Since the POs are located in the pericardial sinus just outside the heart (Maynard, 1960) and are known to release cardioactive hormones (Cooke and Sullivan, ...
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The Journal of Experimental Biology 198, 109–116 (1995) Printed in Great Britain © The Company of Biologists Limited 1995
PHYSIOLOGICAL EFFECTS OF TWO FMRFamide-RELATED PEPTIDES FROM THE CRAYFISH PROCAMBARUS CLARKII MARTHA SKERRETT*, AMY PEAIRE, PATRICIA QUIGLEY AND A. JOFFRE MERCIER† Department of Biological Sciences, Brock University, St Catharines, Ontario, Canada L2S 3A1 Accepted 23 August 1994
Summary The present study examined the effects of two recently identified neuropeptides on crayfish hearts and on neuromuscular junctions of the crayfish deep abdominal extensor muscles. The two peptides, referred to as NF1 (Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2) and DF2 (Asp-ArgAsn-Phe-Leu-Arg-Phe-NH2), increased the rate and amplitude of spontaneous cardiac contractions and increased the amplitude of excitatory junctional potentials (EJPs) in the deep extensors. Both effects were dosedependent, but threshold and EC50 values for the cardiac effects were at least 10 times lower than for the deep extensor effects. The heart responded equally well to three sequential applications of peptide in any given preparation, but the responses of the deep extensors appeared to decline with successive peptide applications. The results support the hypothesis that these two neuropeptides act as
neurohormones to modulate the cardiac and neuromuscular systems in crayfish. Quantal synaptic current recordings from the deep extensor muscles indicate that both peptides increase the number of quanta of transmitter released from synaptic terminals. Neither peptide elicited a measurable change in the size of quantal synaptic currents. NF1 caused a small increase in muscle cell input resistance, while DF2 did not alter input resistance. These data suggest that DF2 increases EJP amplitudes primarily by increasing transmitter release, while the increase elicited by NF1 appears to involve presynaptic and postsynaptic mechanisms. Key words: synaptic modulation, FMRFamide, crustacean, neuromuscular, cardiac, neurohormone, crayfish, Procambarus clarkii.
Introduction Since the neuropeptide FMRFamide (Phe-Met-Arg-PheNH2) was originally isolated from the bivalve mollusc Macrocallista nimbosa (Price and Greenberg, 1977), a large family of FMRFamide-related peptides (FaRPs) has been shown to be widely distributed throughout the animal kingdom (Greenberg and Price, 1983; Price and Greenberg, 1989). Such peptides are reported to elicit numerous physiological effects, such as cardioexcitation and/or cardioinhibition (e.g. Li and Calabrese, 1987; Cuthbert and Evans, 1989; Mercier and Russenes, 1992; Painter and Greenberg, 1982; Watson et al. 1984), modulation of intestinal muscle (e.g. Holman et al. 1986; Robb et al. 1989; Groome et al. 1992), modulation of exoskeletal muscle contraction (e.g. Evans and Myers, 1986; Mercier et al. 1990; Pasztor and Golas, 1993) and modulation of chemical synaptic transmission (e.g. Watson et al. 1984; Walther and Schiebe, 1987; Mercier et al. 1990). In crustaceans, the pericardial organs (POs) appear to contain the highest levels of FMRFamide-like immunoreactivity in the entire nervous system (Kobierski et al. 1987; Krajniak, 1991; Mercier et al. 1991), suggesting that crustacean FaRPs act as
neurohormones. So far, five distinct neuropeptides have been isolated from crustacean POs, one from a brachyuran and four from macrurans. The brachyuran peptide, isolated from the crab Callinectes sapidus, has the sequence GYNRSFLRFamide (Krajniak, 1991). The macruran peptides include two from the lobster Homarus americanus (Trimmer et al. 1987), with the sequences TNRNFLRFamide (F1) and SDRNFLRFamide (F2), and two from the crayfish Procambarus clarkii (Mercier et al. 1993), with the sequences NRNFLRFamide (NF1) and DRNFLRFamide (DF2). These appear to be the predominant FaRPs in crustacean POs, based on radioimmunoassays, and they have in common the carboxy-terminal sequence RXFLRFamide, where X is either Asn or Ser. Effects of crustacean FaRPs on presumptive target organs have been reported briefly in several papers (Mercier et al. 1990, 1993; Krajniak, 1991; Kravitz et al. 1987), but a thorough investigation of their physiological effects and sites of action has been lacking. The primary aim of the present study was to investigate the possible physiological actions of the crayfish peptides NF 1 and DF2; an accompanying paper (Worden et al.
*Present address: School of Biological Sciences, Flinders University, Bedford Park, South Australia 5042, Australia. †Author for correspondence.
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1995) examines the physiological effects of the related lobster peptide F1. Since the POs are located in the pericardial sinus just outside the heart (Maynard, 1960) and are known to release cardioactive hormones (Cooke and Sullivan, 1982; Kravitz et al. 1980), the heart is an obvious candidate as a physiological target. In addition, most of the neurohormones released from the POs modulate chemical synaptic transmission and/or muscle tension (e.g. Battelle and Kravitz, 1978; Cooke and Sullivan, 1982; Kravitz et al. 1980). The results indicate that both NF1 and DF2 are cardioexcitatory, that they enhance synaptic transmission at a neuromuscular synapse and that the latter effect results, at least in part, from actions on presynaptic nerve terminals. Materials and methods Male and female crayfish (Procambarus clarkii) were obtained commercially and were maintained in aerated freshwater tanks at 15 ˚C on a mixed vegetable diet. The animals measured approximately 6–9 cm in length, weighing between 3 and 5 g. Approximately 90 crayfish were used. Cardiac experiments Crayfish hearts were dissected as described by Mercier and Russenes (1992) and were placed in a wax-bottomed chamber. The chamber was perfused with normal crayfish saline of the following composition (in mmol l21): Na+, 205; Cl2, 242.3; K+, 5.3; Ca2+, 13.5; Mg2+, 2.5; Hepes, 5.0 (van Harreveld, 1936). Saline pH was adjusted to 7.4 prior to use. The bath was perfused at a rate of 3 ml min21. Peptides were applied by changing the perfusate to a solution containing a selected peptide concentration. Cardiac contractions were recorded using a tension transducer attached to the sternal artery. Signals were displayed on a Grass model 7B polygraph and were also stored on VHS tape using a Neurocorder VHS adaptor. Recorded signals were replayed through an analog-to-digital converter (TL-1-125 interface, Axon Instruments, Inc.) for subsequent analysis. Only hearts with an initial rate of between 15 and 55 contractions min21 were used. Neuromuscular experiments The deep abdominal extensor muscles were exposed by cutting away the lower half of the isolated abdomen and all the flexor musculature, as described by Parnas and Atwood (1966). Segments 2, 3, 4 and 5 were pinned ventral side uppermost in a Sylgard-lined dish. The preparation was perfused with a lowCa2+, high-Mg2+ saline (Mercier and Atwood, 1989) to prevent muscle twitching. The perfusate contained (in mmol l21): Na+, 200.7; K+, 5.4; Ca2+, 6.8; Mg2+, 12.3; Cl2, 244.3; Hepes, 5 (pH 7.4) and was maintained at 13–15 ˚C. Peptides were applied in the same manner as in the cardiac experiments (see above). Experiments on EJPs were performed at 13–15 ˚C; the temperature was maintained within 1 ˚C in any given experiment using a refrigerated bath circulator. As in previous studies (Mercier and Atwood, 1989; Mercier
et al. 1990, 1993), an excitatory axon in the third segment was stimulated and EJPs were recorded from the L1 muscle in segment 4. The nerves to the medial muscles in segment 4 were cut to prevent muscle twitches. The axon was stimulated at 0.1 Hz, which minimized both synaptic depression and facilitation (Mercier and Atwood, 1989). EJPs were recorded using a WPI model Cyto 721 electrometer. For analysis, the signals were digitized with an Axon Instruments TI-1 DMA interface and were analyzed at a sample rate of 10 kHz using a program (provided by Dr M. Charlton, University of Toronto) that averaged EJPs and calculated their peaks. Except where indicated, all EJP amplitudes were corrected for non-linear summation (Martin, 1955), assuming a reversal potential of +11.5 mV (Onodera and Takeuchi, 1975). Excitatory postsynaptic currents (EPSCs) were recorded with ‘macropatch’ recording electrodes with tip openings of 10 mm diameter, as described elsewhere (Mercier and Atwood, 1989). Recording sites were found by randomly probing the surface of the muscle fiber and gently pressing the electrode onto the surface to improve the seal between the electrode and the muscle. Damaged sites were recognized by the high rate of spontaneous release and were not used. The small synaptic currents were detected using a virtual ground circuit consisting of an inverter with gain. Input resistances were measured using two intracellular electrodes. Current was injected through a WPI model 721 electrometer and membrane potential changes were recorded with a WPI model 7040 electrometer. Input resistance was determined from the slope of voltage versus current plots. The preparation was bathed in low-Ca2+, high-Mg2+ saline and the temperature was maintained at 7.5–9 ˚C. Lowering the temperature slowed the time course of transmitter release so that individual quantal currents were easier to count (Zucker, 1973). In seven of seven trials with NF1 and four of seven trials with DF2, 3 mmol l21 Mn2+ was added to the bath to lower transmitter release. This increased the percentage of failures of release to about 60–80 %, which made counting individual quanta more reliable. EPSCs were acquired and analyzed using the same program as for EJPs. The quantal content (m) was determined for every fifty stimuli before and throughout peptide exposure, using the equation: m = ∑n/N , where n is the number of quanta per stimulus and N is the total number of stimuli. Statistical significance was determined using a Wilcoxon signed-rank test for two correlated samples (Ferguson, 1971). Chemicals Peptides NF1 and DF2 were synthesized by T. S. Chen of the Biotechnology Service Centre, Banting and Best Institute, Toronto, Canada. All other chemicals were obtained from BDH Inc. Results Both NF1 and DF2 increased the rate and amplitude of
Effects of FMRFamide-related peptides on crayfish
111
DF2 1 mN 20 s
NF1 Fig. 1. Sample recordings of cardiac contractions, showing the effects of NF1 and DF2, each at a concentration of 1028 mol l21. Each peptide was applied throughout the time indicated by the horizontal bar.
200
A
Change in rate (%)
175 150
NF1 DF2
125 100 75 50 25 0 10−11 10−10
10−9
10−8
10−7
10−6
10−5
10−6
10−5
[Peptide] (mol l−1)
Change in amplitude (%)
spontaneous cardiac contractions (Fig. 1). The inotropic effect (i.e. the increase in amplitude) and the chronotropic effect (i.e. the increase in rate) were completely reversed when the peptides were washed out of the recording chamber, usually within 10–20 min. Logarithmic dose–response curves (Fig. 2) were constructed with cardiac responses expressed as the percentage change in heart rate or in contraction amplitude between a 5 min period prior to peptide application and the peak response during a 5 min exposure to the peptide. Doses were administered in order of increasing concentrations, and each was followed by a wash-out period of 30 min before the next peptide application. NF1 and DF2 showed nearly identical dose–response curves, with threshold concentrations between 10210 and 1029 mol l21. Although the responses were somewhat variable at 1027 mol l21, this peptide concentration appears to be sufficient to elicit maximal inotropic and chronotropic effects. The cardiac effects were fully reversible and repeatable within individual preparations. Fig. 3 illustrates the changes in amplitude and frequency of contractions induced by three successive applications of 1028 mol l21 NF1 in six preparations. In each experiment, the peptide was applied three times for 10 min each, with a 30 min saline rinse between applications. All three applications were equally effective at increasing the rate and amplitude of contractions, and complete recovery occurred during each rinse. Similar results were obtained with six preparations exposed to 1028 mol l21 DF2 (data not shown). To investigate effects on chemical synaptic transmission, EJPs were recorded in one of the phasic abdominal extensor muscles, muscle L1, in response to stimulation of an excitatory axon, axon 3 (Parnas and Atwood, 1966; see Materials and methods). Both peptides increased EJP amplitudes within 5 min (Fig. 4). In about half the preparations examined, recovery was virtually complete after 30 min of washing in saline, but some preparations showed very little recovery after 30 min of washing.
B 225 200 175 150 125 100 75 50 25 0 −25 10−11 10−10
10−9
10−8
10−7
[Peptide] (mol l−1) Fig. 2. Log dose–response curves for the effects of NF1 and DF2 on the frequency (A) and amplitude (B) of cardiac contractions. For NF1, N=4 in all cases. For DF2, N=6, 7, 7, 8 and 7, respectively, for the points from left to right in A and in B. Error bars in this and all other figures indicate ±1 S.E.M.
M. SKERRETT AND % of initial contraction amplitude
300
OTHERS 7
A
EJP amplitude (mV)
112
200
100
5 4 3 2
NF1
NF1
NF1
1 0 S 300
20
40
60
80
100
Time (min)
0
% of initial heart rate
6
P
S
P
S
P Fig. 5. Effect of repeated applications of NF1 on EJP amplitude. In this example, a single neuromuscular preparation was exposed to 231027 mol l21 NF1 during the times indicated by the horizontal bars. Each point is the mean of six EJPs elicited at a stimulus frequency of 0.1 Hz.
DF2 NF1
B
200
100
0 S
P
S
P
S
P
Fig. 3. Effects of repeated applications of 1028 mol l21 NF1 on the amplitude (A) and frequency (B) of cardiac contractions. NF1 was applied three consecutive times to each of six preparations, with a 30 min washout period between each application. All data are expressed as a percentage of the amplitude or frequency recorded before the first peptide application. Each peptide response is the maximal rate or amplitude recorded during a 5 min peptide application. P, peptide application; S, saline control. Values are means + S.E.M.
NF1 5 mV 10 ms
DF2 0 min
4 min
Fig. 4. Sample EJPs recorded from abdominal extensor muscle L1 before (0 min) and 4 min after applications of 1027 mol l21 NF1 and 1027 mol l21 DF2. Each trace is an averaged signal from six consecutive EJPs and has not been corrected for non-linear summation. The recordings were obtained from two different preparations.
Resting potentials fluctuated by only a few millivolts following peptide application, hyperpolarizing in some cases and depolarizing in others; no consistent effect on resting potential was observed. Treatment with 1027 mol l21 NF1 caused the input resistance of muscle cells to increase by 16±3.1 % (N=5 preparations; P0.05). Unlike cardiac responses, effects on EJP amplitude tended to decline with successive peptide applications. An example of this is illustrated in Fig. 5, which shows results from one preparation that was exposed three times to 231027 mol l21 NF1. In three other preparations, EJP amplitudes increased by an average of 47±18 % in response to an initial application of 1027 mol l21 NF1, but two subsequent applications of the same peptide concentration increased EJPs by only 18±6.4 % and 9.1±5.1 %. Because such responses declined with repeated peptide applications, logarithmic dose–response curves for the effects on synaptic transmission (Fig. 6) were constructed using experiments in which peptides were applied only once to each preparation. The threshold for a significant increase in EJP amplitude was between 1028 and 1029 mol l21. The data for NF1 suggest that the maximally effective concentration is near 1026 mol l21, while data for DF2 do not. However, large variations in the responses at 1027 and 1026 mol l21 made it difficult to determine whether maximal effectiveness had been attained. An increase in dose of DF2 and NF1 from 1027 to 1026 mol l21 increased EJP amplitudes by about 40–60 %. Thus, percentage changes in EJP amplitude were more modest than changes associated with cardiac effects and they occurred at higher peptide concentrations. Quantal synaptic currents were recorded in order to determine whether peptide modulation of EJP amplitudes reflected a change in transmitter release. As in other studies (e.g. Johnson and Wernig, 1971; Mercier and Atwood, 1989;
Effects of FMRFamide-related peptides on crayfish 70
DF2 NF1
60
% change in EJP amplitude
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50 40 30 20 10 0 −10
10−11 10−10 10−9
10−8
10−7
10−6
10−5
[Peptide] (mol l−1) Fig. 6. Log dose–response curves for the effects of NF1 and DF2 on EJP amplitudes. For DF2, N=5 in all cases; for NF1, N=4 in all cases except 10 27 mol l21, where N=8.
Table 1. Effects of DF2 and NF1 on mean quantal content Mean quantal content Peptide NF1 DF2
Saline
Peptide
N
Significance*
0.16±0.05 0.48±0.09
0.24±0.05 0.84±0.16
7 7
P0.05). Discussion The present data support the view that the heart and peripheral neuromuscular synapses are physiological targets
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Table 2. Comparison of EC50 values of four FMRFamiderelated peptides from crustacean pericardial organs
A
EC50 (mol l−1)
Saline Peptide
% of observations
60
40
Peptide
Heart rate
Cardiac amplitude
EJP amplitude
NF1 DF2 F1 F2
2×10−9* 6×10−9* 2×10−9† 7×10−10†
4×10−10* 3×10−9* 3×10−9† 2×10−10†
2×10−8* 7×10−8* 2×10−8‡ 3×10−8‡
*Data from present study; †data from Mercier and Russenes (1992); ‡data from Mercier et al. (1990). 20
0 0 100
1
2
3
B
% of observations
80
60
40
20
0 0
1 Number of quanta
2
Fig. 8. Effects of DF2 (A) and NF1 (B) on frequency distributions of quantal synaptic currents. The data shown in A and B are from two different preparations. In each case, quantal distributions were determined from 150 responses over a 5 min period before applying peptide (open columns) and from 400 responses over a 15 min period after applying peptide (filled columns). The peptide concentration was 531027 mol l21 in each case. In these examples, DF2 increased the mean quantal content from 0.15 to 0.65, and NF1 increased quantal content from 0.32 to 1.16.
for modulation by FMRFamide-related peptides in crayfish. Both of these presumptive targets are sensitive to low concentrations of peptides NF1 and DF2, although there are some differences between their response characteristics. The heart is slightly more sensitive than are the neuromuscular synapses and it appears to respond more reliably to repeated peptide applications.
Logarithmic dose–response curves for the effects of NF1 and DF2 are very similar to those reported previously for effects of lobster peptides F1 and F 2 on EJPs in crayfish phasic extensor muscles (Mercier et al. 1990), on crayfish heart (Mercier and Russenes, 1992) and on heart, muscle and neuromuscular synapses of lobster (Worden et al. 1995). EC50 values, estimated from Figs 2 and 6 and from previously published figures (Mercier and Russenes, 1992; Mercier et al. 1990), are listed in Table 2. In general, the EC50 values are about 10–100 times lower for cardiac effects than for effects on EJP amplitude. All four peptides are almost equally effective in enhancing synaptic transmission. EC50 values for cardiac effects are also very similar for the four peptides, which should not be surprising since their amino acid sequences are so alike. Our data are consistent with those of Krajniak (1991), who compared the effects of several peptide analogs on Callinectes sapidus hearts and concluded that full receptor activation would require at least a heptapeptide consisting of the FLRFamide core with Asn immediately adjacent to it. All four peptides used in the present study satisfy this criterion. FaRP amino acid substitutions in the other positions made little difference to peptide effectiveness in Callinectes sapidus (Krajniak, 1991), except that lobster peptide F1 appeared to be slightly more potent than the others. The present data suggest that F2 may be slightly more potent than the other peptides. This may reflect subtle differences in the selectivity of different FaRP receptors. Three lines of evidence suggest that the heart is a primary target organ for peptides NF1 and DF2. First, the heart is physically closest to the site of release, since the peptides are localized in neurosecretory cells within the pericardial sinus (Mercier et al. 1991, 1993). Thus, physiologically effective concentrations are probably reached most rapidly in the pericardial sinus. Second, the threshold for cardiac effects (between 10 210 and 10 29 mol l21) is very close to the level of FMRFamide-like immunoreactive material reported for lobster hemolymph (10211 to 10210 mol l21, Kobierski et al. 1987). In fact, the hemolymph level may be four times higher than that reported, since the radioimmunoassay is less sensitive to crustacean FaRPs than to FMRFamide (Kobierski et al. 1987). Third, when the peptides are presented repeatedly, cardiac responses are nearly equal each time. Thus, the heart maintains
Effects of FMRFamide-related peptides on crayfish its sensitivity and would presumably respond even if the peptides were released in periodic bursts. The neuromuscular junctions associated with the abdominal extensor muscles represent another potential target for modulation by NF1 and DF2. Since threshold concentrations for modulating the EJPs are ten times higher than for cardiac effects, these synapses may require larger amounts of peptide to be secreted before they respond. Since none of the abdominal nerve roots of crayfish contains FMRFamide-like immunoreactivity except for the intestinal nerve, which supplies the hindgut (Mercier et al. 1991), NF1 and DF2 could only reach the abdominal muscles after circulation and dilution in the hemolymph. The decrease in the sensitivity of the neuromuscular synapses to repeated peptide applications has implications for the physiological actions of NF1 and DF 2 at this target site. If the peptides are released into the hemolymph at concentrations sufficient to modulate these synapses, subsequent release of the same peptides within 30 min would presumably be less effective. Repeated secretion of the peptides at intervals of 30 min or less would be expected to elicit progressively smaller effects at the neuromuscular junctions, while the heart would respond strongly each time. The neuromuscular synapses are able to respond to NF1 and DF2 only if their hemolymph concentrations have been negligible for several hours. Such response characteristics are not unique to the phasic extensors, since Worden et al. (1995) report similar desensitization of lobster neuromuscular synapses in response to peptide F1. The ability of NF1 and DF2 to increase quantal content demonstrates that both peptides increase transmitter release from the synaptic terminals. Neither peptide altered the amplitude of quantal synaptic currents, which suggests that the peptides do not change the sensitivity of postsynaptic receptors to neurotransmitter released at the synapses. Although DF2 did not affect input resistance in the postsynaptic cells, NF1 did. The increased input resistance (16 % at 1027 mol l21), however, is not large enough to account for the full effect on EJP amplitude. Thus, NF1 increases the size of EJPs by postsynaptic and presynaptic means. Since DF2 affects neither input resistance nor quantal size, its ability to increase EJP amplitude appears to result from presynaptic mechanisms alone. Interestingly, F2, a lobster peptide homologous to DF2, does not substantially alter input resistance in crayfish muscle cells, although F1, the homolog of NF1, increases input resistance by 15 % (Mercier et al. 1990). This suggests that postsynaptic receptors responsible for the change in input resistance exhibit specificity for an asparagine residue in both the heptapeptide and the octopeptide. Worden et al. (1995), however, report that F1 does not change input resistance in the lobster dactyl opener muscle. This may reflect differences among the receptors of different muscles or of different species. The dramatic effects on heart rate suggest very strongly that both peptides act on the cardiac ganglion, which establishes the cardiac rhythm and drives the myocardial cells via its motoneurons (Maynard, 1960). The inotropic actions of the
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peptides might reflect enhancement of transmitter release from these motoneurons onto the myocardial cells, as at the neuromuscular junctions of the phasic extensors. In addition, the peptides might alter the contractility of the myocardial cells directly. Postsynaptic effects, such as enhancement of muscle contraction and tonus, were not examined in the present study; however, Worden et al. (1995) provide evidence that the lobster peptide F1 acts postsynaptically to enhance muscle tension in the lobster dactyl opener muscle. A third possibility is that the inotropic effect might stem from an increase in motoneuron impulse frequency, which could enhance the postsynaptic response through summation and facilitation. Further work is needed to identify the sites of action of the peptides and to elucidate the intracellular messengers that mediate their effects. This work was supported by a grant to A.J.M. from the Natural Sciences and Engineering Research Council of Canada. We thank Dr M. K. Worden for stimulating discussions and helpful comments on the manuscript. References BATTELLE, B. A. AND KRAVITZ, E. A. (1978). Targets of octopamine action in the lobster: cyclic nucleotide changes and physiological effects in haemolymph, heart and exoskeletal muscle. J. Pharmac. exp. Ther. 205, 438–448. COOKE, I. M. AND SULLIVAN, R. E. (1982). Hormones and neurosecretion. In The Biology of Crustacea, vol. 3 (ed. D. E. Bliss, H. L. Atwood and D. C. Sandeman), pp. 205–290. New York: Academic Press. CUTHBERT, B. A. AND EVANS, P. D. (1989). A comparison of the effects of FMRFamide-like peptides on locust heart and skeletal muscle. J. exp. Biol. 144, 395–415. EVANS, P. D. AND MYERS, C. M. (1986). Peptidergic and aminergic modulation of insect skeletal muscle. J. exp. Biol. 124, 143–176. FERGUSON, G. A. (1971). Statistical Analysis in Psychology and Education, pp. 324–325. New York: McGraw Hill. GREENBERG, M. J. AND PRICE, D. A. (1983). Invertebrate neuropeptides: native and naturalized. A. Rev. Physiol. 45, 271–288. GROOME, J. R., DE TSCHASCHELL, M. AND WATSON, W. H. (1992). Peptidergic regulation of the Limulus midgut. J. comp. Physiol. A 170, 630–643. HOLMAN, G. M., COOK, B. J. AND NACHMAN, R. J. (1986). Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut. Comp. Biochem. Physiol. 85C, 324–333. JOHNSON, E. W. AND WERNIG, A. (1971). The binomial nature of transmitter release at the crayfish neuromuscular junction. J. Physiol., Lond. 18, 757–767. KOBIERSKI, L. A., BELTZ, B. S., TRIMMER, B. A. AND KRAVITZ, E. A. (1987). FMRFamide-like peptides of Homarus americanus: distribution, immunocytochemical mapping and ultrastructural localization in terminal varicosities. J. comp. Neurol. 266, 1–15. KRAJNIAK, K. G. (1991). The identification and structure–activity relations of a cardioactive FMRFamide-related peptide from the blue crab Callinectes sapidus. Peptides 12, 1295–1302. KRAVITZ, E. A., GLUSMAN, S., HARRIS-WARRICK, R. M., LIVINGSTONE,
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