muscle tension. Evans (198 1) went on further to differentiate the class 2 receptor into presynaptic OA,, and post synaptic OAze receptors. Subsequently, both classes of OA receptors have been identified in other peripheral tissue preparations where OA containing neurons are known to be present. For example, the lantern of the firefly Photuris versicolar and the oviducts of the locust Locusta migratoria are innervated by DUM neruons acting via OA class 2 receptors (Christensen et al., 1983; Nathanson, 1985a; Lange and Orchard, 1984; Orchard and Lange, 1985, 1986). Furthermore, Orchard and Lange (1986) also demonstrated that the OA, receptors were linked to adenylate cyclase and mediated an increase in intracellular cyclic AMP levels. Evans (1984c) suggested that OA, receptors may use Ca2+ as a second messenger. Recently, we described a 5-HT,-like receptor mediating relaxation of the foregut of Schistocerca gregaria (Banner et al., 1987b). The tissue was also relaxed by OA but the maximum effect of this amine
*Author to whom correspondence
should be addressed. 333
was less than 50% of the maximum response caused by 5-hydroxytryptamine (5-HT). Here, we report on the further characterization of this distinct OA receptor by the use of selective agonists and antagonists and present evidence that it is of the OA, class. MATERIALS AND
METHODS
Adult locusts (Schisrocercagregaria)of both sexes, reared in laboratory culture, were used throughout this study. The details of the dissection of the isolated foregut, the salines used, the methods used to maintain these preparations in uitroand the methods used to record the tissue length have been described in a previous paper (Banner et al., 1987a). Dose-response curves were constructed for octopamine (OA), synephrine, noradrenaline (NA), dopamine (DA) and histamine (HA) at concentrations ranging from lo-* M-10-r M. ?he.effects of the antagonists: mianserin (IO-‘M-lo-‘M). cvnrohentadine (lo-‘M-5 x 10m5M). mepyramine (5 x 10Y5M), 1 yohimbine (10e6 M-10e4 M); metiamide (5 x lo-5 M) and ketanserin (10d5 M) on the tissue’s responses to applied OA and synephrine were investigated, the foregut being incubated with an antagonist for 20min prior to retesting the effects of an agonist. OA-induced responses were also investigated in the presence of verapamil (lOms M), caffeine (10e6 M) and cadmium ions (10-s M). Mianserin, cyproheptadine, metiamide and ketanserin were gifts from Organon Laboratories Ltd, Sandoz Ltd, Smith, Kline & French Ltd and Janssen Pharmaceuticals Ltd respectively. All other drugs and reagents were obtained from the Sigma Chemical Company and all compounds were dissolved in Clark Insect Ringer solution.
RESULTS The effects of agonists Application of OA at concentrations ranging from lo-’ M to lo-’ M caused dose dependent relaxation of foreguts isolated from Schistocerca gregaria with an ED% value of 7 x lo-’ M. The maximum response occurred at 5 x 10m6 M with significant tachyphylxis (Fig. 1) at higher doses. Similarly, synephrine caused
S. E. BANNERet al.
334
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:8
75
t a
50
:
25
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tissue relaxation at concentrations ranging from IO-’ M to 2 x lo-’ M in a dose dependent manner with an EDDY value of 2 x 10m6M. The maximum response to synephrine (Fig. 1) occurred at 2 x 10m5M and the agonist proved to have an intrinsic activity identical to that of OA. By comparison, DA at lOAs M induced only 20% of the maximum tissue relaxation caused by OA while, NA and HA were without effect (Fig. 2) on the isolated foregut. The effects of antagonists
Figure 3 shows that mianserin (10-7M-10-5M) caused non-competitive inhibition of OA-induced relaxation of the tissue. Thus, in the presence of lo-’ M mianserin the EDGE value for OA increased from 1.4 x 10m6M to 3.6 x 10e6 M with a 21% reduction in the response. At 10e6 M, mianserin caused a 50% reduction in the maximum response to OA and an increase in the EDGE value from 1.4 x 10m6M to lo-‘M. At lo-‘M, mianserin caused an 84% reduction in the tissue’s response to OA. Both mepyramine and cyproheptadine were also found to non-competitively antagonise OA-induced relaxation. For example, mepyramine (Fig. 4) at 5 x IO-‘M caused 56% inhibition of the maximum response to OA while the same concentration of cyproheptadine (Fig. 5) caused a 46% reduction in
DA 12
---L---_
M
f
;
-5
LO$$ONCENTRATION
Fig. I. Dose-response curves of OA (A) and synephrine (a) on the locust foregut in vitro. Each point is the mean of 69 replicates (ISEM).
t
-7
.
-5 OCTOPAMINE
-4 M
Fig. 3. Dose-response curves of OA in the presence of mianserin IO-’ M (A), 10m6M (O), 5 x 1O-6 M (0) and lo-’ M (0). The 5-HT control dose-response curve is to left of that in the presence of IO-’ M mianserin (A). Each point is the mean of 6-18 replicates. Standard errors (typically 5515% of mean) have been omitted in the interests of clarity.
the maximum response to the agonist. However, yohimbine (10m4M) caused a 30% reduction in the maximum response to OA but had no significant effect on the ED,, value. Finally, ketanserin and metiamide at doses as high as 5 x 10e5 M had no antagonistic effect on OA-mediated tissue relaxation. Both mianserin (10m6M) and cyproheptadine (5 x IO-’ M) reduced the maximum evoked response to synephrine by 64% and 52% respectively (Fig. 6). Yohimbine (10~6M-10~4 M) had no effect on the responses of the tissue to application of synephrine. The effects of caffeine, verapamil and cadmium ions
The phosphodiesterase inhibitor caffeine (10e6 M) was found to potentiate OA-induced tissue relaxation shifting the dose response curve to the left and reducing the EDGE value from 10S6 M to 5.6 x IO-’ M and increasing the maximum response to 220% of the control value (Fig. 7). In the presence of mianserin (10m6M) this potentiating effect of caffeine was totally abolished, the ED,, value being increased to 3 x lo-‘M and the maximum response being restored to the original control value (Fig. 7). Ketanserin (lo-* M) had no effect on the caffeine-
r;ii-__ 0 A
1
10
3 x &=M -~--~-t_-._--L----.‘--~~
~_.._~
&A
-------
-5
M
-i‘
;
Fig. 2. The effect of DA, S-HT, OA, NA and HA on the isolated foregut of Schistocercagregaria. The arrows mark the time of addition of doses of these amines. Calibration (1 cm and 30 set) applies to all traces.
Octopamine,
-0
LOQ,,CONCENTRATION
-4
-5
-6
-7
OCTOPAMINE
receptor in locust foregut
-8
335
-7
-6
LOQ,oCONCENTRATION
M
-5 SYNEPHRINE
-4 M
Fig. 4. The dose-response curve for OA in the absence (A) and presence (a) of mepyramine (5 x 10m5 M) on the locust foregut. Each point is the mean of 618 replicates (+ SEM).
Fig. 6. The dose-response curve for synephrine in the absence (0) and presence of mianserin (10m6 M) (0) and cyproheptadine (5 x IO-‘M) (A) on the locust foregut. Each point is the mean of 6 replicates (+SEM).
potentiated response. Verapamil and cadmium ions at concentrations as high as 1 mM had neither inhibitory nor potentiating effects on the actions of OA.
physiologically-distinct classes of OA receptors in the extensor-tibiae neuromuscular preparation. He concluded that OA, receptors were preferentially antagonised by chlorpromazine, phentolamine and yohimbine while mianserin and cyproheptadine were more effective at OA, receptors. The latter were sub-divided into OA,, and OA,, on the basis of the relative potency of the antagonists mianserin, cyproheptadine and metoclopramide with all three molecules being less potent at the OA,, receptor. Subsequent work (Evans, 1984a) showed that OA, receptors could be blocked by the histamine H, antagonist mepyramine but not by the H, antagonist metiamide. Therefore, the OA receptors mediating relaxation of the foregut of S. gregariu would appear to belong to the OA, sub-type because the actions of OA and synephrine are blocked by mianserin, mepyramine and cyproheptadine while yohimbine, metiamide and ketanserin had no significant effect. However, it is difficult to further differentiate them into either OA,, or OA,, receptor sub-types because the relative potency of mianserin and cyproheptadine in this tissue does not equate with that described by Evans (1981) for the extensor-tibiae preparation. Thus, for class 2A receptors, Evans (1981) reported that mianserin and cyproheptadine had IC,, values of 1 PM and 2 p M respectively whereas at class 2B receptors the values were 20 p M and 50 p M respectively. However, in this study we found that while mianserin had an rc,, value of 1 PM, thereby suggesting that the receptor could belong to class 2A, the value for cyproheptadine (50 PM) matched that expected for Evans’ class 2B site.
DISCUSSION
The results presented above show that octopamine and its naturally occurring N-methylated derivative, synephrine, have identical pharmacological profiles on the isolated foregut of Schistocerca gregaria. Thus, both of these biogenic amines had similar affinities and intrinsic activities for the receptor sites in the tissue and were affected equally by the antagonists used. A similar situation has been reported for the extensor-tibiae preparation from S. gregaria (Evans, 1981) and the oviducal muscle of Locusta migratoriu (Orchard and Lange, 1986). By comparison, the amines NA and HA had little or no effect on the foregut while the actions of DA were much weaker than those of OA. However, the indoleamine 5hydroxytryptamine (5-HT) was a far more powerful relaxant of the tissue than OA (Banner et al., 1986a) thereby suggesting that OA could be acting as a weak agonist at 5-HT receptors especially as the effects of both amines were antagonised by mianserin. Subsequent work (Banner et al., 1986b) showed the effects of 5-HT, but not those of OA, could be blocked by the 5-HT, antagonist ketanserin. Furthermore, differences in the kinetics of antagonism by mianserin against OA and 5-HT suggested that the two amines exerted their effects on the tissue by activating two distinct receptors. Using a wide range of agonists and antagonists, Evans (1981) described three pharmacologicallyand
-7 LOQ,,CONCENTRATION
OCTOPAMINE
M
Fig. 5. The dose-response curve for OA in the absence (A) and presence of yohimbine (10m4M) (0) and cyproheptadine (5 x lo-‘M) (A) on the locust foregut. Each point is the mean of 69 replicates (+SEM).
-6 LOG,oCONCENTRATION
-5
-3
-4 OCTOPAMINE
M
Fig. 7. The dose-response curve for OA in the absence (A) and presence of caffeine (10m6 M) (A) and caffeine (10m6 M) plus mianserin (10m6 M) (0) on the locust foregut. Each point is the mean of 610 replicates (+SEM).
336
S. E. BANNER et al.
It is interesting to speculate on the nature of the 0A2 receptors found in the foregut and other visceral and skeletal tissues of the locust. Thus, while HA has no effect on the isolated foregut and, unlike OA, does not induce cyclic AMP accumulation in extensortibiae preparation (Evans, 1984a), molecules, such as mianserin, cyproheptadine and mepyramine, with high affinity for mammalian HA H, receptors (Leysen and Gommeren, 1986) appear to be potent antagonists of the actions of OA in S. gregaria. By comparison, ketanserin, yohimbine and metiamide have markedly lower affinity for mammalian H, binding sites/receptors. Therefore, it is tempting to speculate that the insect OA receptors and mammalian H, receptors may have pharmacological and even structural similarities. In both the extensor-tibiae (Evans, 1984a) and oviduct neuromuscular (Orchard and Lange, 1986) preparations it has been shown that OA and synephrine cause an increase in cyclic AMP levels by an interaction with OA, receptors which feature a pharmacological profile similar to that described in the foregut. The potentiation of OA-induced foregut relaxation by the phosphodiesterase inhibitor caffeine provides further evidence for the hypothesis that OA, receptors are linked to adenylate cyclase and so utilise cyclic AMP as their secondary messenger. The connection between OA-induced tissue relaxation and adenylate cyclase was confirmed by the observation that the OA, antagonist mianserin blocked the potentiation caused by preincubation of the tissue with caffeine and the failure of caffeine to potentiate 5-HT-induced relaxation (Banner and Scale, personal observation). Evans (1984c) suggested that activation of OA, receptors may lead to an increase in intracellular calcium levels. Consequently, the possibility that the OA receptors mediating relaxation of the foregut may bring about their effect by controlling calcium fluxes was investigated by preincubating the tissue with the Ca*+ channel blockers verapamil (Kohlhardt and Mnich, 1978) and cadmium ions (Cooper and Manalis, 1984). However, both substances failed to inhibit the OA-mediated response thereby providing additional evidence that these receptors are not of the OA, type. The results of this study have shown that OA may have a significant role in the control of foregut motility. However, it is difficult to assess the relative importance of this amine because its actions are far less powerful than either proctolin or S-HT which have been postulated (Banner et al., 1987a) to be responsible for providing the primary motor drive of this tissue. However, it is suggested from this study that the tissue contains an adenylate cyclase-linked OA, receptor which, when activated, mediates tissue relaxation.
REFERENCES Axelrod J. and Saavedra J. M. (1977) Octopamine. Nature 265, 501-504.
Banner S. E., Cattell K. J. and Osborne R. H. (1986a) Characterisation of a S-hydroxytryptamine receptor in the foregut of the locust Schistocerca gregaria. Br. J. Pharmac. 89, 565P.
Banner S. E., Cattell K. J. and Osborne R. H. (1986b) Effects of antagonists on S-HT and octonamine recenters in the foregut of Schistocerca gregaria. B;. J. Pharmai. 89, 825P.
Banner S. E., Osborne R. H. and Cattell K. J. (1987a) The pharmacology of the isolated foregut of the locust Schistocerca gregaria-I. The effect of a range of putative neurotransmitters. Coma. Biochem. Phvsiol. 88C. 131-138. Banner S. E., Osborne R. H. and Cattell K. J. (1987b) The pharmacology of the isolated foregut of the locust Schisiocerca gregaria-II. Characteri&ion of a 5-HT,-like receptor. Comp. Biochem. Physiol. 88C, 139-144. Christensen T. A., Sherman T. G., McCaman R. E. and Carlson A. D. (1983) Presence of octopamine in firefly photomotor neurones. Neuroscience 9, 183-189. Cooper C. P. and Manalis R. S. (1984) Cadmium: effects on transmitter release at the frog neuromuscular junction. Eur. J. Pharmac. 99, 251-256.
David J. C. and Coulon J. E. (1985) Octopamine in invertebrates and vertebrates. A review. Prog. Neurobioi. 24, 141-185.
Evans P. D. (1981) Multiple receptor types for octopamine in the locust. J. Physiol. 318, 99-122. Evans P. D. (1984a) A modulatory octopaminergic neurone increases cyclic nucleotide levels in locust skeletal muscle. J. Physiol. 348, 307-324.
Evans P. D. (1984c) Studies on the mode of action of octopamine, 5-hydroxytryptamine and proctolin on a myogenic rhythm in the locust. J. exp. Biol. 110,231-251. Evans P. D. and O’Shea M. (1977) An octopaminergic neuron modulates neuromuscular transmission in the locust. Nature 270, 257-259. Evans P. D. and O’Shea M. (1978) The identification of an octopaminergic neuron and the modulation of a myogenie rhythm in the locust. J. exp. Bioi. 73, 235-260. Hicks T. P. (1977) The possible role of octopamine as a synaptic transmitter: a review. Can. J. Physiol. Pharmac. 55, 137-152.
Kolhardt M. and Mnich Z. (1978) Studies on the inhibitory effect of verapamil on the slow inward current in mammalian ventricular myocardium. J. Molec. Cell. Cardioi. 10, 1037-1052.
Lange A. B. and Orchard I. (1984) Some pharmacological properties of neuromuscular transmission in the oviduct of the locust Locusta migratoria. Archs Insect Biochem. Physioi. 1, 231-242. Leysen J. E. and Gommeren W. (1986) Drug receptor dissociation time, new tool for drug research: receptor binding affinity and drug receptor dissociation profiles of serotonin-S,, dopamine-D,, histamine-H, antagonists and opiates. Drug Dwel. R&. 8, 119-131.. Nathanson J. A. (1985a) Characterisation of octonamine sensitive adenylate cyclase: elucidation of a class of’potent and selective octopamine-2 receptor agonists with toxic effects in insects. Proc. natn. Acad. Sci. U.S.A. 82, 599-603.
Acknowledgements-SEB
is supported by SERC Research Studentship. We are grateful for financial support from Shell Research Ltd.
Orchard I. (1982) Octopamine in insects: neurotransmitter, neurohormone and neuromodulator. Can. J. 2001. 60, 659669. Orchard I. and Lange A. B. (1985) Evidence for octopaminergic modulation of insect visceral muscle. J. Neurobiol. 16, 171-181. Orchard I. and Lange A. B. (1986) Pharmacological profiles of octopamine receptors on the lateral oviducts of the locust Locusta migratoria. J. Insect Physiol. 32, 141-145.
Physiol.
Vol. 92C, No. 2, pp. 333-336, 1989
0306-4492/89$3.00+ 0.00 0 1989PergamonPressplc
Printed in Great Britain
THE PHARMACOLOGY OF THE ISOLATED FOREGUT OF THE LOCUST SCHISTOCERCA GREGARIA-III. IDENTIFICATION OF AN OCTOPAMINE, RECEPTOR S. E. BANNER, R. H. OSBORNE,* M. J. WALKER and K. J. CATTELL Department of Science, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol BS16 lQY, UK. Telephone (0272) 656261 (Received 20 April 1988) Abetmet-1. Cktopamine (OA) (lO-7-lO-5 M) relaxed isolated foreguts. 2. The effects of OA were mimicked by synephrine. 3. Mianserin (IC, = 1 y M), cyproheptadine (rcso = 50 PM) and mepyramine (ICY= 50 PM) were non competitive antagonists of OA whereas yohimbine had no significant effect. 4. Caffeine-induced (1 PM) potentiation of OA-induced relaxation was antagonised by mianserin (1 PM). 5. It is concluded that OA relaxes the locust foregut by activating an adenylate cyclase-linked OA, receptor.
INTRODUCTION Octopamine (OA) is now considered to be an important biogenic amine in arthropods (for reviews see Axelrod and Saavedra, 1977; Hicks, 1977; and David and Coulon, 1985) where its functions seem to be comparable to those of noradrenaline and adrenaline in mammals. In insects, OA has been shown to act as a neurotransmitter, neurohormone and neuromodulator (Orchard, 1982; Evans, 1984a). The effects of OA on insect skeletal muscle preparations have been studied extensively (Evans and O’Shea, 1977, 1978). For example, two classes of OA receptors were described physiologically and pharmacologically by Evans (1981) using the extensor-tibiae preparation of the locust Schistocerca gregaria. These were designated OA class 1, which mediated the frequency of myogenic rhythm, and OA class 2 which modulated the rate of relaxation of muscle tension. Evans (198 1) went on further to differentiate the class 2 receptor into presynaptic OA,, and post synaptic OAze receptors. Subsequently, both classes of OA receptors have been identified in other peripheral tissue preparations where OA containing neurons are known to be present. For example, the lantern of the firefly Photuris versicolar and the oviducts of the locust Locusta migratoria are innervated by DUM neruons acting via OA class 2 receptors (Christensen et al., 1983; Nathanson, 1985a; Lange and Orchard, 1984; Orchard and Lange, 1985, 1986). Furthermore, Orchard and Lange (1986) also demonstrated that the OA, receptors were linked to adenylate cyclase and mediated an increase in intracellular cyclic AMP levels. Evans (1984c) suggested that OA, receptors may use Ca2+ as a second messenger. Recently, we described a 5-HT,-like receptor mediating relaxation of the foregut of Schistocerca gregaria (Banner et al., 1987b). The tissue was also relaxed by OA but the maximum effect of this amine
*Author to whom correspondence
should be addressed. 333
was less than 50% of the maximum response caused by 5-hydroxytryptamine (5-HT). Here, we report on the further characterization of this distinct OA receptor by the use of selective agonists and antagonists and present evidence that it is of the OA, class. MATERIALS AND
METHODS
Adult locusts (Schisrocercagregaria)of both sexes, reared in laboratory culture, were used throughout this study. The details of the dissection of the isolated foregut, the salines used, the methods used to maintain these preparations in uitroand the methods used to record the tissue length have been described in a previous paper (Banner et al., 1987a). Dose-response curves were constructed for octopamine (OA), synephrine, noradrenaline (NA), dopamine (DA) and histamine (HA) at concentrations ranging from lo-* M-10-r M. ?he.effects of the antagonists: mianserin (IO-‘M-lo-‘M). cvnrohentadine (lo-‘M-5 x 10m5M). mepyramine (5 x 10Y5M), 1 yohimbine (10e6 M-10e4 M); metiamide (5 x lo-5 M) and ketanserin (10d5 M) on the tissue’s responses to applied OA and synephrine were investigated, the foregut being incubated with an antagonist for 20min prior to retesting the effects of an agonist. OA-induced responses were also investigated in the presence of verapamil (lOms M), caffeine (10e6 M) and cadmium ions (10-s M). Mianserin, cyproheptadine, metiamide and ketanserin were gifts from Organon Laboratories Ltd, Sandoz Ltd, Smith, Kline & French Ltd and Janssen Pharmaceuticals Ltd respectively. All other drugs and reagents were obtained from the Sigma Chemical Company and all compounds were dissolved in Clark Insect Ringer solution.
RESULTS The effects of agonists Application of OA at concentrations ranging from lo-’ M to lo-’ M caused dose dependent relaxation of foreguts isolated from Schistocerca gregaria with an ED% value of 7 x lo-’ M. The maximum response occurred at 5 x 10m6 M with significant tachyphylxis (Fig. 1) at higher doses. Similarly, synephrine caused
S. E. BANNERet al.
334
0
:8
75
t a
50
:
25
Ip 0 -5 LOt3,0CONCENTRATION
AQONIST
M
tissue relaxation at concentrations ranging from IO-’ M to 2 x lo-’ M in a dose dependent manner with an EDDY value of 2 x 10m6M. The maximum response to synephrine (Fig. 1) occurred at 2 x 10m5M and the agonist proved to have an intrinsic activity identical to that of OA. By comparison, DA at lOAs M induced only 20% of the maximum tissue relaxation caused by OA while, NA and HA were without effect (Fig. 2) on the isolated foregut. The effects of antagonists
Figure 3 shows that mianserin (10-7M-10-5M) caused non-competitive inhibition of OA-induced relaxation of the tissue. Thus, in the presence of lo-’ M mianserin the EDGE value for OA increased from 1.4 x 10m6M to 3.6 x 10e6 M with a 21% reduction in the response. At 10e6 M, mianserin caused a 50% reduction in the maximum response to OA and an increase in the EDGE value from 1.4 x 10m6M to lo-‘M. At lo-‘M, mianserin caused an 84% reduction in the tissue’s response to OA. Both mepyramine and cyproheptadine were also found to non-competitively antagonise OA-induced relaxation. For example, mepyramine (Fig. 4) at 5 x IO-‘M caused 56% inhibition of the maximum response to OA while the same concentration of cyproheptadine (Fig. 5) caused a 46% reduction in
DA 12
---L---_
M
f
;
-5
LO$$ONCENTRATION
Fig. I. Dose-response curves of OA (A) and synephrine (a) on the locust foregut in vitro. Each point is the mean of 69 replicates (ISEM).
t
-7
.
-5 OCTOPAMINE
-4 M
Fig. 3. Dose-response curves of OA in the presence of mianserin IO-’ M (A), 10m6M (O), 5 x 1O-6 M (0) and lo-’ M (0). The 5-HT control dose-response curve is to left of that in the presence of IO-’ M mianserin (A). Each point is the mean of 6-18 replicates. Standard errors (typically 5515% of mean) have been omitted in the interests of clarity.
the maximum response to the agonist. However, yohimbine (10m4M) caused a 30% reduction in the maximum response to OA but had no significant effect on the ED,, value. Finally, ketanserin and metiamide at doses as high as 5 x 10e5 M had no antagonistic effect on OA-mediated tissue relaxation. Both mianserin (10m6M) and cyproheptadine (5 x IO-’ M) reduced the maximum evoked response to synephrine by 64% and 52% respectively (Fig. 6). Yohimbine (10~6M-10~4 M) had no effect on the responses of the tissue to application of synephrine. The effects of caffeine, verapamil and cadmium ions
The phosphodiesterase inhibitor caffeine (10e6 M) was found to potentiate OA-induced tissue relaxation shifting the dose response curve to the left and reducing the EDGE value from 10S6 M to 5.6 x IO-’ M and increasing the maximum response to 220% of the control value (Fig. 7). In the presence of mianserin (10m6M) this potentiating effect of caffeine was totally abolished, the ED,, value being increased to 3 x lo-‘M and the maximum response being restored to the original control value (Fig. 7). Ketanserin (lo-* M) had no effect on the caffeine-
r;ii-__ 0 A
1
10
3 x &=M -~--~-t_-._--L----.‘--~~
~_.._~
&A
-------
-5
M
-i‘
;
Fig. 2. The effect of DA, S-HT, OA, NA and HA on the isolated foregut of Schistocercagregaria. The arrows mark the time of addition of doses of these amines. Calibration (1 cm and 30 set) applies to all traces.
Octopamine,
-0
LOQ,,CONCENTRATION
-4
-5
-6
-7
OCTOPAMINE
receptor in locust foregut
-8
335
-7
-6
LOQ,oCONCENTRATION
M
-5 SYNEPHRINE
-4 M
Fig. 4. The dose-response curve for OA in the absence (A) and presence (a) of mepyramine (5 x 10m5 M) on the locust foregut. Each point is the mean of 618 replicates (+ SEM).
Fig. 6. The dose-response curve for synephrine in the absence (0) and presence of mianserin (10m6 M) (0) and cyproheptadine (5 x IO-‘M) (A) on the locust foregut. Each point is the mean of 6 replicates (+SEM).
potentiated response. Verapamil and cadmium ions at concentrations as high as 1 mM had neither inhibitory nor potentiating effects on the actions of OA.
physiologically-distinct classes of OA receptors in the extensor-tibiae neuromuscular preparation. He concluded that OA, receptors were preferentially antagonised by chlorpromazine, phentolamine and yohimbine while mianserin and cyproheptadine were more effective at OA, receptors. The latter were sub-divided into OA,, and OA,, on the basis of the relative potency of the antagonists mianserin, cyproheptadine and metoclopramide with all three molecules being less potent at the OA,, receptor. Subsequent work (Evans, 1984a) showed that OA, receptors could be blocked by the histamine H, antagonist mepyramine but not by the H, antagonist metiamide. Therefore, the OA receptors mediating relaxation of the foregut of S. gregariu would appear to belong to the OA, sub-type because the actions of OA and synephrine are blocked by mianserin, mepyramine and cyproheptadine while yohimbine, metiamide and ketanserin had no significant effect. However, it is difficult to further differentiate them into either OA,, or OA,, receptor sub-types because the relative potency of mianserin and cyproheptadine in this tissue does not equate with that described by Evans (1981) for the extensor-tibiae preparation. Thus, for class 2A receptors, Evans (1981) reported that mianserin and cyproheptadine had IC,, values of 1 PM and 2 p M respectively whereas at class 2B receptors the values were 20 p M and 50 p M respectively. However, in this study we found that while mianserin had an rc,, value of 1 PM, thereby suggesting that the receptor could belong to class 2A, the value for cyproheptadine (50 PM) matched that expected for Evans’ class 2B site.
DISCUSSION
The results presented above show that octopamine and its naturally occurring N-methylated derivative, synephrine, have identical pharmacological profiles on the isolated foregut of Schistocerca gregaria. Thus, both of these biogenic amines had similar affinities and intrinsic activities for the receptor sites in the tissue and were affected equally by the antagonists used. A similar situation has been reported for the extensor-tibiae preparation from S. gregaria (Evans, 1981) and the oviducal muscle of Locusta migratoriu (Orchard and Lange, 1986). By comparison, the amines NA and HA had little or no effect on the foregut while the actions of DA were much weaker than those of OA. However, the indoleamine 5hydroxytryptamine (5-HT) was a far more powerful relaxant of the tissue than OA (Banner et al., 1986a) thereby suggesting that OA could be acting as a weak agonist at 5-HT receptors especially as the effects of both amines were antagonised by mianserin. Subsequent work (Banner et al., 1986b) showed the effects of 5-HT, but not those of OA, could be blocked by the 5-HT, antagonist ketanserin. Furthermore, differences in the kinetics of antagonism by mianserin against OA and 5-HT suggested that the two amines exerted their effects on the tissue by activating two distinct receptors. Using a wide range of agonists and antagonists, Evans (1981) described three pharmacologicallyand
-7 LOQ,,CONCENTRATION
OCTOPAMINE
M
Fig. 5. The dose-response curve for OA in the absence (A) and presence of yohimbine (10m4M) (0) and cyproheptadine (5 x lo-‘M) (A) on the locust foregut. Each point is the mean of 69 replicates (+SEM).
-6 LOG,oCONCENTRATION
-5
-3
-4 OCTOPAMINE
M
Fig. 7. The dose-response curve for OA in the absence (A) and presence of caffeine (10m6 M) (A) and caffeine (10m6 M) plus mianserin (10m6 M) (0) on the locust foregut. Each point is the mean of 610 replicates (+SEM).
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S. E. BANNER et al.
It is interesting to speculate on the nature of the 0A2 receptors found in the foregut and other visceral and skeletal tissues of the locust. Thus, while HA has no effect on the isolated foregut and, unlike OA, does not induce cyclic AMP accumulation in extensortibiae preparation (Evans, 1984a), molecules, such as mianserin, cyproheptadine and mepyramine, with high affinity for mammalian HA H, receptors (Leysen and Gommeren, 1986) appear to be potent antagonists of the actions of OA in S. gregaria. By comparison, ketanserin, yohimbine and metiamide have markedly lower affinity for mammalian H, binding sites/receptors. Therefore, it is tempting to speculate that the insect OA receptors and mammalian H, receptors may have pharmacological and even structural similarities. In both the extensor-tibiae (Evans, 1984a) and oviduct neuromuscular (Orchard and Lange, 1986) preparations it has been shown that OA and synephrine cause an increase in cyclic AMP levels by an interaction with OA, receptors which feature a pharmacological profile similar to that described in the foregut. The potentiation of OA-induced foregut relaxation by the phosphodiesterase inhibitor caffeine provides further evidence for the hypothesis that OA, receptors are linked to adenylate cyclase and so utilise cyclic AMP as their secondary messenger. The connection between OA-induced tissue relaxation and adenylate cyclase was confirmed by the observation that the OA, antagonist mianserin blocked the potentiation caused by preincubation of the tissue with caffeine and the failure of caffeine to potentiate 5-HT-induced relaxation (Banner and Scale, personal observation). Evans (1984c) suggested that activation of OA, receptors may lead to an increase in intracellular calcium levels. Consequently, the possibility that the OA receptors mediating relaxation of the foregut may bring about their effect by controlling calcium fluxes was investigated by preincubating the tissue with the Ca*+ channel blockers verapamil (Kohlhardt and Mnich, 1978) and cadmium ions (Cooper and Manalis, 1984). However, both substances failed to inhibit the OA-mediated response thereby providing additional evidence that these receptors are not of the OA, type. The results of this study have shown that OA may have a significant role in the control of foregut motility. However, it is difficult to assess the relative importance of this amine because its actions are far less powerful than either proctolin or S-HT which have been postulated (Banner et al., 1987a) to be responsible for providing the primary motor drive of this tissue. However, it is suggested from this study that the tissue contains an adenylate cyclase-linked OA, receptor which, when activated, mediates tissue relaxation.
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Acknowledgements-SEB
is supported by SERC Research Studentship. We are grateful for financial support from Shell Research Ltd.
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