Sites and modes of action of proctolin and the FLP F₂ on lobster cardiac muscle

For the neurogenic decapod crustacean heart the burst rate and intra-burst spike frequency of the intrinsic cardiac ganglion determines, in the first instance, heart rate and beat amplitude, respectively(Hallett, 1970; Van der Kloot, 1970; Anderson and Cooke, 1971). The myocardium does not possess its own pacemaker and each systole is a brief tetanus (Yazawa et al.,1999).

The basic heart rhythm is modified by extrinsic nervous and hormonal inputs(Wilkens, 1995, 1999). A variety of aminergic and peptidergic cardioactive hormones directly influence heart function and may also affect heart function indirectly via actions in the central nervous system. For those hormones with chronotropic effects the site of action is the cardiac ganglion. Those that produce inotropic effects can act at the level of the cardiac ganglion to change burst characteristics that will alter the level of myocardial depolarization and they may act directly on the myocardium to change its contractile response to a burst. The neuropeptide hormones proctolin (PR) (RLYPT; Sullivan,1979) and the FRLFamide-like peptides (FLP) F₁(TNRNFLRFamide) and F₂ (SDRNFLRFamide)(Trimmer et al., 1987) have chronotropic and inotropic effects on decapod hearts(Wilkens and Mercier, 1993; Saver and Wilkens, 1998). It is assumed that each class of peptide binds to separate myocardial receptors since there is no homology between PR and the FLPs.

PR and F₂ act directly on a variety of crustacean, chelicerate and insect skeletal, cardiac and visceral muscles to increase tonus, the amplitude of contractions and in some cases induce or increase the frequency of myogenic contractions (Lange et al.,1987; Lange, 1988; Griffiths, 1990; Mercier et al., 1991, 1997, 2003; Wilkens and Mercier, 1993; Groome et al., 1994; Skerrett et al., 1995; Worden et al., 1995; Fuse and Orchard, 1998; Saver and Wilkens, 1998; Saver et al., 1998). These peptides appear to exert their actions by binding to sarcolemmal receptors and, in the case of PR, a variety of different signal transduction pathways have been identified in different animals and tissues. PR has been shown to increase Na⁺ efflux by activating Ca²⁺ channels in barnacle muscle (Nowaga and Bittar, 1985; Bittar and Nowaga, 1989). PR increases Na⁺ conductance in lobster cardiac ganglion neurons(Freschi, 1989). PR decreases K⁺ conductance in isopod skeletal muscle and in lobster cardiac ganglion neurons (Erxleben et al.,1995; Sullivan and Miller,1984). In locust skeletal muscle PR decreases K⁺conductance via a G protein mediated pathway;(Walther et al., 1998). The mode of action of F₂ is much less well understood(Mercier et al., 2003). Furthermore, PR and FLP effects on a variety of arthropod neuromuscular synapses and muscles requires external Ca²⁺ (cockroach, Wegener and Nassel, 2000;crab, Rathmayer et al., 2002;limulus, Watson and Hoshi,1985; crayfish, Wilcox and Lange, 1995; Bishop et al.,1991). This diversity of hormone-induced effects may represent true differences in response from animal to animal or it may merely reflect the focus of the different investigations.

It is known that PR increases contractile force in semi-isolated and intact crab and lobster heart (Wilkens and Mercier, 1993; Saver and Wilkens, 1998; Wilkens and Kuramoto, 1998). This could arise from actions of the peptide on the cardiac ganglion or directly on the myocardium. In the present study, we have used the valve muscles of the six ostia of the lobster heart (the orbicularis ostii muscle or OOM) as a model system(Yazawa et al., 1999) to study of the mode(s) of action of PR and F₂ on the myocardium itself. We have already reported on the role of sarcolemmal and sarcoplasmic reticular(SR) Ca²⁺ fluxes in excitation-contraction coupling in this ostial muscle preparation (Shinozaki et al.,2002). We now report on hormonal effects on Ca²⁺dynamics at the level of the sarcolemma and SR. The time course of the peptide-induced responses point to the possible role of second messengers, but this will be the subject of a future study.

Ostial valve muscles (OOM) dissected from lobster (Homarus americanus H. Milne Edwards 1837) hearts, as previously described(Yazawa et al., 1999), were used on the same day or up to five days when stored at 4°C in saline. During experimentation an OOM was pinned in a narrow Sylgard-lined chamber(1.5 ml) and superfused with saline at 2 ml min^-1 at 12-13°C. The lobster saline used for dissection and perfusion contained (in mmol l^-1) 530 NaCl, 10.7 KCl, 18 CaCl₂, 24.6 MgSO₄, 6 Tris buffer, equilibrated with air and adjusted to pH 7.6 at 20°C. Ostia were electrically stimulated via a pair of laterally placed Ag-AgCl electrodes by train stimulation of 1 or 2 ms pulses delivered at 20-30 Hz, 300 ms train duration and train rate of 0.05 or 0.1 train s^-1. Stimulus trains produced tetani(Yazawa et al., 1999). All dissections complied with the guidelines of the Canadian Council for Animal Care.

Membrane potential was measured by hanging or dog-leg shaped(Fedida et al., 1990)microelectrodes filled with 3 mol l^-1 KCl, tip resistance of 10-30 MΩ. Hyperpolarizing 5-15 nA current pulses of 100 ms were used to estimated the input resistance of the fibers. Force was measured isometrically by means of a Pixie (Endevco Corp., San Juan Capistrano, CA) or SensoNor AE801(AME, Sensonor, Horten, Norway) force transducer mounted on a micromanipulator(Yazawa et al., 1999). The transducer was moved to pull slack out of the valve leaflets. The force recorded by this initial stretch is referred to as basal tonus. Data were displayed on an oscilloscope and chart recorder during initial experiments and on an analog-to-digital data acquisition system (Chart4; ADInstruments,Toronto, Ontario, Canada) for later experiments.

Several drugs which are known to modify components of excitation-contraction coupling were tested both for their intrinsic effects on resting tension and active force development, and for their ability to modify the effects of PR (Sigma, St Louis, MO, USA) and F₂ (gift from Ian Orchard, Toronto). We used drugs that act at the sarcolemma L-type Ca²⁺ channels (nifedipine and verapamil; Sigma) and Cd²⁺(added to normal saline) and a T-channel blocker mibefradil dihydrochloride(Hoffmann-LaRoche Ltd, Basel, Switzerland). Two drugs that affect the SR were also tested (ryanodine and caffeine, Sigma). Ryanodine stock (5 mmol l^-1 in water) was diluted to 10 μmol l^-1, a concentration known to lock the ryanodine receptor (RYR, SR Ca²⁺-release channel) in a state of sub-maximal conductance and blocks Ca²⁺-induced Ca²⁺ release (CICR). It took 50 min exposure to ryanodine to reach a steady state where force was reduced by 82%from control. Caffeine (10 μmol l^-1) was applied to cause Ca²⁺ release from the SR (Lea,1996). Caffeine (dissolved in saline) directly gates the ryanodine sensitive SR Ca²⁺ release channels. Joro spider toxin (JSTX; Wako Pure Chemical Industries, Osaka, Japan) was used to block glutamate postsynaptic receptors. PR and F₂, when applied during the perfusion of one of the modified salines, were diluted in that modified saline. Hormones and other drugs were added by switching the perfusion pump source from control saline to one containing the desired concentration of hormone.

Both peptide hormones PR and F₂ caused increases in basal tonus(contracture) and the amplitude of tetani in a concentration-dependent manner. During continuous perfusion for a minimum of 4 min at threshold concentration(PR, 1-10 pmol l^-1; F₂, 10-100 pmol l^-1) each peptide augmented tetani with little or no effect on basal tonus(Fig. 1A,B). When the two peptides were applied together, each at its threshold concentration, the amplitude of tetani was greater (20.2%, N=3) than that during exposure to either peptide alone (PR 11.6%, F₂ 17.3%). At higher concentration each peptide produced a concentration-dependent contracture(with rare exceptions as illustrated in Fig. 1E) in addition to augmenting tetani following the contracture(Fig. 1C,D). Contracture occurred at peptide concentrations as low as 10 pmol l^-1 and 0.1 nmol l^-1 for PR and F₂, respectively. During a contracture the tetani were reduced, although the amplitude from the pre-trial baseline was up to sixfold greater than that prior to the hormone application. Both the rate of onset and of recovery of the peptide effects on tetani and contractures were similar (Fig. 1). Tetani remained augmented for up to 25 min beyond the contracture period following a 30 s exposure to a peptide. The amplitude and duration of the contracture and the period of time during which tetani were enhanced were proportional to the concentration and duration of the peptide application. During continuous perfusion the contracture lasted as long as the peptide was present, up to 40 min in lobster OOM (data not shown) and up to 15 min following 30 s exposure to a peptide. To limit the duration of responses,peptides were usually applied for 30 s, but this required much higher bath concentrations for any response, typically 1 μmol l^-1 compared with <1 nmol l^-1 during prolonged applications. Both peptides increased both the rate of contractile force development and relaxation(Table 1). On average, the responses to F₂ were greater and of longer duration than those to PR when both were applied at the same concentration(Fig. 1E).

Glutamic acid (10 mmol l^-1) also induced strong contractures,but, in contrast to responses to peptides, tetani following the contracture were depressed for up to 15 min (Figs 1F and 8D) and the rate of onset and recovery of the effect of glutamate was always rapid.

Occasionally an OOM showed spontaneous oscillations in membrane potential and force following PR or F₂ (Figs 1D and 2). These oscillations could disappear in a few minutes, as in the traces shown, or occasionally persist for hours and lead to a small contracture. OOM which displayed continuous large amplitude oscillations were discarded.

The resting membrane potential of fibers varied from -30 to -80 mV(Table 2). Threshold concentrations of PR and F₂ that augmented tetani but did not produce contracture (1 pmol l^-1 and 10 pmol l^-1,respectively) did not change the resting membrane potential. Fibers were depolarized by up to 45 mV during exposure to peptide concentrations that produced contracture (Fig. 2, Table 2). The duration of the depolarization corresponded to that of the contracture. The resting membrane potential, but not amplitude of the tetanus, returned to near control level by the end of the contracture period.

The input resistance (R_input) of fibers exposed to PR,F₂ or glutamic acid did not change significantly from the control values at the time of peak contracture or during the period of augmented tetani that followed. (Fig. 2, Table 2).

Since the distal portions of the motoneurons of the cardiac ganglion and their presynaptic terminals on the OOM could have been depolarized by the electrical stimuli, it was necessary to determine whether the evoked tetani or the hormone responses were due, at least in part, to activation of the nerve terminals. Perfusing OOM with JSTX (5 μmol l^-1, 10 min minimum)blocked glutamic acid-induced (10 mmol l^-1) contractures, but did not alter the amplitude of stimulated tetani in control saline nor did it prevent PR- and F₂-induced contracture and enhanced tetani (data not shown).

Dopamine (N=4) and 5-hyroxytryptamine (5-HT or serotonin, N=8) (0.1 μm to 1.0 mmol l^-1) exert strong chronotropic effects on intact crab and lobster hearts(Wilkens and Mercier, 1993; Wilkens and McMahon, 1994; Wilkens and Kuramoto, 1998),presumably by their actions on the cardiac ganglion. Neither amine affected basal tonus or tetani of OOM (data not shown).

The amplitude of tetani in saline was reduced at[Ca²⁺]_o 2 mmol l^-1, but not at[Ca²⁺]_o >6 mmol l^-1 (data not shown). The absolute magnitude of the peptide-enhanced tetani, where amplitude was normalized relative to control conditions, also decreased with reduced[Ca²⁺]_o. PR had no effect at[Ca²⁺]_o 2 mmol l^-1 while F₂augmented the tetani. At higher [Ca²⁺]_o both peptides increased tetani (Fig. 3).

Short exposure to PR at 1 nmol l^-1 and higher caused an increase in [Ca²⁺]_i and force following each depolarization compared with control conditions (Fig. 4), while PR at 10 nmol l^-1 and 1 μmol l^-1 caused a sustained increase in resting[Ca²⁺]_i and a dose-dependent contracture. Unfortunately,F₂ was not tested at the time of the fura-2-based [Ca]_imeasurements.

The force/pCa²⁺ relationship during relaxation of tetani is assumed to reflect the Ca²⁺-sensitivity of the contractile apparatus (Ashley et al.,1993). The force/pCa curves for drug-free conditions and after addition of PR are illustrated in Fig. 5. The recovery portion of these curves is shifted to the right following exposure to PR at both 0.01 (data not shown) and at 1 μmol l^-1.

The increased [Ca²⁺]_i following hormone treatment could come from either or both an increased influx across the sarcolemma and an increased storage and release from the SR. We tried to distinguish between these two possibilities by applying PR or F₂ in the presence of the sarcolemmal L-type Ca²⁺ channel blockers Cd²⁺,nifedipine, and verapamil and the T-type Ca²⁺ blocker mibefradil,as well as in Na⁺-free saline, which blocks the Na⁺/Ca²⁺ exchanger. The efficacy of the peptides was also tested during treatment with ryanodine, which blocks SR Ca²⁺release, and caffeine, which stimulates the release of SR Ca²⁺.

Verapamil (N=3) and nifedipine (N=2) alone (each at 0.1 mmol l^-1) caused a 10% attenuation of tetani (P<0.05, N=5). These L-type Ca²⁺ channel blockers reduced but did not prevent the peptide-induced contracture followed by increased tetani (10 nmol l^-1 to 1 μmol l^-1; Fig. 6A,B). Contracture amplitude in the presence of verapamil and nifedipine was reduced in three experiments and slightly increased in three other trials compared with that in normal saline. Cd²⁺ (1 mmol l^-1) substantially attenuated tetani (70-95%), but did not block peptide-induced contracture(Fig. 6C). These blockers did not prevent caffeine-induced (10 mmol l^-1) contracture (data not shown). When the peptides were applied a threshold concentration, verapamil(0.1 mmol l^-1) and Cd²⁺ (10 μmol l^-1)attenuated the tetani by 10% for PR and 26% for F₂ (three trials, N=2). Mibefradil had no effect on OOM or on peptide-induced responses. Na⁺-free saline caused contracture. Na⁺-free saline reduced the amplitude of, but did not block, peptide contractures(Fig. 7). Peptide induced contractures were also observed in unstimulated OOM(Fig. 8B,C).

SR Ca²⁺ sequestration could account for both the slow onset and recovery of the peptide-induced potentiation of tetani. The increase in[Ca²⁺]_i during a contracture could, by up-regulating the SR Ca²⁺-ATPase pumps (SERCA, Timmerman, in Ashley et al., 1993), increase the amount of releasable Ca²⁺ from the SR and facilitate subsequent tetani. As expected, prolonged electrical stimulation potentiated the brief tetani after the period of prolonged stimulation(Fig. 8A). This potentiation decayed in less than a minute. To study the effects of Ca²⁺sequestration by the SR, we applied pulses of caffeine (10 mmol l^-1, 30 s) before and immediately after PR- and F₂-induced contractures (Fig. 8B,C). The caffeine-contractures following the peptide-induced contracture were indeed augmented by 32% by PR and 54% by F₂. The caffeine-induced contracture following the glutamic acid-induced contracture(Fig. 8D) was 11% smaller than the one before.

Continuous perfusion of caffeine (10 mmol l^-1), which opens the RYR of the SR continuously so that sequestered Ca²⁺ immediately returns to the cytosol, depleted the SR Ca²⁺ store in about 8 min and this dramatically reduced or abolished tetani(Fig. 9). In the presence of caffeine PR and F₂ induced faster and stronger contractures(Fig. 9). OOM recovered completely after their return to normal saline, and tetani recovered rapidly following a peptide contracture when caffeine had been withdrawn prior to the end of the peptide contracture (Fig. 9B).

We have previously shown that sarcolemmal Ca²⁺ entry induces Ca²⁺-induced Ca²⁺ release (CICR, Shinozaki et al., 2002). Ryanodine (10 μmol l^-1, a concentration that locks SR Ca²⁺ release channels in the open state) by itself caused an elevation in [Ca²⁺]_i and an increase in basal tonus (0 time levels in Fig. 10C,E). Continuous treatment with ryanodine eliminated most of the PR-augmented increase in [Ca²⁺]_i and force during stimulation, but it did not prevent the dose-dependent PR-induced increase in[Ca²⁺]_i and contracture. Ryanodine treatment abolished caffeine-induced contracture (data not shown).

The effects of PR and F₂ on force generation by the OOM are similar, even though they are structurally distinct molecules. We argue that each of these hydrophilic peptides binds to distinct OOM sarcolemmal receptors and their effects are partially additive. Our hypotheses are: (1) that at low concentration the peptides increase the current through voltage-gated L-type Ca²⁺ channels; and (2) that at higher peptide levels they directly or indirectly activate different sarcolemmal Ca²⁺ entry mechanisms via a lower affinity receptor. At the time of the fluorescence studies of [Ca²⁺]_i we only tested for the effect of PR,but we assume that F₂ produces similar Ca²⁺ responses,because the force responses to these two peptides are so similar.

At threshold concentration each neuropeptide increases tetani without a change, or at most a small increase, in basal tonus. We argue below that these effects arise from peptide modulation of the voltage-gated Ca²⁺channels. The threshold concentration found here was as low as or lower than the values reported in the literature(Mercier et al., 2003; Kobierski et al., 1987; Erxleben et al., 1995; Lange, 2002). The response to threshold concentrations of the peptides combined was less than the sum of the responses to the individual peptides. The lack of full additive effects would be expected if each peptide activated the same or a similar pathway. The relatively slow onset and recovery, requiring several minutes, of the heightened tetani may suggest involvement of second messenger pathways. Conversely, SR sequestration of Ca²⁺ that enters the cells owing to the peptides effects must play an important buffering role in the time course of the response because caffeine exposure dramatically accelerated the contractures (Fig. 9).

At higher concentration each peptide produces a prominent contracture possibly by activating a lower affinity receptor-mediated Ca²⁺entry mechanism. During contractures, the tetani are reduced. Since[Ca²⁺]_i is elevated during the contracture period, the smaller tetani probably indicate that the fibers are near the upper limit of their [Ca²⁺]_i and of their force generating ability. Since the responses last a long time after a brief exposure to peptide there may be strong receptor binding and a slow off rate. Similar response durations are seen in Aniculus (hermit crab) hearts (T.Y., unpublished). These effects on tonus and tetani may be widespread in arthropod muscles (force in Limulus myocardium, Grome et al., 1994; force and Ca²⁺ in barnacle, Griffiths, 1990;lobster heart, Wilkens and Mercier,1993; crab skeletal muscle, Mercier and Wilkens, 1985;FLPs reviewed by Mercier et al.,2003). Receptor desensitization to prolonged exposure to PR is minimal.

PR and F₂ at contracture-inducing concentrations depolarize the muscle cells by up to 45 mV from resting membrane potential as low as -80 mV. The magnitude and duration of this depolarization is proportional to the contracture and may be responsible for it. Similar results to PR have been found in Aniculus (hermit crab) and Bathynomus (isopod)heart (T.Y., unpublished).

PR increases the [Ca²⁺]_i following each depolarizing stimulus train. The potentiated tetani following PR exposure could arise directly from increased Ca²⁺ entry or indirectly by enhancing Ca²⁺ entry as a consequence of decreasing K⁺ currents. We did not attempt to measure or modify potassium currents, but PR can reduce K⁺ currents in neurons of the cardiac ganglion(Sullivan and Miller, 1984)and in other muscles (locust, Walther et al., 1998; isopod, Erxleben et al., 1995).

The rate of rise and fall of force of the tetani is increased in the presence of PR and F₂. PR, and presumably F₂, increases the rate of Ca²⁺ accumulation in and removal from the cytoplasm. Increased SR Ca²⁺ loading would, in turn, increase the rate of Ca²⁺ release as a result of an increased diffusion gradient from the SR into the sarcoplasm. Increased [Ca²⁺]_i may increase the activity of the SERCA, an action that would increase the rate of removal (Shinozaki et al.,2004).

Some OOM exhibited spontaneous or myogenic membrane potential and force oscillations. The peptides and glutamic acid often induced periods of myogenic oscillation, similar to such peptide-induced myogenic activity observed in other arthropod muscles (shrimp, Meyrand and Marder, 1991; cockroach, Fuse and Orchard, 1998; Limulus, Watson and Hoshi,1985; locust, Evans,1984; Steele et al.,1997). Peptide-induced membrane potential oscillations were always associated with these mechanical oscillations. This observation allows for a plethora of mechanisms that we have not pursued during this study.

We felt it necessary to eliminate the possibility that the peptides were augmenting tetani by increasing neurotransmitter release from nerve terminals. PR should increase presynaptic transmitter release since it increases Na⁺ and decreases K⁺ conductances(Sullivan and Miller, 1984; Walther et al., 1998; Freschi, 1989; Golowasch and Marder, 1992; Erxleben et al., 1995) and FLPs increase transmitter release from lobster and crayfish motoneuron terminals(Worden et al., 1995; Mercier et al., 2003). The cardiac ganglion motoneurons appear to be glutamatergic in lobsters(Anderson, 1973) and a variety of other crustaceans (see Sakurai and Yamagishi, 1996; Yazawa et al., 1998). Field stimulation near the threshold for muscle activation by electrodes parallel to the muscle is unlikely to stimulate nerve terminals. Furthermore, JSTX blocks responses to bath applied glutamic acid but does not reduce electrically evoked tetani or the responses to PR or F₂. This indicates that the evoked tetani arise from direct stimulation of the muscle fibers rather than from activation of the nerve terminals and that PR and F₂ are acting directly on the myocytes rather than modulating transmitter release (also in crayfish, Quigley and Mercier,1997).

The input resistance of fibers was little changed from control both during the contracture (glutamic acid, PR and F₂) and during the subsequent period of augmented tetani by PR or F₂. That glutamic acid fails to alter R_input is puzzling since it is known to depolarize insect and other crustacean muscle fibers and decrease input resistance (Usherwood, 1967;Aonuma et al., 1998). During prolonged exposure of insect muscle to glutamic acid the initial fall in R_input recovers toward the control level during the first minute of exposure, presumably due to desensitization of the glutamate receptors(Usherwood, 1967). Some FLPs modestly increase the input resistance in deep abdominal extensor muscles of crayfish while others do not, and they do not alter input resistance in lobster opener muscle or Cancer borealis muscles associated with the stomach (reviewed by Mercier et al.,2003). Presumably, the effects of the peptides on intracellular calcium could be produced either by increasing Ca²⁺ conductance and/or decreasing K⁺ conductance. If both occur simultaneously, it is possible that the net effect would be little change in input resistance. Although this may not be a parsimonious explanation, there is no reason to exclude it. Activation or inactivation of membrane carriers can lead to substantial changes in input resistance (e.g. Spanswick, 1972). The main carrier-mediated transport systems that may be affected by the peptides,Ca²⁺-ATPase and the Na⁺/Ca²⁺ exchanger,transport Ca²⁺ out of the cell. Inhibition of these carriers by the peptides would increase input resistance in the absence of any other conductance changes; however, it does not appear that the peptides block the Na⁺/Ca²⁺ exchanger since they still cause contracture in Na⁺-free saline. We have not ruled out that the peptides may enhance Na/Ca²⁺ exchange and thereby induce Ca²⁺ entry into the cell. This puzzle is the subject of ongoing study.

External Ca²⁺ is required for PR-induced effects on arthropod muscles (Wilcox and Lange,1995; Wegener and Nassel,2000; Rathmayer et al.,2002). Reducing [Ca²⁺]_o reduces tetani in OOM (Shinozaki et al., 2002)and reduces the responses to PR and F₂. At the lowest[Ca²⁺]_o, tested here, tetani were reduced by 50% and the contraction-enhancing effect of PR, but not F₂, was almost totally eliminated.

At threshold concentration, PR and F₂ appear to increase only voltage-sensitive Ca²⁺ entry into the cell since there is no increase in tonus. This is consistent with previous studies on insect muscle(Wegener and Nassel, 2000)This observation would impute the L-type Ca²⁺ channel as the high affinity receptor. This increased Ca²⁺ current will augment CICR from the SR and increase Ca²⁺ uptake by the SR by increasing the activity of SERCA. We have shown previously(Yazawa et al., 1999) that tetani of the OOM are initiated by action potentials of up to ∼60 mV,which is sufficient to activate crustacean voltage-Ca²⁺ current dependent (Tazaki and Cooke,1986) and hence induce Ca²⁺ release from the SR(Shinozaki et al., 2002). The peptides would increase the rate of rise, the rate of recovery and the amplitude of tetani by increasing CICR and SERCA.

At higher concentration PR causes an increase in the Ca²⁺transient and the resting [Ca²⁺]_i, membrane depolarization and a contracture. These contractures are not significantly reduced by the I_Ca(v) antagonists Cd²⁺, nifedipine and verapamil (Tazaki and Cooke,1986; Maunier and Goblet,1987; Shinozaki et al.,2002). The shift of the force pCa curves suggests a decreased sensitivity to Ca²⁺_i after exposure to PR; however, it is recognized that shifts during hormone exposure may also indicate that the hormone is having effects in addition to those controlling[Ca²⁺]_i alone(Brustle et al., 2001). The responses of tetani to higher concentrations of F₂ are similar to those to PR and, although we did not measure intracellular [Ca²⁺]in the presence of F₂, we assume that there are parallel changes in[Ca²⁺]_i. A sustained Ca²⁺ influx may account for the depolarization that accompanies the contracture. However, we have no data that would help identify the system that carries this Ca²⁺entry. These peptides do not seem to cause contracture by blocking the sarcolemmal Na⁺/Ca²⁺ exchanger (see above). It is also unlikely that the peptides induce contracture by enhancing depolarization dependent Na⁺ entry and subsequent Na/Ca²⁺ exchange,because their effect is still present in the absence of stimulation.

The peptide-induced contracture and increase in[Ca²⁺]_i presumably is caused by a sarcolemmal Ca²⁺ influx because it was not blocked by blocking CICR with ryanodine (Rousseau et al.,1987; Sitsapesan et al.,1991; Hwang et al.,1987) or depleting SR Ca²⁺ stores by caffeine. Both caffeine and ryanodine dramatically attenuates tetani as has also been observed in insect muscle (Wegener and Nassel, 2000), but do not prevent peptide induced contracture indicating that the peptide induced induced sarcolemmal Ca²⁺ influx is enough to directly activate contraction in the absence of SR Ca²⁺ release.

Caffeine-induced contractures are greater following a PR or F₂contracture than before. Since caffeine directly stimulates SR Ca²⁺release (Bianchi, 1962; Lea, 1996), the larger caffeine contractures following peptide pulses indicate that the releasable SR Ca²⁺ pool has been increased, similar to the potentiation of tetani following a prolonged tetanus. The increased influx of Ca²⁺ during peptide contractures is likely to lead to increased uptake of Ca²⁺by the SR via SERCA, thereby increasing the releasable pool of SR Ca²⁺. This would also explain the augmented tetani after the contracture; however, glutamic acid-induced contracture, which should increase Ca²⁺ influx, does not augment the tetani. The difference between the effect of the peptides and the effect of glutamate may reside in differences of the degree of filling of the SR and the degree of desensitization of the contractile filaments. [Ca²⁺]_irises quickly and falls rapidly in a stepwise fashion with glutamic acid. Such a [Ca²⁺]_i increase is known to increase the EC₅₀ of the F-pCa relationship(Shinozaki et al., 2004). If the EC₅₀ decreases slowly after a stepwise decrease in[Ca²⁺]_i without a decline of SR Ca²⁺ release,one may expect an undershoot of force, as is seen following glutamic acid contracture. Other neuropeptide-induced processes may be involved; for example, PR may modulate other intracellular components such as the phosphorylation of a 30 kDa protein associated with thin filaments in an isopod (Brustle et al.,2001).

Continuous perfusion with caffeine saline depletes the SR of Ca²⁺ and eliminates tetani; however, this accelerates and enhances peptide-induced contractures. This is consistent with the proposal that the low affinity receptor effect of each peptide is to enhance net Ca²⁺entry into the cells and that normal SR Ca²⁺ sequestration blunts the response to peptide-induced Ca²⁺ entry.

The force-pCa relationships show that PR augments force by increasing the[Ca²⁺]_i. However, the sensitivity of the contractile components to Ca²⁺ is actually reduced at this time. This decrease in myofibrillar responsiveness to Ca²⁺ following a period of elevated [Ca²⁺]_i, induced here by PR treatment, has also been observed following prolonged tetanic stimulation of OOM(Shinozaki et al., 2004). In mammalian cardiac muscle such [Ca²⁺]_i increase is known to cause decreases in pH and protein phosphorylation, both of which decrease Ca²⁺ sensitivity of the contractile apparatus(Hoerter et al., 1986).

In conclusion, our data are consistent with the hypothesis that the high affinity inotropic effects of proctolin and F₂ arise from modulation of voltage-gated Ca²⁺ channels and the low affinity effects are mediated by activation of ligand-gated Ca²⁺transporters in the sarcolemma. Even though the responses may be reduced in amplitude by some sarcolemmal Ca²⁺ channel blockers, the contracture and augmentation of tetani is still present and qualitatively similar to the responses in normal saline. Continuing studies are investigating the possibility that these peptide hormones may modulate intracellular signal transduction second messenger pathways. Overall, in vivo the dual effects of PR and F₂ to increase heart rate by acting on the cardiac ganglion (Wilkens and Mercier,1993; Wilkens and Kuramoto,1998) and to increase contractile force will have a dramatic effect on cardiac output.

We thank E.-M. Gutknick and P. Weber for advice on the use of mibefradil. This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (J.L.W.), the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada(H.E.D.J.t.K.).