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Interaction between octopamine and proctolin on the oviducts of Locusta migratoria
Journal of Insect Physiology 46 (2000) 809–816 www.elsevier.com/locate/jinsphys
Interaction between octopamine and proctolin on the oviducts of Locusta migratoria David A. Nykamp, Angela B. Lange
*
Department of Zoology, University of Toronto at Mississauga, 3359 Mississauga Rd., Mississauga, Ont., Canada, L5L 1C6 Received 29 June 1999; accepted 24 August 1999
Abstract The biogenic amine octopamine and the pentapeptide proctolin are two important neuroactive chemicals that control contraction of the oviducts of the African locust Locusta migratoria. The physiological responses and signal transduction pathways used by octopamine and proctolin have been well characterized in the locust oviducts and this therefore provides the opportunity to examine the interaction between these two pathways. Octopamine, via the intracellular messenger adenosine 3⬘,5⬘-cyclic monophosphate (cyclic AMP), inhibits contraction of the oviducts, while proctolin, via the phosphoinositol pathway, stimulates contraction. We have examined the physiological response of the oviducts to combinations of octopamine and proctolin and also looked at how combinations of these affect one of the main intracellular mediators of the octopamine response, namely cyclic AMP. It was found that application of octopamine to the oviducts led to a dose-dependent reduction in tonus of the muscle and also a decrease in the amplitude and frequency of spontaneous phasic contractions. Octopamine-induced relaxation was enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Octopamine was also able to inhibit proctolin-induced contractions of the oviducts in a dose-dependent manner. A 10⫺9 M proctolin-induced contraction was inhibited by 83% in the presence of 10⫺5 M octopamine, and was completely inhibited in the presence of 10⫺5 M octopamine plus 5×10⫺4 M IBMX. Octopamine led to a dose-dependent increase in cyclic AMP content as measured by radioimmunoassay. In the presence of 10⫺9 M proctolin, this octopamine-induced increase in cyclic AMP was reduced by as much as 60%. Proctolin also caused a dose-dependent decrease in the cyclic AMP elevation produced by 5×10⫺6 M octopamine. These results indicate that octopamine and proctolin can antagonize each other’s physiological response when added in combination, and that proctolin is able to modulate the response of the oviducts to octopamine by influencing cyclic AMP levels. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cyclic AMP; Insect; Locust; Second messenger; Signal transduction
1. Introduction The control over contraction of the oviducts of the African migratory locust Locusta migratoria has been well characterized physiologically. Two of the main neuromodulators that affect oviduct contractions are the biogenic amine octopamine and the pentapeptide proctolin. Both of these are released onto the oviducts from nerve terminals (Orchard and Lange, 1987), but may also have
* Corresponding author. Tel.: +1-905-828-3972; fax: +1-905-8283792. E-mail address: [email protected] (A.B. Lange).
a hormonal effect, after being released into the haemolymph. Octopamine works primarily to inhibit contractions of the oviduct muscle by decreasing the tonus and has been shown to inhibit neurally-evoked contractions of the oviducts (Orchard and Lange, 1985; Lange and Tsang, 1993). Proctolin on the other hand, is a strong stimulant of contraction of the oviducts, and increases the frequency and amplitude of spontaneous phasic contractions, and results in a sustained basal contraction (Lange et al., 1986, 1987; Lange, 1992). The mechanism of contraction of this insect visceral muscle, as well as the intracellular signal transduction pathways used by octopamine and proctolin, have been more clearly elucidated in recent years (see review by Lange and Nykamp, 1996).
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Octopamine is a biogenic amine that is found in two dorsal unpaired median (DUM) neurons of the VIIth abdominal ganglion which innervate the oviducts (Orchard and Lange, 1985). Application of octopamine to the oviducts, or electrical stimulation of the DUM neurons results in an inhibition of phasic contractions and a decrease in the tonus of the muscle (Orchard and Lange, 1985; Kalogianni and Pfluger, 1992). Octopamine binds to octopamine 2B type receptors on the membrane (Orchard and Lange, 1986) and activates a stimulatory guanine nucleotide-binding protein (G protein) in the membrane (Nykamp and Lange, 1998). The G protein in turn activates adenylate cyclase, resulting in an increase in intracellular adenosine 3⬘,5⬘-cyclic monophosphate (cyclic AMP). Cyclic AMP may then activate a number of potential proteins that result in relaxation of the muscle (Lange and Nykamp, 1996). Termination of the response may be anticipated to occur through detachment of octopamine from the receptor and hydrolysis of cyclic AMP by a phosphodiesterase in the oviduct muscle cells. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), potentiates the increase in cyclic AMP content in the cells and mimics some of the physiological effects of octopamine on the oviducts (Lange and Orchard, 1986). Proctolin, on the other hand, is an excitatory neuromodulator of the oviducts. Application of proctolin to the oviducts increases the frequency and amplitude of myogenic contractions and results in a sustained basal contraction of the oviducts at concentrations greater than 10⫺11 M (Lange et al., 1986, 1987). Proctolin appears to act via the phosphoinositol pathway, increasing the intracellular calcium concentration from both extracellular and intracellular calcium stores (Lange et al., 1987; Lange, 1988; Wilcox and Lange, 1995). It has also been shown that oviduct muscle contraction is a calmodulin-mediated event (Nykamp et al., 1994). Calcium binds to, and activates calmodulin, which can then activate calmodulindependent proteins. One of these proteins is probably a myosin light chain kinase which phosphorylates myosin light chains, allowing actin–myosin cross-bridge cycling and force generation in the muscle (Lange and Nykamp, 1996). It is likely that proctolin and octopamine act in conjunction with each other to modulate contraction of the oviducts in many of its important physiological functions. Such an interaction would be advantageous to the animal, allowing finer control over muscle contraction. In this paper the physiological responses of the oviducts to octopamine was more fully elucidated, and the response of the oviducts to combinations of octopamine and proctolin were investigated. In addition, we examined how octopamine and proctolin alter cyclic AMP content in the oviducts, and discuss some of the possible cross-talk mechanisms which may occur between these two pathways.
2. Materials and methods 2.1. Animals Locusta migratoria were reared at 30°C under crowded conditions using a 12 h light:12 h dark regime and fed on fresh wheat seedlings and bran. 2.2. Physiological studies The ovaries of 4–6 week old adult locusts, with oviducts attached, were dissected through a midventral incision of the abdomen under physiological saline (composition 150 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2 mM MgCl2, 4 mM NaHCO3, 5 mM HEPES pH 7.2, 90 mM sucrose, and 5 mM trehalose). The preparation was placed in a trough, containing 600 µl of saline, moulded into the base of a Sylgard-lined dissecting dish. The posterior end of the common oviduct was attached using fine thread to an AE875 miniature force transducer (Aksjeselskapet Mirko-Elektronikk, Oslo, Norway), and the upper part of the lateral oviducts was attached to the base of the trough using minuten pins. The preparation was mounted at an angle of 25° to the horizontal by its attachment to the force transducer and held under a tension of approximately 200 mg. The bioactive chemicals were applied by replacing 300 µl of the saline with 300 µl of saline containing twice the desired concentration of the drug. The response to the application of the drug was recorded and then the preparation was washed until the response returned to baseline. Octopamine was examined for its ability to inhibit spontaneous or proctolin-induced contractions. For proctolin-induced contractions, the inhibiting effects of octopamine were quantified by measuring the amplitude of the tonic contraction, when it had reached plateau level, and was expressed as a percentage relative to the amplitude of a tonic contraction produced by a standard 10⫺9 M proctolin dose. For basal tonus changes, the relaxation of basal tonus of the oviducts was seen as a drop in the baseline of the chart recorder trace after application of octopamine or octopamine in the presence of 5×10⫺4 M IBMX. This change in baseline was expressed as a percent change in basal tonus relative to the maximum change in basal tonus which was set as 100%. 2.3. Cyclic AMP determination Cyclic AMP content of the oviduct muscle of 8–12 day old adult virgin female locusts was examined. The innervated areas of the lateral oviducts which lie between the ovaries and the common oviduct were dissected under physiological saline. These lateral oviducts were incubated individually for varying times in 100 µl saline containing varying concentrations of octopamine,
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proctolin and IBMX. Experiments were terminated by the addition of 500 µl of boiling 0.05 M sodium acetate buffer (pH 6.2) to the tubes. The contents of the tubes were then boiled for a period of 5 min. The samples were sonicated for 15 s with a Branson Sonifier 250 (VWR, Toronto, Ontario) at setting 3. The homogenates were centrifuged for 15 min at 10,000g. Cyclic AMP levels were assayed in duplicate with a suitable aliquot of the supernatant using a modified commercially available radioimmunoassay kit (Dupont Canada, Mississauga, Ontario). The remaining pellets were dissolved in 50 µl of 0.5 M sodium hydroxide for 2 h at 50°C for subsequent protein determination by the method of Bradford (1976) using bovine gamma-globulin as standard. Values are expressed as pmol cyclic AMP per mg protein. 2.4. Chemicals Proctolin was obtained from Peninsula Laboratories (Belmont, CA, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
3. Results The physiological response of the locust oviducts to the biogenic amine octopamine and to octopamine in the presence of the phosphodiesterase inhibitor IBMX are shown in Fig. 1. Octopamine reduced the tonus of the muscle in a dose-dependent manner and also reduced the amplitude and frequency of the spontaneous phasic contractions of the oviducts (Fig. 1a). Octopamine had a threshold of 10⫺9 M and a maximal effect at 10⫺5 M. The relaxing effect of octopamine on the oviduct muscle was reversed upon washing with physiological saline. Results are presented in a dose–response curve (Fig. 1b), as the percentage change in basal tonus relative to the maximal effects of octopamine for each preparation (generally between 10⫺5 M and 10⫺4 M octopamine). When octopamine was added to the oviducts in the presence of the phosphodiesterase inhibitor IBMX at 5×10⫺4 M, the reduction in the tonus of the muscle was enhanced, thereby shifting the octopamine dose– response curve to the left. For example, at 10⫺8 M octopamine there was a reduction in tonus of the muscle of 27% compared with a 60% reduction in the presence of 5×10⫺4 M IBMX. Generally, maximum relaxation occurred at 10⫺5 M octopamine, while 10⫺4 M octopamine was less effective in relaxing the oviduct muscle (especially in the presence of IBMX). Incubation of the oviducts in 5×10⫺4 M IBMX alone had no effect on the tonus of the muscle. The effects of octopamine on proctolin-induced contractions of the oviducts are shown in Fig. 2. Addition of proctolin to the oviducts at 10⫺9 M resulted in a sustained increase in tonus as well as an increase in the
Fig. 1. The physiological effect of octopamine on the basal tonus of the oviducts of Locusta migratoria. (a) Octopamine (OA) was applied to the oviducts at varying concentrations (10⫺9 to 10⫺5 M) at the upward triangles. Oviduct contractions were monitored by a force transducer and recorded on a chart recorder. (b) Dose–response curve of the effects of octopamine (쐌) and octopamine+5×10⫺4 M IBMX (왖) on the basal tonus of the oviducts of Locusta migratoria. Percent change in basal tonus is measured relative to the maximum change in basal tonus. Bars indicate standard error of the mean of 10–12 preparations.
frequency and amplitude of phasic contractions of the muscle, which were reversible upon washing with physiological saline (Fig. 2a). When octopamine was applied to the oviducts concurrently with 10⫺9 M proctolin, there was a dose-dependent decrease in the amplitude of the proctolin-induced contractions. The proctolin-induced contractions were also delayed in onset in a dose-dependent manner. For example, when 10⫺5 M octopamine was added to 10⫺9 M proctolin, there was a 1-min delay before any increase in tonus of the muscle was seen (Fig. 2a). Also, with concentrations of octopamine greater than 10⫺7 M, there was an initial drop in tonus of the muscle, as well as a decrease in the amplitude of phasic contractions. A dose–response relationship for the effect of octopamine on the amplitude of 10⫺9 M proctolin-induced contractions is shown in Fig. 2b. Octopamine had a threshold of about 10⫺8 M and inhibited the proctolin-induced contraction by 83% at 10⫺5 M (Fig. 2b). When IBMX (5×10⫺4 M) was added concurrent with octopamine, the amplitude of the 10⫺9
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Fig. 2. The effect of octopamine on proctolin-induced contractions of the oviducts of Locusta migratoria. (a) 10⫺9 M proctolin (PROC) was applied to the oviducts alone and in the presence of varying concentrations of octopamine (OA). Upward solid triangles indicate application and downward open triangles indicate washing with physiological saline. (b) Dose–response curve showing the effects of octopamine (쐌) and octopamine+5×10⫺4 M IBMX (왖) on 10⫺9 M proctolininduced contractions of the oviducts. Symbols represent percentage of maximum contraction relative to a standard 10⫺9 M proctolin-induced contraction, and bars indicate standard error of the mean of 10–12 preparations.
M proctolin-induced contractions were reduced further (Fig. 2b). At the lower octopamine concentrations (10⫺11 to 10⫺8 M), the contractions were reduced by 25–30% compared with those with octopamine alone. With higher concentrations of octopamine (10⫺7 to 10⫺6 M), there was a shift of the curve to the left and the proctolininduced contractions were eventually completely inhibited in the presence of 5×10⫺6 M octopamine. Although IBMX, when added alone, had no effect on the tonus of the muscle, it did partially inhibit proctolininduced contractions of the oviducts. When 5×10⫺4 M IBMX was added to the oviducts in the presence of proctolin, there was a 19% decrease in the amplitude of the proctolin-induced contractions (Fig. 3). When the oviducts were pre-incubated for 15 min in 5×10⫺4 M IBMX prior to the addition of proctolin, the proctolin-
Fig. 3. The effect of IBMX on proctolin-induced contractions of the oviducts of Locusta migratoria. Bars indicate percentage of the maximum contraction relative to a 10⫺9 M proctolin-induced contraction. Standard error bars of the mean of eight preparations are shown. Solid bar indicates that the preparation was preincubated in 5×10⫺4 M IBMX for 15 min prior to the addition of 10⫺9 M proctolin in the presence of 5×10⫺4 M IBMX. The proctolin response returned to normal following washing of the oviducts with physiological saline.
induced contractions were reduced further. The effects of IBMX on the proctolin-induced contractions were fully reversible upon washing the preparation with physiological saline (Fig. 3). Following physiological characterization of the oviducts to combinations of octopamine and proctolin, measurements of the intracellular messenger cyclic AMP were carried out in the presence of octopamine, proctolin and IBMX. Cyclic AMP content of the oviduct muscle was determined by radioimmunoassay and was calculated as pmol cyclic AMP per mg protein. Initially, the effects of IBMX by itself on cyclic AMP levels of the oviducts were determined. Incubation of the oviducts in 5×10⫺4 M IBMX for periods ranging from 1 to 120 min resulted in a time-dependent increase in cyclic AMP levels in the oviducts (Fig. 4a). After a 2 h incubation, there was an eight-fold increase in cyclic AMP levels from 9.3 pmol/mg protein at 0 min (control) to 74 pmol/mg protein at 120 min (Fig. 4a). Incubation of the oviducts in various concentrations of IBMX for 10 min also resulted in a dose-dependent increase in cyclic AMP levels above 10⫺5 M IBMX (Fig. 4b). Incubation of the oviducts in octopamine and 5×10⫺4
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Fig. 5. Dose–response curve of the effect of octopamine and octopamine in the presence of 10⫺9 M proctolin on cyclic AMP content in the oviducts of Locusta migratoria. Oviducts were incubated in various concentrations of octopamine (䊐) and octopamine+10⫺9 M proctolin (쐌) in the presence of 5×10⫺4 M IBMX for 5 min. Cyclic AMP levels were determined by radioimmunoassay and are reported as pmol cyclic AMP per mg protein. Bars indicate standard error of the mean of five preparations.
Fig. 4. The effect of IBMX on cyclic AMP levels in the oviducts of Locusta migratoria. (a) The effect of time of incubation of the oviducts in 5×10⫺4 M IBMX on cyclic AMP content. Lateral oviducts were dissected under physiological saline and incubated for various periods of time from 1 min to 2 h in 5×10⫺4 M IBMX. Reactions were stopped and the cyclic AMP content of the oviducts determined by radioimmunoassay as described in Section 2. (b) Dose–response curve showing the effect of IBMX on cyclic AMP levels of the locust oviducts. Oviducts were incubated for 10 min in various concentrations of IBMX. Bars indicate standard error of the mean of five preparations.
M IBMX resulted in a large dose-dependent increase in cyclic AMP in the oviducts (Fig. 5). The response plateaued at 10⫺5 M octopamine where there was a greater than 20-fold increase in cyclic AMP levels over control levels. When 10⫺9 M proctolin was added with octopamine, the curve was shifted to the right at concentrations of octopamine greater than 10⫺7 M and thus resulted in a lesser increase in the cyclic AMP compared with
octopamine alone. The greatest effect was at 10⫺5 M octopamine, which alone elevated cyclic AMP levels to 487 pmol/mg protein but was reduced to 190 pmol/mg in the presence of 10⫺9 M proctolin (Fig. 5). Proctolin was less effective in reducing cyclic AMP at an octopamine concentration of 10⫺4 M. By varying the concentration of proctolin at a single concentration of octopamine, it was also determined that proctolin dose-dependently decreased the amount of cyclic AMP produced by octopamine in the oviducts. The effects of varying concentrations of proctolin by itself and proctolin in the presence of 5×10⫺6 M octopamine (and 5×10⫺4 M IBMX) on cyclic AMP levels are shown in Fig. 6. Proctolin itself had little effect on cyclic AMP levels at 10⫺11 M and 10⫺10 M. However, there was an increase in cyclic AMP levels from 14 pmol/mg protein in the control to 37 pmol/mg protein with 10⫺8 M proctolin. When the alpha-adrenergic receptor antagonist, phentolamine (10⫺5 M), was added in the presence of 10⫺8 M proctolin, this increase in cyclic AMP was not seen (Fig. 6, inset). When varying concentrations of proctolin were added to 5×10⫺6 M octopamine there was a dose-dependent lowering in the levels of cyclic AMP from 627 pmol/mg protein with 10⫺11 M proctolin to 315 pmol/mg with 10⫺8 M proctolin (Fig. 6). Thus, at proctolin concentrations of 10⫺9 to 10⫺8 M, there was a 50% decrease in the amount of cyclic AMP produced with 5×10⫺6 M octopamine alone.
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Fig. 6. Dose–response curve of the effect of proctolin and proctolin in the presence of 5×10⫺6 M octopamine on cyclic AMP content in the oviducts of Locusta migratoria. Oviducts were incubated in various concentrations of proctolin (䊐) and proctolin+5×10⫺6 M octopamine (쐌) in the presence of 5×10⫺4 M IBMX for 5 min. Cyclic AMP levels were determined by radioimmunoassay and are reported as pmol cyclic AMP per mg protein. Bars indicate standard error of the mean of five assays. Inset: effect of 10⫺8 M proctolin (Proc) and proctolin in the presence of the 10⫺5 M phentolamine (Phen) on cyclic AMP levels in the oviducts of Locusta migratoria. Bars indicate standard error of the mean of 10 preparations.
4. Discussion Octopamine is believed to bind to octopamine 2B receptors on locust oviduct muscle cell membranes and to initiate a signal transduction cascade leading to an increase in cyclic AMP in the oviducts (see Lange and Nykamp, 1996). Application of octopamine reduces the tonus of oviduct muscles, reduces the frequency and amplitude of spontaneous phasic contractions and reduces neurally-evoked contractions (Orchard and Lange, 1985; Lange and Tsang, 1993). A termination of the physiological response to octopamine would be expected to occur through activity of a phosphodiesterase to hydrolyse the intracellular cyclic AMP. Evidence for this was obtained with the use of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), whereby IBMX was capable of enhancing the relaxation induced by octopamine on the oviducts, shifting the dose–response curve to the left (Fig. 1). At the same time, IBMX amplified the octopamine-induced increase in intracellular cyclic AMP greater than 20-fold over control levels, compared with a three-fold increase in the absence of IBMX (Fig. 5) (Lange and Orchard, 1986). This increase in cyclic AMP accumulation was paralleled by a decrease in tonus of the muscle of about 30% compared with the effects of octopamine alone (Fig. 1).
At higher concentrations of octopamine this enhancement of the relaxation in the presence of IBMX was no longer seen, implying that a maximal physiological relaxation of the muscle had been reached (Fig. 1). In fact, 10⫺4 M octopamine was slightly less effective than 10⫺5 M octopamine in reducing the tonus of the muscle, paralleling the peak of cyclic AMP production at 10⫺5 M octopamine (Fig. 5). IBMX, alone, had no effect on the basal tonus of the muscle. This may be expected as IBMX only resulted in a relatively small increase in cyclic AMP when applied to the oviducts alone (Fig. 4). With regard to induced contractions of the oviducts, proctolin produced an increase in phasic contractions and a sustained increase in basal tonus of the muscle. Application of octopamine in the presence of proctolin resulted in a dose-dependent decrease in the amplitude of the proctolin-induced contractions (Fig. 2). The threshold of the octopamine effect on proctolin occurred at a concentration of 10⫺8 M (Fig. 2b). This is reasonable since concentrations of octopamine less than 10⫺8 M produce very little cyclic AMP elevation in the oviducts (Lange and Orchard, 1986). When 5×10⫺4 M IBMX was added concurrent with octopamine, it enhanced the inhibition of the proctolin-induced contraction, and, at an octopamine concentration of 5×10⫺6 M, the 10⫺9 M proctolin-induced contraction was completely abolished. Although application of IBMX alone had no effect on basal tonus of the oviducts, it was able to reduce 10⫺9 M proctolin-induced contractions by 20% (Fig. 3). Similarly, IBMX has been shown to be able to inhibit neurally-evoked contractions of the oviducts (Lange and Orchard, 1986). This indicates that at least part of the 25–30% inhibition of the proctolin-induced contraction seen in Fig. 2b is due to IBMX, while the further inhibition, even at the lower concentrations of octopamine (10⫺11 to 10⫺9 M), indicates a synergistic effect of octopamine and IBMX in reducing the proctolin-induced contractions. In the presence of IBMX, one would expect that the breakdown of cyclic AMP would be inhibited, and there would be a buildup of cyclic AMP over time in the muscle cells. Measurement of cyclic AMP levels in the presence of IBMX over a period of 2 h indicated this to be the case (Fig. 4). Also, it was seen that when the oviducts were pre-incubated in 5×10⫺4 M IBMX for a period of 15 min, the 10⫺9 M proctolin-induced contraction was reduced by a further 15% (Fig. 3). In order for this buildup of cyclic AMP to occur, it would seem that there must be some adenylate cyclase activity. It has been hypothesised that this could be the result of a basal level of spontaneously released octopamine onto the oviducts that would result in G protein activation and adenylate cyclase stimulation (Nykamp and Lange, 1998). This is supported by data that show that in the presence of the alpha-adrenergic receptor antagonist phentoalmine, which is known to block the octopamine receptor, IBMX
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does not alter cyclic AMP levels in the oviducts (Nykamp and Lange, 1998). Although it appears that a simple additive effect of the physiological results of octopamine and proctolin could be occurring, it would seem likely that a more complex interaction of the signal transduction pathways would be more efficient and allow for a greater flexibility of physiological responses. For example, it is probable that octopamine and proctolin may be working either hormonally and/or neurally, being released from octopaminergic and proctolinergic neurons to modulate contractions of the oviducts during such varied physiological events as ovulation, egg retention, egg laying and haemolymph circulation (Lange and Nykamp, 1996). A possible mechanism of action for reduction of proctolin-induced contractions by octopamine or IBMXinduced increases in cyclic AMP could be through an alteration of the proctolin signal transduction pathway. Proctolin increases intracellular calcium (Lange et al., 1987; Lange, 1988; Wilcox and Lange, 1995), and it is thought that this leads to activation of myosin light chain kinase, resulting in myosin light chain phosphorylation and thus contraction of the oviducts (Nykamp et al., 1994; Lange and Nykamp, 1996). There is some evidence that this phosphorylation may be reduced in the presence of octopamine. Phosphorylation studies, using homogenates of oviduct muscle, have shown a decrease in phosphorylation of a myosin light chain sized band when octopamine is added to proctolin stimulated tissue (Nykamp, unpublished observations). A possible mechanism for this type of action could be through a cyclic AMP-dependent protein kinase (PKA). In turkey gizzard for example, PKA has been reported to phosphorylate myosin light chain kinase, which results in a decrease in myosin light chain kinase activity and contraction (Conti and Adelstein, 1981). We further investigated the effects of proctolin on the main second messenger involved in the octopamine response, namely cyclic AMP. Proctolin was found to have the ability to reduce cyclic AMP levels produced by octopamine in the presence of IBMX. Proctolin was most effective at 10⫺6 to 10⫺5 M octopamine, reducing cyclic AMP levels by up to 60% (Fig. 5). This indicates that proctolin is altering the ability of octopamine to generate cyclic AMP and thus is likely affecting the octopamine signal transduction pathway. This type of interaction could be occurring through G-protein subunit interaction between the two pathways. For example, there is mounting evidence in tracheal smooth muscle that Gi can inhibit adenylate cyclase activity directly, as well as attenuate the activity of Gs which is responsible for activating adenylate cyclase (Pyne and Pyne, 1993). Further study is necessary to ascertain whether a mechanism such as this is occurring in the oviducts. If the pathways are interacting in a manner such as this, then the response should be dose dependent. We saw that the
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ability of proctolin to reduce cyclic AMP was greatest at concentrations of 10⫺9 M and above, while being less effective at lower concentrations (Fig. 6). Also, it seems that with higher concentrations of octopamine (10⫺4 M), proctolin is less effective in reducing cyclic AMP. It was also noted that proctolin, when added to the oviducts, increased cyclic AMP levels (Fig. 6). Since this increase was blocked in the presence of the alpha-adrenergic receptor antagonist phentolamine (Fig. 6, inset), it was likely not due to proctolin itself but was probably due to octopamine being released onto the oviducts from DUM neuron endings in response to the proctolin-induced contractions. It seems that cross-talk regulation between these two pathways has important functions in controlling the fine tuning of force development in the locust oviducts. Thus, as well as altering the release of neurotransmitters and neurohormones, there appears to be important postreceptor interactions that are integrated to allow for flexibility of contraction during physiological functions of the oviducts.
Acknowledgements This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.
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Orchard, I., Lange, A.B., 1986. Pharmacological profile of octopamine receptors on the lateral oviducts of the locust, Locusta migratoria. Journal of Insect Physiology 32 (8), 741–745. Orchard, I., Lange, A.B., 1985. Evidence for octopaminergic modulation of an insect visceral muscle. Journal of Neurobiology 16 (3), 171–181. Pyne, N.J., Pyne, S., 1993. Cellular signal pathways in tracheal smooth muscle. Cellular Signalling 5 (4), 401–409. Wilcox, C.L., Lange, A.B., 1995. Role of extracellular and intracellular calcium on proctolin-induced contractions in an insect visceral muscle. Regulatory Peptides 56, 49–59.
Interaction between octopamine and proctolin on the oviducts of Locusta migratoria David A. Nykamp, Angela B. Lange
*
Department of Zoology, University of Toronto at Mississauga, 3359 Mississauga Rd., Mississauga, Ont., Canada, L5L 1C6 Received 29 June 1999; accepted 24 August 1999
Abstract The biogenic amine octopamine and the pentapeptide proctolin are two important neuroactive chemicals that control contraction of the oviducts of the African locust Locusta migratoria. The physiological responses and signal transduction pathways used by octopamine and proctolin have been well characterized in the locust oviducts and this therefore provides the opportunity to examine the interaction between these two pathways. Octopamine, via the intracellular messenger adenosine 3⬘,5⬘-cyclic monophosphate (cyclic AMP), inhibits contraction of the oviducts, while proctolin, via the phosphoinositol pathway, stimulates contraction. We have examined the physiological response of the oviducts to combinations of octopamine and proctolin and also looked at how combinations of these affect one of the main intracellular mediators of the octopamine response, namely cyclic AMP. It was found that application of octopamine to the oviducts led to a dose-dependent reduction in tonus of the muscle and also a decrease in the amplitude and frequency of spontaneous phasic contractions. Octopamine-induced relaxation was enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Octopamine was also able to inhibit proctolin-induced contractions of the oviducts in a dose-dependent manner. A 10⫺9 M proctolin-induced contraction was inhibited by 83% in the presence of 10⫺5 M octopamine, and was completely inhibited in the presence of 10⫺5 M octopamine plus 5×10⫺4 M IBMX. Octopamine led to a dose-dependent increase in cyclic AMP content as measured by radioimmunoassay. In the presence of 10⫺9 M proctolin, this octopamine-induced increase in cyclic AMP was reduced by as much as 60%. Proctolin also caused a dose-dependent decrease in the cyclic AMP elevation produced by 5×10⫺6 M octopamine. These results indicate that octopamine and proctolin can antagonize each other’s physiological response when added in combination, and that proctolin is able to modulate the response of the oviducts to octopamine by influencing cyclic AMP levels. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cyclic AMP; Insect; Locust; Second messenger; Signal transduction
1. Introduction The control over contraction of the oviducts of the African migratory locust Locusta migratoria has been well characterized physiologically. Two of the main neuromodulators that affect oviduct contractions are the biogenic amine octopamine and the pentapeptide proctolin. Both of these are released onto the oviducts from nerve terminals (Orchard and Lange, 1987), but may also have
* Corresponding author. Tel.: +1-905-828-3972; fax: +1-905-8283792. E-mail address: [email protected] (A.B. Lange).
a hormonal effect, after being released into the haemolymph. Octopamine works primarily to inhibit contractions of the oviduct muscle by decreasing the tonus and has been shown to inhibit neurally-evoked contractions of the oviducts (Orchard and Lange, 1985; Lange and Tsang, 1993). Proctolin on the other hand, is a strong stimulant of contraction of the oviducts, and increases the frequency and amplitude of spontaneous phasic contractions, and results in a sustained basal contraction (Lange et al., 1986, 1987; Lange, 1992). The mechanism of contraction of this insect visceral muscle, as well as the intracellular signal transduction pathways used by octopamine and proctolin, have been more clearly elucidated in recent years (see review by Lange and Nykamp, 1996).
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Octopamine is a biogenic amine that is found in two dorsal unpaired median (DUM) neurons of the VIIth abdominal ganglion which innervate the oviducts (Orchard and Lange, 1985). Application of octopamine to the oviducts, or electrical stimulation of the DUM neurons results in an inhibition of phasic contractions and a decrease in the tonus of the muscle (Orchard and Lange, 1985; Kalogianni and Pfluger, 1992). Octopamine binds to octopamine 2B type receptors on the membrane (Orchard and Lange, 1986) and activates a stimulatory guanine nucleotide-binding protein (G protein) in the membrane (Nykamp and Lange, 1998). The G protein in turn activates adenylate cyclase, resulting in an increase in intracellular adenosine 3⬘,5⬘-cyclic monophosphate (cyclic AMP). Cyclic AMP may then activate a number of potential proteins that result in relaxation of the muscle (Lange and Nykamp, 1996). Termination of the response may be anticipated to occur through detachment of octopamine from the receptor and hydrolysis of cyclic AMP by a phosphodiesterase in the oviduct muscle cells. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), potentiates the increase in cyclic AMP content in the cells and mimics some of the physiological effects of octopamine on the oviducts (Lange and Orchard, 1986). Proctolin, on the other hand, is an excitatory neuromodulator of the oviducts. Application of proctolin to the oviducts increases the frequency and amplitude of myogenic contractions and results in a sustained basal contraction of the oviducts at concentrations greater than 10⫺11 M (Lange et al., 1986, 1987). Proctolin appears to act via the phosphoinositol pathway, increasing the intracellular calcium concentration from both extracellular and intracellular calcium stores (Lange et al., 1987; Lange, 1988; Wilcox and Lange, 1995). It has also been shown that oviduct muscle contraction is a calmodulin-mediated event (Nykamp et al., 1994). Calcium binds to, and activates calmodulin, which can then activate calmodulindependent proteins. One of these proteins is probably a myosin light chain kinase which phosphorylates myosin light chains, allowing actin–myosin cross-bridge cycling and force generation in the muscle (Lange and Nykamp, 1996). It is likely that proctolin and octopamine act in conjunction with each other to modulate contraction of the oviducts in many of its important physiological functions. Such an interaction would be advantageous to the animal, allowing finer control over muscle contraction. In this paper the physiological responses of the oviducts to octopamine was more fully elucidated, and the response of the oviducts to combinations of octopamine and proctolin were investigated. In addition, we examined how octopamine and proctolin alter cyclic AMP content in the oviducts, and discuss some of the possible cross-talk mechanisms which may occur between these two pathways.
2. Materials and methods 2.1. Animals Locusta migratoria were reared at 30°C under crowded conditions using a 12 h light:12 h dark regime and fed on fresh wheat seedlings and bran. 2.2. Physiological studies The ovaries of 4–6 week old adult locusts, with oviducts attached, were dissected through a midventral incision of the abdomen under physiological saline (composition 150 mM NaCl, 10 mM KCl, 4 mM CaCl2, 2 mM MgCl2, 4 mM NaHCO3, 5 mM HEPES pH 7.2, 90 mM sucrose, and 5 mM trehalose). The preparation was placed in a trough, containing 600 µl of saline, moulded into the base of a Sylgard-lined dissecting dish. The posterior end of the common oviduct was attached using fine thread to an AE875 miniature force transducer (Aksjeselskapet Mirko-Elektronikk, Oslo, Norway), and the upper part of the lateral oviducts was attached to the base of the trough using minuten pins. The preparation was mounted at an angle of 25° to the horizontal by its attachment to the force transducer and held under a tension of approximately 200 mg. The bioactive chemicals were applied by replacing 300 µl of the saline with 300 µl of saline containing twice the desired concentration of the drug. The response to the application of the drug was recorded and then the preparation was washed until the response returned to baseline. Octopamine was examined for its ability to inhibit spontaneous or proctolin-induced contractions. For proctolin-induced contractions, the inhibiting effects of octopamine were quantified by measuring the amplitude of the tonic contraction, when it had reached plateau level, and was expressed as a percentage relative to the amplitude of a tonic contraction produced by a standard 10⫺9 M proctolin dose. For basal tonus changes, the relaxation of basal tonus of the oviducts was seen as a drop in the baseline of the chart recorder trace after application of octopamine or octopamine in the presence of 5×10⫺4 M IBMX. This change in baseline was expressed as a percent change in basal tonus relative to the maximum change in basal tonus which was set as 100%. 2.3. Cyclic AMP determination Cyclic AMP content of the oviduct muscle of 8–12 day old adult virgin female locusts was examined. The innervated areas of the lateral oviducts which lie between the ovaries and the common oviduct were dissected under physiological saline. These lateral oviducts were incubated individually for varying times in 100 µl saline containing varying concentrations of octopamine,
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proctolin and IBMX. Experiments were terminated by the addition of 500 µl of boiling 0.05 M sodium acetate buffer (pH 6.2) to the tubes. The contents of the tubes were then boiled for a period of 5 min. The samples were sonicated for 15 s with a Branson Sonifier 250 (VWR, Toronto, Ontario) at setting 3. The homogenates were centrifuged for 15 min at 10,000g. Cyclic AMP levels were assayed in duplicate with a suitable aliquot of the supernatant using a modified commercially available radioimmunoassay kit (Dupont Canada, Mississauga, Ontario). The remaining pellets were dissolved in 50 µl of 0.5 M sodium hydroxide for 2 h at 50°C for subsequent protein determination by the method of Bradford (1976) using bovine gamma-globulin as standard. Values are expressed as pmol cyclic AMP per mg protein. 2.4. Chemicals Proctolin was obtained from Peninsula Laboratories (Belmont, CA, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
3. Results The physiological response of the locust oviducts to the biogenic amine octopamine and to octopamine in the presence of the phosphodiesterase inhibitor IBMX are shown in Fig. 1. Octopamine reduced the tonus of the muscle in a dose-dependent manner and also reduced the amplitude and frequency of the spontaneous phasic contractions of the oviducts (Fig. 1a). Octopamine had a threshold of 10⫺9 M and a maximal effect at 10⫺5 M. The relaxing effect of octopamine on the oviduct muscle was reversed upon washing with physiological saline. Results are presented in a dose–response curve (Fig. 1b), as the percentage change in basal tonus relative to the maximal effects of octopamine for each preparation (generally between 10⫺5 M and 10⫺4 M octopamine). When octopamine was added to the oviducts in the presence of the phosphodiesterase inhibitor IBMX at 5×10⫺4 M, the reduction in the tonus of the muscle was enhanced, thereby shifting the octopamine dose– response curve to the left. For example, at 10⫺8 M octopamine there was a reduction in tonus of the muscle of 27% compared with a 60% reduction in the presence of 5×10⫺4 M IBMX. Generally, maximum relaxation occurred at 10⫺5 M octopamine, while 10⫺4 M octopamine was less effective in relaxing the oviduct muscle (especially in the presence of IBMX). Incubation of the oviducts in 5×10⫺4 M IBMX alone had no effect on the tonus of the muscle. The effects of octopamine on proctolin-induced contractions of the oviducts are shown in Fig. 2. Addition of proctolin to the oviducts at 10⫺9 M resulted in a sustained increase in tonus as well as an increase in the
Fig. 1. The physiological effect of octopamine on the basal tonus of the oviducts of Locusta migratoria. (a) Octopamine (OA) was applied to the oviducts at varying concentrations (10⫺9 to 10⫺5 M) at the upward triangles. Oviduct contractions were monitored by a force transducer and recorded on a chart recorder. (b) Dose–response curve of the effects of octopamine (쐌) and octopamine+5×10⫺4 M IBMX (왖) on the basal tonus of the oviducts of Locusta migratoria. Percent change in basal tonus is measured relative to the maximum change in basal tonus. Bars indicate standard error of the mean of 10–12 preparations.
frequency and amplitude of phasic contractions of the muscle, which were reversible upon washing with physiological saline (Fig. 2a). When octopamine was applied to the oviducts concurrently with 10⫺9 M proctolin, there was a dose-dependent decrease in the amplitude of the proctolin-induced contractions. The proctolin-induced contractions were also delayed in onset in a dose-dependent manner. For example, when 10⫺5 M octopamine was added to 10⫺9 M proctolin, there was a 1-min delay before any increase in tonus of the muscle was seen (Fig. 2a). Also, with concentrations of octopamine greater than 10⫺7 M, there was an initial drop in tonus of the muscle, as well as a decrease in the amplitude of phasic contractions. A dose–response relationship for the effect of octopamine on the amplitude of 10⫺9 M proctolin-induced contractions is shown in Fig. 2b. Octopamine had a threshold of about 10⫺8 M and inhibited the proctolin-induced contraction by 83% at 10⫺5 M (Fig. 2b). When IBMX (5×10⫺4 M) was added concurrent with octopamine, the amplitude of the 10⫺9
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Fig. 2. The effect of octopamine on proctolin-induced contractions of the oviducts of Locusta migratoria. (a) 10⫺9 M proctolin (PROC) was applied to the oviducts alone and in the presence of varying concentrations of octopamine (OA). Upward solid triangles indicate application and downward open triangles indicate washing with physiological saline. (b) Dose–response curve showing the effects of octopamine (쐌) and octopamine+5×10⫺4 M IBMX (왖) on 10⫺9 M proctolininduced contractions of the oviducts. Symbols represent percentage of maximum contraction relative to a standard 10⫺9 M proctolin-induced contraction, and bars indicate standard error of the mean of 10–12 preparations.
M proctolin-induced contractions were reduced further (Fig. 2b). At the lower octopamine concentrations (10⫺11 to 10⫺8 M), the contractions were reduced by 25–30% compared with those with octopamine alone. With higher concentrations of octopamine (10⫺7 to 10⫺6 M), there was a shift of the curve to the left and the proctolininduced contractions were eventually completely inhibited in the presence of 5×10⫺6 M octopamine. Although IBMX, when added alone, had no effect on the tonus of the muscle, it did partially inhibit proctolininduced contractions of the oviducts. When 5×10⫺4 M IBMX was added to the oviducts in the presence of proctolin, there was a 19% decrease in the amplitude of the proctolin-induced contractions (Fig. 3). When the oviducts were pre-incubated for 15 min in 5×10⫺4 M IBMX prior to the addition of proctolin, the proctolin-
Fig. 3. The effect of IBMX on proctolin-induced contractions of the oviducts of Locusta migratoria. Bars indicate percentage of the maximum contraction relative to a 10⫺9 M proctolin-induced contraction. Standard error bars of the mean of eight preparations are shown. Solid bar indicates that the preparation was preincubated in 5×10⫺4 M IBMX for 15 min prior to the addition of 10⫺9 M proctolin in the presence of 5×10⫺4 M IBMX. The proctolin response returned to normal following washing of the oviducts with physiological saline.
induced contractions were reduced further. The effects of IBMX on the proctolin-induced contractions were fully reversible upon washing the preparation with physiological saline (Fig. 3). Following physiological characterization of the oviducts to combinations of octopamine and proctolin, measurements of the intracellular messenger cyclic AMP were carried out in the presence of octopamine, proctolin and IBMX. Cyclic AMP content of the oviduct muscle was determined by radioimmunoassay and was calculated as pmol cyclic AMP per mg protein. Initially, the effects of IBMX by itself on cyclic AMP levels of the oviducts were determined. Incubation of the oviducts in 5×10⫺4 M IBMX for periods ranging from 1 to 120 min resulted in a time-dependent increase in cyclic AMP levels in the oviducts (Fig. 4a). After a 2 h incubation, there was an eight-fold increase in cyclic AMP levels from 9.3 pmol/mg protein at 0 min (control) to 74 pmol/mg protein at 120 min (Fig. 4a). Incubation of the oviducts in various concentrations of IBMX for 10 min also resulted in a dose-dependent increase in cyclic AMP levels above 10⫺5 M IBMX (Fig. 4b). Incubation of the oviducts in octopamine and 5×10⫺4
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Fig. 5. Dose–response curve of the effect of octopamine and octopamine in the presence of 10⫺9 M proctolin on cyclic AMP content in the oviducts of Locusta migratoria. Oviducts were incubated in various concentrations of octopamine (䊐) and octopamine+10⫺9 M proctolin (쐌) in the presence of 5×10⫺4 M IBMX for 5 min. Cyclic AMP levels were determined by radioimmunoassay and are reported as pmol cyclic AMP per mg protein. Bars indicate standard error of the mean of five preparations.
Fig. 4. The effect of IBMX on cyclic AMP levels in the oviducts of Locusta migratoria. (a) The effect of time of incubation of the oviducts in 5×10⫺4 M IBMX on cyclic AMP content. Lateral oviducts were dissected under physiological saline and incubated for various periods of time from 1 min to 2 h in 5×10⫺4 M IBMX. Reactions were stopped and the cyclic AMP content of the oviducts determined by radioimmunoassay as described in Section 2. (b) Dose–response curve showing the effect of IBMX on cyclic AMP levels of the locust oviducts. Oviducts were incubated for 10 min in various concentrations of IBMX. Bars indicate standard error of the mean of five preparations.
M IBMX resulted in a large dose-dependent increase in cyclic AMP in the oviducts (Fig. 5). The response plateaued at 10⫺5 M octopamine where there was a greater than 20-fold increase in cyclic AMP levels over control levels. When 10⫺9 M proctolin was added with octopamine, the curve was shifted to the right at concentrations of octopamine greater than 10⫺7 M and thus resulted in a lesser increase in the cyclic AMP compared with
octopamine alone. The greatest effect was at 10⫺5 M octopamine, which alone elevated cyclic AMP levels to 487 pmol/mg protein but was reduced to 190 pmol/mg in the presence of 10⫺9 M proctolin (Fig. 5). Proctolin was less effective in reducing cyclic AMP at an octopamine concentration of 10⫺4 M. By varying the concentration of proctolin at a single concentration of octopamine, it was also determined that proctolin dose-dependently decreased the amount of cyclic AMP produced by octopamine in the oviducts. The effects of varying concentrations of proctolin by itself and proctolin in the presence of 5×10⫺6 M octopamine (and 5×10⫺4 M IBMX) on cyclic AMP levels are shown in Fig. 6. Proctolin itself had little effect on cyclic AMP levels at 10⫺11 M and 10⫺10 M. However, there was an increase in cyclic AMP levels from 14 pmol/mg protein in the control to 37 pmol/mg protein with 10⫺8 M proctolin. When the alpha-adrenergic receptor antagonist, phentolamine (10⫺5 M), was added in the presence of 10⫺8 M proctolin, this increase in cyclic AMP was not seen (Fig. 6, inset). When varying concentrations of proctolin were added to 5×10⫺6 M octopamine there was a dose-dependent lowering in the levels of cyclic AMP from 627 pmol/mg protein with 10⫺11 M proctolin to 315 pmol/mg with 10⫺8 M proctolin (Fig. 6). Thus, at proctolin concentrations of 10⫺9 to 10⫺8 M, there was a 50% decrease in the amount of cyclic AMP produced with 5×10⫺6 M octopamine alone.
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Fig. 6. Dose–response curve of the effect of proctolin and proctolin in the presence of 5×10⫺6 M octopamine on cyclic AMP content in the oviducts of Locusta migratoria. Oviducts were incubated in various concentrations of proctolin (䊐) and proctolin+5×10⫺6 M octopamine (쐌) in the presence of 5×10⫺4 M IBMX for 5 min. Cyclic AMP levels were determined by radioimmunoassay and are reported as pmol cyclic AMP per mg protein. Bars indicate standard error of the mean of five assays. Inset: effect of 10⫺8 M proctolin (Proc) and proctolin in the presence of the 10⫺5 M phentolamine (Phen) on cyclic AMP levels in the oviducts of Locusta migratoria. Bars indicate standard error of the mean of 10 preparations.
4. Discussion Octopamine is believed to bind to octopamine 2B receptors on locust oviduct muscle cell membranes and to initiate a signal transduction cascade leading to an increase in cyclic AMP in the oviducts (see Lange and Nykamp, 1996). Application of octopamine reduces the tonus of oviduct muscles, reduces the frequency and amplitude of spontaneous phasic contractions and reduces neurally-evoked contractions (Orchard and Lange, 1985; Lange and Tsang, 1993). A termination of the physiological response to octopamine would be expected to occur through activity of a phosphodiesterase to hydrolyse the intracellular cyclic AMP. Evidence for this was obtained with the use of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), whereby IBMX was capable of enhancing the relaxation induced by octopamine on the oviducts, shifting the dose–response curve to the left (Fig. 1). At the same time, IBMX amplified the octopamine-induced increase in intracellular cyclic AMP greater than 20-fold over control levels, compared with a three-fold increase in the absence of IBMX (Fig. 5) (Lange and Orchard, 1986). This increase in cyclic AMP accumulation was paralleled by a decrease in tonus of the muscle of about 30% compared with the effects of octopamine alone (Fig. 1).
At higher concentrations of octopamine this enhancement of the relaxation in the presence of IBMX was no longer seen, implying that a maximal physiological relaxation of the muscle had been reached (Fig. 1). In fact, 10⫺4 M octopamine was slightly less effective than 10⫺5 M octopamine in reducing the tonus of the muscle, paralleling the peak of cyclic AMP production at 10⫺5 M octopamine (Fig. 5). IBMX, alone, had no effect on the basal tonus of the muscle. This may be expected as IBMX only resulted in a relatively small increase in cyclic AMP when applied to the oviducts alone (Fig. 4). With regard to induced contractions of the oviducts, proctolin produced an increase in phasic contractions and a sustained increase in basal tonus of the muscle. Application of octopamine in the presence of proctolin resulted in a dose-dependent decrease in the amplitude of the proctolin-induced contractions (Fig. 2). The threshold of the octopamine effect on proctolin occurred at a concentration of 10⫺8 M (Fig. 2b). This is reasonable since concentrations of octopamine less than 10⫺8 M produce very little cyclic AMP elevation in the oviducts (Lange and Orchard, 1986). When 5×10⫺4 M IBMX was added concurrent with octopamine, it enhanced the inhibition of the proctolin-induced contraction, and, at an octopamine concentration of 5×10⫺6 M, the 10⫺9 M proctolin-induced contraction was completely abolished. Although application of IBMX alone had no effect on basal tonus of the oviducts, it was able to reduce 10⫺9 M proctolin-induced contractions by 20% (Fig. 3). Similarly, IBMX has been shown to be able to inhibit neurally-evoked contractions of the oviducts (Lange and Orchard, 1986). This indicates that at least part of the 25–30% inhibition of the proctolin-induced contraction seen in Fig. 2b is due to IBMX, while the further inhibition, even at the lower concentrations of octopamine (10⫺11 to 10⫺9 M), indicates a synergistic effect of octopamine and IBMX in reducing the proctolin-induced contractions. In the presence of IBMX, one would expect that the breakdown of cyclic AMP would be inhibited, and there would be a buildup of cyclic AMP over time in the muscle cells. Measurement of cyclic AMP levels in the presence of IBMX over a period of 2 h indicated this to be the case (Fig. 4). Also, it was seen that when the oviducts were pre-incubated in 5×10⫺4 M IBMX for a period of 15 min, the 10⫺9 M proctolin-induced contraction was reduced by a further 15% (Fig. 3). In order for this buildup of cyclic AMP to occur, it would seem that there must be some adenylate cyclase activity. It has been hypothesised that this could be the result of a basal level of spontaneously released octopamine onto the oviducts that would result in G protein activation and adenylate cyclase stimulation (Nykamp and Lange, 1998). This is supported by data that show that in the presence of the alpha-adrenergic receptor antagonist phentoalmine, which is known to block the octopamine receptor, IBMX
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does not alter cyclic AMP levels in the oviducts (Nykamp and Lange, 1998). Although it appears that a simple additive effect of the physiological results of octopamine and proctolin could be occurring, it would seem likely that a more complex interaction of the signal transduction pathways would be more efficient and allow for a greater flexibility of physiological responses. For example, it is probable that octopamine and proctolin may be working either hormonally and/or neurally, being released from octopaminergic and proctolinergic neurons to modulate contractions of the oviducts during such varied physiological events as ovulation, egg retention, egg laying and haemolymph circulation (Lange and Nykamp, 1996). A possible mechanism of action for reduction of proctolin-induced contractions by octopamine or IBMXinduced increases in cyclic AMP could be through an alteration of the proctolin signal transduction pathway. Proctolin increases intracellular calcium (Lange et al., 1987; Lange, 1988; Wilcox and Lange, 1995), and it is thought that this leads to activation of myosin light chain kinase, resulting in myosin light chain phosphorylation and thus contraction of the oviducts (Nykamp et al., 1994; Lange and Nykamp, 1996). There is some evidence that this phosphorylation may be reduced in the presence of octopamine. Phosphorylation studies, using homogenates of oviduct muscle, have shown a decrease in phosphorylation of a myosin light chain sized band when octopamine is added to proctolin stimulated tissue (Nykamp, unpublished observations). A possible mechanism for this type of action could be through a cyclic AMP-dependent protein kinase (PKA). In turkey gizzard for example, PKA has been reported to phosphorylate myosin light chain kinase, which results in a decrease in myosin light chain kinase activity and contraction (Conti and Adelstein, 1981). We further investigated the effects of proctolin on the main second messenger involved in the octopamine response, namely cyclic AMP. Proctolin was found to have the ability to reduce cyclic AMP levels produced by octopamine in the presence of IBMX. Proctolin was most effective at 10⫺6 to 10⫺5 M octopamine, reducing cyclic AMP levels by up to 60% (Fig. 5). This indicates that proctolin is altering the ability of octopamine to generate cyclic AMP and thus is likely affecting the octopamine signal transduction pathway. This type of interaction could be occurring through G-protein subunit interaction between the two pathways. For example, there is mounting evidence in tracheal smooth muscle that Gi can inhibit adenylate cyclase activity directly, as well as attenuate the activity of Gs which is responsible for activating adenylate cyclase (Pyne and Pyne, 1993). Further study is necessary to ascertain whether a mechanism such as this is occurring in the oviducts. If the pathways are interacting in a manner such as this, then the response should be dose dependent. We saw that the
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ability of proctolin to reduce cyclic AMP was greatest at concentrations of 10⫺9 M and above, while being less effective at lower concentrations (Fig. 6). Also, it seems that with higher concentrations of octopamine (10⫺4 M), proctolin is less effective in reducing cyclic AMP. It was also noted that proctolin, when added to the oviducts, increased cyclic AMP levels (Fig. 6). Since this increase was blocked in the presence of the alpha-adrenergic receptor antagonist phentolamine (Fig. 6, inset), it was likely not due to proctolin itself but was probably due to octopamine being released onto the oviducts from DUM neuron endings in response to the proctolin-induced contractions. It seems that cross-talk regulation between these two pathways has important functions in controlling the fine tuning of force development in the locust oviducts. Thus, as well as altering the release of neurotransmitters and neurohormones, there appears to be important postreceptor interactions that are integrated to allow for flexibility of contraction during physiological functions of the oviducts.
Acknowledgements This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.
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