Archives of Insect Biochemistry and Physiology 5:285-295 (1987) Mode of Action of Proctolin on Locust Visceral Muscle Angela B. Lange, Ian Orchard, and William Lam Department of Zoology, University of Toronto, Toronto, Ontario, Canada Proctolin increases the frequency and amplitude of myogenic contractions and results in a sustained contraction of the oviducts of Locusta migratoria. The possible mode of action of proctolin receptors on this visceral muscle has been investigated. Calcium-free saline, containing either 20 m M magnesium ions or 100 pM EGTA, inhibited myogenic contractions, lowered basal tension, and abolished all the effects of proctolin following a 20 min incubation. These effects were reversible upon washing with normal saline. Similar results were obtained with normal saline containing 10 m M cobalt ions. Nifedipine at 50 pM lowered basal tension, abolished myogenic contractions, and reduced the proctolin-induced sustained contraction by 42-62% at 0.5 nM proctolin and by 33-37% at 5 n M proctolin. Similar results were obtained with 100 pM verapamil. Proctolin was still capable of eliciting considerable contractions (25-67% of controls) in preparations depolarized with 100 m M potassium saline. The removal of calcium from the highpotassium saline reversibly abolished the potassium-induced contraction and reversibly blocked the action of proctolin. Nifedipine was ineffective in blocking the action of proctolin in high-potassium saline. Neither cyclic AMP levels nor cyclic GMP levels of the lateral oviducts were elevated by proctolin in the presence of a phosphodiesterase inhibitor. The results indicate that proctolin mediates its effects via an influx of external calcium ions. This calcium appears t o enter through two channels, a voltage-dependent channel and a receptor-operated channel. Cyclic nucleotides do not appear to be involved i n the action of proctolin in this visceral muscle. Key words: calcium, calcium antagonists, cyclic nucleotides INTRODUCTION The importance of the pentapeptide proctolin as a neuroactive substance in insects and crustacea has now been firmly established with the discovery Acknowledgments: This work was supported by the Natural Sciences and Engineering Research Council of Canada. Received January 2,1987; accepted April 3,1987. Address reprint requests to Angela B. Lange, Department of Zoology, University of Toronto, Toronto, Ontario, Canada M55 1Al. 0 1 9 8 7 Alan R. Liss, Inc. 286 Lange, Orchard, and Lam that proctolin acts as a modulator of both visceral and skeletal muscle [1-7]. Thus proctolin-containing neurons innervate a variety of muscles in insects and crustacea, in which proctolin has been implicated as a cotransmitter along with a more conventional transmitter such as glutamate [1,3]. The proctolin released from such neurons may have direct or indirect actions, inducing contraction by a direct action on the muscle andlor indirectly by modulating the synaptic actions of the transmitter with which it is coreleased [1,3,8]. The mode of action of proctolin in these preparations is not that of a conventional neurotransmitter, since proctolin induces only minor changes in membrane potential and may result in decreases in muscle membrane conductance. Various mechanisms by which proctolin may result in contractions have been suggested, including second messengers such as cyclic AMP  or phosphoinositols [S], receptor-operated calcium channels [lo], and voltage-dependent calcium channels that are open at the resting potential [ll]. Clearly, proctolin has diverse modes of action requiring a study of the mode of action of proctolin receptors for each preparation in which proctolin is believed to have a physiological role. This is illustrated for different species of cockroach, in which agents that elevate cyclic AMP levels may potentiate the action of proctolin on the hindgut of Peeriplunetu but inhibit the action of proctolin on the hindgut of Leucophaea . Recently, we demonstrated that the oviducts of the locust, Locustu migrutoria, receive innervation from proctolin-containing neurons . Proctolin appears to be a cotransmitter in these motoneurons, since the amplitude of EJPs" recorded from the muscle are sensitive to glutamate but not to proctolin, and proctolin results in only a minor change in muscle membrane potential 1121. The contractile properties of this visceral muscle are, however, very sensitive to proctolin, with as little as 50 pM proctolin capable of increasing the amplitude of neurally evoked contractions, increasing the frequency and amplitude of myogenic contractions, and increasing the basal tonus as indicated by a sustained contraction . Larger doses of proctolin induce correspondingly greater effects. The present study investigates the possible mode of action of proctolin receptors on locust oviducts by examining the effects of calcium-free solutions, calcium channel antagonists, and high-potassium saline on proctolininduced contractions. The effects of proctolin on cyclic AMP and cyclic GMP content of the oviducts have also been studied. The results are discussed in relation to other preparations in which the mode of action of proctolin has been examined. MATERIALS AND METHODS Adult female L. migrutoriu were reared under crowded conditions at 30°C on a 12 h light:l2 h dark regime and fed on freshly grown wheat and bran. The ovaries, with oviducts attached, were dissected through a midventral *Abbreviations: EJPs = excitatory junction potentials; IBMX = 3-isobutyl-I-methylxanthine; RIA = radioirnmunoassay. Mode of Action of Proctolin 287 incision of the abdomen under physiological saline (150 mM NaCI, 10 mM KCI, 4 mM CaC12, 2 mM MgCI2, 4 mM NaHC03, 5 mM HEPES pH 7.2, 90 mM sucrose, and 5 mM trehalose). The preparation was placed in a trough, containing 1ml of saline, molded into the wax base of a dissecting dish. The posterior end of the common oviduct was attached using fine thread to an AE875 miniature force transducer (Aksjeselskapet Mikro-Elektronikk, Oslo, Norway), and the upper part of each lateral oviduct was attached to the base of the trough using minuten pins. The preparation was mounted at an angle of 30" to the horizontal by its attachment to the force transducer and held under a tension of approximately 200 mg. Proctolin (Peninsula Laboratories, Inc., Belmont, CA) was applied by replacing 500 pl of the saline with 500 pl of the appropriate concentration in saline. The concentrations given reflect the final concentration in the bath. Proctolin was washed off the preparation when maximum basal contraction had been reached. The effect of modified salines on the proctolin-induced contractions was examined. Calcium-free, high-magnesium saline was made by removing the CaCI2 from the saline and increasing the MgCI2concentration to 20 mM. High-potassium (100 mM) saline was made by replacing 90 mM of the NaCl with 90 mM KCI. Cobalt chloride (10 mM), verapamil hydrochloride (100 pM), and EGTA (100 pM) were added directly to the normal saline. Nifedipine was made as a stock solution (10 mM) in acetone and then diluted with saline to 50 pM, giving a final acetone concentration of 0.5%. This acetone concentration did not affect the preparation in any noticeable manner. For studies involving cyclic AMP and cyclic GMP, lateral oviducts were incubated for 10 min in 50 pM IBMX containing the appropriate concentrations of proctolin. The reaction was terminated as described previously 1141, and cyclic AMP or cyclic GMP was measured by means of commercially available RIA kits (New England Nuclear, Boston, MA). Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. RESULTS As was previously demonstrated [7,13], locust oviducts are myogenically active as judged by the persistence of phasic contractions when isolated from the central nervous system (see Fig. 1).Proctolin increases the frequency of myogenic contractions and also results in a dose-dependent, sustained contraction, which develops into large phasic contractions during wash-off (Fig. 1). Incubation with a calcium-free saline containing 20 mM magnesium chloride resulted (in some preparations) in an initial small increase in both basal tension and amplitude of myogenic contraction. However, in all preparations, these myogenic contractions were eventually abolished, and there was a relaxation in basal tension (Fig. 1).Proctolin was still capable of inducing a sustained contraction after 7 min in this saline (Fig. lA), although the amplitude of this contraction was no longer dose-dependent. As can be seen from Figure lA, with increasing concentrations of proctolin, the response diminished, possibly indicating a depletion of calcium. Myogenic contractions were not induced by proctolin in this saline. The effects of calcium-free, high- 288 Lange, Orchard, and lam A A A 0.5 1 5 A A A A A A 0.5 1 5 0.5 1 5 nM B Calcium-free. high magnesium L !A A 5 A A A 55 5 nM Fig. 1. Effects of a calcium-free, high-magnesium (20 mM) saline on proctolin-induced contractions of locust oviduct. The dose of proctolin in molarity is shown at the bottom of each trace. Preparations were washed in saline between each dose of proctolin (applied at arrowheads) except following the second application of 5 nM proctolin in B when calcium was added in the continual presence of proctolin. These traces are typical of several experiments. A The effects of three doses of proctolin before, during, and after perfusion with the calciumfree, high-magnesium saline. Perfusion with this saline resulted in an initial increase in myogenic activity, which was followed by inhibition of myogenic contractions and a lowering of baseline tension. Proctolin was still active after 7 min in this saline, but the response was no longer dose-dependent. The effects were reversible. B: A longer incubation in calciumfree, high-magnesium saline (20 rnin) abolished the effect of proctolin (applied at arrowheads). Readdition of calcium in the continued presence of 5 nM proctolin immediately restored the action of proctolin. Following a further wash, the response was similar to that obtained at the start of the experiment. Scale bars = 100 mg, 3 min. magnesium saline were fully reversible (Fig. 1A). Following a 20 min incubation, however (Fig. 1B), this calcium-free saline abolished all the effects of proctolin. Reintroduction of calcium in the presence of proctolin immediately restored the response to proctolin, which recovered to its full magnitude within minutes (Fig. 1B). Results similar to those shown in Figure 1A and B were obtained using calcium-free saline containing 100 ph4 EGTA (not shown). To examine further the role of extracellular calcium in the action of proctolin, we looked at the effects of calcium-channel antagonists. Addition of 10 Mode of Action of Proctolin 289 mM cobalt chloride to normal saline resulted in a relaxation of basal tension and inhibition of myogenic contractions and abolished all the effects of proctolin (Fig. 2). The antagonistic action of cobalt did not require the extensive incubation required for calcium-free, high magnesium saline. The effects of cobalt were completely reversible upon washing in normal saline (Fig. 2). The results with cobalt support the idea that proctolin mediates its effects via an influx of extracellular calcium and argues against the notion that calcium is needed for receptor binding. In contrast to the action of cobalt, the voltage-dependent calcium-channel antagonist nifedipine was less effective. Nifedipine at 50 pM resulted in a 4262% inhibition of the response to 0.5 nM proctolin and 33-37% inhibition of the response to 5 nM proctolin (Fig. 3). Nifedipine is an "open"-channel blocker and was applied during wash-off from a proctolin dose of 5 nM. Results similar to those shown in Figure 3 were achieved using the voltage- b 1 h III A A A A A A 0.5 1 0.5 1 0.5 1 nM Fig. 2. Effects of 10 mM cobalt choride on proctolin-induced contractions. Dose of proctolin (applied at arrowheads) shown at bottom of trace in molarity. Preparation was washed between each proctolin application. Cobalt ions inhibited myogenic contractions, lowered basal tension, and inhibited the action of proctolin. The effect was reversible. Typical example shown. Scale bars = 100 mg, 3 min. I A A A 0.5 1 5 5 0 uM n i f e d i p i n e I A A A 0.5 1 5 nM Fig. 3. Effects of nifedipine on proctolin-induced contractions. Preparation was washed following each dose of proctolin (applied at arrowheads; shown in molarity). Nifedipine inhibited myogenic contractions and reduced the amplitude of the proctolin-induced contraction. Typical example shown. Scale bars = 100 mg, 3 min. 290 Lange, Orchard, and Lam dependent calcium antagonist verapamil. At 100 pM of verapamil, the proctolin-induced contraction was reduced by only 39% in response to 0.5 nM proctolin and by only 18% in response to 5 nM proctolin despite extensive incubation (not shown). These results indicate that, whereas extracellular calcium is required for the action of proctolin, calcium-channels that are not voltage-dependent mediate much of the effect of proctolin. To test for the presence of receptoroperated channels, we examined the influence of high-potassium saline on the action of proctolin. High-potassium saline (100 mM) abolished myogenic contractions and resulted in a sustained contraction of locust oviducts, which was followed by a slow relaxation toward a plateau level. The time to reach this plateau varied considerably between preparations, and in some it took up to 2 h. Proctolin was still capable of inducing a sustained contraction in high-potassium saline during the plateau phase (Fig. 4). The amplitude of these contractions varied between 37% and 67% of controls with 0.5 nM proctolin and between 25% and 31% of controls with 5 nM proctolin. The proctolin-induced contractions in high-potassium saline were calcium-dependent. Calcium-free saline containing 20 mM magnesium chloride rapidly abolished the contraction resulting from high potassium and blocked the action of proctolin (Fig. 5). Reintroduction of calcium immediately restored the effect of high potassium, These results indicate that proctolin can act on preparations depolarized by high-potassium saline and that its actions are still dependent on extracellular calcium ions. High-potassium saline induced a sustained contraction, which was greatly reduced in the presence of 50 pM nifedipine (Fig. 6). Even in preparations 1 High potassium A A A A 0.5 1 0.5 1 nM Fig. 4. Effects of high-potassium (100 mM) saline on proctolin-induced contractions. Highpotassium saline resulted in a sustained contraction, which was followed by a slow relaxation towards a plateau level. Proctolin was still capable of inducing a contraction in high-potassium saline. Doses of proctolin, shown in molarity, were applied at arrowheads, and the preparation was washed following each application of proctolin. Typical example shown. Scale bars = 100 mg, 3 min. Mode of Action of Proctolin I I High p o t a s s i u m Calcium-free, 291 high magnesium A A 1 1 nM Fig. 5. Effects of calcium-free, high-magnesium saline on proctolin-induced contractions in high-potassium saline. Calcium-free, high-magnesium saline abolished the contraction induced by high-potassium saline and also abolished the proctolin-induced contractions. Proctolin was applied at arrowheads (dose given in molarity). Preparation was washed following each dose of proctolin. Typical example shown. Scale bars = 100 mg, 3 min. 1 High p o t a s s i u m 50 A A A 0.5 1 5 uM nifedipine A A 0.5 1 A 5 nM Fig. 6. Effects of nifedipine on proctolin-induced contractions in high-potassium saline. Nifedipine did not abolish the proctolin-induced contractions obtained in high-potassium saline. Proctolin was applied at arrowheads (dose given in molarity). Preparation was washed following each application of proctolin. Typical example shown. Scale bars = 100 mg, 3 min. 292 Lange, Orchard, and Lam incubated in high-potassium saline containing 50 pM nifedipine, proctolin resulted in a sustained contraction ranging between 37% and 71% of that obtained in normal saline (Fig. 6). The action of proctolin appears to be due in part to voltage-dependent calcium channels and in part to a system not dependent on depolarization, ie, receptor-operated channels. To examine further the actions of proctolin, we looked at the second messengers cyclic AMP and cyclic GMP. In experiments involving cyclic AMP, the octopamine antagonist phentolamine (5 pM) was included to avoid any possible elevation of cyclic AMP stimulated by any contraction-induced release of octopamine [see 151. As was anticipated from earlier studies in which elevated levels of cyclic AMP were linked to relaxation of basal tension , proctolin at 1nM failed to result in an elevation in cyclic AMP over basal levels (control 17.0 k 1.7 pmollmg protein, n = 4; experimental 14.5 k 3.5 pmollmg protein, n = 5). Similarly, proctolin failed to elevate cyclic GMP even when tested at 100 nM (control 3.64 & 0.8 pmollmg protein, n = 4; experimental 4.5 k 1.0 pmollmg protein, n = 6). Clearly, activation of proctolin receptors does not mediate an elevation in either cyclic AMP or cyclic GMP. DISCUSSION The application of proctolin to the oviduct musculature of the locust results in a dose-dependent increase in frequency and amplitude of myogenic contractions and a sustained contraction. The results of the present study suggest that proctolin mediates its effects via an influx of extracellular calcium. Thus both the myogenic contractions and the proctolin-induced sustained contractions are reversibly abolished in calcium-free saline containing either high magnesium or EGTA and by normal saline containing cobalt ions. It should be pointed out, however, that the myogenic contractions are more rapidly abolished by such treatments than are the proctolin-induced sustained contractions. The implication of this is that the myogenic contractions may require a greater influx of calcium and therefore may be more sensitive to calcium-free solutions. Alternatively, there could be two pools of calcium involved in mediating the changes in contraction, with a freely accessible pool involved in the myogenic contractions and a loosely bound pool involved in the proctolin-induced sustained contractions. In support of the latter possibility are the results with repeated doses of proctolin following a short wash in calcium-free solutions. In these experiments, increasing doses of proctolin resulted in a weaker response, implying a gradual depletion of calcium from a less accessible pool. However, a longer incubation (20 min) in calcium-free solutions was capable of eliminating all of the effects of proctolin. Readdition of calcium in the presence of proctolin resulted in an immediate return of the response, indicating that proctolin facilitates the reentry of calcium when these loosely bound pools have been depleted. Similar results were obtained by Cook and Holman [lo] and Holman and Cook  for the oviduct and hindgut of the cockroach Leucophaea rnaderae. In these preparations, a 7 min incubation in calcium-free saline was capable of reducing the proctolin-induced sustained contraction by about 95%. In both prep- Mode of Action of Proctolin 293 arations, 100 pM lanthanum ions was capable of abolishing all the effects of proctolin. A similar calcium dependence of proctolin action has been reported in locust hindgut , cockroach hyperneural muscle [lq,and lobster walking leg opener muscle [ll]. Interestingly, in contrast to these reports, is the observation by Worden and O’Shea  that the direct contractile effect of proctolin on the extensor tibialis muscle of locust hindleg was not dependent on the presence of extracellular calcium. In addition, Evans  suggested that the increase in frequency of a myogenic rhythm in the locust extensor tibialis muscle in response to proctolin may not be mediated by increases in intracellular calcium levels. Extensive investigations on vertebrate smooth muscle [see, eg, 19/20] indicate that there may be two types of membrane channels for calcium mediating the effects of stimulatory substances. One such channel may be voltage-dependent and lead to an influx of calcium when the membrane is depolarized. The other channel is a receptor-operated channel, controlled or operated by a receptor for the stirnulatory substance. These receptor-operated channels may or may not be linked to an intracellular second messenger, but, once again, attachment of the stimulatory substance to the receptor leads to an influx of calcium. The existence of similar channels in insect visceral muscle has been postulated by Cook and Holman [lo] for cockroach hindgut. Thus proctolin was capable of mediating a substantial contraction in hindgut muscle that had been depolarized by high-potassium saline. Furthermore, manganese ions, which are voltage-dependent calcium-channel blockers, reduced proctolin-induced contractions by only 40% at 10 nM proctolin. In a similar way, locust oviducts appear to possess two types of calcium channels; whereas both channels are blocked by cobalt ions, one channel may be voltage-dependent, since some of the response is blocked by nifedipine, whereas the other may be receptor-operated and still be capable of being opened in a preparation depolarized by high-potassium saline. The contribution of each channel to calcium influx appears to be dependent on the dose of proctolin used. At 0.5 nM proctolin, 4O-6O0h of the proctolininduced contraction is due to the voltage-dependent channel, whereas, at 5 nM proctolin, only 30% is due to this channel. The presence of a receptoroperated channel may explain earlier results obtained with locust oviducts . In this study, it was found that neurally evoked EJPs were not affected by proctolin and that proctolin resulted in only a minor depolarization of membrane potential. This depolarization, however, was dose-dependent, with 1.6 nM proctolin producing a 2.8 mV depolarization and 6 nM resulting in a 4.3 mV depolarization. As can be seen from the present work, these concentrations of proctolin result in a considerable sustained contraction. Clearly, small depolarizations may open voltage-dependent calcium channels [see 111 and contribute to the contraction induced by proctolin, with the remaining contraction produced by receptor-operated calcium channels. Similar results have been obtained in other preparations when the effects of proctolin on membrane potential have been examined. Thus proctolin has either no effect or only a minor effect on membrane potential and conductance of coxal depressor muscle of cockroach [l], tonic flexor muscle of crayfish abdomen , hyperneural muscle of cockroach [lq, and leg opener 294 lange, Orchard, and lam muscle of lobster [ll]. In the case of the hyperneural muscle of cockroach, Hertel and Penzlin  considered the depolarization and decrease in membrane conductance induced by proctolin to be due to a calcium-dependent reduction of the potassium outward current. In the present study, proctolin was found to have no effect on the content of cyclic AMP or cyclic GMP in the locust oviducts. The results with cyclic AMP are not surprising; we have previously shown that elevated levels of cyclic AMP produce a relaxation of this muscle . This is, however, still an interesting observation in that proctolin responses are potentiated by agents that elevate cyclic AMP levels in the myogenic bundle of locust extensor tibialis muscle  and Periplaneta hindgut . On the other hand, a recent report  found that proctolin did not elevate levels of cyclic nucleotides in the extensor tibialis muscle of locust but did stimulate metabolism of inositol phosphates. 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