Peptide-Induced Ca2+ Movements in a Tonic Insect Muscle: Effects of Proctolin and Periviscerokinin-2

Christian Wegener1,2 and Dick R. Nässel1

 1Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden; and  2Institute of General Zoology and Animal Physiology, Friedrich-Schiller-University, D-07743 Jena, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Wegener, Christian and Dick R. Nässel. Peptide-Induced Ca2+ Movements in a Tonic Insect Muscle: Effects of Proctolin and Periviscerokinin-2. J. Neurophysiol. 84: 3056-3066, 2000. Although most of the characterized insect neuropeptides have been detected by their actions on muscle contractions, not much is known about the mechanisms underlying excitation-contraction coupling. Thus we initiated a pharmacological study on the myotropic action of the peptides periviscerokinin-2 (PVK-2) and proctolin on the hyperneural muscle of the cockroach Periplaneta americana. Both peptides required extracellular Ca2+ to induce muscle contraction, and a blockage of sarcolemmal Ca2+ channels by Mn2+ or La3+ inhibited myotropic effects. The peptides were able to induce contractions in dependence on the extracellular Ca2+ concentration in muscles depolarized with high K+ saline. A reduction of extracellular Na+, K+, or Cl- did not effect peptide action. Nifedipine, an L-type Ca2+-channel blocker, partially blocked the response to both peptides but to a much lesser extent than contractions evoked by elevated K+. Using calcium imaging with fluo-3, we show that proctolin induces an increase of the intracellular Ca2+ concentration. In calcium-free saline, no increase of the intracellular Ca2+ concentration could be detected. The inhibiting effect of ryanodine, thapsigargin, and TMB-8 on peptide-induced contractions suggests that Ca2+ release from the sarcoplasmic reticulum plays a major role during peptide-induced contractions. Preliminary experiments suggest that the peptides do not employ cyclic nucleotides as second messengers, but may activate protein kinase C. Our results indicate that the peptides induce Ca2+ influx by an activation or modulation of dihydropyridine-sensitive and voltage-independent sarcolemmal Ca2+ channels. Ca2+-induced Ca2+ release from intracellular stores, but not inositol trisphosphate-induced Ca2+ release, seems to account for most of the observed increase in intracellular Ca2+. Additionally, both peptides were able to potentiate glutamate-induced contractions at threshold concentrations.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Most of the numerous insect neuropeptides known to date can induce stimulatory or inhibitory responses in different types of muscle (see Gäde 1997; Holman et al. 1991; Schoofs et al. 1997). Nevertheless our knowledge about the mechanisms of peptide-induced excitation-contraction coupling in insect muscles is very limited. Most studies have been made on the actions of proctolin. Proctolin (RYLPT) is found in different arthropods and has myostimulatory actions or potentiates neurally evoked contractions in a variety of arthropod muscles (see Orchard et al. 1989). There is compelling evidence that proctolin in insect muscles activates the phospholipase C (PLC) pathway leading to production of the second messengers 1,4,5-inositoltrisphosphate (InsP3) and diacylglycerol (DAG) (Baines et al. 1990; Hinton and Osborne 1995, 1996; Lange 1988; Mazzocco-Manneval et al. 1998). Proctolin also increases muscle membrane resistance through a reduction of the resting K+ conductance (Baines et al. 1996; Erxleben et al. 1995; Hertel and Penzlin 1986; Hertel et al. 1997; Walther et al. 1998). The action of proctolin is dependent on the presence of extracellular Ca2+ (Cook and Holman 1985; Hertel and Penzlin 1986; Hinton et al. 1998; Lange et al. 1987; Penzlin 1994; Washio and Koga 1990) and comprises an influx of Ca2+ into the muscle (Baines and Downer 1991; Dunbar and Huddart 1982; Wilcox and Lange 1995). A proctolin-induced increase of the intracellular Ca2+ concentration ([Ca2+]i) has been demonstrated for barnacle muscle fibers (Bittar and Nwoga 1989). An involvement of both voltage-dependent and non-voltage-dependent Ca2+ channels during proctolin action has been proposed for the oviduct of Locusta migratoria (Lange et al. 1987) and the hindgut of the cockroach Leucophaea maderae (Cook and Holman 1985). It is, however, not clear to what extent proctolin-induced muscle contractions in insects depend on intracellular Ca2+ mobilization by InsP3 or by Ca2+-induced Ca2+ release (CICR). There is also little data available on the myotropic mode of action of other insect neuropeptides. Thus we initiated the present pharmacological study on the action of proctolin on muscle contractions and calcium movements and made a comparison to the actions of another myotropic cockroach peptide, periviscerokinin-2.

Periviscerokinins (PVKs) are abundant myotropic neuropeptides in the abdominal neurosecretory system of the cockroach Periplaneta americana and are stored in the abdominal perisympathetic organs (Eckert et al. 1999; Predel et al. 1995, 1998; Wegener et al. 1999). The two known PVKs (PVK-1: GASGLIPVMRNamide, and PVK-2: GSSSGLISMPRVamide) both have myotropic effects on the heart, segmental vessels, and the hyperneural muscle (HNM) of P. americana (Eckert et al. 1999; Predel et al. 1995, 1998).

We chose the HNM of P. americana to study peptide action because the HNM is sensitive to both PVKs and proctolin (Hertel and Penzlin 1986; Penzlin 1994; Predel et al. 1995, 1998) and has ideal properties for pharmacological studies (see Moss and Miller 1988). Also, as we will show, the HNM is suitable for Ca2+ monitoring using Ca2+-sensitive dyes. This property enabled us to investigate for the first time the effect of proctolin on [Ca2+]i in an intact insect muscle. The close proximity of the PVK-2-containing abdominal perisympathetic organs to the HNM (see Klass 1999) suggests that, on release from these neurohemal organs, PVK-2 activates the muscle in vivo. Evidence for a physiological role of proctolin on the HNM of P. americana is that proctolin-immunoreactive nerve fibers innervate the muscle (Witten and O'Shea 1985).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Insects

American cockroaches (P. americana) were reared under a 16-h-light, 8-h-dark photoperiod at a constant temperature of 28°C. Adult males were used throughout the experiments. They had free access to water and were fed dog food pellets and occasionally fruits.

Chemicals

Methoxy-verapamil, tetraethylammonium (TEA), MDL-12,330-A, ryanodine, and TMB-8 were from RBI (Natick, MA). The acetoxymethyl ester form of fluo-3 (fluo-3 AM) was from TEF Labs (Austin, TX). All other chemicals were purchased from Sigma (Stockholm). PVK-2 was a kind gift of Reinhard Predel (Jena).

Preparation

For both bioassay and Ca2+ imaging, the HNM was prepared according to Predel et al. (1994). The abdominal sternites 3 to 8 were taken away and the HNM was removed along with the 2nd and 9th sternite, to which the muscle is attached. The ventral nerve cord was removed and the preparation was cleaned from attached fat body and tracheae.

Bioassay

For measurement of isotonic contractions, the HNM was mounted in a glass chamber filled with 2.5 ml continuously aerated cockroach saline [containing (in mM) 140 NaCl, 5 KCl, 5 CaCl2, 1 MgCl2, 5 glucose, and 9.2 HEPES; pH 7.25]. During the application of 3-isobutyl-1-methylxaanthine (IBMX), H-7, Rp-cAMPS, MDL-12,330, phorbol 12-myristate 13-acetate (TPA), and cyclic nucleotide analogues, the saline additionally contained 1% DMSO, which by itself had no effect on peptide-induced muscle contraction (n = 6). A small amount of NaOH was used to solubilize EGTA prior to the preparation of an appropriate stock solution in saline. Application of stock solution aliquots did not change the pH in the bathing saline. One end of the muscle was attached to the lever of a photoelectric transducer, the other was fixed at the bottom of the chamber. The jacketed saline reservoirs and glass chamber were adjusted to 28°C using a heating circulator (MP-5A; Julabo, Seelbach, Germany). A detailed description of the bioassay is given by Penzlin (1994).

After mounting in the glass chamber, the muscle was allowed to relax for 20 min under a tension of approximately 0.5 mN. Further elongation was prevented by a stopping lever. During the pharmacological experiments, peptide and drug applications, usually in aliquots of 25 µl, followed the scheme shown in Fig. 1. Contractions were simultaneously recorded using a flatbed recorder (Goerz Servogor 124, Wr. Neudorf, Austria) and the software LabView 5.0 (National Instruments, Austin, TX) on a Macintosh G3 computer with built-in PCI-MIO-16E-4 card (National Instruments, Austin, TX).



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Fig. 1. Scheme of peptide and drug applications used in bioassay experiments. Peptides were applied at the times indicated (down-arrow ) and washed out 3 min later. Drugs were applied 10 min before the "test" peptide application (up-arrow ).

In Na+-, Ca2+- or K+-free solutions, the ions were substituted by an osmotically equivalent amount of choline. For high K+ or Li+ solutions, an osmotically equivalent amount of Na+ was omitted. For reduced Cl- solution, NaCl was substituted by an osmotically equivalent of the sodium salt of isethionic acid.

Ca2+ imaging

Loading of the HNM with the fluorescent Ca2+ indicator fluo-3 (Minta et al. 1989) was performed as follows: the HNM was incubated for 1 h at room temperature in darkness in 1 ml continuously aerated saline containing 20 µM fluo-3. To reduce dye leakage out of the muscle, 2 mM probenecid was included in the saline. Before adding fluo-3 to the saline, an appropriate amount of the dye was taken from a stock solution in dry DMSO and mixed with 1 µl 15% pluronic F-127 in dry DMSO to prevent dye precipitation. After loading, the muscle was washed three times with 1 ml saline containing 2 mM probenecid and mounted in an open Plexiglas chamber with a capacity of 2 ml by fixing the attached sternites to the silicone elastomer (Sylgard, Dow Corning, Midland, MI) lining of the chamber. Solutions were continuously aerated and gravity-fed to the chamber at a flow-rate of approximately 3 ml/min. The muscle was viewed with an Zeiss Axioplan 2 microscope equipped with a Zeiss water immersion objective (Achroplan 20x/NA 0.5). The light source was a HBO 100 W lamp (Zeiss, Jena, Germany). A normal filter combination for fluorescein (FITC) fluorescence was used. Images were captured with a chilled CCD camera (C4742-95, Hamamatsu, Japan) at a frequency of 0.33 or 1 picture/s and stored and analyzed on a Macintosh PowerPC 8600 using Openlab 2.06 (Improvision, Coventry, UK). Regions of interest were selected and relative fluo-3 fluorescence was measured. If necessary, regions of interest were adjusted on a frame-to-frame basis to keep track of the muscle movements. Fluorescence intensity is presented as relative gray scale intensity after subtraction of the acellular background. Individual muscles were used for a single proctolin application only.

Statistics

Data were evaluated with StatView 5.0 (SAS Institute, Cary, NC) using a Wilcoxon signed-rank test. Differences in contraction before and after drug application (compare Fig. 1) were defined to be significant when P(control 1/control 2) > 0.1, P(control 2/drug) < 0.05. Data are presented in percentages as means ± SE of control 1.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Proctolin and PVK-2 induce long-lasting tonic contractions

Proctolin and PVK-2 induced long-lasting tonic contractions of the HNM when applied at 10 nM (Fig. 2, B and C). These contractions were reproducible even after several applications of the same peptide concentration, which allowed a quantification of the contractile effect of the peptides. Usually it took around 10-20 s after peptide application until the muscle started to contract. Likewise, relaxation after peptide wash-out was not immediate, and some preparations needed several minutes to return to the resting state (see Fig. 2C). Peptide-induced contractions were different from contractions evoked by depolarization with 100 mM K+. Application of high-K+ saline resulted in an immediate strong contraction of the HNM without any plateau-phase (Fig. 2). On saline wash, the muscle returned immediately to the resting length.



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Fig. 2. Contractions of the hyperneural muscle (HNM) induced by depolarization with saline containing 100 mM K+ (A), 10 nM proctolin followed by a depolarization with 100 mM K+ (B), and 10 nM PVK-2 followed by a depolarization with 100 mM K+ (C). black-triangle, application of peptide/100 mM K+; black-down-triangle , wash with normal saline. The difference between high-K+- and peptide-induced contractions is not due to the preparation but to different mode of actions of high K+ and the peptides.

Peptide-induced contractions are dependent on extracellular Ca2+ but not on extracellular Na+, K+, or Cl-

Ca2+-free saline containing 2 mM of the Ca2+-chelator EGTA completely suppressed the myotropic action of both peptides (n = 6). This effect was not reversible, even after several washes with normal saline. Incubating the muscle in Na+-free saline (n = 6) or Cl--reduced saline (50 mM Cl-, n = 4) neither altered the basal tonus of the HNM nor significantly changed peptide-induced contractions. Incubation of the muscle in K+-free saline led to a higher basal tonus, but PVK-2 was still able to induce contractions similar to the controls in normal saline, i.e., peptide-induced contractions were additive to the effect of omitting K+ (n = 8). Proctolin-induced contractions were slightly but significantly decreased in K+-free saline and were not additive to the effect of omitting K+ (Penzlin 1994).

Sarcolemmal Na+/Ca2+ exchanger is active during peptide-induced contractions

The HNM relaxed normally after peptide-induced contractions when washed with Na+-free saline. However, after a subsequent 20 min in Na+-free saline, peptide-induced contractions were significantly increased (Fig. 3A). After washing with normal saline, the peptide response returned to the level of the first control. This indicated the presence of a Na+/Ca2+ exchange mechanism that counterbalances the peptide-induced increase in [Ca2+]i. Therefore we tested the effect of benzamil, a blocker of the Na+/Ca2+ exchanger in the sarcolemmal membrane. At 50 µM, benzamil significantly increased proctolin-induced contractions to 166% (n = 6) and PVK-2-induced contractions to 173% (n = 5) of the control (Fig. 3B). This reversible effect of benzamil suggests the presence of a Na+/Ca2+ exchanger in the sarcolemmal membrane of the HNM.



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Fig. 3. The effect on contractile responses to proctolin () and PVK-2 () of washing with Na+-free saline after peptide-application (A, n = 7/6), benzamil, a blocker of the Na+/Ca2+ exchanger (B, n = 6/5), effect of 5 µM nifedipine, a L-type Ca2+ channel blocker (C, n = 8/7), and effect of 2 mM Cs+, a blocker of inward rectifying K+ channels (D, n = 6). *, significant differences between control 2 and test application (P < 0.05).

Effect of K+ channel blockers on peptide-induced contractions

The resting potential of insect muscle fibers is primarily determined by K+ (Pichon and Ashcroft 1985; Walther et al. 1998). In many cells, inward rectifying K+ channels regulate the resting membrane potential and can be blocked by extracellular application of Cs+. At 2 mM, Cs+ attenuated proctolin-induced contractions significantly to 81% (n = 6) and PVK-2-induced contractions not significantly to 86% (n = 6) of the controls (Fig. 3D). The unspecific K+ blocker 4-amino pyridine (4-AP) produced a transient twitch-like contraction of the muscle when directly applied at 1 mM but did not reduce PVK-2-induced contractions (n = 6). The same was found for proctolin (Hertel and Penzlin 1986). Also TEA, another unspecific K+ blocker, had no significant effect on contractions evoked by proctolin (n = 7) or PVK-2 (n = 9) when applied at 1 mM.

Effect of Ca2+ channel blockers on peptide-induced contractions

The dependence of peptide-induced contractions on extracellular Ca2+ indicated that the contractile effect is due to an influx of Ca2+ into the HNM. This was substantiated by the complete block of peptide-induced contractions by 2 mM Mn2+ or 100 µM La3+. Both ions are unspecific Ca2+ channel blockers. Their effect was not reversible after several washes with saline without these ions (n = 6).

In body-wall muscles of larval Drosophila melanogaster, the main Ca2+ channel current consists of a dihydropyridine (DHP)-sensitive ("L-type") and an amiloride-sensitive ("T-type") component (Gielow et al. 1995). Voltage-dependent L-type Ca2+ channels are also present in crustacean muscles (Araque et al. 1998; Erxleben and Rathmayer 1997). Therefore we used the DHP nifedipine and the phenylalkylamine (PAA) methoxy-verapamil, another antagonist of vertebrate L-type channels, and amiloride to investigate if similar channels are involved during peptide-induced Ca2+ influx of the HNM.

Even at 100 µM, methoxy-verapamil had no inhibitory effect on PVK-2-induced contractions (n = 6). At the same concentration, verapamil also did not prevent proctolin-induced contractions (Penzlin 1994). Nifedipine at 5 µM reduced peptide-evoked contractions significantly to 72% of the controls for PVK-2 (n = 7) and to 56% for proctolin (n = 8, Fig. 3C). The effect of nifedipine was, however, more pronounced on high-K+ (100 mM)-induced contractions, which were significantly reduced to an average of 20% of the control with some contractions being completely blocked (n = 6). At 60 µM nifedipine, PVK-2-induced contractions were significantly reduced to 65% (n = 6), which is comparable to the effect at 5 µM. Both proctolin and PVK-2 were also able to further contract the muscle during a depolarization with 100 mM K+ and in presence of 5 µM nifedipine. The effect of nifedipine was partially reversible (Fig. 3C).

Amiloride had no significant effect at a concentration of 100 µM on either PVK-2- or proctolin-induced contractions (n = 6).

Proctolin and PVK-2 can induce muscle contractions in depolarized muscles

The much more pronounced effect of nifedipine on high-K+-induced contractions than on peptide-induced contractions suggested that the peptides use mechanisms other than or additional to membrane depolarization to induce Ca2+ influx. Since depolarization of the HNM by an incubation in saline with high (100 mM) K+ resulted in a near-to-maximum contraction (Fig. 2) of the muscle, it was not possible to test in normal saline whether peptide-induced contractions are additive to high-K+-induced contractions. To circumvent this problem, we first depolarized the muscle with 25 or 100 mM K+ in nominal Ca2+-free saline (without EGTA) and then increased the extracellular Ca2+ concentration stepwise. Following a wash with 10 ml high-K+, Ca2+-free saline, the same procedure was repeated but in presence of 10 nM of the peptides. Most preparations reacted with a short twitch-like contraction and an immediate relaxation to the depolarization with high K+ in nominally Ca2+-free saline. With introduction of extracellular Ca2+, muscle contractions reappeared. In general, the contractile response to high K+ under this experimental regime was strongly reduced compared with that in normal Ca2+-containing saline. A possible explanation for this effect could be that the voltage-dependent Ca2+ channels became activated by the introduction of high-K+, nominally Ca2+-free saline and that their activity decreased with time and was already diminished when extracellular Ca2+ was stepwise added after 3 min. This would result in a smaller Ca2+ influx and hence a smaller contraction and is also consistent with only transient contractions being evoked by high K+ in normal saline. Proctolin or PVK-2, however, induced normal or even increased tonic contractions in high K+-containing saline. These contractions first increased with increasing extracellular Ca2+ concentration before becoming smaller or unchanged at higher extracellular Ca2+ concentrations (Fig. 4).



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Fig. 4. Proctolin and periviscerokinin-2 (PVK-2) are able to induce contractions of depolarized muscles. Muscles were depolarized with either 25 or 100 mM K+ in nominally Ca2+-free saline, and then the extracellular Ca2+ concentration was increased in a stepwise manner. After a wash with 5 mM K+ in nominally Ca2+-free saline, the same procedure was repeated in presence of either 10 nM proctolin or PVK-2 (n = 3 for each combination).

Intracellular Ca2+ is mobilized during peptide action

To test whether intracellular Ca2+ from the sarcoplasmic reticulum is released during peptide action, we used TMB-8, an unspecific blocker of intracellular Ca2+ release. TMB-8 had a strong inhibitory effect on the myotropic action of both peptides (Fig. 5A). Proctolin-induced contractions were significantly reduced to about 20% (n = 5) and PVK-2-induced contractions to 40% of the controls (n = 5). Thapsigargin, a blocker of the sarcoplasmic Ca2+-ATPase (see Treiman et al. 1998), also reduced peptide-induced contractions significantly (Fig. 5B). These results indicated that both peptides mobilize Ca2+ from the sarcoplasmic reticulum. Therefore we tested the effect of the ryanodine receptor (RyR) blocker ryanodine on peptide-induced contractions.



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Fig. 5. Effects on proctolin () and PVK-2 response () of drugs influencing intracellular Ca2+ release. A: effect of 1 mM TMB-8, an unspecific blocker of intracellular Ca2+ release (n = 5). B: effect of 1 µM thapsigargin, a blocker of the Ca2+-ATPase of the sarcoplasmic reticulum (n = 6). C: effect of 10 µM ryanodine, a ryanodine receptor antagonist (n = 6). *, significant differences between control 2 and test application (P < 0.05). The effect of thapsigargin was not reversible since thapsigargin binding to the sarcoplasmic Ca2+-ATPase is practically irreversible (see Treiman et al. 1998).

Ryanodine at 10 µM suppressed proctolin-induced contractions significantly to 50% (n = 6) and PVK-2-induced contractions to 60% of the control (Fig. 5C, n = 6). Increasing the ryanodine concentration to 100 µM did not result in a further suppression. Proctolin-induced contractions were significantly reduced to 51% (n = 5), those of PVK-2 to 63% (n = 5).

Functional ryanodine receptors are present in the HNM

The preceding results suggest that intracellular Ca2+ release via RyR is functionally important for peptide-induced contractions. To show that functional RyR are present in the HNM, we used caffeine, a RyR agonist. When applied at 20 mM, caffeine produced a short twitch-like contraction (Fig. 6). In most preparations, this twitch-like contraction was followed by a long-lasting tonic or phasic contraction (Fig. 6). The twitch-like contraction could be completely blocked by 10 µM ryanodine in most preparations (n = 6). Application of 20 mM caffeine in Ca2+-free Ringer containing 2 mM EGTA caused only twitch-like contractions, a tonic phase was never observed (n = 6). Therefore the tonic component appears to be dependent on extracellular Ca2+ influx.



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Fig. 6. Caffeine- and phorbol 12-myristate 13-acetate (TPA)-induced muscle contractions. A: at 20 mM, the ryanodine receptor agonist caffeine induced a short twitch-like contraction that was most often followed by a long-lasting contraction that could be phasic (to the left) or tonic (to the right). B: at 5 µM, the PKC activator TPA induced tonic contractions that were slower but comparable to peptide-induced contractions. black-triangle, drug application; black-down-triangle , wash with normal saline.

Preliminary evidence for an activation of the PLC signaling pathway by proctolin and PVK-2

Activation of PLC leads to production of the second messengers InsP3 and diacylglycerol (DAG). Whereas InsP3 induces Ca2+ release from the sarcoplasmic reticulum, DAG activates protein kinase C (PKC). Since proctolin activates the PLC pathway in several insect muscles, we tested the effect of Li+, an inhibitor of several enzymes in the phosphoinositol cycle, and the unspecific PKC blocker H-7 (Hidaka et al. 1984) on peptide-induced contractions. It has been proposed that application of Li+ leads either to an accumulation or a reduction of InsP3 and DAG (Nahorski et al. 1991). When applied at 50 mM 20 min before peptide application, Li+ caused a slight but significant increase of proctolin-induced contractions to 109% of the control (n = 9, Fig. 7A). PVK-2-induced contractions were not significantly increased to 105% (n = 8, Fig. 7A).



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Fig. 7. A: effect of Li+, a blocker of several enzymes of the phosphoinositide cycle on responses to proctolin (, n = 9) and PVK-2 (, n = 8). B: effect of H-7, an unspecific PKC blocker (n = 7/6). *, significant differences between control 2 and test application (P < 0.05).

At 10 µM, H-7 significantly reduced proctolin-induced contractions to 87% (n = 7) and PVK-2-induced contractions to 86% (n = 6) of the control (Fig. 7B), suggesting that a protein kinase is involved in the action of both peptides. The phorbol ester TPA can activate PKC and thus mimics the action of DAG. When TPA was applied at 1-5 µM, it caused a tonic contraction of the muscle that was slower but very similar to peptide-induced contractions (Fig. 6B). Like peptide-induced contractions, the myotropic action of TPA could be completely blocked by 100 µM La3+ (n = 4). The effect of TPA was not reversible: the muscle remained in its tonic state even after longer periods of washing.

Cyclic nucleotides seem not to be involved in the action of proctolin and PVK-2

Since H-7 also blocks the protein kinases A and G (Hidaka et al. 1984), it seemed possible that also cyclic nucleotides could mediate the myotropic effect of the peptides. Therefore we tested the effect of several chemicals known to interfere with cyclic nucleotide signaling pathways.

The phosphodiesterase blocker IBMX at 100 µM had no effect on contractions evoked by PVK-2 (n = 9) or proctolin (n = 6). The specific protein kinase A blocker Rp-cAMPS at 100 µM did not reduce PVK-2-induced contractions (n = 4). The adenylate cyclase blocker MDL-12,330 A always evoked tonic contractions when applied at 10-50 µM (n = 7) and did not alter proctolin- or PVK-2-induced contractions at lower drug concentrations (MDL-12,330 A alone at lower concentrations did not induce HNM contractions). The membrane permeable cAMP analogue 8-bromo-cAMP, which is resistant to phosphodiesterase degradation, had no contractile effect when applied at 0.2-1 mM for up to 40 min (n = 5). Also the membrane permeable cGMP analogue db-cGMP had no contractile effect on the HNM when applied at 100-200 µM for 40 min together with 100 µM IBMX (n = 4).

Threshold levels of proctolin and PVK-2 potentiate glutamate-induced contractions

The HNM is antagonistically controlled by excitatory and inhibitory neurons running in the transverse nerves 1-7 and the ramus hyperneuromuscularis of the nerve VIII A/1 of the terminal ganglion (Penzlin 1994). Glutamate is probably the main excitatory neurotransmitter at the HNM (Moss and Miller 1988). We tested the effect of proctolin and PVK-2 on glutamate-induced contractions of the HNM. Both peptides, applied at threshold level of 0.5 nM together with 100 µM glutamate, significantly potentiated contractions compared with the effect of 100 µM L-glutamate alone to 202 ± 38% for proctolin and 257 ± 61% for PVK-2 (n = 5 for each combination). Control 3 of the glutamate-induced contractions remained increased even in the absence of the peptides (168 ± 52 and 242 ± 59% following proctolin and PVK-2 application, respectively), although this effect was only significant for PVK-2.

Ca2+ imaging

The Ca2+-sensitive dye fluo-3 has often been used for Ca2+ imaging in muscles (e.g., Pagala and Taylor 1998; Somlyo et al. 1992; Vergara et al. 1991) and was applied to investigate proctolin-induced changes of [Ca2+]i in the HNM. One advantage of fluo-3 is its large rise in fluorescence on Ca2+ binding, which makes the fluo-3 signal relatively insensitive to movement artifacts and relatively free of noise (Caputo et al. 1994).

After loading with fluo-3, the fluorescence intensity was not evenly distributed throughout the HNM due to the varying muscle thickness. We therefore selected homogenous regions of interest within which fluorescence intensity was measured. At higher proctolin concentrations, muscle movements could not be completely eliminated due to elastic elements within the HNM. We thus had to determine whether an increase in fluorescence, caused by an increasing dye concentration due to muscle shortening, interfered with the fluorescence signal caused by increasing [Ca2+]i during peptide action. For this, we monitored the effect of 100 nM proctolin on the fluo-3 signal in muscles immobilized with hypertonic saline containing 300 mM sucrose. Hypertonic sucrose salines do not significantly change membrane properties and intracellular Ca2+ release (Gallagher and Huang 1997; Lamb et al. 1993) and did not affect the signal of Ca2+-sensitive dyes in frog muscle fibers at up to two times normal tonicity (Parker and Zhu 1987; Taylor et al. 1975). Incubation of the HNM in hypertonic saline resulted in a transient decrease of the fluo-3 signal (Fig. 8D). Proctolin was able to induce an increase of the fluo-3 signal that was similar in shape and intensity to that induced in normal saline and that was reversible by washing with hypertonic saline (Fig. 8D, n = 2). This showed that movement artifacts are negligible and that fluo-3 can be used to monitor peptide-induced relative changes in [Ca2+]i in the HNM. However, the onset and time course of the proctolin-induced increase of [Ca2+]i were considerably delayed in hypertonic saline.



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Fig. 8. Measurements of [Ca2+]i in the HNM with the Ca2+-sensitive dye fluo-3. Typical traces of the fluo-3 signal after 1 nM (A), 10 nM (B), or 100 nM (C) proctolin application (n = 5 for each concentration). In B, the numbered bars 2-6 indicate the time at which images A2-6 in Fig. 10 were captured. The relative fluorescence intensity is not truly comparable between different examples. D: the effect of 100 nM proctolin on a HNM in hypertonic saline containing 300 mM sucrose (n = 2). Due to the long monitoring time, we had to store data during the experiment, which caused the gap in the curve.

Typical traces of the fluo-3 signal during stimulation with 1, 10, or 100 nM proctolin in normal saline are shown in Fig. 8, A-C (n = 5 for each concentration). After application of 1 nM proctolin, the increase in fluorescence intensity was sometimes only transient even in the presence of the peptide. The increase of fluorescence caused by 10 nM proctolin (Fig. 10) lasted until the peptide was washed out. Following an initial peak, the fluorescence intensity invariantly declined and stabilized on a somewhat lower level that returned to the basic level after peptide wash-out. Proctolin at 100 mM consistently caused an increase in fluorescence intensity that did not decline but slightly increased until the peptide was washed out. As with peptide-induced contractions, there was a delay between the application of proctolin and the onset of the proctolin-induced increase of [Ca2+]i. This increase was fast at 10 and 100 nM proctolin, with a slope independent of the applied proctolin concentration. The increase seems therefore to be caused by intracellular Ca2+ release rather than Ca2+ influx.

In Ca2+-free saline containing 2 mM EGTA, 100 nM proctolin was unable to produce a detectable increase in fluorescence intensity, whereas 2 mM caffeine produced a strong signal (Fig. 9A, n = 5). The time course of high-K+-induced changes in fluorescence intensity differed from that induced by proctolin (Fig. 9B). A steep initial increase was followed by a steady decline, no plateau phase could be detected (n = 4).



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Fig. 9. Measurements of Ca2+ levels in the HNM with the calcium-sensitive dye fluo-3. A: in Ca2+-free saline containing 2 mM EGTA, 100 nM proctolin was unable to induce a detectable rise in fluo-3 fluorescence intensity. Subsequent application of 2 mM caffeine, however, produced a strong [Ca2+] increase, demonstrating that the sarcoplasmic reticulum was functionally intact and not devoid of Ca2+ (n = 5). B: depolarization with 100 mM K+ produced a strong increase in [Ca2+]i without a plateau-phase (n = 4).

Figure 10 shows a typical example of a Ca2+ imaging experiment using fluo-3.


    DISCUSSION
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Action potentials in arthropod muscles are in general Ca2+ dependent but Na+ independent (Ashcroft 1981; Deitmer and Rathmayer 1976; Fatt and Ginsborg 1958; Hagiwara and Naka 1964; Hencek and Zachar 1977; Washio 1972), whereas action potentials in nerves are Na+ dependent. The ability of proctolin and PVK-2 to induce contractions in Na+-free saline thus suggests that both peptides act postsynaptically at the muscle membrane. Neurally evoked contractions of the HNM disappeared completely in Na+-free saline, whereas proctolin was still able to induce contractions in the absence of Na+ (Hertel and Penzlin 1986; Penzlin 1994).

The myotropic effects of proctolin and PVK-2 on the HNM are completely dependent on an influx of extracellular Ca2+. This is consistent with the complete dependence of proctolin-induced contractions on extracellular Ca2+ influx in other insect muscles (Cook and Holman 1985; Hertel and Penzlin 1986; Hinton et al. 1998; Lange et al. 1987; Penzlin 1994; Washio and Koga 1990). We showed that peptide-induced Ca2+ entry is not through amiloride-sensitive voltage-dependent (T-type-like) channels, but at least partially through nifedipine-sensitive/verapamil-insensitive Ca2+ channels that most likely are voltage dependent since also high-K+-induced contractions were inhibited by nifedipine. Similarly it has been shown that the DHP-sensitive voltage-dependent Ca2+ channels of larval Drosophila melanogaster body-wall muscles are relatively insensitive to verapamil (Gielow et al. 1995). For P. americana muscles, only PAA binding sites that were DHP sensitive have so far been characterized (Skeer and Sattelle 1993; Skeer et al. 1992). Our findings suggest that also a DHP-sensitive but (relatively) PAA-insensitive Ca2+ channel exists in sarcolemmal membranes of P. americana. Also in the Schistocerca gregaria foregut, up to 1 mM verapamil had no effect on proctolin-induced contractions but dose-dependently reduced acetylcholine-induced contractions (Hinton et al. 1998). Verapamil was also much less effective than nifedipine in reducing proctolin-induced contractions of the oviduct of L. migratoria (Lange et al. 1987). Taken together with the findings in D. melanogaster, it seems thus likely that a DHP-sensitive, PAA-insensitive voltage-dependent Ca2+ channel is a general feature of insect muscle membranes.

An involvement of DHP-sensitive voltage-dependent Ca2+ channels in the HNM seems to be in conflict with the finding that the HNM is not electrically excitable (Hertel and Penzlin 1986; Miller and Adams 1974). Recently, however, Monterrubio and co-workers (2000) described "silent" nifedipine-sensitive Ca2+ channels that occur in low density in the tonic abdominal flexor muscles of the crustacean Atya lanipes. These muscles, like the HNM, are not electrically excitable. Under voltage-clamp conditions, Ca2+ inward currents were too small to measure in response to depolarizing currents but seem to be sufficient to induce CICR. This is probably due to the close proximity of the silent Ca2+ channels to the sarcoplasmic reticulum in the dyad (Monterrubio et al. 1999).

The ability of PVK-2 and proctolin to induce contractions in depolarized muscles suggests that the mode of action of both peptides comprises additional mechanisms besides activation of voltage-dependent Ca2+ channels. This is also suggested by the differences between peptide-induced and high-K+-induced contractions and increases of [Ca2+]i and the more pronounced inhibiting effect of nifedipine on high-K+-induced contractions than on peptide-induced contractions. An activation of non-voltage-dependent Ca2+ channels by proctolin was suggested by the fact that proctolin was able to induce contractions of high K+-depolarized cockroach hindgut (Cook and Holman 1980, 1985), and locust oviduct (Lange et al. 1987) which could not be blocked by nifedipine (Lange et al. 1987). Due to a lack of specific antagonists of receptor-operated channels, we could not obtain direct pharmacological evidence for an activation of receptor-operated channels during peptide action. An alternative explanation for the myotropic effect of the peptides on depolarized muscles is that the peptides up-modulate Ca2+ channels that were activated by high-K+ depolarization. In crayfish, proctolin markedly increased the opening probability of Ca2+ channels in depolarized patches of the tonic flexor muscle (Bishop et al. 1991); several neuropeptides enhance the DHP-sensitive L-type current of the accessory radula closer muscle of the mollusc Aplysia californica (Brezina et al. 1994).

Although influx of extracellular Ca2+ is a prerequisite for the myotropic effect of the peptides on the HNM, the peptide-induced [Ca2+]i increase appears largely to be caused by intracellular Ca2+ release via RyR as suggested by the inhibiting effect of TMB-8, thapsigargin, and ryanodine and the slope of the proctolin-induced [Ca2+]i increase. This resembles the situation in crab muscle fibers, where extracellular Ca2+ is essential for mechanical activity, but Ca2+ release from the sarcoplasmic reticulum is required to induce contractions (Mounier and Goblet 1987). Ryanodine receptors have been characterized for P. americana muscle membranes (Lehmberg and Casida 1994; Schmitt et al. 1996, 1997), and the effects of caffeine on contraction and [Ca2+]i in Ca2+-free saline show that they are also functional in the HNM. A peptide-induced depolarization (by non-voltage-dependent Ca2+ influx?) could activate DHP-sensitive Ca2+ channels and CICR as described for A. lanipes by Monterrubio and co-workers (2000). This is suggested by the presence of dyad structures associated with the transverse tubular system of the HNM (Miller and Adams 1974), the demonstrated involvement of DHP-sensitive Ca2+ channels, the small proctolin-induced depolarization (Hertel and Penzlin 1986), and the proctolin-induced increase of the input resistance of the HNM membrane, which increases membrane excitability (Hertel and Penzlin 1986). The observation of proctolin-induced Ca2+ waves in some preparations suggests that peptide-induced Ca2+ influx may trigger local CICR that then spreads over the whole muscle fiber.

Agonists that increase InsP3 can induce smooth muscle contractions and raise [Ca2+]i also in Ca2+-free extracellular medium containing 2 mM EGTA (e.g., Itoh et al. 1992; Kasuya et al. 1989). In the absence of extracellular Ca2+ or after a block of sarcolemmal Ca2+ channels, proctolin and PVK-2 were not able to contract the HNM. Proctolin at 100 mM was also not able to elevate [Ca2+]i in the absence of extracellular Ca2+, although 100 nM proctolin elicits a near maximal contraction of the HNM and a strong increase of the fluo-3 signal when applied in the presence of extracellular Ca2+. The increase of [Ca2+]i and resulting contractions induced by caffeine in Ca2+-free saline showed that the lack of effect of proctolin in Ca2+-free saline was not caused by a prior depletion of stored Ca2+ in the sarcoplasmic reticulum. Although we cannot rule out that InsP3-induced Ca2+ release occurs at higher [Ca2+]i than was present during our experiments, our findings still suggest that InsP3-induced Ca2+ mobilization is not a key mechanism during peptide action on the HNM. This is in contrast to the models of proctolin action on the oviduct of L. migratoria (Lange and Nykamp 1996) and the foregut of S. gregaria (Hinton et al. 1998), which suggest a role of InsP3-induced Ca2+ mobilization. Our findings are, however, in agreement with experiments on the locust hindleg extensor tibia muscle with the proctolin analog [Afb (p-NO2)2]-proctolin (Baines et al. 1996). [Afb (p-NO2)2]-proctolin was equipotent to proctolin in the ability to raise InsP3 but was about 1,000 times less efficacious than proctolin in evoking contractions and to reduce the resting potassium conductance. Thus the contractile effect of proctolin is obviously not linked to InsP3 production in the hindleg extensor tibia muscle. Perhaps there are differences in the importance of InsP3-induced Ca2+ mobilization during proctolin action between "visceral" insect muscles such as the oviduct or foregut and "skeletal" insect muscles such as the HNM and the hindleg extensor tibiae muscle.



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Fig. 10. Typical example of a Ca2+ imaging experiment using fluo-3. A1: the field of view after mounting of the muscle. A trachea is visible. For fluorescence measurements, a region of interest in the middle of the upper muscle band was chosen. A2-6: Ca2+ images before and during application of 10 nM proctolin. Images shown here were recorded at times corresponding to those indicated in Fig. 8B.

Although we used a range of drugs influencing cyclic nucleotide signaling pathways, no evidence was found for an involvement of cyclic nucleotides in proctolin- or PVK-2-induced contractions. Thus the myoinhibitory effect of the unspecific protein kinase blocker H-7 appears to be due to an inhibition of PKC. This assumption is supported by the tonic contractions of the HNM evoked by the PKC activator TPA that could be blocked by La3+ similar to peptide-induced contractions. A role of PKC in proctolin-induced contractions was also proposed for the oviduct of L. migratoria (Lange and Nykamp 1996) and the foregut of S. gregaria (Hinton et al. 1998) but rejected for the mandibular closer muscle of L. migratoria (Baines and Downer 1992). Clearly further research is needed to clarify the role of PKC during proctolin- and PVK-2-induced contractions.

The most likely primary excitatory transmitter at the HNM is L-glutamate (Moss and Miller 1988). The potentiating effect of proctolin and PVK-2 on glutamate-induced contractions of the HNM suggests a modulatory role of the peptides when present in sub-nanomolar concentrations. This assumption is supported by the potentiating effect of proctolin on neurally elicited contractions of the HNM (Hertel and Penzlin 1986) and the increase in spontaneous HNM activity after application of proctolin and PVK-1 on in situ preparations of the HNM (Predel et al. 1995; S. Marsch, unpublished data). Proctolin is also known to both induce contractions and modulate neurally evoked contractions of the locust oviduct (Noronha and Lange 1997).

In conclusion, we have presented evidence that proctolin and PVK-2 induce contractions of the HNM by an activation of Ca2+ influx through DHP-sensitive and voltage-independent channels in the sarcoplasmic membrane. The main increase of [Ca2+]i is due to CICR triggered by the Ca2+ influx. This mechanism for excitation-contraction coupling in the HNM is thus the same as proposed for crustacean skeletal muscles (Györke and Palade 1993a,b, 1994). InsP3-induced Ca2+ mobilization from the sarcoplasmic reticulum seems not to contribute to the peptide-induced increase of [Ca2+]i. The peptide actions on the muscle at lower concentrations may serve to potentiate neurally evoked contractions.


    ACKNOWLEDGMENTS

We thank M. Axelsson (Göteborg) for help with the LabView software, K. Åkermann (Uppsala) for the kind gift of fluo-3 and helpful suggestions concerning Ca2+ imaging, E. Krause (Jena) for teaching us the bioassay, R. Predel (Jena) for the kind gift of PVK-2, and S. Kreissl (Konstanz), D. Wicher (Jena), and A. Lange (Toronto) for valuable comments on an earlier draft of the manuscript.

This investigation was supported by the Swedish Natural Science Research Council (NFR) (D. R. Nässel). C. Wegener was supported by a predoctoral grant of the German Academic Exchange Service (DAAD; Hochschulsonderprogramm III von Bund und Ländern).


    FOOTNOTES

Address for reprint requests: D. R. Nässel, Dept. of Zoology, Stockholm University, Svante Arrhenius väg 14, SE-106 91 Stockholm, Sweden (E-mail: dnassel{at}zoologi.su.se).

Received 23 February 2000; accepted in final form 22 August 2000.


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