Pharmacological profile of octopamine receptors on the lateral oviducts of the locust, Locusta migratoria

J. Inxcr Physiol. Vol. 32, No. 8, pp. 741-745, 1986

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PHARMACOLOGICAL PROFILE OF OCTOPAMINE RECEPTORS ON THE LATERAL OVIDUCTS OF THE LOCUST, LOCUSTA A4ZGRATORZA IAN ORCHARDand ANGELAB. LANGE Department of Zoology, University of Toronto, Toronto, Ontario, Canada, M5S 1Al

(Received 31 October 1985; revised 20 December t985) Abstract-The

pharmacological properties of octopamine receptors lying on the lateral oviducts of

Locusta migrotoriu have been examined. These receptors mediate an elevation in cyclic AMP indicating

that they may be octopamine-2 receptors. This was further clarified with the use of agonists and antagonists. The a-adrenergic agonists, clonidine and naphazoline, induced small increases in cyclic AMP levels, whereas tolazoline had no effect. Of the formamidine pesticides tested, demethylchlordimeform was found to be a potent, though partial agonist, whereas chlordimeform was a weak agonist. The effects of demethylchlordimeform and D,L-octopamine together were not additive indicating that they may be acting upon the same receptors. A number of amine@ antagonsits were capable of blocking octopamine-mediated elevations in cyclic AMP. The IC,, values indicated a rank order of potency of phentolamine > gramine > metoclopramide > mianserin > cyproheptadine > yohimbine. The rank order on demethylchlordimeformmediated elevations in cyclic AMP was the same for selected antagonists, again indicating that demethylchlordimeform was acting upon the same receptors as octopamine. The pharmacological profile of these octopamine receptors supports their classification as octopamine-2 receptors. Key Word Index: Octopamine-receptors, locust

aminergic agonists and antagonists, formamidines, oviduct,

INTRODUCTION Octopamine is a multifunctional, naturally occurring biogenic amine possessing the properties of a neurotransmitter, neurohormone and neuromodulator in insects (Evans, 1980; Orchard, 1982) and probably other invertebrates (Robertson and Juorio, 1976). Although octopamine appears to be physiologically involved in many insect systems, there are only a limited number of tissues which have been shown to be innervated by identified octopaminergic neurones. These tissues include the lantern of firefly (Christensen et al., 1983), skeletal muscle in the extensortibialis of locusts (Hoyle, 1975; Evans and O’Shea, 1978) and visceral muscle in the oviducts of locusts (Orchard and Lange, 1985). All of these tissues receive innervation from dorsal unpaired median neurones. Many of the physiological functions of octopamine, including those of the above three cases, appear to be mediated by a class of octopamine receptors (octopamine-2-receptors) specifically linked to an adenylate cyclase (Evans, 1984; Nathanson, 1979, 1985; Lange and Orchard, 1986). Thus the physiological actions of octopamine have been shown to be associated with elevated levels of cyclic AMP. Interest in octopamine receptors has grown with the discovery that a novel group of pesticides, the formamidines, mimic the effects of octopamine on a number of tissues (Hollingworth and Murdock, 1980; Evans and Gee, 1980; Nathanson and Hunnicutt, 1981; Singh et al., 1981; Orchard et ai., 1982). The possibility has recently been raised, therefore, that potent and selective octopamine agonists could be useful toxins in invertebrates with low toxicity in

vertebrates (Nathanson, 1985). Critical information on the pharmacological properties of octopamine receptors is however limited to studies examining lantern tissue (Nathanson, 1979) and skeletal muscle (Evans, 1981); tissues rich in octopamine receptors. Recently, we have described a visceral muscle preparation with which to extend our knowledge of octopamine-receptors. Locust oviducts are innervated by dorsal unpaired median neurones, DUMOVI and 2, which are octopaminergic and lie in the VIIth abdominal ganglion (Lange and Orchard, 1984; Orchard and Lange, 1985). Octopamine mediates a relaxation of oviduct muscle tension, an inhibition of myogenic contractions and a reduction in amplitude of neurally evoked contractions. The major physiological effects of octopamine are mimicked by agents which elevate cyclic AMP levels. In addition, direct stimulation of dorsal unpaired median neurones of the oviduct results in an elevation of cyclic AMP in the oviducts (Lange and Orchard, 1986), as too, does exogenous application of octopamine (35-fold). No other naturally-occurring amine has been shown to produce similar effects. Furthermore, release of other endogenous neurotransmitters, via oviducal nerve stimulation, does not lead to an accumulation of cyclic AMP. Thus the elevation in cyclic AMP is due to octopamine and probably not due to an endogenous neurotransmitter which may be released by octopamine. The locust oviduct is therefore an ideal tissue upon which to characterise and compare the pharmacological properties of octopamine-2 receptors with those found in other tissues.

IANORCHARD

142 MATERIALS AND

and ANGELAB.

LANGE

METHODS

All experiments were performed on adult female locusts (days 8-12) obtained from a colony of Locusta migratoria reared at 30°C under crowded conditions. The locusts were fed on fresh wheat seedlings supplemented with bran. The two lateral oviducts from each locust were dissected out and washed in physiologial saline (composition: 150 mM, NaCl; lOmM, KCl; 4mM, CaCl,; 2 mM, MgCl,; 4mM, NaHCO,; 5 mM HEPES, pH 7.2; 90 mM, sucrose; 5 mM, trehalose). Only the most innervated region of each lateral oviduct was used, this being the stretch of oviduct lying between the common oviduct and the origin of the ovaries (see Orchard and Lange, 1985). Individual lateral oviducts were incubated in 100 ~1 saline containing 0.5 mM 3-isobutyl- 1-methylxanthine (IBMX) and the desired concentration of drug(s) under test. All experiments were terminated after 10 min by the addition of 500 ~1 of cold 0.4 N perchloric acid containing 5 mM sodium sulphite. After 15 min at 4°C the tissue was removed by centrifugation and dissolved in 100~1 0.5 N sodium hydroxide for protein determination by the Bio-Rad assay of Bradford (1976) using gamma giobuIin as standard. The supernatant was adjusted to pH 6.2 with 2.5 M KHCO, and cyclic AMP measured in a suitable aliquot, all as previously described (Lange and Orchard. 1986). The following chemicals were prepared as stock solutions (10-l to 10e3M) using suitable solvents (ethanol or water), and diluted with saline prior to use: naphazoline HCl, phentolamine HCl, demethylchlordimeform, chlordimeform (Ciba-Geigy); phenoxybenzamine HCl (Smith, Kline and French); mianserin HCl (Organon); all other chemicals from Sigma. Suitable solvent controls were run, and found to have no effect at their final concentrations (0.001~. 1%). RESULTS

We have previously shown that D,r_-octopamine produces a dose-dependent accumulation of cyclic AMP in the oviducts of days 10-15 adult female locusts (Lange and Orchard, 1986). Similar results were obtained in the present study (Fig. 1) using slightly younger animals (days 8-12), although the basal level of cyclic AMP was found to be higher. E&cts of agonists

The effectiveness of a series of synthetic agonists in increasing cyclic AMP levels in locust lateral oviduct is shown in Table 1. The a-adrenergic agonists, clonidine and naphazoline, induced small increases in cyclic AMP levels, whereas tolazoline had no effect. The formamidine pesticides, chlordimeform (CDM) and demethylchlordimeform (DCDM) have been shown to be octopamine agonists in a variety of preparations (Evans and Gee, 1980; Nathanson and Hunnicutt, 1981; Singh et al., 1981). In locust oviducts, CDM was a weak agonist, being only 3.6% as effective as octopamine at lo-’ M (Table 1). DCDM on the other hand was a strong agonist (approx. 53% as effective as octopamine at 10e5 M). Since there is considerable interest in the formamidines as pesticides we examine their effects more closely. Figure

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1. Dose-response curves for the effects of D,L-OCtOpIIke (OCT), demethylchlordimefor (DCDM) and chlordimeform (CDM) upon cyclic AMP levels in the lateral oviducts of Locusta migraforia.All values obtained in the presence of 5 x 10e4M IBMX, after a 10min incubation. Basal level of cyclic AMP after 10min in IBMX alone was 67.6 + 12.8 pmol/mg protein. Points represent mean + SEM, n = 68.

1 presents dose-response curves for octopamine, DCDM, and CDM. DCDM caused significant accumulation of cyclic AMP at lo-* M and maximal accumulation at about 10-s-10-4 M. Maximal stimulation was consistently 30-47% less than that caused by octopamine. Half-maximal stimulation occurred at approx. 2 x lo-‘M. DCDM may therefore be considered a potent, though partial agonist of these octopamine receptors. CDM was a poor agonist at the ranges tested 10-6-10-4 M). In additivity experiments (Table 2), the accumulation of cyclic AMP induced by octopamine and DCDM together (10e4 M) was approximately equal to that produced by DCDM (10m4M) alone, and therefore only about 55% of that produced by octop-

Table I. Action of agonists on cyclic AMP levels in lateral oviducts of Locustamigrotoria

Drug IBMX D,L-OCtOpUIIiW

Clonidine Naphazoline Tolazoline DCDM CDM

Cyclic AMP (pmol/mg protein) 93.0 f 671 .Of 149.6 f 136.8 f 80.8 5 399.8 5 113.7 +

32.6 134.0 11.8 16.8 32.8 8.12 14.4

Percentage response relative to 10eJ M D,L-OCtOpt3mitX 0 100 9.8 7.6 0 53. I 3.6

Cyclic AMP values are expressed as mean f standard error of fhe mean (n > 5). Drugs tested at IO-‘M in the presena of 5 x lo-‘M IBMX. IBMX, 3-isobutyl-I-merhylxanthine; CDM, Chlordimefonn; DCDM, demethylchIordimefor.

receptors on lateral oviducts

Octopamine-2 Table 2. Effects of formamidines upon octopamine-stimulated mulations of cyclic AMP in lateral oviducts

Drug

accu-

Cyclic AMP (pmol/mg protein)

Percentage response relative to 10m4M o,L-octopamine

84.6 f 21 .O 607.8 &-19.2 359.7 + 71.0 374.3 f 49.1

0 100 52.6 55.4

117.6k21.2 272.9 k 22.6

6.3 36.0

IBMX o,L-octopaminc DCDM D,L-octopamine + DCDM CDM o,L-octopamine + CDM

amine (1O-4 M) alone. The accumulation was therefore not additive and, presumably because of a higher affinity of DCDM for the receptor, the accumulation of cyclic AMP was determined by DCDM. At the high concentration of 10m4M, CDM was an active inhibitor of the octopamine-mediated accumulation of cyclic AMP. The effects of octopamine and CDM together produced an effect which was only 36% that produced by octopamine alone. Eflects of antagonists The effectiveness of a wide range of amine@ antagonists upon the octopamine receptors is shown in Table 3. A number of these antagonists were capable of lowering the basal level of cyclic AMP illustrating that the oviducts in these animals may already have been partially stimulated by octopamine. At lo-’ M, phentolamine, gramine and metoclopramide completely inhibited the effects of 10m6M octopamine, while mianserin was 86% effective. Yohimbine was a poor blocking agent, and chlorpromazine resulted in no inhibition. Indeed, chlorpromazine was found to potentiate the octopaminemediated accumulation of cyclic AMP. The blocking action of the antagonists was competitive with octopamine since the presence. of the drug shifted the dose-response curve for octopamine to the right. The relative effectiveness of the different antagonists was

Drug IBMX Phentolamine Gramine Metoclopramide Mianserin Cyproheptadine Phenoxybenzamine Yohimbine Chlororomazine

Table4. Actionof antagonists on DCDM-stimulated accumulation of cyclic AMP in lateral oviducts

Phentolamine Gramine Cyproheptadine Yohimbine

92.6 78.2 24.7 19.7

compared from dose-response curves in which the concentration of drug was altered whilst maintaining the concentration of octopamine at 10w6M. The IC,, was calculated from the dose-response curves as the concentration of drug required to reduce the response to 10s6 M octopamine by 50%. The rank order of potency was phentolamine > gramine > metoclopramide > mianserin > cyproheptadine > yohimbine. Since the earlier data indicated DCDM to be a potent agonist of the octopamine receptors, the effects of some selected amine@-antagonists upon the action of DCDM was examined. Table 4 reveals that the DCDM-mediated accumulation in cyclic AMP was blocked by the same antagonists that blocked the octopamine receptors, providing further evidence that octopamine and DCDM were acting upon the same receptors.

DISCUSSION

The results of the present investigation extend our knowledge of the pharmacology of octopamine-2 receptors. We have previously shown that in the locust oviduct the increase in cyclic AMP is specific for octopamine, and for the N-methylated derivative of octopamine, synephrine (Lange and Orchard, 1986). Other naturally occurring amines including tyramine, norepinephrine, dopamine and S-hydroxytryptamine are poor agonists. Furthermore artificially elevating cyclic AMP levels mimics the major physiological effects specific to octopamine. We are thus dealing with a tissue rich in octopamine-2 receptors, and possibly devoid of other types of aminergic receptors.

on octopamine-stimulated lateral oviducts

Basal level (pmol/mg protein) 93.4 f 51.5 f 45. I + 48.2 k 49.2 f 17.9 f 81.7 + 70.0 f 80.7 +

Percentage increase relative to 10m6M DCDM

Drug

Drugs tested at IO-‘M in the presence of 5 x IO-‘M IBMX.

Cyclic AMP values expressed as mean f standard error of the mean (n = 3-8). Drugs tested at 10e4M in the presence of 5 x IO-*M IBMX. Abbreviations as in Table 1.

Table 3. Action of antagonists

743

20.9 12.0 6.2 7.5 5.9 15.9 19.1 22.0 13.4

accumulation

of cyclic AMP in

Percentage decrease relative to IO-” M D,L-octopamine 0

100 100 100 86. I 39.0 28.2 6.1 l

IC,”

6.5 x 1.5 x 1.9 x 5.0 x 1.5 x

IO-’ lo-’ lo-* 1om6 10-s

9.6 x IO-’ -

Basal level of cyclic AMP expressed as meanf standarderror of the mean(n 3 5-s). Drugs tested at IO-‘M in the absence (basal level) or presence of 10e6M o,r-octopamine. IBMX (5 x lo-‘M) included in all incubations. IC, represents the concentration of antagonist required to inhibit the effects of 10m6M qwxtopamine by 50%. *Chlorpromazine actually potentiated the actions of D,L-OctOpamine. Due to solubility problems an IC, could not be calculated for phenoxybenzamine.

744

IAN ORCHARDand ANGELAB. LANGE

The most detailed study of octopamine receptors has been made by Evans (1981, 1984), utilising the extensor-tibiae muscle of locusts. Using a wide range of agonists and antagonists, Evans has described the presence of 3 distinct classes of octopamine receptors. Octopamine-1 receptors, which slow the rhythm of myogenic contractions, do not act via cyclic AMP (Evans, 1984). Octopamine-2 receptors, which modulate twitch tension, act via cyclic AMP, and can be further divided into type 2A receptors, lying presynaptically, and type 2B receptors lying postsynaptically (Evans, 1981). The octopamine receptors mediating an elevation in cyclic AMP in locust oviducts are, in general terms. similar to octopamine-2 receptors in insects (Evans, 1981, 1984; Nathanson, 1979). These receptors are blocked by OL -adrenergic receptor antagonists such as phentolamine, whilst being much less sensitive to fl-adrenergic receptor antagonists such as propranolol. Similarly vertebrate a-adrenergic agonists such as clonidine are also agonists (albeit often weak) of octopamine-2 receptors (Evans, 1981, 1984). It is important to re-emphasize however (see Evans, 1984), that although sharing pharmacological properties with /I-adrenergic receptors, the mode of action of octopamine-2 receptors appears different. Alphaadrenergic receptors are believed to act via changes in calcium permeability, activation of cyclic GMP or inhibition of adenylate cyclase (see Bodnaryk, 1985) whereas octopamine-2 receptors appear to activate adenylate cyclase. The receptors described in the present study are clearly different from octopamine-1 receptors and share many of the pharmacological properties of octopamine-2 receptors as described by Evans (198 1, 1984). For example, octopamine-2 receptors appear to be distinguished from octopamine-1 receptors by being linked to adenylate cyclase and by illustrating a higher sensitivity to metoclopramide than to chlorpromazine or yohimbine. Similar properties are revealed by the octopamine receptors on locust oviduct. In addition we have found gramine to be a potent antagonist of octopamine-mediated elevations in cyclic AMP, with an I&, of 1.5 x lo-’ M when compared to 10V6M D,L-octopamine. Gramine also antagonises the octopamine-2 receptors in the glandular lobe of the corpus cardiacum of locusts (Orchard et al., 1983) but does not block octopamine-1 receptors in locust extensor-tibiae muscle (Evans, 1981). As such, therefore, gramine may prove a useful tool in distinguishing between these classes of octopamine receptors. Unfortunately the effects of gramine upon other octopamine-2 receptors have not yet been reported. Octopamine-2A receptors (Evans, 1981) as distinguished by the use of antagonists upon the physiological effects of octopamine, show an order of potency of metoclopramide > mianserin > phentolamine > cyproheptadine > phenoxybenzamine > chlorpromazine > yohimbine (ineffective) whereas 2B receptors are less sensitive and have the order phentolamine > metoclopromide > mianserin > cyproheptadine > chlorpromazine > phenoxybenzamine > yohimbine (ineffective). In determining the effects of antagonists upon octopamine-stimulated levels of cyclic AMP (Evans, 1984) the order of

potency is the same as that shown above for 2B receptors although interestingly yohimbine is partially effective (20% inhibition at 10m4M). Oviduct octopamine-2 receptors mediating an elevation in cyclic AMP are similar to 2B receptors except for the position of chlorpromazine. Thus the rank order for oviduct becomes phentolamine > metoclopromide > mianserin > cyproheptadine > phenoxybenzamine > yohimbine > chlorpromazine (ineffective). The distinction between octopamine receptors in agonist studies is much less clear when their ability to elevate cyclic AMP is examined (Evans, 1984). Synthetic cc-agonists offer weak responses in comparison to their ability to induce physiological effects. Clonidine and naphazoline are less than 10% as effective as octopamine on locust oviduct at concentrations of lo-‘M. A similar result was obtained by Evans (1984) except that octopamine-2A and 2B receptors were also sensitive to tolazoline. Thus it appears that there are differences between the octopamine-2 receptors described by Evans (1981, 1984) and those of the present study, thereby indicating the presence of at least a third class of octopamine-2 receptors. The effects of formamidines upon locust oviduct are similar to those reported upon octopaminesensitive adenylate cyclases (Nathanson and Hunnicutt, 1981; Cole et al., 1983; Davenport et aZ., 1985; Orchard et al., 1982). DCDM is a potent, though partial agonist of oviduct octopamine receptors, being only about 53% as effective as octopamine, yet having a threshold around lo-’ M. The elevation in cyclic AMP induced by DCDM was not additive to that induced by octopamine, and indeed DCDM caused a 45% inhibition of octopamine-stimulation. Further evidence for DCDM acting upon octopamine-receptors was obtained with antagonists. Selected a-adrenergic antagonists antagonised the actions of DCDM to a similar extent as that shown against octopamine. CDM is a weak agonist of octopamine-receptors and at a high concentration acted as an octopamine-antagonist, inhibiting the effect of octopamine by 64%. Similar results were obtained by Nathanson and Hunnicutt (1981) in examining octopamine-sensitive adenylate cyclase in firefly light organ. Their results indicated that CDM was unlikely to be interacting with octopamine receptors and suggested that the inhibitory actions of CDM lay elsewhere in the cascade of events. The agonistic properties of CDM, especially those shown in vivo, are probably due to its conversion to DCDM (see Hollingsworth and Murdock, 1980). The activation of octopamine-sensitive adenylate cyclase in insects and ticks by formamidines has deleterious behavioural and physiological consequences, which probably convey the pesticidal properties to this new class of pesticides. Since tissue levels of octopamine are considerably higher in invertebrates than in vertebrates, and since octopamine-sensitive adenylate cyclases have not been described in vertebrates (see Nathanson, 1985), it seems clear that octopamine receptors should prove to be a useful target site against which to develop specific and safe pesticides. Acknowledgement-This work was supported by the Natural Sciences and Engineering Research Council of Canada,

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